THE 17TH BIENNIAL CONGRESS. Metastasis Research Society. Conference Program Book. August 1st - 5th, 2018 Princeton, USA

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1 THE 17TH BIENNIAL CONGRESS Metastasis Research Society Conference Program Book August 1st - 5th, 2018 Princeton, USA

2 Table of Contents Sponsors Welcome About MRS Committees Directions Venue map, jogging map & housing info. Program at a Glance Agenda Keynote and Award Speakers Abstracts Poster Session I: Abstracts #1 ~ # Poster Session II: Abstracts #Y1 ~ #Y Acknowledgment

3 The MRS gratefully acknowledges the sponsorship of the following; and the academic support of; 2

4 Welcome Message Dear Colleagues and Friends: I am delighted to welcome you to the 17th Biennial Congress of the Metastasis Research Society (MRS) to be held in Princeton, New Jersey from August 1st to 5th, The mission of the MRS is to support progressive research on processes fundamental to metastasis. This includes supporting the exchange of information between researchers, clinicians, industry, and patients globally. We also strive to educate the public about metastatic cancer and raise awareness about insufficient funding for metastatic research worldwide. The 17th MRS conference reflects the commitment of the MRS to these stated goals. In addition to outstanding invited lectures from worldleading experts in different areas of metastasis research and clinical development, more than 130 oral and poster presentations selected from submitted abstract will showcase the latest advance in the field. The conference also offers exciting programs in the Young Investigator Satellite Meeting and the Patient Advocacy Session. In addition, conference attendees will have ample opportunities to interact with editors from leading scientific journals, as well as administrators and scientists from the NIH and the industry. We believe that such interactions will create great synergy to advance metastasis research and accelerate the translation of research findings to better medicine for metastatic cancer patients. We are grateful for Princeton University to serve as the host institution for the conference. Situated in one of the beautiful and idyllic part of the Garden State, Princeton is one of the oldest and most prominent universities in the United States. During your visit to Princeton to attend the MRS conference, I hope you take the opportunity to visit some of the historical landmarks at Princeton and enjoy the networking social events to strengthen the friendship among colleagues in the field. Sincerely, Yibin Kang, Ph.D. President, Metastasis Research Society Co-Chair, The 17th Biennial Congress of the Metastasis Research Society Warner-Lambert/Parke-Davis Professor of Molecular Biology, Princeton University 3

5 Welcome Message Dear All: Welcome to the 17th Biannual Congress of the Metastasis Research Society (MRS)! Thanks to the dedication of the Scientific Program Committee, and together with many outstanding scientists across the globe, we have a superb scientific program (please see the program book and visit bc-introduction). Undoubtedly, the 2018 Congress will highlight exciting progress and creative research in the metastasis research field, while also providing abundant networking opportunities. Cancer metastasis remains to be the number one cause of cancer-related deaths! It challenges us to pursue fundamental basic and translational research and apply the knowledge to develop effective therapies that can be rapidly translated into the clinic to benefit metastatic cancer patients. We are fortunate to be holding the 2018 MRS Congress at a unique time of groundbreaking biomedical research. Our scientific breakthroughs and collaborative efforts will bring the next wave of metastasis research. This is the right time to shape a clear vision and make a huge impact. We will share our discoveries, experiences, ideas, and opportunities, while fostering a network to support our scientific and personal goals. This MRS Biannual Congress will be a scientific feast! Respectfully, Dihua Yu, M.D., Ph.D. President-Elect, Metastasis Research Society Co-Chair, 17th Biannual Congress of the Metastasis Research Society Hubert L. & Olive Stringer Distinguished Chair in Basic Science University of Texas MD Anderson Cancer Center, USA 4

6 About MRS The Metastasis Research Society is an international non-profit organization dedicated to promote the exchange of information and furtherance of research into all aspects of Metastasis, from the molecular bases of tumor cell invasion to clinical management of disseminated cancer. This unique Society commits itself to providing a forum for scientists and clinicians from all over the world with interests in any aspect of the metastatic process. These aims are primarily realized by the organization of major Metastasis Research Society Congresses held every two years, and by publication of high quality, peerreviewed research papers and reviews in the official journals of the Society, Clinical and Experimental Metastasis and Cancer and Metastasis Reviews. Since the first inaugural meeting organized in 1984 by the society's founder, Dr. Hellman, the society has brought together international experts in the field every two years. 15 international conferences have been organized since that inaugural conference. For information regarding the previous meeting please see as follows. Year Location President 1984 London, UK K. Hellmann 1986 Trieste, Italy T. Giraldi 1988 Heidelberg, Germany V. Schirrmacher 1990 Bethesda, US G. Nicolson 1992 Paris, France M. France-Poupon 1994 Washington, DC, US L. Liotta 1996 Ghent, Belgium M. Mareel 1998 San Diego, US W. Stetler-Stevenson 2000 London, UK S. Eccles 2002 Chicago, US A. Raz 2004 Genoa, Italy A. Albini 2006 Tokushima, Japan S. Sone 2008 Vancouver, British Columbia, Canada D. Welch 2010 Philadelphia, PA US P. Steeg 2012 Brisbane, Australia E. Thompson 2014 Heidelberg, Germany J. Sleeman 2016 Chengdu, China Q.H. Zhou, Y.B.Kang 2018 Princeton, USA Y.B. Kang, D.Yu THE 17th INTERNATIONAL BIENNIAL CONGRESS OF THE MRS 5

7 COMMITTEE SCIENTIFIC COMMITTEE Julio Aguirre-Ghiso Salvador Aznar Benitah Thomas Brabletz Mikala Egeblad Jacky Goetz Yibin Kang Dan Landau David Lyden Pere Puigserver Ashani Weeraratna Jedd D. Wolchok Dihua Yu Icahn School of Medicine at Mount Sinai, USA Institute for Research in Biomedicine, Spain University Erlangen, Germany Cold Spring Harbor Laboratory, USA Université de Strasbourg, Faculté de Médecine, France Princeton University, USA Weill Cornell Medicine, USA Weill Cornell Medicine, USA Harvard University, USA The Wistar Institute Melanoma Research Center, USA Memorial Sloan Kettering Cancer Center, USA University of Texas MD Anderson Cancer Center, USA (Listed by alphabetical order of last name) 6

8 COMMITTEE MRS AWARDS SELECTION COMMITTEE Julio Aguirre-Ghiso Salva Aznar Benitah David Cheresh Yibin Kang Joan Massagué Erik Sahai Alana Welm Jing Yang Dihua Yu Icahn School of Medicine at Mount Sinai, USA Institute for Research in Biomedicine, Spain University of California, San Diego, USA Princeton University, USA Memorial Sloan-Kettering Cancer Center, USA The Francis Crick Institute, UK Huntsman Cancer Institute, University of Utah, USA University of California, San Diego, USA University of Texas MD Anderson Cancer Center, USA (listed by alphabetical order of last name) 7

9 COMMITTEE ABSTRACT REVIEW COMMITTEE I (NON-TRAINEES ) Julio Aguirre-ghiso Barbara Fingleton Irwin Gelman Douglas Hurst Yasumasa Kato Conor Lynch John Price Rajeev Samant Jonathan Sleeman Ulrike Stein Dihua Yu Andries Zijlstra Icahn School of Medicine at Mount Sinai, USA Vanderbilt University, USA Roswell Park Comprehensive Cancer Center, USA University of Alabama at Birmingham, USA Ohu University, Japan Moffitt Cancer Center, USA Monash University, Australia University of Alabama at Birmingham, USA University of Heidelberg, Germany Max Delbrück Center for Molecular Medicine, Germany University of Texas MD Anderson Cancer Center, USA Vanderbilt University Medical Center, USA (Listed by alphabetical order of last name) 8

10 COMMITTEE ABSTRACT REVIEW COMMITTEE II (TRAINEES ) Lucia Borriello Leah Cook Thomas Cox Lenka Kyjacova Kristina Marinak Sandra Scherer Adrian Wiegmans Hanqiu Zheng Albert Einstein College of Medicine, USA University of Nebraska Medical Center, USA The Garvan Institute of Medical Research, Australia University of Heidelberg, Germany West Virginia University Cancer Institute, USA University of Heidelberg, Germany QIMR Berghofer Medical Research Institute, Australia Tsinghua University, China (Listed by alphabetical order of last name) 9

11 VANDEV ENT ER AV E. ST. DR. EA ST Lake Carnegie Lowrie House W est Lodge W yman Cottage LOT 19 W yman House 63, 65 & 67 GRA DUAT E COLLEGE Procter Cleveland Tower STOCKTON ST. Princeton Theological Seminary 26 LOT 11 ROUTE 20 6 M ERC ER ST. FOR BES COLLEGE Palmer House LOT LOT College Road Apartments Entrepreneurial H ub LOT 8 11 UNIVERSITY PL. 22 ROCK EFELLER COLLEGE M adison Hamilton Joline 2 71 M ccarter Theatre Berlind Theatre Lewis Arts complex Arts Tower W allace Dance Building & Theater CHA M BERS ST. 48 W awa Foulke Henry Campbell Holder Laughlin Little Palmer Square North Guard Booth Alexander Dillon Gym Garden Theatre Labyrinth Books Scheide M aclean Henry House Caldwell House House Andlinger Center for the Humanities Stanhope Chancellor Nassau Hall Green Brown Bloomberg Prospect House Prospect Gardens Firestone Library MAT HEY Eas t COLLEGE Pyne Cannon M orris on Chapel Green Blair Dickins on CHAPEL DR. 36 Buyers Prospect Clio W hig Dodge House Lockhart W ithers poon M cc osh Speaker Dinner M urray Edwards Theater Cuyler Jones Pyne Stephens Fitness Center Feinberg W right W alker M cc os h Dillon Dillon Health West East Patton W ILS ON COLLEGE LOT Dodge-Os born Fisher Gaus s Spelman Eno Clapp Dinky Bar W HIT M AN Dorms & Kitchen W endell COLLEGE Bloomberg W ilcox and Rock M urley- W u Mag Lab Scully Halls Cargot Pivirotto Hargadon Community Hall Yoselo 1976 Brasserie New South Lauritzen BUT LER Bogle W ilf Baker COLLEGE Scully New Music Princeton Station W est Garage M acm illan Dod 20 0 Elm M cc ormick Art Museum Field Chilled Cogen Water Plant Plant South Guard Cooling Booth Towers LOT 28 Cordis h Lenz Thermal Family Tennis Center Energy Pavilion LOT 23 NASSAU ST. W IT HERSPOON ST. Architecture W oolworth Music Lourie-Love 1912 Pavilion Pavilion Frist Campus Center & Burr M arx Guyot Green Schultz Julis Romo Rabinowitz Thomas Icahn Robertson Campus M o ett Princeton Neuroscience Institute LOT 15 Hoyt Simpson Terrace Center for Jewish Life Peretsman Scully Corwin W allace 20 1 P.U. Press Bendheim ( Former) Fisher Bendheim Finance Tower LOT 10 LOT 26 5 Lewis Library Fine M cdonnell Streicker Bridge LOT 13 Jadwin / 2 Colonial Peyton Friend Center Computer Science Sherrerd M udd Library Tiger CDE Quad Ivy Cottage Frick Architecture Lab LOT 1 Olden House Fields Center 58 Cap & Gown Princeton Stadium Powers Field OLDEN ST. CHANCELLOR WAY A B C D E F G H I J K L M COLLEGE RD. WEST EDWARDS PL. DICKINSON ST. COLLEGE RD. Train Station PYNE DR. Whitman Dining Hall Breakfast and Dinner for Meal Plan Holders ELM DR. FRIST LN. WILLIAM ST. IVY LN. WASHINGTON RD. CHARLT ON LOT 4 Poster LOT 14 LOT Sessions 25 LOT 5 F Engineering Quadrangle C A E M aeder Hall Clois ter Bowen North Garage Bobs t Ferris Thompson Apartments Clarke Field Press Box Clarke Field Campbell Field Finney Frelinghuysen Field Frick Field W eaver Track Chemistry Lab and Field Stadium Jadwin Gym NASSAU ST. B Charter D Andlinger Caldwell and Field Banquet House DeNunzio Pool J PROSPECT AVE. G 87 H ( V on Neumann) 91 LOT 35 LOT MURRAY PL Pros pect Apartments Upper Strubing Field Elementary Particles Lab West Strubing Field LOT 21 Sexton Field FitzRandolph Observatory Elementary Particles Lab East PRINCET ON AV E. ALEXANDER ST. 200 Elm Public Safety Baker Rink Nassau Inn Poe Field Pardee Field Roberts Stadium M ys lik Plummer Poe Field Field Gulick Pavilion Outdoor Activities Lewis Library STADIUM DR. WEST McDonnell Hall Main Conference Facility STADIUM WESTERN WAY Lunch Sessions, Group Photo FACULTY RD. Lot 21 Day Parking 171 LOT BROADM EAD FITZRANDOLPH RD. SPRIN GDALE RD. Springdale Golf Course LOT 16 LOT 17 SOUTH DR. Bedford Field Sherrerd Field at 1952 Stadium ELM LOT 27 LOT 20 DR. Grounds Storage Buildings 26 2 LOT A FACULTY RD University Buildings Non-University Buildings Under Construction N TO 701 CARNEGIE CENTER LOT LOT 31 Helm Lakes ide Garage 20 2 LAKESIDE RD HIBBEN MAGIE RD. Lakeside Garage Overnight Parking LEMONICK CT TAYLOR CT. Lakeside Graduate Housing Shea Rowing Center Class of 1887 Boathouse TO U.S. ROUTE 1

12 207 ft 180 ft via Streicker Bridge shortcut Washington Monument Map data 2018 Google The map shows a jogging route of about 6 miles long that pass through several landmarks at Princeton. Two shortcuts will reduce the length of the route to roughly 4 miles and 2 miles. If weather permits, we will arrange group jogging along this route early in the morning Shortcuts: Olden Ln & University PI Landmarks 5.9 miles 1 h 57 min 500 ft Walk 5.9 miles, 1 h 57 min Jogging Map@ Princeton shortcut Nassau Hall Lourie-Love Walk, Princeton, NJ to Guyot Ln, Princeton, NJ

13 The 17 th Biannual Congress of Metastasis Research Society July 31 August 5, 2018 CONFERENCE HOUSING INFORMATION SHEET CHECK-IN CHECK-OUT Tuesday, July 31, 2018 Any Day 12:00 PM 5:00 PM Public Safety Drop Box Bloomberg Hall 201 Elm Drive 200 Elm Drive Princeton, NJ Princeton, NJ NOTE: We will not be able to accommodate any early arrivals before 12:00 PM on Tuesday, July 31st. If you are arriving to Princeton after 5pm on July 31 st you may pick up your key at Public Safety located at 200 Elm Drive. When departing campus please deposit your key in the drop box located in the lobby of 200 Elm Drive. IMPORTANT PHONE NUMBERS Emergencies - Dial 911 (Call from your cell phone for on-campus emergencies) Princeton University Public Safety - (609) (non-emergencies) Princeton University Conference & Event Services (609) (8:30am-4:30pm Monday-Friday) ACCOMMODATIONS Participants will live in Bloomberg and Scully Halls at Princeton University. Both dorms are located on Elm Drive, a five minute walk from the NJ Transit Train - Princeton (aka Dinky ) Station. All accommodations will be in airconditioned single bedrooms with either shared or hall bathrooms. Linens are provided. Toiletries and hangers are not provided. SMOKING POLICY Princeton University is committed to providing a healthy, smoke-free living environment for all its students. Further, New Jersey law prohibits smoking in all dormitories/annexes, including private student rooms and common areas. Accordingly, smoking is not permitted anywhere in Princeton University dormitories/annexes. Failure to follow the policy will result in disciplinary action. ALCOHOL POLICY Participants are expected to be acquainted with and abide by University regulations regarding the use of alcoholic beverages ( These regulations are designed to be consistent with the laws of the State of New Jersey, and, in general, prohibit the consumption and serving of alcohol beverages by and to persons under 21 years of age. Students are in violation of the alcohol policy under any of the following circumstances: Possession of an open container of alcohol, by any student, in common spaces (lounges, living rooms, game rooms, hallways, entryways, dining areas, courtyards, etc.); 12

14 Possession of any container of alcohol (open or sealed) in common spaces by students under the age of 21; Possession of a keg or a keg tap; Serving, providing, or making available alcohol to persons under 21. The aforementioned violations are regarded more seriously if property is damaged or the privacy of others is infringed upon. The University respects the right to privacy, and its representatives do not enter dormitory rooms in order to enforce this policy without reasonable suspicion that University policies or regulations have been violated. If a violation is determined, all alcohol and equipment used to dispense it are confiscated and not returned. GUEST POLICY All residents must be participants in Princeton University approved programs. Unaffiliated overnight guests are not permitted in the dorms. PARKING Parking is free and permits will be available at registration. If you commit a parking violation you will be subject to, and responsible for, a fine and/or tow. Note: Overnight parking is prohibited on all Princeton Streets. LAUNDRY Free washers and dryers are located in both buildings ON-CAMPUS FOOD A meal plan will be provided for any participant living in the dorm during the conference. Your meal plan includes five breakfasts (starting August 1 st ) and three dinners (no dinner August 4 th or 5 th ). You will be eating in Whitman College Dining Hall. Breakfast is served starting at 7am and dinner is served from 5pm-7:30pm. Lunch is provided each day by the conference at the conference facility. ATHLETIC FACILITIES During the summer, participants will have access to the University s tennis courts, health/fitness room, swimming pool, basketball courts, etc. In order to use these facilities, participants must purchase athletic chits at the Conference Office. Athletic chits are good for one visit to Dillon Gym you may stay as long as you like but once you leave, another chit is required to re-enter. There is a cost of $5.00/chit. PRINCETON UNIVERSITY STORE The U-Store sells a wide range of items, including stationary, sportswear, small appliances, cameras, books and magazines. It also has a pharmacy and a limited grocery section. INTERNET Wireless is available throughout campus via the PUVisitor network. There is not password required to connect. 13

15 August 1 st (Wed) August 2 nd (Thu) August 3 rd (Fri) August 4 th (Sat) August 5 th (Sun) Session 10 Genetic & epigenetic heterogeneity & landscape of metastatic evolution Coffee Hellmann Award Lecture Paget-Ewing Award Lecture Young Investigator Travel Awards Closing Remarks Lunch/Social Hours 2018 MRS Congress Conference at a glance 8-9am 9-10am 10-11am 11-12pm 12-1pm Fidler Award Lecture Session 1 Dormancy & Early dissemination Photo & Coffee Session 2 Susan Komen Clinical Session 4 Metabolic dysregulation & Metastasis Coffee Session 5 TME in metastasis & resistance Lunch Session 7 Advanced imaging & New technologies Coffee Session 8 Metastatic organotropism Lunch pm 2-3pm 3-4pm 4-5pm 5-6pm 6-7pm Registration MRS Board Meeting Susan Komen Patient Advocacy with METAvivor Session 3 Susan Komen Pre-clinical NCI s funding talk Poster Session I (Abstracts 1-65) MRS General Member Meeting Session 6 EMT, Cancer stem cells & Plasticity Poster Session II (Abstracts Y1-Y78) Meet the editors Session 9 Stress, inflammation & immune regulation of metastasis Coffee Young Investigator Award Lecture Concurrent session I & II 7-8pm Opening ceremony & Keynote lecture Social Hours MRS Banquet 8-9pm Networking

16 The 17th Biennial Congress of the Metastasis Research Society YOUNG INVESTIGATOR SATELLITE MEETING (Room: McDonnell Hall A01) Wednesday, August 1, :30-08:30 am Arrival and YISM Registration Room: Brush Gallery 08:30-09:40 am Young Investigator Session 1: Room: McDonnell Hall A01 Chairpersons: Thomas Cox, PhD, Garvan Institute, Sydney, Australia Adrian Wiegmans, PhD, QIMR Berghofer, Brisbane, Australia 08:30-08:40 am Opening and welcome Thomas Cox, PhD, Chair of the Early Career Leadership Committee (ECLC) 08:40-08:55 am An activated, pro-tumor lung microenvironment promotes the outgrowth of breast cancer lung metastases through lung-specific expression of the androgen receptor (Abs #Y8) Jessica Christenson, PhD, University of Colorado-Denver, USA (*) 08:55-09:10 am Inhibition of Hedgehog signaling reprograms the dysfunctional immune microenvironment in breast cancer (Abs #Y21) Ann Hanna, PhD, University of Alabama at Birmingham, USA 09:10-09:25 am Mitochondrial genetics appear to alter immune cell development/trafficking (Abs #Y3) Thomas Beadnell, PhD, University of Kansas Medical Center, USA 09:25-09:40 am Microglia subset revealed by single cell analyses promotes brain metastasis outgrowth through fractalkine receptor-dependent interferon response (Abs #Y20) Ian H. Guldner, University of Notre Dame, USA (*) 09:40-10:05 am Coffee Break and Networking Room: Brush Gallery 10:05-11:50 am Young Investigator Session 2: Room: McDonnell Hall A01 Chairpersons: Lucia Borriello, PhD, Albert Einstein College of Medicine, USA Hanqiu Zheng, PhD, Tsinghua University, China 15

17 10:05-11:05 am Digital pathology - Leveraging image analysis to predict clinical outcome in cancer Andries Zijlstra, PhD, Vanderbilt University Medical Center, USA 11:05-11:20 am DNA Damage response drives chemo-resistance and supports metastasis in triple negative breast cancer (Abs #Y57) Ambber Ward, QIMR Berghofer, Australia (*) 11:20-11:35 am The role of AIB1-delta 4 as a driver of breast cancer metastasis (Abs #Y49) Ghada Sharif, PhD, Georgetown University, USA (*) 11:35-11:50 am Nuclear Aurora-A Kinase: a novel driver of metastasis (Abs #Y59) Kristina Whately, West Virginia University, USA (*) 12:00-01:00 pm Lunch and Meet the Professors Round Table Session Room: Frick Atrium Coordinators: Thomas Cox, PhD, Garvan Institute, Sydney, Australia Adrian Wiegmans, PhD, QIMR Berghofer, Brisbane, Australia Leah Cook, PhD, University of Nebraska Medical Center, USA 01:10-02:55 pm Young Investigator Session 3: Room: McDonnell Hall A01 Chairpersons: Lenka Kyjacova, PhD, University of Heidelberg, Germany Leah Cook, PhD, University of Nebraska Medical Center, USA 01:10-02:10 pm Young Investigator Keynote: From South to North: A journey of self-discovery Soledad Sosa, PhD, Mount Sinai School of Medicine, USA 02:10-02:25 pm Immediate early gene Ier2 stimulates migration and invasion of metastatic melanoma through inducing a senescence-associated secretory phenotype (SASP) (Abs #Y16) Amelia Foss, University of Heidelberg, Germany 02:25-02:40 pm Melanoma phenotype switching and metastatic growth after dissemination (Abs #Y52) Jacinta Simmons, PhD, QIMR Berghofer, Australia 02:40-02:55 pm Deficiency of tumor suppressor Merlin facilitates metabolic adaptation by cooperative engagement of SMAD-Hippo signaling in breast cancer (Abs #Y36) Mateus Mota, University of Alabama, USA (*) 02:55-03:25 pm Coffee Break Room: Brush Gallery 16

18 03:25-04:25 pm Young Investigator Session 4: Room: McDonnell Hall A01 Chairpersons: Sandra Scherer, PhD, Huntsman Cancer Institute, USA Kristina Whately, West Virginia University Cancer Institute, USA 03:25-03:40 pm Elucidating skeletal muscle-derived factors that suppress breast cancer metastasis (Abs #Y9) Sarah Crist, Fred Hutchinson Cancer Research Center, USA (*) 03:40-03:55 pm Extracellular matrix stiffness activates a novel mechanotransduction kinase cascade to promote EMT and tumor metastasis (Abs #Y14) Laurent Fattet, PhD, UCSD - Moores Cancer Center, USA (*) 03:55-04:10 pm Calpain-2 mediated amoeboid reprogramming and dissemination triggered by hypoxia (Abs #Y55) Veronika te Boekhorst, PhD, MD Anderson Cancer Center, USA 04:10-04:25 pm Crosstalk between osteoblasts and breast cancer cells alters breast cancer proliferation through multiple mechanisms (Abs #Y51) Alison Shupp, Thomas Jefferson University, USA (*) 04:25-05:25 pm Young Investigator Session 5: Pre-Clinical Models of Cancer Metastasis -- With an Emphasis on Metastatic Breast Cancer Room: McDonnell Hall A01 Supported by: Theresa s Research Foundation (Sponsor) Chairperson: Speakers: Panel Member: Alana Welm, PhD, Huntsman Cancer Institute, USA Andrew Ewald, PhD, Johns Hopkins University School of Medicine, USA Jeff Rosen, PhD, Baylor College of Medicine, USA Somdutta Roy, PhD, Abbvie Stemcentrx, USA 05:25-05:35 pm YISM Concluding remarks Room: McDonnell Hall A01 Thomas R. Cox, PhD Chair of the Early Career Leadership Council 05:35-06:30 pm Free time Note: (*) Indicates Young Investigators conducting research on breast cancer metastasis. All Young Investigators in this session are partially supported by Travel Awards from Susan G. Komen and Theresa s Research Foundation. 17

19 The 17th Biennial Congress of the Metastasis Research Society Wednesday, August 1, 2018 MAIN SESSIONS 8:30 am-8:30 pm MRS Congress Registration Room: Brush Gallery 01:00-03:00 pm MRS Board Meeting Room: Frick A81 06:45-07:00 pm 17th Biennial Congress Opening Ceremony Room: McDonnell Hall A01 Chairperson: Yibin Kang, PhD, Princeton University, USA Dihua Yu, MD, PhD, University of Texas MD Anderson Cancer Center, USA 07:00-08:00 pm 17th Biennial Congress Keynote Lecture Room: McDonnell Hall A01 Chairperson: Dihua Yu, MD, PhD, UT MD Anderson Cancer Center, USA The Complex Challenge of Treating Metastasis Isaiah J. Fidler, DVM, PhD Professor, Department of Cancer Biology Professor, Department of Neurosurgery UT MD Anderson Cancer Center, USA 08:00-09:15 pm Networking Reception Room: Lewis Library 18

20 Thursday, August 2, :30-09:15 am Fidler Innovation Award Lecture Room: McDonnell Hall A01 Chairperson: Isaiah J. Fidler, DVM, PhD, UT MD Anderson Cancer Center, USA Co-Chair: Dihua Yu, MD, PhD, UT MD Anderson Cancer Center, USA Studying the nature of the seed: unexpected findings and future challenges Christoph Klein, MD, PhD, University of Regensburg 09:15-10:30 am Session 1: Dormancy and early dissemination Room: McDonnell Hall A01 Chairperson: Julio Aguirre-Ghiso, PhD, Icahn School of Medicine at Mount Sinai, USA Co-Chair: Irwin Gelman, PhD, Roswell Park Comprehensive Cancer Center, USA 09:15-09:40 am Analysis of early dissemination reveals a new EMT-linked mechanism of DTC dormancy Julio Aguirre-Ghiso, PhD, Icahn School of Medicine at Mount Sinai, USA 09:40-10:05 am Defining and targeting the mechanisms of cancer cell transit in bone Dorothy Sipkins, MD, PhD, Duke University, USA 10:05-10:17 am In vivo functional screening identifies novel drivers of metastasis in lung cancer (Abs #17) Samrat Kundu, PhD, UT MD Anderson Cancer Center, USA 10:17-10:30 am Impact of intratumoral clonal heterogeneity on immune checkpoint inhibitor response (Abs #38) Anton Wellstein, PhD, Georgetown University, USA 10:30-10:45 am Group Photo Room: Frick Atrium 10:30-11:00 am Coffee Break Room: Frick Atrium 11:00 am-12:40 pm Susan G. Komen Clinical Session 2: Clinical trials advancing metastatic cancer patient care Room: McDonnell Hal A01 Chairperson: Jedd D. Wolchok, MD, PhD, Memorial Sloan Kettering Cancer Center, USA Co-Chair: Barbara Fingleton, PhD, Vanderbilt University, USA 11:00-11:25 am Understanding mechanisms for combination immunotherapy 19

21 Jedd D. Wolchok, MD, PhD, Memorial Sloan Kettering Cancer Center, USA 11:25-11:50 am From the clinic to the lab: Investigating response and resistance mechanisms to immune checkpoint therapy Padmanee Sharma, MD, PhD, UT MD Anderson Cancer Center, USA 11:50-12:15 pm The genomic landscape of ER + metastatic breast cancer Jose Baselga, MD, PhD, Memorial Sloan Kettering Cancer Center, USA 12:15-12:27 pm ONECUT2 is a targetable master regulator of lethal variants of prostate cancer that suppresses the androgen axis (Abs #Y48) Mirja Rotinen, PhD, Cedars-Sinai Medical Center, USA 12:27-12:40 pm Reactive Astrocytic S1P3 signaling modulates blood-tumor barrier permeability in brain metastases (Abs #13) Brunilde Gril, PhD, National Cancer Institute, USA 12:40-02:40 pm Susan G. Komen Patient Advocacy Session for Metastatic Cancers, in collaboration with METAvivor Room: Frick Atrium B02 Chairperson: Irwin Gelman, PhD, Roswell Park Comprehensive Cancer Center, USA 12:40 01:00 pm Lunch (pick-up) & Networking Room: Frick Atrium 01:00 01:05 pm Welcome remarks 01:05 01:20 pm Metastatic Cancer Topic Highlight #1 01:20 01:35 pm Metastatic Cancer Topic Highlight #2 01:35 01:50 pm Metastatic Cancer Topic Highlight #3 01:50 02:05 pm Metastatic Cancer Advocates Highlight 02:05 02:40 pm Panel Discussion with Q&A 02:50-04:30 pm Susan G. Komen Translational Session 3: Targeting metastasis and drug development Room: McDonnell Hall A01 Chairperson: Salvador Aznar Benitah, PhD, Institute for Research in Biomedicine (IRB), Spain 20

22 Co-Chair: Yasumasa Kato, MedScD, PhD, Ohu University, Japan 02:50-03:15 pm Epigenetic memory of dietary fatty acids in metastasis: why what we eat matters Salvador Aznar Benitah, PhD, Institute for Research in Biomedicine (IRB), Spain 03:15-03:40 pm Activation of specific EZH2 drives basal-like breast cancer and lung metastasis Mien-Chie Hung, PhD, UT MD Anderson Cancer Center, USA 03:40-04:05 pm Immune evasion and metastasis in colorectal cancer Eduard Batlle, PhD, IRB, Spain 04:05-04:17 pm Adipocytes drive melanoma progression through FATP proteins (Abs #51) Richard White, MD, PhD, Memorial Sloan Kettering Cancer Center, USA 04:17-04:30 pm Metastatic cancer induces muscle wasting through aberrant zinc transport (Abs #1) Swarnali Acharyya, PhD, Columbia University, USA 04:30-05:00 pm NCI s vision/initiatives funding opportunities in promoting metastasis research Nancy Boudreau, PhD, Tumor Metastasis Branch, National Cancer Institute, USA 05:00-06:50 pm Poster Session I (Abs #1 #70) / Coffee Break Room: Lewis Library 06:00-07:00 pm CEM Editorial Board Meeting Room: Lewis Library 122 Dinner on your own 21

23 Friday, August 3, :30-10:10 am Session 4: Metabolic dysregulation and metastasis Room: McDonnell Hall A01 Chairperson: Pere Puigserver, PhD, Harvard University, USA Co-Chair: Rajeev Samant, PhD, University of Alabama at Birmingham, USA 08:30-08:55 am Mitochondrial Heterogeneity in melanoma metastasis Pere Puigserver, PhD, Harvard University, USA 08:55-09:20 am Molecular & cellular mechanisms underlying metastasis formation Sohail Tavazoie, MD, PhD, Rockefeller University, USA 09:20-09:45 am Hypoxia, metabolism, and metastasis Celeste Simon, PhD, University of Pennsylvania Perelman School of Medicine, USA 09:45-09:57 am Ultrasensitive detection of disseminated tumor cells in intraoperative washings of Robot-Assisted Radical Cystectomy for bladder cancer as a potential prognosis biomarker (Abs #37) Lei Wei, PhD, Roswell Park Comprehensive Cancer Center, USA 09:57-10:10 am BMP4 is a bona-fide breast cancer metastasis suppressor (Abs #3) Robin Anderson, PhD, Olivia Newton-John Cancer Research Institute, Australia 10:10-10:30 am Coffee Break Room: Brush Gallery 10:30 am-12:10 pm Session 5: Tumor and tumor microenvironment (TME) interaction in metastasis and therapeutic resistance Room: McDonnell Hall A01 Chairperson: Ashani Weeraratna, PhD, The Wistar Institute Melanoma Research Center, USA Co-Chair: Andries Zijlstra, PhD, Vanderbilt University, USA 10:30-10:55 am A Wrinkle in Time- How the aging ECM drives metastatic progression Ashani Weeraratna, PhD, The Wistar Institute Melanoma Research Center, USA 10:55-11:20 am Desmoplastic roles in supporting and immunosuppressing pancreatic cancer Edna Cukierman, PhD, Fox Chase Cancer Center, USA 11:20-11:45 am Mesenchymal stromal cells and metastasis Paula Hurley, PhD, Johns Hopkins University School of Medicine, USA 22

24 11:45-11:57 am Transcriptional control of metabolic adaptations in breast cancer metastasis (Abs #52) Subhamoy Dasgupta, PhD, Roswell Park Comprehensive Cancer Center, USA 11:57-12:10 pm Myeloid-derived suppressor cells inhibit T Cell activation through nitrating LCK in mouse cancers (Abs #20) Xin Lu, PhD, University of Notre Dame, USA 12:10 12:40 pm Lunch Pickup Room: Frick Atrium 12:40-02:20 pm MRS General Member Meeting Room: Frick Atrium B02 Chairperson: Yibin Kang, PhD, Princeton University, USA 02:30-04:10 pm Session 6: Epithelial-mesenchymal Transition (EMT), cancer stem cells (CSCs) and plasticity Room: McDonnell Hall A01 Chairperson: Thomas Brabletz, PhD, University Erlangen, Germany Co-Chair: Ulrike Stein, PhD, Max Delbrück Center for Molecular Medicine, Germany 02:30-02:55 pm Cellular plasticity in cancer: Driving force and therapeutic target Thomas Brabletz, PhD, University Erlangen, Germany (EACR designated speaker) 02:55-03:20 pm Cellular senescence and cancer Xiao-Fan Wang, PhD, Duke University, USA 03:20-03:45 pm EMT and cellular pliancy Alain Puisieux, PhD, Centre de Recherche en Cancérologie de Lyon, France 03:45-03:57 pm Immediate early gene Ier2 induces a senescence-associated secretome that includes osteopontin, and which is associated with progression and metastasis in melanoma (Abs #18) Lenka Kyjacova, PhD, University of Heidelberg, Germany 03:57-04:10 pm Genomic analysis of breast cancer mouse models reveals new insights into metastatic biology (Abs #28) Christina Ross, PhD, National Cancer Institute, USA 23

25 04:10-06:10 pm Poster Session II (Abs #Y1 #Y82) / Coffee Break Room: Lewis Library 06:10-08:30 pm Social Hour / Free Time / Dinner on your own Location to be announced at the conference 06:20-08:20 pm MRS Board & Speakers Dinner (by invitation) Room: Garden Room, Prospect House 24

26 Saturday, August 4, :30-09:45 am Session 7: Advanced imaging and new technologies in metastasis research and treatment Room: McDonnell Hall A01 Chairperson: Jacky G. Goetz, PhD, Université de Strasbourg, Faculté de Médecine, France Co-Chair: Erik Thompson, PhD, Queensland University of Technology, Australia 08:30-08:55 am Tracking tumor metastasis at high spatio-temporal resolution Jacky G. Goetz, PhD, Université de Strasbourg, Faculté de Médecine, France 08:55-09:20 am Fine-tuning the stroma in pancreatic cancer to improve response to therapy and target metastasis: insights from intravital imaging Paul Timpson, PhD, The Kinghorn Cancer Centre, Garvan Institute of Medical Research, Australia 09:20-09:32 am Intravital microscopy at single cell resolution reveals the mechanism of cancer cell dissemination and metastasis (Abs #53) Lucia Borriello, PhD, Albert Einstein College of Medicine, USA 09:32-09:45 am Colorectal cancer associated Natural Killer cells are endowed by pro-metastatic and pro-angiogenic phenotype/functions by upregulating the MMP9-TIMP2 and Angiogenin axis (Abs #2) Adriana Albini, PhD, IRCCS Multimedica, Italian Ministry of Health, Italy 09:45-10:05 am Coffee Break Room: Brush Gallery 10:05-11:45 am Session 8: Systemic and local regulation of metastasis organotropism Room: McDonnell Hall A01 Chairperson: David Lyden, PhD, Weill Cornell Medicine, USA Co-Chair: Jonathan Sleeman, PhD, University of Heidelberg, Germany 10:05-10:30 am Nanosized particles promote the systemic effects of cancer metastasis David Lyden, PhD, Weill Cornell Medicine, USA 10:30-10:55 am Pattern recognition receptor and anti-viral signaling in cancer Andy Minn, MD, PhD, University of Pennsylvania, USA 10:55-11:20 am Regulation of breast cancer metastasis organotropism Guohong Hu, PhD, Shanghai Institute of Health Sciences, China 25

27 11:20-11:32 am Understanding and visualizing the role of extracellular matrix remodeling in cancer progression and metastasis (Abs #45) Thomas Cox, PhD, Garvan Institute of Medial Research, Australia 11:32-11:45 am Targeting invadopodia-mediated breast cancer metastasis by using ABL kinase inhibitors (Abs #49) Hava Gil-Henn, PhD, Bar-Ilan University, Israel 11:45 am 12:15 pm Lunch Room: Frick Atrium 12:15-01:45 pm Meet the Editors Session Room: Frick Atrium B02 Chairperson: Jonathan Sleeman, PhD, University of Heidelberg, Germany Editor-in-Chief, Clinical and Experimental Metastasis Aishwarya Sundaram, PhD Nature Communications Alexia-Ileana Zaromytidou, PhD Nature Cell Biology Anna Dart, PhD Nature Reviews Cancer Harmony Turk, PhD Cancer Cell I-Mei Siu, PhD Cancer Discovery Javier Carmona, PhD Nature Medicine Melina Casadio, PhD, Journal of Cell Biology (listed by alphabetical order of first name) 02:00-03:40 pm Session 9: Stress, inflammation, and immune regulation of metastasis Room: McDonnell Hall A01 Chairperson: Mikala Egeblad, PhD, Cold Spring Harbor Laboratory, USA Co-Chair: Conor Lynch, PhD, Moffitt Cancer Center, USA 02:00-02:25 pm Neutrophil extracellular traps formed during inflammation promote metastasis by extracellular matrix remodeling Mikala Egeblad, PhD, Cold Spring Harbor Laboratory, USA 02:25-02:50 pm Metabolic dependencies of tumor-host interaction Eileen White, PhD, Cancer Institute of New Jersey, USA 02:50-03:15 pm Cancer and its host, a story of corruption Ilaria Malanchi, PhD, The Francis Crick Institute, UK 26

28 03:15-03:27 pm RON kinase: a therapeutic target for cancer-induced bone destruction (Abs #Y15) Jaime Fornetti, PhD, University of Utah, USA 03:27-03:40 pm Controlling and conquering incurable metastatic cancer by targeting the major tumor-driving K-RAS/SIAH pathway activation to stratify patients, identify resistant tumor clones, quantify therapy efficacy, forecast tumor relapse, and predict patient survival in the neoadjuvant setting (Abs #33) Amy Tang, PhD, Eastern Virginia Medical School, USA 03:40-04:00 pm Coffee Break Room: Brush Gallery 04:00-04:40 pm Young Investigator Award Lecture Room: McDonnell Hall A01 Chairperson: Yibin Kang, PhD, Princeton University, USA New insights into early-stage bone colonization of disseminated tumor cells Xiang Zhang, PhD, Baylor College of Medicine, USA 04:50-06:02 pm Concurrent Session I Room: Frick B02 Chairperson: Alana Welm, PhD, The University of Utah, USA Co-Chair: David Croucher, PhD, Garvan Institute of Medical Research, Australia 04:50-05:02 pm Factor XIIIA-expressing inflammatory monocytes promote lung squamous cancer through fibrin cross-linking (Abs #26) Chad Pecot, PhD, University of North Carolina, USA 05:02-05:14 pm Subcellular specific targeting of JNK as a novel anti-metastatic therapy in triple negative breast cancer (Abs #54) David Croucher, PhD, Garvan Institute of Medical Research, Australia 05:14-05:26 pm Understanding mitochondrial heterogeneity and metastatic potential in microengineered tumor models (Abs #29) Keyue Shen, PhD, University of Southern California, USA 05:26-05:38 pm Estradiol induces BDNF/TrkB signaling to promote brain metastasis of triplenegative breast cancer (Abs #57) Diana Cittelly, PhD, University of Colorado, USA 05:38-05:50 pm Metastasis characteristics of breast cancer patient-derived xenografts (Abs #14) 27

29 Chuck Harrell, PhD, Virginia Commonwealth University, USA 05:50-06:02 pm Bcl-xL promotes metastasis via a novel nuclear function (Abs #12) Nancy Du, PhD, Weill Cornell Medical College, USA 04:50-06:02 pm Concurrent Session II Room: McDonnel Hall A02 Chairperson: Danny R. Welch, PhD, The Kansas University Medical Center, USA Co-Chair: Jiyang Yu, PhD, St. Jude Children's Research Hospital, USA 04:50-05:02 pm Pleiotriphin and midkine promote metastasis in pre-clinical models of breast cancer (Abs #Y78) Debolina Ganguly, UT Southwestern Medical Center, USA 05:02-05:14 pm Systems biology analysis of single cell transcriptomic profiles identifies master regulators of pediatric liver cancer metastasis selectively in early developing microenvironment (Abs #43) Jiyang Yu, PhD, St. Jude Children's Research Hospital, USA 05:14-05:26 pm Cyclophilin A regulates mammary development, tumorigenesis, and metastatic outgrowth (Abs #11) Charles Clevenger, PhD, Virginia Commonwealth University, USA 05:26-05:38 pm Novel kinase regulators of cancer metastasis: Essential kinases and Epithelial- Mesenchymal Transition (Abs #50) Wenliang Li, PhD, University of Texas Health Science Center at Houston, USA 05:38-05:50 pm Evaluation of Neratinib efficacy and mechanisms of resistance in a new syngeneic model of spontaneous breast cancer brain metastasis (Abs #27) Normand Pouliot, PhD, Olivia Newton John Cancer Research Institute, Australia 05:50-06:02 pm CCL18 promotes invasion and metastasis of hepatocellular carcinoma through Type IV Collagen dependent autocrine signaling (Abs #9) Jingqi Chen, PhD, Guangzhou Medical University, China 06:15-08:00 pm MRS BANQUET DINNER (open to all who registered for a ticket) Room: Frick Atrium 28

30 Sunday, August 5, :30-10:10 am Session 10: Genetic and epigenetic heterogeneity and landscape of metastatic evolution Room: McDonnell Hall A01 Chairperson: Dan Landau, MD, PhD, Weill Cornell Medicine, USA Co-Chair: Douglas Hurst, PhD, University of Alabama at Birmingham, USA 08:30-08:55 am Ultra-sensitive liquid biopsy through genome wide integration for early detection and residual disease monitoring Dan Landau, MD, PhD, Weill Cornell Medicine, USA 08:55-09:20 am Tracing the origins of colorectal cancer metastasis with hypermutable DNA Kamila N. Naxerova, PhD, Harvard University, USA 09:20-09:45 am Evolution of metastases in clear cell renal cell carcinoma Samra Turajlic, MBBS, MRCP, PhD, Royal Marsden Hospital and The Francis Crick Institute, UK (EACR designated speaker) 09:45-09:57 am Purinergic receptor P2X7 in the maintenance of cancer stem cells and neuroblastoma metastasis (Abs #Y33) Poliana Martins, PhD, University of Sao Paulo, Brazil 09:57-10:10 am Tissue-specific mediation of metastatic formation by L-selectin on myeloid cells Abs #6) Lubor Borsig, PhD, University of Zurich, Switzerland 10:10-10:30 am Coffee Break Room: Brush Gallery 10:30-11:15 am Kurt Hellmann Award Lecture Room: McDonnell Hall A01 Chairperson: Dihua Yu, MD, PhD, UT MD Anderson Cancer Center, USA Lessons learned from anti-pd cancer immunotherapy Lieping Chen, MD, PhD, Yale University, USA 11:15 am-12:00 pm Paget-Ewing Award Lecture Room: McDonnell Hall A01 Chairperson: Yibin Kang, PhD, Princeton University, USA Extracellular matrix: a key contributor to tumor progression and metastasis Richard O. Hynes, PhD, FRS, Massachusetts Institute of Technology, USA 29

31 12:00-12:20 pm Young Investigator Travel Awards, etc. Sponsored by Susan G. Komen, Theresa s Research Foundation & Nature Reviews Cancer Room: McDonnell Hall A01 Chairpersons: Conor Lynch, PhD, Moffitt Cancer Center, USA Barbara Fingleton, PhD, Vanderbilt University, USA 12:20-12:30 pm Closing Remarks (Yibin Kang & Dihua Yu) 12:30-02:00 pm Social Hour Lunch Pickup: Brush Gallery (Luggage storage available in McDonnell A01) 30

32 Keynote Lecture The Complex Challenge of Treating Metastasis Isaiah J. Fidler DVM Ph.D. Professor, Department of Cancer Biology Professor, Department of Neurosurgery The University of Texas M.D. Anderson Cancer Center, USA Dr. Fidler is a true pioneer in the field of cancer metastasis. His decades of innovative research has brought major breakthroughs in our understanding of the origins and pathogenesis of cancer metastasis and dramatically changed the clinical management of metastatic disease. One of his significant discoveries was the recognition of subsets within primary tumors that harbored metastatic potential. His demonstration of environmental factors interacting with specific tumor cell attributes provided critical evidence for the seed and soil theory. These findings directly contributed to the current explosion in therapeutic approaches for cancer that target stromal factors. Dr. Fidler s many well-earned accolades include induction as a member of the American Association for Cancer Research (AACR) Academy, and as a Fellow of the American Association for the Advancement of Science (AAAS), Nature Publishing s Lifetime achievement award, and the American Cancer Society Medal of Honor for Basic Research. The MRS considers Dr. Fidler one of the key leaders in the field of metastasis research and we are honored to name one of the society s most prestigious prizes for him. 31

33 Fidler Innovation Awardee Prof. Dr. med. Christoph Klein Chair of Experimental Medicine and Therapy Research University of Regensburg Germany Lecture Title: Studying the nature of the seed: unexpected findings and future challenges Thursday August 2 nd at 8.30am The I.J. "Josh" Fidler Innovation in Metastasis Research Award acknowledges the special contributions of Dr. Fidler, who developed new models, shifted the manner in which metastasis is studied in experimental settings, contributed to our fundamental understanding of tumor progression and heterogeneity and translated laboratory findings into the clinical management of metastatic disease. The award is given to an investigator who has made innovative contributions to metastasis research, thereby influencing the direction -- either technically or conceptually -- of this area of research. Christoph A. Klein studied Medicine in Munich, Toronto and New Orleans. He performed his MD thesis in the laboratories of G. Riethmüller and T.W. Mak when he started to establish single cell technologies for the analysis of single disseminated cancer cells. After his postdoctoral training at the Institute of Immunology, LMU Munich, he was awarded with the BioFuture Award of the German Ministry of Science to establish his research group in In 2006, he became professor for Oncogenomics, and in 2010 Chair of Experimental Medicine and Therapy Research at the University of Regensburg. In conjunction, he was appointed to become head of the Fraunhofer Project Group Personalized Tumor Therapy. In 2011, he was awarded with the Dr Josef Steiner Award, in 2015 with the German Cancer Award and in 2017 with Gerhard Domagk Award for Cancer Research. 32

34 Sue Eccles Young Investigator Awardee Xiang Zhang, Ph.D. Baylor College of Medicine, Texas, USA Lecture Title: New insights into early-stage bone colonization of disseminated tumor cells Friday August 4 th at 4.00pm The MRS board of directors established a Young Investigator Award in 2016 in honor of Dr. Sue Eccles, one of our most accomplished investigators and original supporter of the MRS. This award honors excellent metastasis research performed by members of the MRS who are 40 years old or less and who have established his/her own research group. Eligible work for consideration should have been carried out after postdoctoral training and before tenure. Dr. Xiang Zhang obtained his Ph.D. degree from Columbia University in 2006, and did his postdoctoral training at Memorial Sloan Kettering Cancer Center from 2006 to He was then recruited to the Breast Center of Baylor College of Medicine as a McNair Scholar, and is now a tenured associate professor. His lab focuses on metastatic breast cancer, and has made seminal contributions in two areas: 1) bone metastasis and 2) tumor immunology and immunotherapies. Dr. Zhang is the first or corresponding author of 15 papers published in journals including Cell, Nature, Cancer Cell, Nature Cell Biology, Nature Communications, Genes and Development, PNAS, Genome Research and MCB. He also co-authored another 40 papers published in Cell, Nature, Cancer Cell, Nature Medicine etc. Dr. Zhang s discoveries have made significant impact on our understanding of tumor-microenvironment interactions and the consequent effects on therapies. 33

35 Kurt Hellman Translational Metastasis Research Awardee Lieping Chen M.D. Ph.D. Yale University, Connecticut, USA Lecture Title: Lesson learned from anti-pd cancer immunotherapy Saturday August 5 th am The renowned clinical pharmacologist Kurt Hellmann ( ) was one of the founding fathers of the Metastasis Research Society and an early pioneer in the translation of basic metastasis research findings into clinical application. The Kurt Hellmann Award Lecture honors his outstanding contribution to the field. The award is given to an investigator who has contributed significantly to the translation of metastasis research into clinical practice. Dr. Lieping Chen studies cell membrane proteins that control lymphocyte functions and translates his laboratory findings for the treatment of human diseases including cancer. Dr. Chen did the first proof-of-concept study in 1992 showing that the B7-CD28 family molecules could be the targets for cancer immunotherapy by introducing B7-1 into tumor cells to enhance therapeutic immunity. This study inspired subsequent studies targeting the B7-CD28/CTLA-4 family molecules for the treatment of cancer. Dr. Chen discovered B7-H1 (also called PD-L1) molecule in 1999 and demonstrated the role of PD- L1 in the evasion of immunity in the tumor microenvironment. He singularly established the PD-1/PD-L1 pathway as the target for cancer immunotherapy in He also initiated and helped organize the first-in-man clinical trial of anti-pd-1 monoclonal antibody for treating human cancer in 2006, and developed PD-L1 staining as a biomarker to predict treatment outcome. His discoveries directly led to the development of anti-pd-1/pd-l1 antibody therapy for use in a broad spectrum of human cancers. Dr. Chen s discoveries have revolutionized current oncology practice and cancer treatment. 34

36 Paget-Ewing Awardee Richard O. Hynes Ph.D. Howard Hughes Medical Institute, Massachusetts Institute of Technology, Massachusetts, USA Lecture Title: Extracellular matrix: a key contributor to tumor progression and metastasis Saturday August 5 th am The MRS recognizes excellence in the field of metastasis by awarding the Paget-Ewing Award. The Paget-Ewing Award is named after Sir Stephen Paget and Dr. James Ewing, pioneers in metastasis research in the late 19th and early 20th centuries who proposed the two major theories to explain the organ selectivity of metastasis. The Paget-Ewing Award is the highest honor bestowed by the Metastasis Research Society and honors aperson's scientific excellence and substantial contributions to the understanding and/or control of cancer metastasis. Dr. Richard O. Hynes did his undergraduate work in Biochemistry at Trinity College, Cambridge, UK, and his PhD in Biology at MIT and was a postdoctoral fellow at Imperial Cancer Research Fund in London. Dr. Hynes returned to MIT in 1975 as an Assistant Professor and one of the founding members of the MIT Cancer Center. He is now the Daniel K. Ludwig Professor for Cancer Research at the Koch Institute and Department of Biology at MIT, Investigator of the Howard Hughes Medical Institute and Senior Associate Member of the Broad Institute. Dr. Hynes work over the past 45 years has played a major role in establishing the molecular basis of cell adhesion and its many diverse and important effects on cells both in vitro and in vivo. This molecular understanding forms the basis for development of antibodies and drugs that modulate cell adhesion and are in clinical use against thrombosis, inflammation and autoimmune diseases and under investigation for efficacy against cancers. Molecular understanding of cell-ecm interactions is also being exploited in tissue engineering and regenerative medicine. Most recently the Hynes laboratory has focused on metastasis, particularly the contributions of the ECM in promoting metastasis and has developed methods for systematic characterization and analysis of ECM changes in vivo, clearly implicating ECM proteins in tumor progression. 35

37 ABSTRACTS POSTER SESSION I (NON-TRAINEES ) Abstract # Abstract Title Author 1 Metastatic cancer induces muscle wasting through aberrant zinc transport 2 Colorectal cancer associated Natural Killer cells are endowed by prometastatic and pro-angiogenic phenotype/functions by upregulating the MMP9-TIMP2 and Angiogenin axis 3 BMP4 is a bonafide breast cancer metastasis suppressor In vivo/in silico modeling of metastasis after neoadjuvant angiogenic inhibition for personalized predictions of therapeutic benefit Tissue-specific mediation of metastatic formation by L-selectin on myeloid cells Heat shock factor 1 (HSF1) is associated with metastasis in a wide range of epithelial tumor types Regulation of non-proteolytic ubiquitination in cancer chemoresistance and progression CCL18 Promotes Invasion and Metastasis of Hepatocellular Carcinoma Through Type IV Collagen Dependent Autocrine Signalling Kallikrein-related peptidase 4 induces plasticity and cancer-associated fibroblast features in prostate stromal cells Cyclophilin A regulates mammary development, tumorigenesis, and metastatic outgrowth Bcl-xL promotes metastasis via a novel nuclear function Reactive Astrocytic S1P3 signaling modulates blood-tumor barrier permeability in brain metastases 14 Metastasis characteristics of breast cancer patient-derived xenografts An easy and reliable murine model of bone metastasis by injecting cancer cells through caudal arteries C/EBPδ links IL-6 and HIF-1 signaling to promote breast cancer stem cellassociated phenotypes In Vivo Functional Screening Identifies Novel Drivers of Metastasis in Lung Cancer Immediate early gene Ier2 induces a senescence-associated secretome that includes osteopontin, and which is associated with progression and metastasis in melanoma Acharyya, Swarnali Albini, Adriana Anderson, Robin Benzekry, Sebastien Borsig, Lubor Carpenter, Richard Chan, Chia- Hsin Chen, Jingqi Clements, Judith Clevenger, Charles Du, Nancy Gril, Bruniled Harrell, Chuck Kachimaru, Takahiro Kappusamy, Balamurugan Kundu, Samrat Kyjacova, Lenka 36

38 Abstract # Abstract Title Author Purinergic and Kininergic signaling as potential targets for the development of anti-metastatic treatments Myeloid-Derived Suppressor Cells Inhibit T Cell Activation through Nitrating LCK in Mouse Cancers ZMYND8 acetylation mediates HIF-dependent breast cancer progression and metastasis Molecular and Biochemical Basis of Metastatic Hepatotropism: Novel Insights from New Mouse Model Novel tumor intrinsic vs. extrinsic mechanisms of resistance to chemotherapy in metastatic disease Profiling the distinct tumor microenvironments of lytic and blastic prostate cancer metastases in bone Defining how cooperative interactions between tumor subpopulations promote metastasis Factor XIIIA-Expressing Inflammatory Monocytes Promote Lung Squamous Cancer through Fibrin Cross-Linking Evaluation of Neratinib efficacy and mechanisms of resistance in a new syngeneic model of spontaneous breast cancer brain metastasis Genomic analysis of breast cancer mouse models reveals new insights into metastatic biology Understanding Mitochondrial Heterogeneity and Metastatic Potential in Micro-engineered Tumor Models 30 Fructose Fuels Metabolic Reprogramming of Liver Metastasis Long-term, 3-dimentional spheroid culture: a putative model to study evolution of detached cancer cells in tumor metastasis Dietary Poly Unsaturated Fatty Acids Regulate the Number and Sites of Spontaneous Mammary Tumor Metastasis Controlling and Conquering Incurable Metastatic Cancer by Targeting the Major Tumor-Driving K-RAS/SIAH Pathway Activation to Stratify Patients, Identify Resistant Tumor Clones, Quantify Therapy Efficacy, Forecast Tumor Relapse, and Predict Patient Survival in the Neoadjuvant Settings Epithelial-mesenchymal plasticity in breast cancer model systems; Implications for metastatic progression of clinical breast cancer Patient-derived xenografts (PDX) models of peritoneal metastasis from colorectal cancer to identify novel predictive biomarkers for improved therapy 36 Src-mediated phosphorylation converts FHL1 from tumor Ultrasensitive detection of disseminated tumor cells in intraoperative washings of Robot-Assisted Radical Cystectomy for bladder cancer as a potential prognosis biomarker Impact of intratumoral clonal heterogeneity on immune checkpoint inhibitor response Lameu, Claudiana Lu, Xin Luo, Weibo Neychev, Vladimir Oudin, Madeline Owens, Phillip Pearson, Gray Pecot, Chad Pouliot, Normand Ross, Christina Shen, Keyue Shen, Xiling Shibano, Tomiko Talmadge, James Tang, Amy Thompson, Erik Walther, Wolfgang Wei, ShiaoFan Wei, Lei Wellstein, Anton 37

39 Abstract # Abstract Title Author Secreted cellular Prion protein mediates resistance to doxorubicin and capacity for metastasis in breast cancer Epithelial-mesenchymal plasticity in breast cancer model systems; Implications for metastatic progression of clinical breast cancer Distinctive Roles of MST1 and MST2 in Regulation of Cell Mobility and Cancer Metastasis Deoxyguanosine kinase controls the self-renewal of cancer stem-like cells in lung cancer through an AMPK-YAP1 signaling circuit Systems biology analysis of single cell transcriptomic profiles identifies master regulators of pediatric liver cancer metastasis selectively in early developing microenvironment Hippo/Mst signaling couples metabolic state and immune function of CD8α+dendritic cells for cytotoxic T-cell priming1 Understanding and visualising the role of extracellular matrix remodelling in cancer progression and metastasis Circulating tumor cells recapitulate metastatic tropism and reveal brain metastasis drivers Differential regulation of invadopodia-mediated breast cancer metastasis by FAK family kinases Targeting invadopodia-mediated breast cancer metastasis by using ABL kinase inhibitors Novel Kinase Regulators of Cancer Metastasis: Essential Kinases and Epithelial-Mesenchymal Transition 51 Adipocytes drive melanoma progression through FATP proteins Transcriptional control of metabolic adaptations in breast cancer metastasis Subcellular specific targeting of JNK as a novel anti-metastatic therapy in triple negative breast cancer The metastatic receptor status impact on first-line treatment plans and clinical outcomes for recurrent metastatic breast cancer 56 NEDD9 adaptor protein regulates HER2-driven metastasis in breast cancer Estradiol induces BDNF/TrkB signaling to promote brain metastasis of triple-negative breast cancer Ligand dependent and independent function of IL13Rα2 in breast cancer brain metastasis Epithelial-mesenchymal plasticity in breast cancer model systems; Implications for metastatic progression of clinical breast cancer Extracellular vesicles derived from cancer associated fibroblasts enhanced the gastric cancer invasion in extracellular matrix 61 Role of the neural niche in medulloblastoma metastasis 62 All trans retinoic acid promotes apoptosis and inhibits serous ovarian cancer invasion Wiegmans, Adrian Williams, Elizabeth Xiao, Zhixiong Yang, Shengyu Yu, Jiyang Yu, Jiyang Cox, Thomas Yu, Min Gil-Henn, Hava Gil-Henn, Hava Li, Wenliang White, Richard Dasgupta, Subhamoy Croucher, David Pannell, Allen Pugacheva, Elena Cittelly, Diana Cittelly, Diana Williams, Elizabeth Ishimoto, Takatsugu Neman, Joshua Ricciardelli, Carmela 38

40 Abstract # Abstract Title Author T lymphocytes facilitate brain metastasis of breast cancer by inducing Guanylate-Binding Protein 1 expression Gangliosides ratio is dependent of GRP94 through down-regulation of GM2-AP expression: a new GRP94 role in brain metastasis progression S100A4 in cancer metastasis: Translating Wnt/b-catenin signaling-targeted interventions by repositioned drugs into a clinical phase II trial for metastasis restriction in colorectal cancer Mustafa, Dana Sierra, Angels Stein, Ulrike 66 Metabolic targeting of metastasis in osteosarcoma Ren, Ling Mechanisms of cancer-associated inflammation and breast cancer metastasis Productive interactions with the metastatic microenvironment suppresses mir-193b expression and promotes ovarian cancer metastasis The EMT program is orchestrated by coordinated expression of multiple EMT transcription factors to promote breast cancer metastasis DDR1 regulates the construction of a dormancy-supportive niche by coordinating the assembly of a pro-quiescence ECM proteome Yang, Li Mitra, Anirban Ivanov, Alexey Bravo- Cordero, Jose-Javier 39

41 ABSTRACTS POSTER SESSION I (NON-TRAINEES ) Abstract #1 Metastatic cancer induces muscle wasting through aberrant zinc transport Gang Wang, Anup Biswas, Wanchao Ma, Courtney Coker, Kurenai Tanji and Swarnali Acharyya Department of Pathology and Cell Biology, Institute for Cancer Genetics, Columbia University Medical Center Metastasis contributes to the vast majority of cancer-related mortalities. This can occur through vital organ dysfunction where metastatic cancer cells invade and grow. Metastatic tumors also release factors that can systemically affect tissues that are otherwise cancer-free such as skeletal muscle. More than 80% of metastatic cancer patients experience loss of muscle mass and function known as cachexia. Cachectic patients suffer deterioration of diaphragm and cardiac muscles and often die prematurely due to respiratory and cardiac failure. Cachectic patients become too weak to tolerate standard doses of anti-cancer treatments. Cachexia is therefore an important determinant of patient survival in metastatic cancer patients. Therefore, insights into the specific interventions that could either reverse or prevent cachexia are expected to improve treatment outcome, survival and quality of life in metastatic cancer patients. Using transcriptomic profiling, we identified a metal ion transporter, ZIP14 that was upregulated in cachectic muscles from five independent metastatic models, as well as in metastatic cancer patients. We find that TNF- and TGF- cytokines upregulate ZIP14 in muscles, which in turn results in the accumulation of intracellular zinc. Increased zinc influx in muscle cells degrades myosin heavy chain, a major determinant of muscle mass and function. Importantly, germline ablation or muscle-specific depletion of Zip14 markedly inhibits cancer-induced muscle wasting. Our study demonstrates a novel function of ZIP14 in muscles as a mediator of cachexia. These findings can inform development of therapeutic strategies to prevent cachexia, and improve the survival and quality of life in metastatic cancer patients. Abstract #2 Colorectal cancer associated Natural Killer cells are endowed by pro-metastatic and proangiogenic phenotype/functions by upregulating the MMP9-TIMP2 and Angiogenin axis 1 Antonino Bruno, 1 Barbara Bassani, 2 Giuseppe Pelosi, 2,3 Luigi Boni, 4 Lorenzo Dominioni, 4 Lorenzo Mortara, 1,4 Douglas M. Noonan, 1,5 Adriana Albini. Natural Killer (NK) cells are effector lymphocytes involved in tumor immunosurveillance. In solid malignancies, tumor associated (TANK; peripheral blood) and tumor infiltrating (TINK) NK cells acquire altered phenotype and functions. In previous studies, we reported that NK cells from non-small cell lung cancer can acquire a decidual-like CD56 bright CD16 - VEGF high PlGF high IL-8 + low 40

42 phenotype and functionally and molecularly characterize the TINK and TANKs from blood and tissue samples of colorectal cancer (CRC) patients, compared to NK isolated from control and non-oncologic inflammatory bowel disease patients. NK subset distribution and cytokine profiling were detected by multicolor flow cytometry, using peripheral blood and tissue samples from CRC patients, for surface antigen and cytokine profiling characterization. Conditioned media (CM) from FACS-sorted NKs were used either for secretome profiling, using antibody membrane arrays or in functional in vitro angiogenesis assays. We describe for the first time the CRC derived NK cells express MMP2, MMP9, TIMP1 TIMP2, along with several pro-angiogenic factors (Angiogenin, VEGF, CXCL8). This could be a phenotype relevant to their pro-metastatic capabilities and pro-angiogenic function. STAT-3/STAT-5 activation was observed in TANKs, and inhibition of the STAT5 pathway by pimozide, an antipsychotic drug, attenuated their ability to release TIMP-1, TIMP-2 and MMP-9 and blocked the production of VEGF and Angiogenin. Taken together, our data support the hypothesis that a combination of MMP-inhibitors and immunotherapy might be consider as a valid approach the contrast the pro-metastatic/proangiogenic activity of NK cells in colorectal cancer patients. We also propose to use olive derived polyphenols, endowed with anti-inflammatory/anti-angiogenic activities, to repolarized the CRC altered TINK and TANKs. Abstract #3 BMP4 is a bonafide breast cancer metastasis suppressor Eckhardt BL, Redfern A, Sloan EK, Cao Y, Parker BS, Ueno N, Anderson RL Metastasis is the major cause of death in breast cancer patients, largely due to the poor efficacy of existing therapies. Here we report that bone morphogenetic protein-4 (BMP4) blocks metastasis in animal models of breast cancer and predicts improved survival in patients. In preclinical models of spontaneous metastasis, we demonstrate that BMP4 acts as an autocrine mediator to modulate a range of known metastasis regulating genes, including SMAD7, via activation of canonical BMP-SMAD signaling. Restored BMP4 expression in metastatic mammary tumour lines blocks metastasis and increases survival by sensitizing cancer cells to anoikis, thereby reducing the number of circulating tumor cells. Knockdown of its downstream mediator SMAD7, reverses the protection against metastasis afforded by BMP4. Silencing of BMP4 in poorly metastatic lines enhanced their metastatic capacity in mice. Finally, administration of recombinant BMP4 markedly reduces spontaneous metastasis to lung and bone. Collectively, these findings demonstrate that BMP4 can modulate the metastatic potential of breast cancer, without impacting on primary tumor growth. As such, we propose that BMP4 is a bonafide breast cancer metastasis suppressor. A high throughput screen for small molecules that mimic the activity of BMP4 is underway. In a cohort of 535 breast cancer samples, we show that BMP4 and SMAD7 are prognostic for improved recurrence-free survival and overall survival in breast cancer patients, indicating the 41

43 importance of canonical BMP4 signaling in the suppression of metastasis and highlighting new avenues for therapy against metastatic disease. Abstract #4 In vivo/in silico modeling of metastasis after neoadjuvant angiogenic inhibition for personalized predictions of therapeutic benefit Benzekry S. 1, Nicolò C. 1, Mastri M. 2, Tracz A. 2, Ebos J. ML 2 Sunitinib is a drug with anti-angiogenic activity used in the treatment of patients with metastases from renal cell carcinoma or gastrointestinal tumors. However, despite clear efficacy in reducing established tumor growth, recent preclinical studies have shown limited, or even opposing, efficacies in preventing metastatic spread [1, 2]. In this work, we evaluated a previously validated mechanistic mathematical model of metastasis [3] to describe primary tumor and metastatic dynamics in response to neoadjuvant antiangiogenic treatment in clinically relevant mouse models of spontaneous metastatic breast and kidney cancers that develop after surgical removal of orthotopically implanted primary tumors. The data of more than 380 mice receiving either vehicle or sunitinib in the neoadjuvant (presurgical) setting according to different schedules was analyzed. The experimental datasets comprise measurements of primary tumor and metastatic burden kinetics as well as pre-surgical molecular and cellular biomarkers, including vascular cell Ki67 and CD31 expression, circulating tumor cells (CTCs) and myeloid derived suppressor cell counts (MDSCs). Estimation of the mathematical model's parameters was performed using a mixed-effects population approach. Population fits obtained modeling the effect of treatment only on primary tumor growth described well the experimental data of all the treated groups considered, suggesting a negligible effect of the neo-adjuvant treatment on early metastatic spread and growth. When inserting in the model the available biomarkers as covariates, measurements of Ki67+/CD31+, CTCs and granulocytic MDSCs were found significantly correlated with a specific model parameter expressing the metastatic aggressiveness of the tumor. Together, our mathematical model confirms a differential effect of sunitinib on primary (localized) tumors compared to secondary (metastatic) disease. Our results suggest that CTCs and MDSCs might help in predicting metastatic potential and provide a biologically-based computational model integrating these biomarkers into personalized predictions of metastatic benefit of pre-operative treatments. Abstract #6 Tissue-specific mediation of metastatic formation by L-selectin on myeloid cells Borsig L, Glaus Garzon JF, Protsyuk D 42

44 Bone marrow-derived myeloid cells are the major population of cells recruited to the metastatic niche, which promote metastasis. L-selectin is an adhesion molecule on leukocytes that facilitates the initial rolling and capture of leukocytes on activated endothelium. Here we wanted to determine the function of L-selectin in metastatic progression. L-selectin-dependent monocyte recruitment to the metastatic lungs promoted metastasis. Reduced myeloid cell recruitment in the absence of L-selectin correlated with lower cytokine levels. A depletion of circulating monocytes diminished metastasis both in wild-type and L-selectin deficient mice, while an adoptive transfer of these cells restored lung metastasis. We show that spontaneous lung metastasis was reduced; whereas liver metastasis was increased in the absence of L-selectin. Further analysis revealed altered cytokine expression that resulted in altered myeloid-derived cell recruitment in the pre-metastatic liver as determined by Ly6C and Ly6G analysis. Adoptive transfer of wild-type cells reduced liver metastasis. Taken together, we present a mechanism for L-selectin-mediated leukocyte recruitment that exerts specific roles depending on metastatic sites. Abstract #7 Heat shock factor 1 (HSF1) is associated with metastasis in a wide range of epithelial tumor types Carpenter RL, Cao S, Ray H Metastasis remains a significant clinical problem as >90% of cancer deaths from solid tumors are attributable to metastasis. Despite the importance of metastasis, there remains few therapeutic options for metastatic patients. Previously published data by our lab indicate that heat shock factor 1 (HSF1) is an important mediator of epithelial-to-mesenchymal transition (EMT) in HER2- positive breast cancer. HSF1 is a transcription factor whose physiological function is the master regulator of the heat shock response. HSF1 has, in recent years, been shown to be activated in several other solid tumor types including ovarian, colon, skin, and liver cancers. Therefore, the association of HSF1 with EMT and metastasis in other breast tumor subtypes and tumors from these other organ sites was assessed. In order for HSF1 to be transcriptionally active, it requires a multistep activation. Therefore, a gene expression signature was generated using known HSF1 target genes that were altered with knockdown of HSF1. This HSF1 activity signature was significantly associated with metastasis-free survival in multiple solid tumor types including breast, ovarian, lung, skin, pancreas, and prostate. Additionally, gene set enrichment analysis (GSEA) revealed that tumors with high HSF1 activity were enriched for EMT signatures in most tumor types analyzed, including breast cancer confirming our previous findings. Furthermore, tumors with high HSF1 activity were enriched for multiple signatures predictive for metastasis. Lastly, a therapeutic strategy was employed to inhibit AKT and HSF1 using small molecular inhibitors and this successfully reduced primary breast tumor growth in vivo, but also delayed metastasis and enhanced overall survival. Together, these data suggest HSF1 transcriptional activity is strongly associated with EMT and metastasis across many epithelial tumor types. Further investigation into the HSF1-regulated genes important for this process is currently underway. 43

45 Abstract #8 Regulation of non-proteolytic ubiquitination in cancer chemoresistance and progression Chia-Hsin (Lori) Chan 1 *, Hong-Jen Lee 1, Diane Ruan 1, Suchandrima Saha 1 Basal-like breast cancer (BLBC) is a heterogeneous cancer subtype that harbored enriched cancer stem cell (CSC) populations in tumors. Conventional chemotherapy is used as a systematic treatment for BLBC at the time, but it spares the CSC population, which is known to contribute to cancer recurrence after the initial treatment. Therefore, identification of the core molecular pathway that control CSC activity and expansion is important for the development of effective cancer therapeutics for BLBC. Intriguingly, several genes essential for CSCs and their mediated tumor progression and spread are in common with genes controlling epithelial-mesenchymal transition (EMT). EMT is driven by upregulation and stabilization of a group of transcription factors. Ubiquitination (Ub) is a versatile regulatory signal. In contrast to the canonical K48-Ub pathways that have been known to lead to protein degradation, K63-Ub is a newly recognized regulatory post-translational modification important for activating protein function and its mediated signal pathways. My laboratory now has recently reported that K63-Ub is an important posttranslational modification that controls Twist activation (1, 2). Twist. Twist is a transcription factor not only generates CSCs from differentiated cancer cells through EMT, but also plays a vital role in maintaining cancer stemness via crosstalk with the stem cell regulator, Bmi1. Through systematic screen and biochemical characterization, we identified an ubiquitin ligase namely RNF8 as a novel Twist activator by promoting its K63-Ub. We revealed that RNF8 is commonly amplified/overexpressed (~57%) in BLBC with overexpression associated with poor patient outcomes. Aside from regulating CSCs, our preliminary data suggest that RNF8 is required for the growth and spread of differentiated cancer cells. These findings together implicate, for the first time, RNF8 s new function in cancer progression and treatment resistance. Abstract #9 CCL18 Promotes Invasion and Metastasis of Hepatocellular Carcinoma Through Type IV Collagen Dependent Autocrine Signaling Zhang H 1,2, Hou K 1,2, Zhu B 1,2, Hu H 1,2, Qian BZ 3, Chen J 1,2 1. Department of Medical Oncology, The Second Affiliated Hospital, Guangzhou Medical University, Guangzhou, People s Republic of China 2 Translational Medicine Center, The Second Affiliated Hospital, Guangzhou Medical University, Guangzhou, People's Republic of China. 3. MRC Centre for Reproductive Health & Edinburgh Cancer Research UK Centre, University of Edinburgh, Edinburgh, United Kingdom Hepatocellular carcinoma (HCC) is the most common form of primary liver cancer and the third leading cause of cancer-related death in the world. Macrophage infiltration has been shown to 44

46 correlate with HCC progression and poor prognosis. However the molecular mechanism of macrophage promotion of HCC is largely unknown. Here we show that expression of macrophage CC chemokine 18 (CCL18) in patient HCC samples correlates with advanced disease, distal metastasis and poor survival. Mechanistically, CCL18 induces Snail transcription factor mediated epithelial to mesenchymal transition (EMT) of hepatoma cells through a type IV collagen dependent autocrine signaling. This leads to increased invasiveness, migration and anchorage independent growth of hepatoma cell in vitro. Silencing of either PITPNM3, a functional receptor of CCL18 or collagen IV abrogates the tumour promoting function of CCL18 in vitro. Importantly, exogenous CCL18 promote liver and lung colonization of hepatoma cells in vivo, which is abrogated by collagen IV knockdown in hepatoma cells. Furthermore, in patient samples, HCC collagen IV expression correlates significantly with macrophage CCL18 expression, which also correlates with advanced disease, distal metastasis and poor survival. Together our studies illustrate a novel molecular mechanism of HCC progression and metastasis that couples tumour microenvironmental cues with tumour cell intrinsic autocrine signaling. This CCL18/collagen IV pathway may represent important therapeutic target in treating advanced HCC. Abstract #10 Kallikrein-related peptidase 4 induces plasticity and cancer-associated fibroblast features in prostate stromal cells Kryza T 1, Fuhrman-Luck R 1, Stephens C 1, Lawrence M 2, Hooper J 3, Dong Y 1, Risbridger G 2, and Clements J 1. Kallikrein-related peptidase 4 (KLK4) is over-expressed in prostatic intraepithelial neoplasia (PIN) lesions, highlighting its possible involvement in the initiation steps of prostate cancer. Transcriptomic analysis demonstrates that KLK4 could modulate signaling pathways in prostatederived stromal cells, notably, protease-activated receptor (PAR1)-associated signaling. We hypothesize that KLK4 is involved in activation of stromal cells surrounding prostate epithelium through activating PAR1. PAR1 expression in prostate-derived cell lines was assessed, and KLK4-mediated activation of PAR1 in stromal cells confirmed. Normal prostate fibroblast WPMY1cells, primary prostate fibroblasts and primary cancer associated fibroblasts were treated with KLK4 and qrt-pcr performed for PAR1-regulated genes known to be involved in stromal cell activation; PAR1- specific sirna and a PAR1 inhibitor served as negative controls. KLK4-mediated regulation of selected targets was confirmed at the protein level. The effect of KLK4 on WPMY1 cell proliferation and production of the fibroblast activation marker, alpha-sma, was assessed. Finally, the secretome of stromal cells treated with KLK4 was analyzed using a protein array and its pro-angiogenic activity was evaluated by the tubulogenesis assay. 45

47 WPMY1 cell-produced PAR1 was activated by KLK4, inducing an intracellular calcium-flux and modulating the production of factors (FGF1, TAGLN) associated with fibroblast activation and plasticity at both the gene and protein level. This deregulation was inhibited by a PAR1-specific inhibitor or sirna. As with a PAR1-activating peptide, KLK4 increased WPMY1 cell proliferation and expression of the fibroblast activation marker, alpha-sma. Moreover, KLK4 treatment modulated the WPMY1 secretome inducing an upregulation of different growth factors and cytokines known to play key roles during prostate cancer progression. As KLK4 is over-expressed in PIN lesions, which can precede, or be associated with, prostate cancer, and as it can activate prostate-derived fibroblasts, a required step for prostate cancer initiation, attenuating KLK4 activity in non-cancerous prostate lesions may limit prostate cancer initiation. Abstract #11 Cyclophilin A regulates mammary development, tumorigenesis, and metastatic outgrowth Hakim S, Hedrick SE, Clevenger CV The prolyl isomerase cyclophilin A (CypA) regulates the Jak2/Stat5 pathway, which is necessary for mammary differentiation and the pathogenesis of breast cancer. We assessed the role of this isomerase during mammary gland development and erbb2-and PyMT-driven tumorigenesis. Genetic deletion of CypA resulted in delayed mammary gland morphogenesis and differentiation with corresponding decrease in Jak2/Stat5 activation; mammary gland cross-transplantation confirmed this defect was epithelial in nature. Analysis of mammary stem and progenitor populations revealed significant disruption of epithelial maturation. Loss of CypA in the erbb2 transgenic mouse model revealed a marked increase in mammary tumor latency that correlated with decreased Stat5 activation, associated gene expression, and reduced epithelial cell proliferation. Similarly, the PyMT model demonstrated inhibition of tumorigenesis, with significant delays in the outgrowth of hyperplasia and adenoma. Treatment of ER+ and ER-, GFPtagged, breast cancer xenografts with inhibitors of CypA, namely CsA and NIM811, inhibited primary tumor growth inducing central tumor cell death. Interestingly, while individual GFPpositive T47D and MDA-231 cells were observed in xenograft lymph nodes, the outgrowth of macro-metastasis was completely inhibited. These results demonstrate an important role for CypA in the regulation of Jak2/Stat5-mediated biology in mammary epithelium, identifying this isomerase as a novel target for therapeutic intervention, as a chemo-preventive, and as an inhibitor of metastatic outgrowth. Abstract #12 Bcl-xL promotes metastasis via a novel nuclear function Li S., Choi S., Zhang T., Du N. 46

48 Bcl-xL, a negative regulator of mitochondria-mediated apoptosis, is frequently overexpressed in cancer cells. Bcl-xL has long been known for its function in regulating apoptosis during embryonic development and in pathological conditions. Any role that Bcl-xL might play in tumor metastasis has been ascribed to its anti-apoptotic function; i.e. Bcl-xL may increase metastasis by lending survival advantage to the tumor cells during the course of metastasis. However, we demonstrated that Bcl-xL s metastatic function is independent of its canonical anti-apoptotic activity and instead requires a novel nuclear function in cancer. We found that Bcl-xL promotes migration, even when cells are defective in mitochondria-mediated apoptosis. Consistently, BclxL mutants lacking anti-apoptotic activity can still promote migration, induce epithelialmesenchymal transition (EMT), upregulate TGFβ, and increase migration and invasion of pancreatic neuroendocrine tumor cells and breast cancer cells. Importantly, our data demonstrated that forcible localization of Bcl-xL outside the nucleus impairs its metastatic function. Bcl-xL is a transmembrane protein on the mitochondria membrane in healthy cells. The transmembrane domain of Bcl-xL prevents its nuclear import, which suggests that Bcl-xL enters the nucleus by an active mechanism through specific carriers in cancer cells. We hypothesized that unique transporters likely exist so as to transport this normally mitochondria membranebound Bcl-xL protein to the nucleus. By using immunoprecipitation in combination with mass spectrometry, we identified a few novel interacting partners of Bcl-xL. We are investigating whether they transport Bcl-xL into the nucleus to promote metastasis. Abstract #13 Reactive Astrocytic S1P3 signaling modulates blood-tumor barrier permeability in brain metastases Gril B, Paranjape AN, Woditschka S, Hanson J, Wu X, Kloc W, Izycka-Swieszewska E, Duchnowska R, Pęksa R, Biernat W, Jassem J, Nayyar N, Brastianos PK, Peer CJ, Figg WD, Pauly GT, Robinson C, Difilippantonio S, Bialecki E, Metellus P, Schneider PJ, Steeg PS. Breast cancer brain metastases (BCBM) are incurable. The blood-brain barrier (BBB) is a multicellular dynamic structure regulating exchanges between the blood and the central nervous system. As cancer cells colonize the brain, the BBB evolves into a blood-tumor barrier (BTB) and remains a potent barrier to drug penetration, contributing to poor efficacy of brain chemotherapy. Identifying the molecular underpinnings of BBB/BTB permeability will lead to the development of efficacious treatments. METHODS: Three mouse models of BCBM were utilized: a triple negative (231-BR) and two HER2 overexpressing (SUM190-BR, JIMT-1-BR) subtypes. Paracellular permeability of the BTB was assessed using 3kDa Texas Red dextran (TRD). Combining fluorescent imaging techniques with laser capture microdissection, highly vs. poorly permeable metastases were dissected. Gene expression profiling was performed on extracted RNA. RESULTS: Only 10% of the metastatic lesions harbored a TRD diffusion correlating with chemotherapy efficacy (i.e. highly permeable metastases). Several genes were differentially expressed between highly and poorly permeable metastases. Sphingosine-1-phosphate receptor 47

49 3 (S1P3) showed a 2.4-fold increase (P=0.02) in RNA expression in highly permeable lesions. S1P3 protein was overexpressed by metastasis-activated astrocytes in the highly permeable lesions in the three models (231-BR, P=0.034; JIMT-1-BR, P=0.010 and SUM190-BR, P=0.0162). S1P3+ astrocytes were detected in human brain metastasis craniotomies, validating the clinical relevance of our findings. The functional role of astrocytic S1P3 in BTB permeability was assessed in mice with an S1P3 antagonist administered for four days at 10mg/kg twice a day. S1P3 antagonist decreased the diffusion of TRD by 70% (P=0.016). In vitro mechanistic studies revealed that S1P3 exerted its effect by inducing IL-6 and CCL2 secretion and decreasing adhesive endothelial proteins. CONCLUSION: This study represents an important proof of concept for the BTB permeability modulation through reactive astrocytes. These data may identify new strategies to selectively permeabilize the BTB and enhance chemotherapeutic efficacy. Abstract #14 Metastasis characteristics of breast cancer patient-derived xenografts Alzubi M, Turner T, Sohal S, Olex A, Dozmorov M, Harrell JC Patient-derived xenografts (PDX) have been shown to maintain the majority of gene expression and morphological characteristics that were found in the patient. Their utility as models for breast cancer genetic studies, as well as drug treatment studies, has been well defined, but their usefulness as models to study metastatic disease is less documented. In these studies, we acquired 14 breast cancer PDX models that classified as triple negative breast cancer, estrogen receptor positive, or HER2+. We first transduced the models with a lentivirus encoding green fluorescent protein and luciferase, then expanded the lines as mammary tumors; immunohistochemistry confirmed their receptor status. Since it was unclear which experimental approach was the most efficient at generating metastases to the brain, lung, or liver, we tested several to define the metastatic tropism of each PDX. Spontaneous metastases from primary tumors after surgical resection inconsistently yielded metastases to organs other than the lung. Tail-vein injections and intracardiac injections were more consistent at generating metastases to the brain, lung, liver, and usually metastases were found in multiple organs. With all approaches, tropisms to certain organs were observed, and these varied across different PDXs. RNAsequencing of the tumors and metastases allowed us to decipher genetic changes that occurred during the metastatic process in both the cancer cells (human transcripts) and organs being invaded by the human cancer cells (mouse transcripts). Integration of the PDX data into the TCGA data showed the PDX tumors and metastases maintained their intrinsic subtype status. Singlecell suspensions derived from mammary tumors were tested in short-term suspension culture to identify effective therapeutics, and in a BRCA mutant PDX, we could cure the liver and lung metastases, but not brain metastases with cyclophosphamide and carboplatin. These ongoing studies highlight the usefulness of PDX models for metastasis research. Abstract #15 48

50 An easy and reliable murine model of bone metastasis by injecting cancer cells through caudal arteries Kuchimaru T, Miyabara H, Minegishi M, Kadonosono T, Kizaka-Kondoh S Bone is one of the most common sites of metastasis for various primary tumors. However, a promising therapy to prevent bone metastasis is currently unavailable. This deficiency emphasizes the need for new animal models to accelerate translational studies of bone metastasis. During past two decades, intra-cardiac (IC) injection has been the gold standard to develop bone metastasis in mice by injecting cancer cells into the left ventricle to disseminate them to the whole body including bone marrow tissue via the arterial bloodstream, which eventually develop into metastatic colonies in the bone and other organs. Although the current murine model of bone metastasis using IC injection successfully recapitulates the process of bone metastasis, further progress in the study of bone metastasis requires a new model to circumvent some limitations of this model including requirement of high technical proficiency and inefficient delivery of cancer cells to the bone marrow. Here, we present a new murine model of bone metastasis achieved by injecting cancer cells through the intra-caudal arteries (CA). This model does not require high technical proficiency, delivers cancer cells to bone marrow of hind limbs with much higher efficiency than IC injection, and greatly shortens the period of overt bone metastasis development. Moreover, CA injection barely caused acute death of mice after injecting cancer cells, enabling us to inject a larger number of cancer cells to further accelerate the development of bone metastasis with a wide variety of cancer cell lines. Our model may open a new avenue for understanding the bone metastatic processes and development of drugs preventing bone metastasis and recurrence. Abstract #16 C/EBPδ links IL-6 and HIF-1 signaling to promote breast cancer stem cell-associated phenotypes Kuppusamy Balamurugan 1, Daniel Mendoza-Villanueva 1, Shikha Sharan 1, Glenn H. Summers 2, Lacey E. Dobrolecki 3, Michael T. Lewis 3, and Esta Sterneck 1 To significantly improve outcomes for cancer patients, we must understand the mechanisms regulating stem-like cancer cells, which have been implicated in metastasis and treatment resistance. The transcription factor C/EBPδ can exhibit pro- and anti-tumorigenic activities, but the mechanisms of its differential functions are poorly understood. Here, we identify a role for breast cancer cell intrinsic C/EBPδ in promoting phenotypes associated with cancer stem cells (CSC). While C/EBPδ expression is not abundant in most metastatic breast cancers, our data support a pro-tumorigenic role of C/EBPδ when expressed in subsets of tumor cells and/or through transient activation by the tumor microenvironment or loss of substrate adhesion. Using genetic mouse models and human breast cancer cell lines, we show that deletion or depletion of C/EBPδ reduced expression of stem cell factors and stemnness markers, sphere formation and self-renewal, along with growth of tumors and established experimental metastases in vivo. C/EBPδ is also known as a mediator of the innate immune response that is enhanced by hypoxia 49

51 and interleukin-6 (IL-6) signaling, which play important roles in cancer progression. Our mechanistic data reveal C/EBPδ as a link that engages two positive feed-back loops, in part through IL-6 receptor gene as a novel C/EBPδ target, thus amplifying IL-6 and HIF-1 signaling. This study provides a molecular mechanism for the synergism between conditions in cancer progression and is positioned like a rheostat between microenvironmental signals and executioners, C/EBPδ and mechanisms that impinge on its activity may be ideal targets to combat cancer cell stemness. Abstract #17 In Vivo Functional Screening Identifies Novel Drivers of Metastasis in Lung Cancer Kundu ST 1, Grzeskowiak CL 2, Fradette J 1, Rodriguez LB 1, Gibson LA 1, Creighton CJ 2, Scott KL 2, Gibbons DL 1 Lung cancer is the foremost cause of cancer associated mortality worldwide, which primarily results from its extreme propensity for refractory distant metastases. Albeit years of research invested to identify underlying mechanisms, novel genetic aberrations driving pro-oncogenic and pro-metastatic activity remain an elusive target in the quest of precision oncology. To specifically identify such oncogenic driver genes, we used a well-defined syngeneic animal model of KRASmutant lung adenocarcinoma to conduct an oncogenomics-guided in vivo gain-of-function screen of 217 genes selected by a multi-level cross-species comparison of our published high confidence transcriptome data from genetically-engineered mouse models and genomic data of human lung cancers from TCGA, focusing on elevated gene expression and/or gene amplification. Our novel In vivo screening identified several driver genes whose expression promoted tumor metastasis to the lung in mice, among which were both known (e.g. MYC) and several novel (e.g. GATAD2B, TMEM106B, GNAS) oncogenic and metastatic drivers. We extensively validated and mechanistically characterized some of the top metastasis driver genes for their individual in vivo metastatic driver functions. We identified TMEM106B as a primary potent driver of lung cancer metastasis, whose ectopic expression could significantly promote the synthesis of enlarged vesicular lysosomes that are laden with elevated levels of active cathepsins. In a TFEB (Transcription factor EB) dependent manner, TMEM106B could modulate the expression of a large subset of lysosomal genes of the CLEAR (Coordinated lysosomal expression and regulation) pathway in lung cancer cells and human patient samples. We also observed that TMEM106Binduced lysosomes undergo calcium dependent exocytosis thereby releasing increased levels of lysosomal cathepsins necessary for TMEM106B-mediated cancer cell invasion and metastasis which could be prevented upon systemic inhibition of cathepsins in vivo. Further, in TCGA lung adenocarcinoma data sets, 19% patients show genomic amplification and elevated expression of TMEM106B, which also predicts for significantly low disease free and overall survival for patients whose tumors harbor these events. Therefore, we have successfully performed a high value functional in vivo screen where we have elucidated several novel metastatic drivers of lung adenocarcinoma like TMEM106B, with high clinical relevance, making then top candidates for targeted therapy. 50

52 Abstract #18 Immediate early gene Ier2 induces a senescence-associated secretome that includes osteopontin, and which is associated with progression and metastasis in melanoma Kyjacova L 1, Rönsch K 1,2, Wallbaum S 2, Dukovic-Schultz S 2, Scherer SD 2, Neeb A 2, Grau N 2, Cremers N 2, Scholl I 2, Elton J 1, Utikal J 3,4, and Sleeman JP 1,2* Immediate early response 2 (Ier2) is rapidly and transiently upregulated in response to a variety of extracellular stimuli and stresses, and may act as a transcriptional regulator. We have previously shown that Ier2 can be constitutively expressed in a variety of cancer types, promotes migration and invasiveness of cancer cells in vitro, fosters metastasis in experimental animals, and is associated with poor survival in colorectal cancer patients. Here we report that Ier2 is also aberrantly expressed in melanoma, and correlates with poor overall survival of melanoma patients. To understand mechanistically the role of Ier2 in melanoma, we established RheoSwitch murine melanoma B16-F10 and Ret cell lines with inducible expression of Ier2. Prolonged stimulation of Ier2 led to enhanced migration and invasion of melanoma cells in vitro. Remarkably, a p53/p21-driven senescent-like phenotype was also induced stochastically in a subpopulation of cells, as evidenced by i) typical flattened cellular morphology, ii) elevated activity of senescence-associated beta-galactosidase, and iii) the presence of a characteristic senescence-associated secretory phenotype (SASP). Importantly, transcriptomic analysis of Ier2- overexpressing melanoma cells and proteomic analysis of their conditioned media revealed that the SASP contains a number of potential metastasis-promoting pro-inflammatory cytokines and chemokines. The most prominent of these secreted factors was osteopontin, a matricellular phosphoglycoprotein whose expression is associated with an aggressive phenotype and poor prognosis in melanoma patients. Importantly, linear regression analysis of immunohistochemically-stained tissue microarrays demonstrated a positive correlation between Ier2 and osteopontin expression in primary melanomas and distant metastases. Furthermore, both Ier2 and osteopontin were strongly expressed in specimens with active p53/p21 signaling, highlighting the clinical relevance of our findings. Together, our data link the novel metastasispromoting transcription regulator Ier2 to premature senescence-dependent osteopontin secretion, suggesting that targeting the Ier2-driven SASP may provide novel avenues for preventing melanoma metastasis. Abstract #19 Purinergic and Kininergic signaling as potential targets for the development of anti-metastatic treatments Ulrich, H; Martins PCM, Glaser T, Coutinho F, Lameu, C. The axis (SDF-1) / CXCR4 plays a key role in bone marrow metastasis, however, blocking this axis does not prevent the metastatic process of various types of tumor, including neuroblastoma. The 51

53 major agonists of purinergic signaling and kallikrein-kinin systems, ATP and bradykinin (BK), respectively, are released during radiotherapy and chemotherapy by the bone marrow, while SDF-1 levels are decreased due to the highly proteolytic environment. Based on this, we suggest that the inflammatory molecules released by damaged bone marrow cells during chemotherapy and radiotherapy would be potential pro-metastatic factors that orchestrate the seeding of tumor cells in the bone marrow along with SDF-1. Most importantly, we accumulate evidence of the interaction between BK, ATP and SDF-1 signaling pathways in promoting metastasis. We have shown that both ATP and BK increase the invasiveness of neuroblastoma cells and improve the responsiveness of neuroblastoma cells to low concentration of SDF-1. P2X7 receptor signaling is involved with many functions, among them cell death and, controversially, cell survival and increased proliferation of cancer cells, depending on which isoform, the full-length (P2X7A) or truncated (P2X7B), is activated. We have gathered evidence that BK causes an imbalance between P2X7A and P2X7B expression thus favoring proliferation and metastasis of neuroblastomas cells rather than cell death. In short- and long-term murine model of xenotransplantation of human neuroblastoma, BK has increased significantly neuroblastoma metastasis to the bone marrow. However, mice receiving the antagonist of the P2X7 receptor, had metastasis significantly diminished in both groups. Collectively, our studies provide data for the clinical relevance of BK, as well as of purinergic receptor P2X7, making them interesting candidates to develop anti-cancer drugs. Abstract #20 Myeloid-Derived Suppressor Cells Inhibit T Cell Activation through Nitrating LCK in Mouse Cancers Feng S, Cheng X, Lu X, Zhang L, Chaudhary S, Frederickson C, Champion M, Zhao R, Cheng L, Deng H, Lu X Potent immunosuppressive mechanisms within the tumor microenvironment contributes to the resistance of aggressive human cancers to immune checkpoint blockade (ICB) therapy. One of the main mechanisms for myeloid-derived suppressor cells (MDSCs) to induce T cell tolerance is through secretion of reactive nitrogen species (RNS), which nitrates tyrosine residues in proteins involved in T cell function. However, so far very few proteins with specific nitration sites have been identified. Here, using a transgenic mouse model of prostate cancer and a syngeneic cell line model of lung cancer, we applied a new nitroproteomic approach based on chemical derivation of 3-nitrotyrosine and identified that lymphocyte-specific protein tyrosine kinase (LCK), an initiating tyrosine kinase in the T cell receptor signaling cascade, is nitrated at Tyr394 by MDSCs. LCK nitration inhibited T cell activation, leading to reduced interleukin 2 (IL2) production and proliferation. In human T cells with defective endogenous LCK, wild type, but not nitrated LCK, rescued IL2 production. We documented elevated 3-nitrotyrosine signals in clinical samples of castration-resistant prostate cancer (CRPC). In a mouse model of ICB-resistant CRPC by prostate-specific deletion of Pten, p53 and Smad4, we showed that an ICB therapy, composed of anti-programmed cell death 1 (PD1) and anti-cytotoxic-t-lymphocyte-associated protein 4 (CTLA4) antibodies, elicited strong anti-crpc efficacy when combined with a RNS neutralizing 52

54 agent. Together, these data identify a previously unknown mechanism of T cell inactivation by MDSC-induced protein nitration and illuminate a clinical path hypothesis for combining ICB with RNS-reducing agents in the treatment of CRPC. Abstract #21 ZMYND8 acetylation mediates HIF-dependent breast cancer progression and metastasis Chen Y, 1 Zhang B, 1 Bao L, 1 Jin L, 1 Yang M, 1 Peng Y, 1 Kumar A, 2 Wang JE, 1 Wang C, 1 Zou X, 1 Xing C, 2,3,4 Wang Y, 1,5 and Luo W. 1,6 Metastatic breast cancer is lethal and currently incurable. Accumulating evidence indicates that altered epigenetic reprogramming contributes to breast cancer metastasis. However, the role of the epigenetic reader in breast cancer metastasis remains poorly understood. We recently identified the epigenetic reader ZMYND8 as a novel HIF target gene in breast cancer cells. ZMYND8 is highly expressed in metastatic breast tumors and is correlated with poor clinical outcomes in patients with breast cancer. Genetic deletion of ZMYND8 decreases breast cancer cell colony formation, migration, and invasion in vitro, and inhibits breast tumor growth and metastasis to the lungs in mice by decreasing HIF-dependent angiogenesis and cell survival. We further showed that ZMYND8 interacts with HIF- and HIF- and enhances elongation of the global HIF-induced oncogenic genes by increasing recruitment of BRD4 and subsequent release of paused RNA polymerase II in breast cancer cells. ZMYND8 acetylation at lysine 1007 and 1034 by p300 is required for HIF activation and breast cancer progression and metastasis. These findings uncover a primary epigenetic mechanism of HIF activation and HIF-mediated breast cancer progression, and discover a possible molecular target for the diagnosis and treatment of breast cancer. This work was supported by the Susan G. Komen (CCR ), the NIH (R00CA168746, R00NS078049, and R35GM124693), the CPRIT (RR140036, RP170671), the Welch Foundation (I and I ), and the American Cancer Society/UTSW Simmons Cancer Center (ACS-IRG ). Abstract #22 Molecular and Biochemical Basis of Metastatic Hepatotropism: Novel Insights from New Mouse Model Neychev V 1, Gara SK 2, Zhang L 3, Yan C 4, Kebebew E 5 Background: Liver metastases are the major cause of death from a variety of advanced cancers. Yet, the pathophysiological, molecular, and biochemical mechanisms of metastatic hepatotropism remain poorly understood. 53

55 Materials and Methods: Mouse model using human follicular thyroid cancer cell lines, FTC133 and FTC236, with distinctive patterns of lung and liver colonization, respectively, was generated. Organ-specific metastases were evaluated by bioluminescence imaging, in vivo. Comprehensive comparative analysis of injected cell lines and their liver(li) and lung(lu) xenograft clonal descendants was performed, including: cell proliferation, migration, and invasion; genome-wide gene expression(gwe); cellular metabolism, mitochondrial activity, and energy state. Results: Mice injected with FTC236 through tail veins developed significantly more hepatic (15 ± 1.7) than lung (9 ± 1.6) metastases (p=0.0027), and significantly more liver lesions than FTC133 injected animals (15 ± 1. 7 vs 8 ± 2.1, respectively (p=0.0049)). There was no difference in proliferation of injected cell lines and their liver (FTC133-Li and FTC236-Li) and lung (FTC133-Lu and FTC236-Lu) metastatic descendants. FTC236 and FTC133-Li cells were significantly more migratory and invasive than FTC133 and FTC133-Lu cells. O2 consumption and extracellular acidification rates of hepatotropic FTC236 (29.6±11.3pmol/min and 14±4.0mpH/min; p=0.001) and FTC133-Li cells (46.2±6.7pmol/min and 21±3.5mpH/min; p=0.001) were significantly lower than FTC133 cells (125.8±10.8pmol/min and 28.1±3.9mpH/min). Hepatotropic cells were able to switch on and off their mitochondrial function, burning glucose and fructose aerobically, while utilizing mannose and glutamine anaerobically or for synthesis. GWE analysis showed a molecular signature associated with hepatotropism of FTC236 and FTC133-Li cells, including seven underexpressed (POSTN, XKR4, ITGA11, NPR3, TNP1, CFI, and CEACAM1) and two over-expressed (CLDN1 and TM4SF1) genes. Conclusions: FTC236 and FTC133-Li cells possess unique molecular and biochemical traits that may explain, at least in part, the underlying mechanisms of successful admission and adaptation to highly selective liver metabolic microenvironment and sinusoidal O2 gradient. Abstract #23 Novel tumor intrinsic vs. extrinsic mechanisms of resistance to chemotherapy in metastatic disease Madeleine J. Oudin 1, Lucie Barbier 1, Tatiana Kosciuk 1, Miles A. Miller 2, Oliver Jonas 1, Jeff Wyckoff 1, Douglas A. Lauffenburger 1,2, Frank B. Gertler 1 For patients with metastatic triple negative breast cancer (TNBC), where metastasis occurs in 30% of patients, treatment options are limited to chemotherapy, given the lack of any effective targeted therapies approved for this subtype. However, the response is rate low and the time to relapse is short. While most chemotherapeutic drugs target proliferating cells, we set out to investigate the effect of these drugs on metastatic cells and identify metastasis-specific mechanisms of resistance. In preliminary experiments, mice with triple-negative xenograft tumors and known metastatic disease were treated with Taxol and Doxorubicin, two drugs commonly used to treat metastatic TNBC patients. Both drugs significantly slowed tumor growth; however, Taxol, but not Doxorubicin, treatment reduced cell motility within the primary tumor as measured by intravital imaging. These data support the idea that chemotherapeutics can have 54

56 different effects on growth and motility in vivo, and that effects on tumor size might not correlate with changes in cell motility. We first focused on identifying tumor-intrinsic mechanisms of resistance to chemotherapy. We found that cells expressing the pro-invasive isoform of the actin regulatory protein Mena are resistant to the taxane paclitaxel, but not to the widely used DNA damaging agents doxorubicin or cisplatin. Furthermore, paclitaxel treatment does not attenuate growth of Mena INV -driven metastatic lesions or reduce metastatic burden. Mechanistically, Mena isoform expression alters the ratio of dynamic and stable microtubule populations in paclitaxel-treated cells, while also increasing MAPK signaling in response to paclitaxel treatment. Our data indicate that highly metastatic cells may respond differently to chemotherapeutic drugs than non-metastatic ones. Second, we investigated the role of tumor extrinsic factors on chemoresistance of metastatic cells, focusing on the extracellular matrix, which is known to drive local invasion and metastasis. We found that in vitro, plating TNBC cells on collagen renders cells more resistant to Doxorubicin, but not Taxol. In vivo, both Taxol and Doxorubicin treatments led to increases in collagen deposition. Our data suggest that chemotherapeutic treatments can therefore lead to changes in tumor ECM amount and can also change how cells respond to tumor ECM. Further studies are underway to identify the role of the matrix in chemoresistance. Together, these data indicate that chemotherapeutic drugs, commonly used to treat metastatic disease, have different effects on cell growth vs. cell migration, and that chemotherapy can lead to the generation of pro-metastatic environments. Abstract #24 Profiling the distinct tumor microenvironments of lytic and blastic prostate cancer metastases in bone Provera MD 1, Straign D 1, Smith EE 1, Edgerton SM 1, Van Bokhoven A 1, Lucia MS 1, Owens P 1,2 The bone represents the most common metastatic site for prostate cancer and can be classified into three distinct pathology subtypes: Lytic, Blastic and indeterminate. Only recently has the exploration of distinct metastatic sites undergone characterization for the changes caused by the tumor and the surrounding stromal microenvironment. We have identified a cohort of tissue samples from bone that contain metastatic prostate cancer that have been decalcified and Formalin-Fixed Paraffin-Embedded (FFPE). These tissues have been successfully utilized for immunohistochemical staining as well as isolation of RNA and DNA for profiling of both changes in the tumor and microenvironment. We have found a diverse set of myeloid cell populations and lymphoid cells in both lytic and blastic types of prostate cancer metastases. Specific changes in the bone identify key cellular populations that may yield to appropriate targeting of metastatic disease based on distinct pathologic subtype of lytic or blastic phenotypes in metastatic prostate cancer are being identified. Molecular pathology has been an excellent complement to the 55

57 diagnosis, treatment and management of primary tumors, which should be extended to patients with metastatic lesions. Abstract #25 Defining how cooperative interactions between tumor subpopulations promote metastasis Jill M. Westcott, Sharon Camacho, Molly Huysman and Gray W. Pearson The cooperative relationships between distinct tumor subpopulations are a critical yet poorly understood property of heterogeneity within tumors. A lack of techniques for defining how discrete tumor cell variants interact has impeded our understanding of collaborative tumor cell interactions. We have addressed this challenge by developing methods for sophisticated liveimaging and functional interrogation of tumor population dynamics in organotypic culture systems and primary tumor models. With this approach we have uncovered a new symbiotic relationship between tumor subpopulations that promotes a transition from benign to malignant growth by inducing the collective invasion of cohesive groups of cells. Through quantitative analysis of intrinsic invasive heterogeneity within cell communities, we discovered an epigenetically distinct subpopulation of breast cancer trailblazer cells that has an enhanced ability to initiate collective invasion. Importantly, sibling opportunist cells can invade through paths in the ECM created by a minority subpopulation of trailblazer cells. Our functional dissection of this new form of tumor cell cooperation has revealed fresh approaches for improving the accuracy of patient diagnosis and uncovered previously unrecognized therapeutic targets with associated sensitivity signatures. We now find that slow-cycling trailblazer cells promotes the opportunistic invasion of a distinct subpopulation of highly proliferative cells that lack autonomous invasive ability. Through gene expression analysis and functional testing across multiple genotypes we found that reduced expression of the transcription factor p63 is necessary for cells to convert to a trailblazer state in organotypic culture and orthotopic tumor models. Further investigation revealed that p63 suppresses genes that are essential to form a unique type of actin-rich protrusion that is specifically required to initiate collective invasion, but not other forms of cell movement. Notably, the reduction in p63 expression that is necessary for cells to convert to a trailblazer state also causes the collateral inactivation of pro-growth signaling pathways. Indeed, trailblazer cells have a reduced growth rate relative to sibling opportunist cells in our preliminary studies using in vitro and in vivo tumor models. Together, our results suggest a new way in which heterogeneity promotes tumor development by revealing how the cancer hallmarks of proliferation and autonomous invasion can be distributed across distinct populations and shared in a synergistic relationship that promotes disease progression. Abstract #26 56

58 Factor XIIIA-Expressing Inflammatory Monocytes Promote Lung Squamous Cancer through Fibrin Cross-Linking Porrello A, Leslie PL, Harrison EB, Gorentla BK, Kattula S, Ghosh SK, Azam SH, Holtzhausen A, Chao YL, Hayward MC, Waugh TA, Bae S, Godfrey V, Randell SH, Oderup C, Makowski L, Weiss J, Wilkerson MD, Hayes DN, Earp HS, Baldwin AS, Wolberg AS, Pecot CV Lung cancer is the leading cause of cancer-related deaths worldwide, and lung squamous carcinomas (LUSC) represent about 30% of cases. Molecular aberrations in lung adenocarcinomas have allowed for effective targeted treatments, but corresponding therapeutic advances in LUSC have not materialized. Using computational analyses of The Cancer Genome Atlas (TCGA) dataset, we identified a subset of LUSC tumors characterized by dense infiltration of inflammatory monocytes (IMs) and poor survival. Multiplex immunohistochemistry for CD14/CCR2/pan-cytokeratin revealed that dual-positive CD14+/CCR2+ cells predominately reside in the stromal LUSC compartment. Using immunogenomics, we found that amongst 9 immune subsets expressing CD14, IMs had the strongest relationship with CD14 expression and poor LUSC survival. With novel, immunocompetent metastasis models, we demonstrated that tumor cell derived CCL2-mediated recruitment of IMs is driven by TNF -mediated activation of the NF B pathway. Furthermore, tumor production of CCL2 is necessary and sufficient for LUSC metastasis. Pharmacologic inhibition of IM recruitment with a potent CCR2 inhibitor (PF ) had substantial anti-metastatic effects, which was not due to inhibition of tumorassociated macrophages (TAMs) but rather by blockade of IMs in the blood and tumor microenvironment. Notably, in contrast to TAMs, we show that IMs express high levels of Factor XIIIA (FXIIIA). We demonstrate that FXIIIA is packaged into podosome-like structures in IMs, and when in contact with fibrin the IMs are capable of rapidly creating fibrin cross-linkages. We show that FXIIIA-mediated IM fibrin cross-linking creates a scaffold for LUSC cell invasion, migration and metastases. Consist with this observation, we found clinical LUSC samples containing extensive cross-linked fibrin in the microenvironment correlated with poor relapse-free survival. Given the rapidly evolving landscape of precision immune-oncology, these findings identify IMs as a novel context-specific vulnerability of LUSC and provide an important insight into the mechanisms through which this immune cell type determines a poor prognosis. Abstract #27 Evaluation of Neratinib efficacy and mechanisms of resistance in a new syngeneic model of spontaneous breast cancer brain metastasis Nagpal A 1, Redvers RP 1, Denoyer D 1, Ayton SJ 2, de Araujo H 3, Fuentes M 1, Mouhtouris E 1, Anderson R 1, Loi S 4, David S 4 and Pouliot N 1. The introduction of targeted therapies has significantly improved survival outcomes in patients with human epidermal growth factor 2 (HER2) breast cancer. However, resistance almost always develops, leading to treatment failure. Moreover, effective treatment of brain metastases that occur in up to 50% of patients remains a challenge. Progress in identifying more effective treatments against therapy-resistant brain-metastatic HER2 breast cancer, has been hampered 57

59 by the lack of robust and clinically relevant preclinical models. We describe a new Balb/c syngeneic model of spontaneous breast cancer brain metastasis (TBCP- 1) that lacks expression of hormone receptors but naturally expresses high levels of HER2. This model gives rise to a high incidence of spontaneous brain metastases from orthotopic tumours or experimental brain metastases following intra-cardiac inoculation. TBCP-1 cell proliferation is inhibited by HER2-targeting tyrosine kinase inhibitors (TKI), but not by anti-estrogens, indicating phenotypic and functional similarities to human HER2 breast cancer. In this model, we present new data on the efficacy of Neratinib, an irreversible TKI targeting HER1, HER2 and HER4. Resistant variants (TBCP-1NR) developed by long-term exposure to Neratinib showed complete lack of response to Neratinib in vitro and in vivo, allowing us to investigate mechanisms of resistance and to test new combination therapy against treatment resistant HER2 breast cancer. Using transcriptomic analyses (RNAseq) and functional validation in vitro, we identified a new mechanisms by which Neratinib induces cell death termed ferroptosis in TBCP-1 or in human SKBR3 cells but fails to induce this response in TBCP-1NR variants. Importantly, pharmacological inhibition of αvβ3 integrin synergistically inhibited proliferation and overcame resistance to ferroptotic death in resistant variants. To our knowledge, TBCP-1 is the only model that fully recapitulates the spontaneous spread of HER2 breast cancer to brain in immune-competent hosts and provides a unique tool to identify novel therapeutics and biomarkers. Abstract #28 Genomic analysis of breast cancer mouse models reveals new insights into metastatic biology Ross, C. Breast cancer is a leading cause of cancer related mortality in American women, surpassed only by lung cancer. While localized breast cancer is highly survivable, 30% of patients progress to metastatic disease, which has a survival rate of 25%. Current treatments are based on primary tumor biology, but metastatic biology is distinct and often heterogeneous. Creating metastasis specific therapy is challenging as metastatic tissue is rarely resected from patients, and the biology is often confounded by treatment. In addition, no standards exist for sampling lesions postmortem. Thus, there is little human tissue available to research disseminated cancers. To overcome this, we used metastatic breast cancer mouse models to identify metastatic gene expression programs and genomic alterations. We sampled metastatic and paired tumor tissue from 4 genetically engineered models with spontaneous tumors across 5 strains, 4 common injection mouse models, and 7 cultured cell lines. Together this study included 160 mice and 330 samples, which were analyzed by RNA-sequencing, exome-sequencing, and whole genome sequencing. 58

60 RNA-sequencing revealed metastases from orthotopic models had increased immune signaling when compared to tail vein injection, the extent of which was cell line dependent. In addition, transcriptomes of metastases from orthotopic models increased developmental gene programs, whereas those from genetically engineered mice heightened immune signaling pathways. Common metastatic gene expression programs across all models included nicotine degradation, cellular response to Viagra, and diabetes signaling. Intriguingly, treatment of orthotopic models with a low dose of Viagra or diabetes medications significantly reduced lung metastasis. Finally, RNA-sequencing data also revealed that metastases express genes specific to the secondary organ, such as lung surfactant proteins or liver-specific factor ADH1. Exome-sequencing of 48 paired samples identified 58 metastasis-specific single nucleotide variants. Kras and Shc1, members of the Ras pathway, were mutated in 17% of metastases. Orthotopic injection of cells harboring Kras G12D increased pulmonary metastasis, and validation of additional mutations is ongoing. We are also currently analyzing exome-sequencing data for large chromosomal gains and losses in metastatic tissue. This study has identified several targetable pathways and unique aspects of metastatic biology that may be readily targeted clinically. Abstract #29 Understanding Mitochondrial Heterogeneity and Metastatic Potential in Micro-engineered Tumor Models Begum HM, Nemes K, Ando Y, Yu M, Shen K Altered energy metabolism is an emerging hallmark of cancer cells. Interactions in tumor microenvironment (TME) have been shown to reprogram cancer metabolism and promote metastasis. A heterogeneous pattern of redox state and glucose uptake in tumor biopsies has been identified with metabolic imaging, which cannot be explained by oxygenation alone. Owing to the difficulty of investigating this phenomenon in situ, the exact nature of metabolism-altering interactions in the TME remains unclear. We use our previously reported micropatterned tumorstromal assay (μtsa) (Shen et al., Nat Commun, 2014) that recapitulates spatially resolved intercellular cross talk in the TME to investigate the metabolic changes in tumor stromal interactions in breast cancer. Using fluorescent markers for mitochondrial membrane potential (Δψm), we discovered distinct spatial distributions of Δψm in micropatterns of MCF-7 monoculture and MCF-7/fibroblastic stromal cell co-cultures. We created a series of micropatterns and stromal conditions to investigate the effects and mechanisms of stromal cells and mechanical constraints on the observed differences in Δψm and cancer metabolism in the micro-patterns. Gene expression analysis revealed down-regulated oxidative phosphorylation and mitochondrial biogenesis at the edges of monoculture and co-culture μtsa compared to total mechanical confinement. A significant increase in YAP/TAZ activity was observed in cancer cells at the tumor boundary in both monoculture and co-culture μtsa, which correlates with enhanced Δψm. Inhibition of YAP/TAZ pathway totally abrogated the spatial distribution of Δψm. We further demonstrated that sorted MDA-MB-231 cells based on Δψm result in different metastatic burdens in a xenograft model in vivo. Our findings suggest that the mechanical constraints imposed by the surrounding tissue or stromal cells in TME are essential in regulating cancer metabolism and metastatic potential. We are further investigating the signaling elements 59

61 of the mechano-transduction and tumor-stromal communication to find new targets for treating and preventing cancer metastasis. Abstract #30 Fructose Fuels Metabolic Reprogramming of Liver Metastasis Bu P, Chen K, Xiang K, Johnson C, Herman M, Hsu D, Xu G, Shen X Cancer metastasis accounts for the majority of cancer-related deaths and remains a clinical challenge. Metastatic cancer cells generally resemble cells of the primary cancer, but they may be influenced by the microenvironment of the organs they colonize. Based on meta-analysis of extensive clinical datasets and in vivo orthotopic-metastasis models, we show that colorectal, breast, and pancreatic cancer cells undergo metabolic reprogramming after they metastasize and colonize the liver, a key metabolic organ. In particular, metastatic cells in the liver up-regulate the enzyme aldolase B (ALDOB) and ketohexokinase (KHK), which enhances fructose metabolism and provides fuel for major pathways of central carbon metabolism, nucleotide synthesis, and lipid synthesis during tumor cell proliferation. Fructose consumption is also shown to influence surrounding hepatic cells to create a more amicable milieu to metastatic tumor cells. Targeting the fructose metabolism enzymes or reducing dietary fructose significantly reduces liver metastatic growth but has little effect on the primary tumor. Our findings suggest that metastatic cells can take advantage of reprogrammed metabolism in their new microenvironment, especially in a metabolically active organ such as the liver. Manipulation of involved pathways may impact the course of metastatic growth. Furthermore, our upcoming collaboration with Pfizer enables us to test their newly developed, the first potent and specific small molecule inhibitor against the fructose enzymes in our patient-derived preclinical liver metastasis models. Abstract #31 Long-term, 3-dimentional spheroid culture: a putative model to study evolution of detached cancer cells in tumor metastasis Shibano T, Nakano T, Kanai Y, Yoshimoto T, Amano Y, Matsubara D, Murakami T, Endo S, and Niki T Lung adenocarcinoma is a set of heterogeneous diseases with diverse driver mutations, such as EGFR, KRAS, ALK, HER2, BRAF, RET, etc. We have used a panel of lung cancer cell lines representing these diversities and characterized them both in vitro and in vivo (Matsubara et al. Am J Pathol 2010; J Thorac Oncol 2010, 2012; Cancer Sci 2013, 2016; Nakano et al. PlosONE 2017). So far, we have succeeded in creating metastatic models of EGFR-mut (HCC4006, HCC827, PC3), KRAS-mut (A549, H441, H358), ALK-fusion (H2228), and RET-fusion (LC-2/ad). In this study, we sought to elucidate the molecular changes that may occur during evolution of detached cancer cells. We subjected our cell panels to long-term, 3-dimentional spheroid culture in detachment up to 3 months, and obtained sublines A549-FL and H441-FL. These sublines show no changes in 60

62 growth in ordinary culture as compared to parental cells, but they gained prominent grow potential in detached culture condition and high metastatic capabilities in vivo. mrna expression profiling, Western blotting, and immunohistochemistry data demonstrated that these FL sublines showed features of EMT (upregulation of vimentin and ZEB1), altered glucose metabolism (upregulation of GLUT-1), altered cell cycle control (downregulation of p27), and signs of stress (upregulation of phospho-p38). Also, genomic analysis revealed gene amplification of c-myc in H441-FL. This system allowed us to study the time-dependent changes of these molecular evolutions in culture. Thus, long-term, 3-dimentional spheroid culture may be a useful model to study the evolution of detached cancer cells that may occur in tumor metastasis. Abstract #32 Dietary Poly Unsaturated Fatty Acids Regulate the Number and Sites of Spontaneous Mammary Tumor Metastasis Khadge S, Thiele G, Sharp J, McGuire T, Klassen L, and Talmadge J Dietary consumption of long-chain (LC) omega (ω)-3 fatty acids (FAs) before or during mammary gland development can suppress murine mammary carcinogenesis. However, the regulatory role of dietary ω-6 and LC-ω-3 FAs on progression and metastasis has been little studied. In these studies, mice were pair fed either a LC-ω-3 diet (fish oil) or an isocaloric, isolipidic ω-6 liquid diet (Liber-DeCarli) for 16 weeks (from 10 weeks of age) prior to orthotopic injection of 4T1 mammary tumor cells. Tumors size, metastatic lesions, and the tumor microenvironment were analyzed at autopsy on day 35 post injection and in another group survival was assessed. These studies showed that consumption of LC-ω-3FA resulted in significantly slower tumor growth and fewer pulmonary metastases (mets). We also analyzed mets in organs where 4T1 tumors metastasize less commonly including significantly fewer cardiac, renal, ovarian and bone mets in mice fed the ω-3 diet relative to the ω-6 group, as well as, significantly prolonged survival. Immunohistochemical analysis of the tumor microenvironment revealed a significantly lower number of proliferating tumor cells (Ki-67), CD31 + vessels in oxic tumor areas and an increased incidence of apoptotic tumor cells (TUNEL) in mice fed an ω-3 diet. Neovascularization was directly correlated with intra-tumoral macrophage (F4/80 + ) infiltration and inversely with T-cell (CD3 + ) infiltration, which was significantly higher in the tumors from the ω-3 group resulting in a lower neutrophil (elastase) to T-cell ratio (NLR). The NLR was directly correlated with the tumor size and T-cell infiltration was directly correlated with apoptotic tumor cells. Collectively, our data suggest that dietary LC-ω-3FA prolongs survival from mammary tumors by suppressing tumor growth, metastasis, and regulating metastasis sites. Potential mechanisms include decreased tumor proliferation and increased tumor cell apoptosis, and reduced inflammatory cell infiltration (macrophages and neutrophils), angiogenesis and increased CD3 + lymphocyte tumor infiltration. Abstract #33 61

63 Controlling and Conquering Incurable Metastatic Cancer by Targeting the Major Tumor-Driving K-RAS/SIAH Pathway Activation to Stratify Patients, Identify Resistant Tumor Clones, Quantify Therapy Efficacy, Forecast Tumor Relapse, and Predict Patient Survival in the Neoadjuvant Settings Tang, A.H. 1, Lee, M.P. 1, Lee. C.D. 1, Lafever, A.C. 1, Siewertsz Van Reesema, L.L. 1, Collier, A.L. 1, Iyer, A.S. 1, Van Sciver, R.E. 1, Svyatova, E. 1, Kanda, K.F. 1, Winston, J.S. 2, Allen, C.A. 3, Chang, D.Z. 4, Petersen, G.M. 5, Hoefer, R.A Eastern Virginia Medical School, Norfolk, VA; 2 Sentara Norfolk General Hospital, Norfolk, VA; 3 Sentara Healthcare, Virginia Beach, VA; 4 Virginia Oncology Associates, Newport News, VA; 5 Mayo Clinic, Rochester, MN; 6 Sentara Surgery Specialists, Newport News, VA Human Seven-In-Absentia Homologue (SIAH) is a major tumor vulnerability in metastatic cancer: Guided by Drosophila RAS studies, we found that SIAH is the most downstream signaling gatekeeper indispensible for proper K-RAS/EGFR/HER2 pathway activation that drives uncontrolled tumor growth, therapy-resistance, and systemic metastasis in human cancer. Supported by its extraordinarily evolutionary conservation, SIAH E3 ligase is ideally positioned to serve as a logical drug target for developing new anticancer and anti-metastasis therapy. We have shown that anti-siah-based anti-k-ras/erbb strategy is highly effective in inhibiting both tumorigenesis and metastasis of well-established, super-large, and late-stage pancreatic cancer and triple negative breast tumors in a xenograft model in vivo. Through these preclinical studies, SIAH has emerged as a major tumor vulnerability against intractable metastatic cancer. In combination with current standard therapies, anti-siah therapy can be added to save more patients with therapy-refractory, relapsed and metastatic diseases in the future. SIAH is a therapy-responsive, prognostic and predictive biomarker in metastatic cancer: We found that the therapy-induced molecular changes in SIAH expression can be used to differentiate partial responders who will stay in remission from the ones who will relapse, identify chemo-resistant tumor clones, and predict patient survival at neoadjuvant settings. Loss of SIAH expression correlates with effective therapy, K-RAS/EGFR/HER2 pathway inactivation, tumor regression, and good prognosis. In contrast, persistent SIAH expression correlates with ineffective therapy, K-RAS/EGFR/HER2 pathway activation, tumor progression, cancer dissemination, and poor prognosis. SIAH outperform ER, PR, HER2 and Ki67 in forecasting tumor relapse and predicting patient survival in metastatic breast cancer. The prognostic value of SIAH alone is comparable to the best clinical predictors (LN status, tumor size, tumor grade, pathological stage, and molecular subtypes). By developing the K-RAS/SIAH pathway-centered prognostic biomarkers, we hope to stratify patients, assist oncologists to make better treatment decisions to save more lives in the clinic. 62

64 Abstract #34 Epithelial-mesenchymal plasticity in breast cancer model systems; Implications for metastatic progression of clinical breast cancer Hassan S 1,2, *, Foroutan M 3,4 *, Blick T 1,2, Williams E 1,2,5, Le A 3,6, Tachtsidis A 3,7, Gunasinghe D 3,7, Hugo H 1,2,7, Waltham M 3,7, Soo E 3,7, van Denderen B 7, Dowling A 8, Dobrovic A 5,9, Gilles C 10, Davis M 4,11#, Thompson E 1,2,3# Epithelial-mesenchymal plasticity (EMP) can provide carcinoma cells with the invasive ability to leave the primary tumour, enter into the circulation as circulating tumour cells (CTCs), arrive at a distant organ and ultimately form a metastatic deposit. Basal A, triple-negative, MDA-MB-468 human breast cancer cells exhibit mesenchymal change in response to EGF or hypoxia in vitro. In vivo, they exhibit spontaneous epithelial mesenchymal transition (EMT) at the stromal periphery and in the hypoxic region at the necrotic interface. Vascular inclusions at the primary site or in lymph node and lung metastases were predominantly epithelial, however CTCs exhibited a mixed phenotype with both epithelial and mesenchymal gene expression, a finding confirmed in the ED-03 BC PDX system. Mesenchymal enforcement through shrna suppression of E-cadherin or mir-200c knockdown dramatically reduced growth rate in vitro and in vivo, promoted constitutive mesenchymal change, and stimulated lymph node colonisation. We have also bioinformatically selected an EMP-sensitive gene set predicted to have low/no expression in blood cells and reasonably high levels of expression in breast cancer (BC) cells, for tandem-nested RT-qPCR assay of clinical samples. EMP sensitivity of this gene set was validated in public BC cell line data. We have assessed publically-available BC and BC-CTC gene expression data, as well as cohorts of early stage and metastatic breast cancers in which CTCs have been enriched using a combination EpCAM/EGF receptor-directed immunobead approach or microfluidics using the Clearbridge Fx spiral system. Strongly positive cases are rare, but exhibit a predominantly epithelial signature. 63

65 Molecular characterisation of BC CTCs may provide avenues for novel therapeutic approaches that can be developed and tested in cell line and PDX xenograft models. This work was supported by the NHMRC # and the National Breast Cancer Foundation (NBCF#CG EMPathy Breast Cancer Network; NBCF#PF ; NBCF / Cancer Australia #628720). Abstract #35 Patient-derived xenografts (PDX) models of peritoneal metastasis from colorectal cancer to identify novel predictive biomarkers for improved therapy Pachmayr E 1, Brzezicha B 2, Büttner B 2, Rau B 3, Stein U, Walther W 1 Peritoneal metastasis (PM) of colorectal cancer (CRC) constitutes a terminal tumor stage with limited therapy options. To improve therapeutic success, identification of appropriate prognostic and predictive markers is essential. Access to in vivo models of PM will improve the evaluation of chemosensitivity as well as identification of novel biomarkers and therapeutic targets. Here we generated the first patient derived xenograft (PDX) models of PM from CRC to test chemotherapy response and to analyze predictive biomarkers, such as the Wnt signaling target S100A4. For PDX establishment surgical tumor specimens of 48 patients were transplanted subcutaneously onto NOG mice and later transferred to NMRI nu/nu mice for further passaging. Of those, 23 PDX have stably engrafted. Thirteen PDX models thereof were characterized in more detail. All PDX retained the histological appearance of original tumor samples. They showed individual growth characteristics reflected by range of tumor doubling times and replacement of human stroma by mouse stroma. Chemosensitivity of PDX models was analyzed by testing a panel of conventional and targeted drugs. In chemosensitivity testing the PM PDX responded to different degrees to the drugs. Overall, for the routinely used drugs 5-fluorouracil, oxaliplatin and bevacizumab only limited response was seen. Best response was seen for irinotecan. Cetuximab, bevacizumab and erlotinib generated only limited response. Biomarker expression was analyzed in tumor tissues via immunohistochemistry and real-time RT-PCR and correlated to treatment response of the PDX. Of the biomarkers tested, expression of the metastasis-promoting gene S100A4 correlated best to chemotherapy response of the PDX. The novel PDX models of PM from CRC reflect key characteristics of the primary tumors including individual response to chemotherapy. They provide a useful platform for identification and validation of novel predictive biomarkers, such as S100A4 and chemosensitivity testing for improved therapy of CRC-derived PM. Abstract #36 64

66 Src-mediated phosphorylation converts FHL1 from tumor suppressor to tumor promoter Xiang Wang,1* Xiaofan Wei,1* Yang Yuan,1 Qingrui Sun,1 Jun Zhan,1 Jing Zhang,1 Yan Tang,1 Feng Li,1 Lihua Ding,2 Qinong Ye,2 and Hongquan Zhang1 Background: FHL1 has been recognized for a long time as a tumor suppressor protein that associates with both the actin cytoskeleton and the transcriptional machinery. We present in this study a paradigm that phosphorylated FHL1 functions as an oncogenic protein by promoting tumor cell proliferation. Methods: Coimmunoprecipitations (co-ips) were used to detect protein inreraction. Confocal microscopy was applied to observe protein colocalization. Cell proliferation was analyzed by crystal violet. Cell cycle analysis was determined by flow cytometry. Xenograft tumor formation were observed in mice. Protein expression on human tissue sections were examined by immunohistochemical staining. Results: The cytosolic tyrosine kinase Src interacts with and phosphorylates FHL1 at Y149 and Y272, which switches FHL1 from a tumor suppressor to a cell growth accelerator. Phosphorylated FHL1 translocates into the nucleus, where it binds to the transcription factor BCL AF1 and promotes tumor cell growth. Importantly, the phosphorylation of FHL1 is increased in tissues from lung adenocarcinoma patients despite the down-regulation of total FHL1 expression. Kindlin-2 was found to interact with FHL1 and recruit FHL1 to focal adhesions. Kindlin-2 competes with Src for binding to FHL1 and suppresses Srcmediated FHL1 phosphorylation. Conclusion: Collectively, we demonstrate that FHL1 can either suppress or promote tumor cell growth depending on the status of the sites for phosphorylation by Src. Abstract #37 Ultrasensitive detection of disseminated tumor cells in intraoperative washings of Robot - Assisted Radical Cystectomy for bladder cancer as a potential prognosis biomarker Wei L, Hussein AA, Ma Y, Azabdaftari G, Ahmed Y, Wong LP, Hu Q, Luo W, Cranwell VN, Bunch BL, Kozlowski JD, Singh PK, Glenn ST, Johnson CS, Liu S, Guru KA Disseminated tumor cells (DTCs), either occurred before- or during- surgery, can result in cancer metastases and recurrences. In bladder cancer, pelvic recurrence following radical cystectomy is a major cause of mortality. Detection and quantification of DTCs are challenging due to their rarity. Next-generation sequencing (NGS) provides a robust platform for detecting tumorigenic mutations in a small fraction of cells. In this study, we aim to detect DTCs in blood and pelvic washings (PWs) before-, during- and after- robotic-assisted radical cystectomy (RARC) using NGS. For each patient, somatic mutations were first identified from the tumor using whole-exome sequencing, followed by targeted ultra-deep sequencing (>=10,000X) on a subset of mutations in blood and PWs collected: before RARC, after RARC, after pelvic lymph node dissection, and suction fluid during the procedure. The detected mutations allele frequencies were used to calculate residual cancer cell fraction (CCF) in each sample. 17 patients were initially enrolled, with eight later excluded due to quality issues. In the nine completed patients, DTCs were absent in all pre-surgery-, but present in half (14/26) of the after-surgery- PWs, with CCFs ranging from % (median=0.19%). No DTCs were detected in blood except for one sample at very low CCF (0.02%). Interestingly, a potential association was found between each patient s highest CCF 65

67 in PWs and cancer recurrence status: most (6/7) high-ccf (defined as >0.1%) patients developed recurrence with a median of 5.5 months, and none of the low-ccf (0/2) patients had recurrence after at least 9-month follow-up, which was a better predictor than tumor stage, surgical margin or lymph node status. Although limited by a small number of patients, our proof-of-principle study demonstrated the feasibility using NGS to detect rare DTCs in the intraoperative environment, and showed potential association with recurrence which warrants future larger studies to determine potential clinical implications. Abstract #38 Impact of intratumoral clonal heterogeneity on immune checkpoint inhibitor response Vietsch EE, Martinez SR, Giaccone G, Mock B*, Riegel AT, Wellstein A Cancer cells are subjected to evolutionary selection of clonal populations by immune editing, distinct microenvironment at the local and metastatic sites as well as in response to drug treatment. Here we evaluate how clonal heterogeneity impacts the efficacy of immune checkpoint inhibition. Methods: We established a model of tumor heterogeneity from syngeneic, clonal primary cancer cells isolated from a mutant Kras/P53 mouse pancreatic cancer (KPC). The clones were characterized molecularly and tumors reconstituted from mixes of the clonal cell lines. Results and Discussion: These clonal cells formed invasive and metastatic lesions when grafted into syngeneic hosts. The original tumor and clonal cell lines harbored common mutations in ~100 genes suggesting their common ancestry. Unique mutations in each clonal cell line were used to identify and quantitate the clones in heterogeneous cell pools. The clones showed different levels of MAP kinase signaling, unique morphologies, different growth rates in vitro and tumor growth rates in immune competent mice. Moreover, the sensitivity to ~200 anticancer drugs revealed an up to 25-fold varying in vitro sensitivity of the clones to signal transduction inhibitors and cytotoxic drugs. To our surprise, drug sensitivity of individual clones when included in a heterogeneous cell population was strikingly different from their drug sensitivity when growing on their own. In particular, the sensitivity of clones to an anti-pd1 checkpoint inhibitor was distinct across the clonal cells growing in the heterogeneous mixture. Some clones were resistant and others highly sensitive to the checkpoint inhibition. We will discuss pathways and drivers of resistance in the different subpopulations. Conclusions: We conclude that malignant progression and selection of immune checkpoint inhibitor-sensitive and resistant cancer cell subpopulations is impacted by the crosstalk between clonal cell populations present in heterogeneous tumors and the host environment. 66

68 Abstract #39 Secreted cellular Prion protein mediates resistance to doxorubicin and capacity for metastasis in breast cancer Wiegmans A.P, Saunus J.M, Ham S, Lobb R, Kutasovic J.R, Dalley A.J, Miranda M, Atkinson C, Foliaki S.T, Ferguson K, Niland C, Johnstone C.N, Lewis V, Collins S.J, Lakhani S.R, Al-Ejeh F and Möller A. Anthracyclines are amongst the most effective anti-cancer chemotherapeutics ever developed but produce grueling side-effects in breast cancer patients with increased risk of serious adverse events, and therapy resistance often develops over time. We found that doxorubicin, but not its epimer epirubicin, is sequestered in the extracellular environment by secreted cellular Prion protein (PrP C ), blocking its cytotoxic activity. This PrP C effect was dose-dependent and alleviated by genetic depletion of PrP C or chelation of ionic copper. Investigating the expression of PrP C in breast cancer clinical samples, we found that PrP C could neutralize doxorubicin in both local and systemic environments supporting micro-metastases. Further we found that PrP C expression correlates with poorer response to doxorubicin than to epirubicin and that patients could be stratified for survival with Prion expression based on which anthracycline treatment they received. These findings support the development of chemo-sensitization strategies focused on the PrP C -doxorubicin interaction and suggest that diagnostic PrP C testing could be used to guide the selection of anthracycline-based chemotherapy in breast cancer. Abstract #40 Epithelial-mesenchymal plasticity in breast cancer model systems; Implications for metastatic progression of clinical breast cancer Atack A, Bock N, Hutmacher D, Williams ED Bone is the most common site of metastatic spread in men with prostate cancer. These metastases are typically osteoblastic (based on radiographic appearance), unlike the osteolytic phenotype observed for other solid tumours. Clinically relevant models of osteoblastic metastasis have proven difficult to generate, which is turn has impeded progress in addressing this aspect of the disease. Our study aimed to develop a 3D bioengineered in vitro model for the study of osteoblastic bone metastasis Medical-grade Polycaprolactone based scaffolds were generated using melt electrospinning (0.25mm porosity, 0.4mm thickness). Scaffolds coated with calcium phosphate to improve osteogenic properties, and seeded with mesenchymal stem cells isolated from human bone samples. Once cells reached confluency, mineralisation was induced using osteogenic media (ascorbate-2-phosphate, β-glycerophosphate, dexamethasone). Baseline microct imaging was performed to confirm mineralisation. Human prostate cancer cells, derived from prostate cancer patient derived xenografts, were added to the top of the scaffold. Following 3 weeks of culture, samples were imaged using microct and processed for histopathological analyses. 67

69 MicroCT analysis revealed that prostate cancer PDX induced substantial mineralisation (up to 0.5mm thick) ranging from a density equivalent to cancellous bone to that of dense cortical bone (>1500 Hounsfield Units). Mineralisation was significantly altered by treatment with clinically relevant androgen targeted therapy enzalutamide. By contrast, treatment with endothelin receptor antagonist atrasentan had no effect. These observations are consistent with results obtained in clinical trials where enzalutamide increase time first skeletal related event (AFFIRM) but atrasentan failed to improve outcomes for men with metastatic prostate cancer despite promising preclinical results. We are currently investigating the molecular mechanisms involved in the stimulation of mineralisation induced by the prostate cancer cells to facilitate the development of novel approaches to manage prostate cancer bone metastases. *Supported by a Prostate Cancer Foundation of Australia Movember Revolutionary Team Award. Abstract #41 Distinctive Roles of MST1 and MST2 in Regulation of Cell Mobility and Cancer Metastasis Wang Y, Li J, and Xiao ZX MST1/MST2 plays an critical role in the Hippo pathway in regulation of cell proliferation and apoptosis. However, the precise roles of MST1 and MST2 in tumor development remain elusive. In this study, we show that expression of MST2 is down-regulated in human cancers, PyMT of mouse models and PDX models derived from human breast cancer. Oxidative stress activates MST1/MST2, resulting in inhibition of FOXO transactivation activity and expression of p63, which in turn leads to reduced expression of adhesion molecules including integrin and E-cadherin. CRISPR/Cas9 Knockout of MST2 results in increased cell migration in vitro and tumor metastasis in vivo, while knockout of MST1 leads to scattered cell growth with little effect on cell migration. Analyses of transcriptomes in MST1-KO and MST2-KO cells reveal that MST1 and MST2 regulate a distinctive subset of genes involved in a variety of biological processes, including cell mobility. Together, these results reveal that MST1 and MST2 have related but distinctive function in regulation of cell mobility and cancer metastasis. Abstract #42 Deoxyguanosine kinase controls the self-renewal of cancer stem-like cells in lung cancer through an AMPK-YAP1 signaling circuit Lin S, Gunda V, Sum J, Kang J, Hao J, Chellappan S, Izumi V, Fang B, Koomen J, Singh P, Yang S Metastasis involves the dissemination of tumor cells from primary site to distant organs, and the colonization of disseminated cancer cells to establish distant metastases. Some of the disseminated cancer stem-like cells (CSC) are able to remain in dormancy for years before 68

70 establishing secondary tumor and triggering metastatic recurrence. The accepted paradigm of cancer therapy aims at eradicating rapidly proliferating cancer cells. However, how to eliminate disseminated metastatic cells in dormancy and to prevent metastatic recurrence remains a major challenge in the clinic. Here, we report that the CSC in non-small cell lung cancer (NSCLC) rely on the mitochondrial deoxyguanosine kinase (dgk) to maintain mitochondrial homeostasis and cancer cell stemness. Our data demonstrate that dgk highly overexpressed in lung adenocarcinoma patients and dgk overexpression was robustly correlated with the overall survival of patients. Depletion of dgk in lung cancer cells remarkably reduced the proportion of ALDH+ CSC, inhibited tumor sphere formation in vitro and prevent tumor-initiation ability of CSC in xenograft mouse models. Mechanistically, dgk regulates CSC self-renewal through an AMPK- YAP1 signaling circuit downstream of mitochondrial Oxidative Phosphorylation (OXPHOS).Our data indicate that the non-oncogene addiction of lung adenocarcinoma CSC on dgk is a therapeutic vulnerability that could be exploited to prevent lung cancer metastasis. Abstract #43 Systems biology analysis of single cell transcriptomic profiles identifies master regulators of pediatric liver cancer metastasis selectively in early developing microenvironment Pan Q, Li L, Ju B, Nguyen L, Chi H, Easton J, Zhu L, Yu, J Introduction Hepatoblastoma (HB) is the most common pediatric liver cancer. Five-year survival of HB falls from 70% to below 30% once tumor has metastasized. The rapidly developing tumor microenvironment (TME) plays an essential role in regulating HB metastasis. However, current pediatric cancer models are almost exclusively established in adult TME which has greatly hindered our ability to identify key microenvironmental regulators of pediatric cancer metastasis. We have recently developed a novel HB transplantation model 1 and shown that human HB cell HepG2, while developing tumors at a similar rate in neonatal (NN) and adult (AD) liver, generated lung metastasis exclusively in NN mice. Methods We employed the 10X Genomics single-cell RNA-sequencing (scrna-seq) platform and profiled total 67,498 cells from tumor and TME in the liver and lung from both NN and AD HB models at early and late stages of tumor development. We applied a novel nonlinear-based clustering and network inference framework, scminer 2, to identify cell types and reconstruct cluster-specific interactomes in tumor and TME from our scrna-seq data and then used scnetbid, a single cell version of NetBID 3, to infer hidden drivers of HB metastasis selectively in NN mice. Results Disseminated tumor cells (DTCs) in liver TME were identified and had distinct molecular features from primary bulk tumors, while metastatic tumor cells in lung at late stage were clustered together with primary bulk tumors in liver, indicating that DTCs in lung reprogramed back to bulk liver tumors. Bulk tumors demonstrated significant progression, while DTCs stayed unchanged, from early to late stage. Multiple cell types including cancer associated fibroblasts (CAFs), macrophages and others were identified in liver and lung TME at different stages. Cell 69

71 type-specific network and driver analysis of TME of NN and AD mice identified promising master regulators that drive lung metastasis in NN but not AD TME. Abstract #44 Hippo/Mst signaling couples metabolic state and immune function of CD8α + dendritic cells for cytotoxic T-cell priming 1 Du X, Wen J, Wang Y, Karmaus P, Khatamian A, Tan H, Li Y, Guy C, Nguyen T, Dhungana Y, Neale G, Peng J, Chi H, Yu J CD8α + dendritic cells (DCs) present antigens to CD8 + T cells and play a pivotal role in activating cytotoxic T cell responses to viruses, bacteria and tumors. Although lineage-specific transcriptional regulators of CD8α + DC development have been identified, the molecular pathways that selectively orchestrate CD8α + DC function remain elusive. In order to identify subset-specific regulators between CD8α + and CD8α DCs, we developed a systems biology approach, data-driven Network-based Bayesian Inference of Drivers (NetBID), by integrating transcriptomic, whole proteomic and phosphoproteomic data. Our NetBID analysis reveals a marked enrichment of the activity of Hippo pathway kinases in CD8α + DCs relative to CD8α DCs, although the expression levels of the individual molecules show little changes. For functional validation, we applied genetic models to disrupt Hippo signaling in DCs via the CD11c-Cre deletion system. DC-specific deletion of Mst1/2, but not Lats1/2 or Yap/Taz that mediate canonical Hippo signaling, disrupts homeostasis and function of CD8 + T cells and anti-tumor immunity. Mst1/2-deficient CD8α + DCs are impaired in presenting extracellular proteins and cognate peptides to prime CD8 + T cells, while CD8α DCs lacking Mst1/2 have largely normal function. Mechanistically, compared with CD8α DCs, CD8α + DCs show much stronger oxidative metabolism and critically depend upon Mst1/2 signaling to maintain bioenergetic activities and mitochondrial dynamics for functional capacities. Further, CD8α + DCs selectively express IL-12 that depends upon Mst1/2 and the crosstalk with non-canonical NF-kB signaling. In conclusion, Mst1/2 function as selective drivers of CD8α + DC function by integrating metabolic activity and cytokine signaling. Our results also highlight the power of NetBID in identifying hidden drivers in the immune system, which can be further applied to other biological problems including metastasis. Abstract #45 Understanding and visualising the role of extracellular matrix remodelling in cancer progression and metastasis Cox, Thomas R. 1 70

72 1 The Garvan Institute of Medical Research and The Kinghorn Cancer Centre, Cancer Division, St Vincent s Clinical School, Faculty of Medicine, UNSW Sydney, Sydney, Australia Homeostasis of the extracellular matrix (ECM) is critical for correct organ and tissue function. Both the biochemical and biomechanical properties of the ECM contribute to modulating the behaviour of resident cells and are more than just passive bystanders. In tissue diseases such as cancer, the ECM undergoes significant change. These changes, driven by both tumour and stromal cells, feed into the pathological progression of the disease. Cancer associated fibroblasts (CAFs) are the major ECM remodellers in cancer, and underpin the stromal desmoplastic response of almost all solid tumours. Lysyl oxidases, secreted by both cancer cells and CAFs are a family of secreted copper-dependent enzymes that posttranslationally remodel the ECM through cross-linking collagens and elastin. To date, a functional role for lysyl oxidases has been reported in almost all solid tumours. Our work has shown that CAF-driven, and LOX-mediated ECM remodelling are botg critical in modulating Src, FAK and Akt signalling activation, VEGF-driven angiogenesis, and nfatc1- mediated osteoclastogenesis in both breast and pancreatic cancer. Thus, ECM remodelling plays an important and crucial role in primary tumour growth and invasion; the generation of premetastatic niches; the progression of organ fibrosis and tumour desmoplasia; and can be used to stratify patients that will respond favourably to treatment with already approved clinical drugs, including bisphosphonates, and ROCK, Src, Akt, VEGF and FAK inhibitors. Furthermore, we have developed new approaches to visualise and study ECM remodelling in cancer. We have established a novel in situ decellularisation approach (ISDoT), which allows high-resolution fluorescence and second harmonic imaging, as well as quantitative proteomic interrogation of the 3D structure and spatial organisation of the ECM. Understanding at the molecular level how the changing ECM landscape facilitates tumour progression is an important step in the treatment of cancer. Abstract #46 Circulating tumor cells recapitulate metastatic tropism and reveal brain metastasis drivers Remi Klotz 1,2, Amal Thomas 3, Teng Teng 1,2, Oihana Iriondo 1,2, Lin Li 1,2, Sara Restrepo-Vassalli 4, Sung Min Han 1,2, Alan Wang 1,2, Negeen Izadian 1,2, Matthew MacKay 1,2, Byoung-San Moon 1,5, Sathish Kumar Ganesan 1,2, Grace Lee 1,2, Diane S. Kang 1,2, Charlotte S. Walmsley 6, Michael Press 2,7, Wange Lu 1,5, Janice Lu 2, Dejan Juric 6, Aditya Bardia 6, James Hicks 4, Bodour Salhia 2,8, Andrew D. Smith 3, Min Yu 1,2 Hematogenous metastasis is initiated by a subset of circulating tumor cells (CTCs) shed from primary or metastatic tumors into the blood circulation. Thus, CTCs provide a unique patient biopsy resource to decipher the cellular subpopulations that initiate metastasis and their 71

73 molecular properties. However, one crucial question is whether CTCs derived from patients recapitulate human metastatic disease in an animal model. Here, we show that CTC lines established from breast cancer patients are capable of generating metastases in mice with a pattern recapitulating most major organs from corresponding patients. Organ tropism can also be enriched in CTC-derived metastatic variants with an increased preference for the brain, lung and bone. Genome-wide sequencing analyses of metastatic variants identified semaphorin 4D (SEMA4D) as a possible regulator of tumor cell transmigration through the blood-brain-barrier (BBB) and MYC as a crucial regulator for the adaptation of disseminated tumor cells to the activated brain microenvironment. These data provide the experimental evidence of the promising role of CTCs as a prognostic factor for metastasis. Novel organ tropism-associated markers identified from CTCs can also facilitate the development of potential therapies targeting metastasis initiating cells in circulation. Abstract #48 Differential regulation of invadopodia-mediated breast cancer metastasis by FAK family kinases Alessandro Genna 1, Nikola Lukic 1, Shams Twafra 1, Tomer Meirson 1, George S. Karagiannis 2, Ved P. Sharma 2, Yarong Wang 2, David Entenberg 2, John S. Condeelis 2, and Hava Gil-Henn 1 Metastatic dissemination of cancer cells from the primary tumor and their spread to distant sites in the body is the leading cause of mortality in breast cancer patients. Invasive cancer cells penetrate through the basement membrane and into blood vessels using invadopodia, F-actin rich protrusions with matrix-degrading activity. Focal adhesion kinase (FAK) and proline-rich tyrosine kinase 2 (Pyk2) define a distinct family of non-receptor tyrosine kinases that are highly expressed in invasive cancers, but the molecular mechanism by which these proteins regulate invasion of breast cancer cells to neighboring tissues and their subsequent metastatic dissemination is unclear at present. Using high-throughput protein array screening and bioinformatic analysis, we identified cortactin as a novel substrate and interactor of Pyk2 in invadopodia. Pyk2 colocalizes with cortactin to invadopodia of invasive breast cancer cells, where it mediates epidermal growth factor-induced cortactin tyrosine phosphorylation both directly and indirectly via Src-mediated Abl-related gene (Arg) activation, leading to actin polymerization in invadopodia, extracellular matrix degradation, and tumor cell invasion. Further in vivo metastasis assays and high-resolution intravital imaging revealed that both Pyk2 and FAK regulate tumor cell invasion and consequent in vivo metastatic dissemination, but that they do so via distinct mechanisms. While Pyk2 regulates tumor cell invasion by controlling invadopodium-mediated functions, FAK controls invasiveness of tumor cells by regulating focal adhesion-mediated motility and functions. Further breast cancer patient database analysis revealed that increased mrna co-expression of Pyk2, Arg and cortactin or high expression of both Pyk2 and FAK is associated with significantly lower distant metastasis freesurvival. 72

74 Our data suggest that Pyk2 and FAK may be used to predict the probability of distant metastasis and that inhibition of either or both kinases may potentially reduce invadopodia-dependent functions and consequent breast cancer dissemination. Abstract #49 Targeting invadopodia-mediated breast cancer metastasis by using ABL kinase inhibitors Tomer Meirson 1*, Alessandro Genna 1*, Nikola Lukic 1, Ved P. Sharma 2, Yarong Wang 2, John S. Condeelis 2, and Hava Gil-Henn 1 Metastatic dissemination of cancer cells from the primary tumor and their spread to distant sites in the body is the leading cause of mortality in breast cancer patients. While researchers have identified treatments that shrink or slow metastatic tumors, no treatment that permanently eradicates metastasis exists at present. The ABL family of non-receptor tyrosine kinases, which includes c-abl and the Abl-related gene (Arg) link diverse stimuli from cell surface growth factor and adhesion receptors to signaling pathways controlling cell proliferation, survival, adhesion, migration and invasion. We have previously shown that Arg localizes to invadopodia in breast cancer cells, where it controls actin polymerization, matrix degradation, and consequent tumor cell invasion. Based on our previous findings, we hypothesized that Arg kinase could be used as a therapeutic candidate for inhibition of breast cancer metastasis. Here, we demonstrate that inhibition of ABL family kinases by imatinib, nilotinib, or GNF-5 blocked invadopodia formation and function and consequent in vivo breast cancer invasiveness. ABL kinase inhibitors significantly reduced invadopodium precursor formation as well as cortactin tyrosine phosphorylation and consequent actin polymerization, extracellular matrix degradation, and 3D tumor cell invasion in invadopodia of inhibitor-treated breast cancer cells. Additionally, while primary tumor growth was not affected by ABL kinase inhibitors, in vivo matrix metalloproteinase (MMP) activation, tumor cell invasion, and consequent pulmonary metastasis were severely impaired in breast tumor bearing mice that were treated with ABL kinase inhibitors. Careful proteogenomic analysis of breast cancer patient databases revealed a correlation between increased Arg and cortactin expression to metastatic dissemination and poor patient prognosis. Our findings establish a prognostic value for Arg and cortactin as predictors of metastatic dissemination and suggest that therapeutic inhibition of ABL kinases may be used for blocking breast cancer metastasis. Abstract #50 Novel Kinase Regulators of Cancer Metastasis: Essential Kinases and Epithelial-Mesenchymal Transition 73

75 Li W 1, Sang M 1, Hulsurkar M 1, Su N 1, Ai N 1, Zheng D 1, Song H 1, Watnick R 2, Harlow E 3, Xu J 4, Ittmann M 5, Li M 1 We have employed two types of unbiased screens to survey human kinases for new regulators of cancer metastasis: shrna screens for essential kinases and cdna screens for EMT. GRK3 (Gprotein coupled receptor kinase 3) is one of the essential kinases preferentially in high metastatic cancer cells. It not only is necessary for survival and proliferation of metastatic cancer cells in vitro and in vivo, but also is sufficient to promote prostate cancer progression. GRK3 is overexpressed in human metastatic prostate cancers. Moreover, GRK3 promotes neuroendocrine phenotypes of prostate cancer cells, which is associated with poor prognosis of prostate cancer patients. From our kinase EMT screens, we discovered several potent new EMT regulators, two of which are CDKL2 (cyclin-dependent kinase-like 2) and a mixed lineage kinase ZAK (also called MAP3K20). CDKL2 promotes EMT through activating a ZEB1/E-cadherin/βcatenin positive feedback loop and regulating CD44 mrna alternative splicing to promote conversion of CD24 high cells to CD44 high cells. ZAK promotes EMT phenotypes and apoptosis resistance. Depletion of ZAK in aggressive mesenchymal cancer cells reverses EMT phenotypes and attenuates bone metastasis potential, with little impact on primary tumor growth. Of note, ZAK overexpression is significantly associated with poor survival in a number of TCGA human cancer types. Tissue microarray analysis on human invasive breast carcinoma further supports that ZAK overexpression is an independent poor prognostic factor for overall survival in breast cancers. Through combination with ZAK, prognostic accuracy of other common clinicopathological markers in breast cancer is improved by up to 21%. We are current investigating the mechanisms of action of the new regulators in metastasis and to search for their specific inhibitors as potential new drug candidates. Taken together, these studies demonstrated the power of unbiased functional screens in identifying new regulators and potential drug targets for cancer metastasis. Abstract #51 Adipocytes drive melanoma progression through FATP proteins Zhang M, Di Martino JS, Bowman RL, Campbell NR, Baksh SC, Simon-Vermot T, Kim IS, Haldeman P, Mondal C, Yong-Gonzalez V, Abu-Akeel M, Merghoub T, Jones DR, Zhu XG, Weiss J, Huang R, Arora A, Ariyan CE, Birsoy K, Wolchok JD, Panageas KS, Hollmann TJ, Bravo-Cordero JJ, White RM During metastatic dissemination, tumor cells come into contact with a wide variety of stromal cells in the microenvironment. In melanoma, primary tumors are in direct contact with keratinocytes, but as they progress and then metastasize, they then come into contact with subcutaneous tissues. Although this tissue is largely composed of adipocytes, little is known of the interaction between melanoma cells and adipocytes. Here, we demonstrate that adipocytes in the melanoma microenvironment are a key mediator of melanoma progression. Using a zebrafish model of melanoma, we find that melanoma cells home to adipocyte-rich areas in 57% of cases. Once in contact, the melanoma cells induce lipolysis in the adjacent adipocytes, resulting in the release of fatty acids into the extracellular environment. In turn, the melanoma cells take up these fatty acids through FATP proteins, long chain fatty acid transporters that are aberrantly 74

76 expressed in ~40% of melanomas. Once inside the melanoma cell, these adipocyte derived lipids cause increases in tumor cell proliferation, invasion and metastasis. Global lipiodomic analysis revealed a core set of fatty acids that are increased in the melanoma cell after exposure to adipocytes. One fate of these lipids is utilization in the β-oxidation pathway to synthesize ATP for energy. However, surprisingly, we found that the acetyl CoA produced via β-oxidation can also be shunted and used for acetylation of histones, leading to global changes in H3K9 and H3K27Ac, along with widespread alterations of transcriptional programs in the melanoma cells. Taken together, our data demonstrate a mechanism by which adipocytes promote tumor progression by providing lipids that can alter the epigenetic state of the nearby cancer cells. Abstract #52 Transcriptional control of metabolic adaptations in breast cancer metastasis Anand V 1, O Malley BW 2, Dasgupta S 1 1 Department of Cell Stress Biology, Roswell Park Comprehensive Cancer Center, Buffalo, NY. 2 Department of Molecular and Cellular Biology, Baylor College of Medicine, Houston, TX. Metabolic reprogramming is an essential hallmark of tumor progression and metastasis. Cancer cells use altered metabolic pathways to sustain rapid growth and to overcome enormous stress encountered in tumor microenvironment. Tumor cells constantly alter their metabolic state in response to oncogenic stimuli, nutrient availability, and interaction with immune cells however the precise regulation that precedes the metabolic alteration is poorly understood. We believe information about a tumor cell's metabolic state is integrated into the regulation of transcription, and understanding these checkpoints will allow us to specifically target aggressive tumors. From an unbiased kinome library screen we discovered glycolytic enzyme 6-phosphofructo-2- kinase/fructose-2,6-bisphosphatase 4 (PFKFB4) regulates transcriptional reprogramming by activating the oncogenic steroid receptor coactivator-3 (SRC-3). SRC-3 is a transcriptional coregulator which coactivates estrogen receptor α (ERα) and other transcription factors regulating expression of genes in breast cancer progression. Molecular studies revealed that PFKFB4 enzyme phosphorylates SRC-3 at serine 857 (S857) enhancing its transcriptional activity, whereas either suppression of PFKFB4 or ectopic expression of a phosphorylation-deficient SRC- 3 mutant S857A (SRC-3 S857A ) significantly abolishes SRC-3-mediated transcriptional output. Functionally, PFKFB4-driven SRC-3 activation drives glucose flux towards the pentose phosphate pathway enabling purine synthesis by transcriptionally upregulating the expression of critical enzymes in the pathway. Ablation of SRC-3 or PFKFB4 suppresses in vivo breast tumor growth and prevents metastasis to the lung from an orthotopic setting. PFKFB4 and phosphorylated SRC- 3 levels are increased and correlate in estrogen receptor-positive tumors, whereas, in patients with the basal subtype, PFKFB4 and SRC-3 drive a common protein signature that correlates with the poor survival of patients with breast cancer. These findings suggest that the metabolic enzyme PFKFB4 acts as a critical molecular fulcrum coupling sugar metabolism to transcriptional activation by stimulating coactivator SRC-3 to promote aggressive metastatic tumors. 75

77 Abstract #54 Subcellular specific targeting of JNK as a novel anti-metastatic therapy in triple negative breast cancer Han J, Hastings J, Cadell A, Miladinovic D, O Donnell Y, Parker B, Young A, Oakes S, Cox T, Croucher D. The triple negative breast cancer (TNBC) subtype has a much poorer prognosis than hormone receptor or HER2 positive breast cancer, due in part to a higher relapse rate and frequent metastasis to the pleura, lung, liver and brain. The lack of clinically successful targeted therapies for TNBC means that chemotherapy remains the standard-of-care. To date, inhibitors to targets such as EGFR and c-met have shown promising initial responses, followed by rapid relapse. Therefore, instead of targeting individual molecules at the top of signalling pathways, we hypothesise that inhibiting the key nodes that integrate signalling from many potential oncogenes will be a more efficient strategy. To this end, we have identified JNK as a critical oncogenic signalling node within TNBC cells, which conveys signals from multiple oncogenic pathways to promote proliferation, invasion and metastasis. Due to the necessity of JNK for many normal processes, tumour suppression and chemotherapy response in other tissues, systemically targeting JNK is unlikely to yield successful translation to the clinic. Instead we now demonstrate that two distinct JNK network states exist simultaneously within breast cancer cells, with opposing functional and prognostic roles. Through studies of patient cohorts, and the use of genetically encoded localization-specific JNK inhibitors within three-dimensional cell culture models and in vivo metastasis assays, we show that these two network states are spatially separated into a tumour suppressing, nuclear JNK pool, and an oncogenic, cytosolic JNK pool. By adapting our recently published, interaction-based proteomic platform (Croucher et al., Science Signaling, 2016) we have specifically isolated and characterised the oncogenic, cytosolic JNK complex. We also demonstrate that perturbing this signalling complex can specifically inhibit the oncogenic form of JNK, without disturbing the tumour suppressing function of nuclear JNK activity. This finding therefore opens up novel therapeutic options for targeting this critical oncogenic signalling node in TNBC. Abstract #55 The metastatic receptor status impact on first-line treatment plans and clinical outcomes for recurrent metastatic breast cancer Pannell, T. 76

78 Background: Based on two decades of metastatic breast cancer (MBC) research into the risks and clinical importance of testing the receptor status of metastatic tumors, there is growing consensus that the receptor status should be re-tested and the circumstances under which that re-testing should occur. However, there is little to no evidence that utilizing metastatic tumor receptor status test results improves the clinical outcomes of patients. Materials and Methods: A thorough literature review evaluated the current state of evidence related to altering patient treatment plans based on the re-test results of metastatic tumors, when that status differs from the primary tumor. A retrospective observational study was performed utilizing tumor registry data from the University of Tennessee Cancer Institute, yielding124 complete and relevant recurrent metastatic breast cancer records. Of the 124 patients, 92 had the receptor status of their metastatic tumor evaluated. Of those 92, 14 had receptor status results discordant with their primary tumor. Eight of the 14 patients had their first-line treatment plan based on their primary tumor receptor status; the remaining six had their first-line treatment plan based on the tumor receptor status of their metastatic tumor. Results: The study sample revealed that discordant MBC patients, whose first-line treatment plan was based on their primary tumor rather than their metastatic tumor, had a longer median life expectancy of 40 months with a p-value of.049. To date, no other published research has explicitly made such a comparison. Conclusions: The study outcomes demonstrate that basing first-line treatment plans for MBC patients on the receptor status of the primary tumor instead of the metastatic tumor receptor status extends the life expectancy of patients. A standard of care is proposed to impact national and international guidelines. This research impacts the lives of 6,000 women a year in the US. Abstract #56 NEDD9 adaptor protein regulates HER2-driven metastasis in breast cancer Varvara Kozyreva 1,4, Ryan Ice 1,4, Anna Kiseleva 1, Adham Salkeni 1,3,4, Hyeran Choi 1, and Elena Pugacheva 1,2,4 West Virginia University Cancer Institute 1, Department of Biochemistry 2, Department of Medicine 3, Cancer Cell Biology Program 4. Human epidermal growth factor receptor positive (HER2+) breast cancers have been shown to be responsive to monoclonal antibody therapies, however, due to gained resistance, patients usually relapsed after only a short time. Many HER2+ breast cancers show overexpression of neural precursor cell expressed developmentally down-regulated protein 9 (NEDD9). NEDD9 plays an important role in cell cycle progression, migration, adhesion and invasion. Upregulation of NEDD9 has been shown to be an indicator of poor prognosis in various cancers. We have generated transgenic mouse model with conditional (Cre-inducible) overexpression of NEDD9 cdna. When crossed to MMTV-Cre mice, expressing cre recombinase in mammary luminal 77

79 epithelium, overexpression of NEDD9 does not induce cancer. While overexpression of NEDD9 with oncogene, Her2, results in increased incidence, growth of tumors and metastasis in MMTV- Her2-NEDD9 mouse model. Upregulation of NEDD9 increased incidence of metastases in distant organs nearly two fold. We hypothesize that NEDD9 is critical downstream signaling target of Her2 affecting proliferation and/or survival of tumor cells. We report here that 1) upregulation of NEDD9 is tightly correlate with HER2 expression and required for tumor growth in mouse and human tumor cell lines and patient tissue samples; 2) NEDD9 interacts with HER2 stabilizing active kinase; 3) HER2 phosphorylates NEDD9 to initiate downstream MAPK kinase pathway. Interestingly, overexpression of NEDD9 in HER2+ human breast cancer cells increases sensitivity to HER2 monoclonal antibody therapies (Herceptin), while depletion of NEDD9 leads to resistance. This may allow to uncover novel mechanisms of how HER2+ tumor cells acquire resistance during treatment. We theorize that the NEDD9 expression is required for initiation of HER2-driven tumors, but is also indicative of sensitivity of this tumors to Herceptin. Loss of NEDD9 at later stages or during treatment might be indicative of Herceptin-resistant tumors. Further studies in immunocompetent mouse models of Her2 driven tumorigenesis are need to test this hypothesis. Abstract #57 Estradiol induces BDNF/TrkB signaling to promote brain metastasis of triple-negative breast cancer Contreras-Zarate M 1, Day N 1, Ormond R 2, Borges V 3, Gril B 4, Steeg P 4, Cittelly D 1 Breast cancer brain metastases (BM) affect younger women disproportionally, especially those lacking estrogen-receptor (ER), progesterone-receptor and HER2 (known as triple-negative breast cancer, TNBC). Previous studies showed that pre-menopausal levels of estradiol (E2) promote TNBC BM, though the mechanisms are incompletely understood. The current study suggests a novel mechanism involving E2-dependent upregulation of brain-derived neurotrophic factor (BDNF) and subsequent activation of tumor cell tropomyosin kinase receptor B (TrkB). In vivo, E2 increased experimental BM of 4T1BR5 cells compared to E2-depleted mice (ovariectomized and treated with the aromatase inhibitor letrozole) (64.3 vs. 13%, P=0.0078). E2 upregulated BDNF mrna and protein levels in ER + reactive astrocytes in vitro (2.0 fold, P=0.018); and brains of E2-treated mice showed increased percentage of BDNF+ reactive astrocytes (51.4 ± 11.2% E2 vs 23.7 ± 14.3% OVX, P=0.0002). BDNF-receptor TrkB was expressed in a subpopulation of cells within TNBC brain-trophic cell lines, BM-patient-derived xenografts and breast cancer BM. BDNF and conditioned media from E2-treated astrocytes (CM-E2), activated TrkB and downstream AKT, ERK and PLC- signaling in TNBC cells, increasing their invasiveness and tumor-initiating capability in vitro. The promotion of brain metastases by production of BDNF by E2 activated astrocytes was found to be more complex, involving feedback loops and other receptor tyrosine kinases. In 4T1BR5 cells, there was a positive feedback loop whereby exogenous BDNF induced cancer-cell BDNF translation. Upregulation of cancer-cell-bdnf was required to promote full invasiveness of 4T1BR5 in response to CM-E2, and was observed in E2- treated mice in vivo. Additionally, BDNF cross-activated EGFR in TrkB + EGFR + brain trophic cells, 78

80 suggesting that E2-action through astrocytes induces redundant activation of BDNF/TrkB and EGFR signaling in TNBC. These findings have important therapeutic implications, as they provide a rationale to use E2-depletion therapies or TrkB inhibitors to prevent or delay progression of brain metastasis in younger women. Abstract #58 Ligand dependent and independent function of IL13Rα2 in breast cancer brain metastasis Day N*, Contreras-Zarate M*, Hanna C, Ormond R, Cittelly D IL13Rα2, best known as a decoy receptor for IL-13, has been shown to be upregulated in breast cancer cells with increased metastatic potential to lung and brain. Recent studies suggest that IL- 13 activates signaling downstream of IL13Rα2 to promote metastatic traits in colon cancer, but its functional or clinical relevance in breast cancer brain metastasis (BCBM) remains unknown. These studies assessed whether ligand dependent or independent function of IL13Rα2 contributes to breast cancer brain metastasis. IL13Rα2 was expressed at various levels in braintrophic breast cancer cell lines (231BR, JmT1BR3, 4T1BR5, F2-7), brain-metastasis patient-derived xenografts (BM-PDXs) and a cohort of breast cancer brain metastasis from TN and Her2+ BC subtypes. IL-13 was expressed by reactive astrocytes and a subset of BCBM, suggesting that autocrine and paracrine ligand-dependent IL13Rα2 signaling may influence BCBM. Serumstarved 231BR, 4T1BR5 and JmT1Br3 cells stimulated with exogenous IL-13 showed activation of focal adhesion kinase (Y925FAK), increased migration/invasion and suppressed proliferation. FAK inhibitors blocked IL-13-induced migration/invasion in 231BR and 4T1BR5 cells, suggesting that FAK activation mediates in part the pro-invasive effects of IL-13/IL13Rα2. Stable knockdown of IL-13Rα2 showed that sgil13rα2-231br clones exhibited a reduced ability to migrate (41.7% ± 4.5 vs 71.5% ± 9.7; p=0.001) and invade (49.5% ± 6.5 vs 73.1% ± 2.9; p=0.001) compared to gevcontrol cells. In vivo, 231BR sg IL13Rα2 showed a trend towards reduction in the median number of brain micrometastasis formed after intracardiac injection in NSG mice. Experiments are ongoing to define the downstream mediators of IL-13-dependent and independent IL13Rα2 signaling (RNAseq). These studies suggest that IL13Rα2 plays ligand-dependent and independent pro-metastatic roles in BCBM, and constitutes a suitable candidate for therapeutic target of breast cancer brain metastasis. Abstract #59 Epithelial-mesenchymal plasticity in breast cancer model systems; Implications for metastatic progression of clinical breast cancer Atack A, Bock N, Hutmacher D, Williams ED Institute of Health and Biomedical Innovation and School of Biomedical Sciences, Queensland University of Technology, Queensland, Australia; Translational Research Institute, Queensland, Australia; Australian Prostate Cancer Research Centre Queensland, Australia; 79

81 Bone is the most common site of metastatic spread in men with prostate cancer. These metastases are typically osteoblastic (based on radiographic appearance), unlike the osteolytic phenotype observed for other solid tumours. Clinically relevant models of osteoblastic metastasis have proven difficult to generate, which is turn has impeded progress in addressing this aspect of the disease. Our study aimed to develop a 3D bioengineered in vitro model for the study of osteoblastic bone metastasis Medical-grade Polycaprolactone based scaffolds were generated using melt electrospinning (0.25mm porosity, 0.4mm thickness). Scaffolds coated with calcium phosphate to improve osteogenic properties, and seeded with mesenchymal stem cells isolated from human bone samples. Once cells reached confluency, mineralisation was induced using osteogenic media (ascorbate-2-phosphate, β-glycerophosphate, dexamethasone). Baseline microct imaging was performed to confirm mineralisation. Human prostate cancer cells, derived from prostate cancer patient derived xenografts, were added to the top of the scaffold. Following 3 weeks of culture, samples were imaged using microct and processed for histopathological analyses. MicroCT analysis revealed that prostate cancer PDX induced substantial mineralisation (up to 0.5mm thick) ranging from a density equivalent to cancellous bone to that of dense cortical bone (>1500 Hounsfield Units). Mineralisation was significantly altered by treatment with clinically relevant androgen targeted therapy enzalutamide. By contrast, treatment with endothelin receptor antagonist atrasentan had no effect. These observations are consistent with results obtained in clinical trials where enzalutamide increase time first skeletal related event (AFFIRM) but atrasentan failed to improve outcomes for men with metastatic prostate cancer despite promising preclinical results. We are currently investigating the molecular mechanisms involved in the stimulation of mineralisation induced by the prostate cancer cells to facilitate the development of novel approaches to manage prostate cancer bone metastases. This work was supported by a Prostate Cancer Foundation of Australia Movember Revolutionary Team Award. Abstract #60 Extracellular vesicles derived from cancer associated fibroblasts enhanced the gastric cancer invasion in extracellular matrix Ishimoto T 1, 2, Uchihara T 1, 2, Miyake K 1, 2, Arima K 1, 2, Yasuda T 1, 2, Itoyama R 1, 2, Yoshida N 1, Baba H 1 Background: Cancer-associated fibroblasts (CAFs) enhance tumor progression through secretion of soluble factors. Although we demonstrated that direct-interaction between CAFs and cancer cells confer on gastric cancer (GC) cells the ability to invade extracellular matrix (ECM), the molecular mechanism underlying the invasion mediated by soluble factors from CAFs has not been determined. 80

82 Methods: We have established primary fibroblasts from more than 100 GC patients and collected conditioned medium (CM) of CAFs. Human GC cell lines cultured in normal medium or CAF-CM were examined the morphological changes with or without extracellular matrix (ECM). We isolated extracellular vesicles (EVs) from CAF-CM by ultracentrifugal separation. We performed RNA sequencing of GC cell lines and mass analysis of GC-EVs and CAF-EVs. Results: GC cells treated with CAF-CM or CAF-EVs did not show morphological change on normal coated plates. Notably, we found that GC cells with CAF-CM or CAF-EVs on ECM-coated plates showed unique network formation and high-motility. Given that the morphological change appeared within 1-3 hours, we predicted the stimulation of secreted factor from CAF-CM changed the expression pattern of GC s membrane protein. Mass analysis using isolated CAF-EVs was conducted and revealed that CAF-EVs contained high amount of annexin family protein members. Moreover, we showed that annexin in CAF-EVs is critical for network formation of GC cells. We are now investigating the membrane proteins on GC cells during network formation. Conclusions: These findings highlight the underlying mechanism whereby CAF-EVs in tumormicroenvironment confer GC cells on their invasiveness. Abstract #61 Role of the neural niche in medulloblastoma metastasis Martirosian V, Lin M, TC Chen, De Feyter H, Neman J Medulloblastoma (MB) is the most common and lethal intracranial tumor in children. MB, originating in the cerebellum, is comprised of 4 pathologically diverse subgroups and their genetic diversity makes it particularly difficult to find a common therapeutic target. The tumor microenvironment has increasingly become important in cancer research. The microenvironment of the cerebellum and the cerebrospinal fluid contains GABA, a neurotransmitter which can be processed as a metabolite by neural cells through the GABA shunt comprised of GABA transaminase (ABAT) and succinic semialdehyde dehydrogenase (ALDH5A1). We hypothesize that dysregulated expression of GABA shunt mediators and alteration in GABA metabolism contributes to growth and metastasis of MB tumors. Utilizing foundations of neuroscience and cancer biology we investigated the correlation between GABA metabolism and MB tumorigenesis. Our results show: (A) through magnetic resonance spectroscopy (MRS) analysis, MB xenografts have a GABA metabolic signature profile, (B) administration of GABA increases proliferation and a susceptibility to form epithelial-mesenchymal transition (EMT) positive tumor-spheres in the 4 various MB subgroups, (C) co-culture of MB cells and GABAergic neurons and CSF and GABA-producing choroid plexus cells induced and EMT phenotypes, (D) xenografts of tumor spheres into the CSF of the lateral ventricle induced profound leptomeningeal metastases while cells with knockdown of ABAT were unable to form tumors, and (E) high ABAT: ALDH5A1 ratio is correlated with expression of MB self-replicating cancer stem cell markers and resistance to Cisplatin chemotherapy, (F) targeting MB cells with novel 81

83 small molecule NEO216, which inhibits GABA metabolism, reverses tumor EMT-state. These findings provide evidence that GABA metabolism in medulloblastoma metastatic cells may be imperative for survivability in the CSF and leptomeningeal spread. Furthermore, elucidating the contribution of GABA metabolism to medulloblastoma pathogenesis, independent of subgroup stratification will allow for establishment of a novel common biomarker and discovery of therapies targeting this pathway. Abstract #62 All trans retinoic acid promotes apoptosis and inhibits serous ovarian cancer invasion Lokman NA 1, Ho R 1, Gunasegaran K 1, Bonner WM 1, Oehler MK 1,2 & Ricciardelli C 1 Annexin A2 is increased in serous ovarian cancer and plays an essential role in ovarian cancer invasion and metastasis. In combination with S100A10 annexin A2 plays an important role in the plasminogen activator system regulating plasmin production. The aim of this study was to investigate the potential utility of all-trans retinoid acid (ATRA), an inhibitor of the annexin A2 signalling pathway, as a new therapeutic against ovarian cancer. Survival of serous ovarian cancer cells (OVCAR-3, OV-90 & OAW-28) was significantly decreased by ATRA treatment (1-5µM). ATRA (1µM) also significantly decreased proliferation (Ki67 positivity, p=0.0156) and S100A10 protein levels (p=0.0286), and increased cell apoptosis (cleaved caspase-3 positivity, p=0.0006) in serous ovarian cancer tissues using an ex vivo explant assay. In OAW-28 cells reduced cell survival was associated with a reduction of S100A10 protein levels, S100A10-annexin A2 membrane localization, plasmin generation, motility and invasion. In contrast, ATRA inhibited OV-90 cell survival and invasion but did not affect S100A10 protein levels, plasmin activation or S100A10- annexin A2 membrane localization. These findings suggest that ATRA inhibits serous ovarian cancer proliferation and invasion via both S100A10 dependant and S100A10 independent mechanisms. Our results show that ATRA has promising potential as a novel therapy against advanced stage ovarian cancer that warrants further evaluation. Abstract #63 T lymphocytes facilitate brain metastasis of breast cancer by inducing Guanylate-Binding Protein 1 expression Pedrosa R, Sieuwerts A, Berrevoets C, Zeneyedpour L, Priego N, Valiente M, Luider TM, Debets R, Martens J, Kros JM, Mustafa DA The discovery of genes and molecular pathways involved in the formation of brain metastasis would direct the development of therapeutic strategies to prevent this deadly complication of cancer. By comparing gene expression profiles of Estrogen Receptor negative (ER-) primary breast tumors between patients who developed metastasis to brain and to organs other than brain, we found that T lymphocytes promote the formation 82

84 of brain metastases. To functionally test the ability of T cells to promote brain metastasis, we used an in vitro blood-brain barrier (BBB) model. By co-culturing T lymphocytes with breast cancer cells, we confirmed that T cells increase the ability of breast cancer cells to cross the BBB. Proteomics analysis of the tumor cells revealed Guanylate-Binding Protein 1 (GBP1) as a key T lymphocyte-induced protein that enables breast cancer cells to cross the BBB. The GBP1 gene appeared to be up-regulated in breast cancer of patients who developed brain metastasis. Silencing of GBP1 reduced the ability of breast cancer cells to cross the in vitro BBB model. In addition, the findings were confirmed in vivo in an immunocompetent syngeneic mouse model. Co-culturing of ErbB2 tumor cells with activated T cells induced a significant increase in Gbp1 expression by the cancer cells. Intracardial inoculation of the co-cultured tumor cells resulted in preferential seeding to brain. Moreover, intracerebral outgrowth of the tumor cells was demonstrated. The findings point to a role of T cells in the formation of brain metastases in ERbreast cancers, and provide potential targets for intervention to prevent the development of cerebral metastases. Abstract #64 Gangliosides ratio is dependent of GRP94 through down-regulation of GM2-AP expression: a new GRP94 role in brain metastasis progression Bedia C 1, Badia M 1, Muixí L 2, Levade T 3, Tauler R 4, Sierra A 1,5. GRP94, the luminal ER paralog of HSP90, is an ATP-dependent chaperone involved in the assembly or oligomerization of loaded protein cargo, as well as transport and folding of other proteins. Many cancers over-express GRP94, and in vivo studies have evidenced that this chaperone has a critical function in brain metastasis pathogenesis. Since previous studies have suggested a possible role of GRP94 in lipid metabolism, in this work the lipid changes between GRP94 silenced and control cells were explored through an untargeted approach. As the main result, the levels of two ganglioside species, GM2 and GM3, resulted dysregulated in GRP94 silenced cells. In concrete, elevated levels of GM2 and reduced levels of GM3 were found. Since the hydrolysis of GM2 to produce GM3 is regulated by the enzymatic activity of - hexosaminidase A (HexA), the presence and activity of this enzyme as well as its co-factor GM2- AP were investigated. The HexA activity was decreased in the GRP94 silenced cells and, whereas HexA levels were not changed, the presence of the cofactor GM2-AP was almost abolished, suggesting that this cofactor was responsible for the reduced HexA activity and the ganglioside dysregulation observed. We hypothesize that the GRP94 knockdown impairs the correct folding of the GM2-AP cofactor, whose structure has specific binding sites for the substrate GM2 and the alpha helix of HexA. Gangliosides are glycosphingolipids mainly localized in the outer leaflet of plasma membrane in lipid-enriched microdomains called lipid rafts where they are involved in cell-cell recognition, adhesion and signal transduction. Given the fact that GM2-AP has also been described as a secreted protein that serves as a general intra- and/or inter-cellular lipid transport protein in vivo, changes in GM2-AP levels might have a role in modifying the interaction of brain metastatic cells with other cells and/or the brain microenvironment. 83

85 Abstract #65 S100A4 in cancer metastasis: Translating Wnt/b-catenin signaling-targeted interventions by repositioned drugs into a clinical phase II trial for metastasis restriction in colorectal cancer Stein U (1,2) Walther W (1), Monks A (3), Harris ED (3), Burock S (4), Keilholz U (2,4), Birchmeier W (5), Kobelt D (1,2), Dahlmann M (1,2), Shoemaker RH (6) (1) Experimental and Clinical Research Center, Charité- Universitätsmedizin Berlin, and Max-Delbrück-Center for Molecular Medicine, Berlin, Germany (2) German Cancer Consortium (DKTK), Heidelberg, Germany (3) National Cancer Institute/NIH, Frederick, MD, USA (4) Charité Comprehensive Cancer Center, Berlin, Germany (5) Max-Delbrück-Center for Molecular Medicine, Berlin, Germany (6) National Cancer Institute/NIH, Bethesda, MD, USA Background: Aberrant Wnt/b-catenin activity is an early step in tumorigenesis, leading to tumor progression and metastases. In colorectal cancer (CRC), metastasis critically limits successful therapy, is directly linked to patient survival and counts for about 90% of patient deaths. We identified the metastasis-inducing gene S100A4 as a transcriptional b-catenin target, thereby linking as important for metastasis: Wnt/b-catenin pathway and S100A4-induced motility. We demonstrated S100A4-induced migration and invasion, liver and lung metastases in mice. We showed in primary CRC patient tissue and blood that high S100A4 expression predicts metachronous metastasis formation and reduced patient survival. Aim: Here, we aim at development of drugs targeting S100A4, alone or in combinatorial approaches, thereby restricting cancer metastasis. Methods: We established high-throughput screenings (HTS) of pharmacologically active compound libraries employing an S100A4 promoter-driven reporter thereby identifying small molecule transcriptional S100A4 inhibitors. As proof-of-concept, we applied shrna acting on S100A4 systemically and prevented metastasis in mice. Results: Best HTS hits, e.g. niclosamide, act, at least in part, via intervening in the Wnt/b-catenin pathway, leading to S100A4 downregulation, reduced migration and invasiveness, and most importantly, restricted metastases formation in mice. We are now translating our findings on restricting S100A4-driven metastasis into clinical application. The repositioned FDA-approved anti-helminthic drug niclosamide is currently being tested in a prospective phase II clinical trial to investigate the safety and efficacy of orally applied niclosamide in patients with metachronous or sychronous metastases of CRC progressing after therapy (NIKOLO; NCT ). Our assay for circulating S100A4 transcripts together with proteomics and drug determination via HPLC in patient blood is used to monitor treatment 84

86 success. Our patent: Niclosamide for treatment of cancer metastasis (EP , ) was recently granted. Conclusion: The repositioned FDA-approved drug niclosamide inhibits S100A4-induced metastasis in CRC mouse models and has therapeutic potential for human CRC. Abstract #66 Metabolic targeting of metastasis in osteosarcoma Ren L 1, Huang S 1, Ruiz Rodado V 3, Issaq S 2, Tran Hoang C 1, Wang H 1, Larion M 3, LeBlanc AK 1. 1 Comparative Oncology Program, MIP/CCR/NCI/NIH, Bethesda, MD, USA 2 Pediatric Oncology Branch, CCR/NCI/NIH, Bethesda, MD, USA 3 Metabolomics Section, NeuroOncology Branch, CCR/NCI/NIH, Bethesda, MD, USA Osteosarcoma (OS) is a highly metastatic malignancy of bone affecting ~1000 pediatric and adolescent patients per year in the US. Based on historical data over 80% of patients will develop metastases despite definitive primary tumor management and multi-agent adjuvant chemotherapy. Cancer metastasis is characterized by complex molecular events. The acquisition of these events is primarily believed to result from alterations in gene and protein expression/function. Recent studies have also suggested the role of metabolic alterations, or metabolic reprogramming, that may similarly contribute to cancer metastasis. We have previously demonstrated that OS cellular metabolism, specifically glycolytic capacity and oxidative phosphorylation, is altered in concert with metastatic potential, emblematic of the enhanced bioenergetic demands of metastasis. (Ren et al. Cancer Res. 2012). The present study aims to evaluate the effects of targeting glutamine and/or glucose metabolism with a glutaminase-1 (GLS-1) inhibitor (CB-839) and Metformin in mouse models of OS metastasis. Mice injected with highly metastatic MG63.3 human OS cells were treated with CB- 839 and Metformin, both as single agents and in combination. As single agents, there was a moderate inhibition of metastasis by CB-839 and an induction of metastasis with Metformin. When CB-839 and Metformin treatments were combined, a distinct reduction (>80%) in metastatic burden within the lungs was observed. The inhibition of metastatic progression was observed even when the treatment started after micro metastases were established. A similar result was obtained with a syngeneic murine OS metastasis mouse model. The inhibition of both glutamine and glucose metabolism was confirmed with nuclear magnetic resonance (NMR) spectroscopy in the xenograft tumor tissues with the combination treatment. Extracellular flux analysis of combination-treated OS cells showed a reduction in cellular mitochondrial respiration. Work is underway to explore, through 13 C-NMR flux analysis, the mechanistic underpinnings of this potential anti-metastatic approach in anticipation of clinical translation to patients. 85

87 Abstract #67 Mechanisms of cancer-associated inflammation and breast cancer metastasis Pang, Y, Achyut, BR, Yan, HY, Ishii, H, Hollander, C, Yang, L Inflammation has been identified as one of the hallmarks of cancer. However, what roles cancerassociated inflammation (CAI) play in metastatic process and the critical underlying mechanisms have yet to be investigated. Intertwined with the metastasis biology is the longstanding challenge in our understanding of both anti- and pro- tumor functions of TGF-β. Our studies demonstrate an important anti-inflammation function of epithelial TGF-β signaling, which is critical for the anti-tumor properties of TGF-β. We further identify a metastasis-promoting function of myeloid TGF-β signaling that is key in host immune suppression and type II inflammatory response. Recent immunogenomics analyses of TCGA (33 cancer types and 10,000 tumor specimens) identify six different immune landscapes of cancers. One of which is TGF-β dominant. We anticipate our work provide new understanding and insight for immune resistance and relapse, as well as therapeutic options. Abstract #68 Productive interactions with the metastatic microenvironment suppresses mir-193b expression and promotes ovarian cancer metastasis Mitra A Productive reciprocal interactions between ovarian cancer cells with the microenvironment of the site of metastasis is essential for successful metastatic colonization. micrornas have been well established to play critical roles in various stages of cancer progression, including metastasis. However, the role of the signals from the microenvironment in regulating key micrornas in metastasizing cancer cells needs to be studied. Using an organotypic 3D culture model mimicking the human omentum, one of the principal sites of ovarian cancer metastasis, we have identified the microenvironment-induced downregulation of a tumor suppressor microrna mir-193b in the metastasizing ovarian cancer cells. The direct interaction of the ovarian cancer cells with the mesothelial cells covering the surface of the omentum caused DNMT1 mediated methylation and decreased expression of mir-193b. This was mediated through an induction of TGF and MAPK signaling in the cancer cells by the mesothelial cells in the metastatic microenvironment. The decrease in mir-193b promoted metastatic colonization of the omentum through increased invasion and proliferation and in a mouse xenograft model of ovarian cancer metastasis. The functional effects of mir-193b were mediated at least partly through the concomitant increased expression of its target urokinase-type plasminogen activator (upa). Our findings link paracrine signals from the microenvironment with the regulation of a key microrna in cancer cells that is essential for the initial steps of ovarian cancer metastatic colonization. Targeting mir-193b would be a promising approach to treat ovarian cancer metastasis. 86

88 Abstract #69 The EMT program is orchestrated by coordinated expression of multiple EMT transcription factors to promote breast cancer metastasis Joseph B. Addison 1,$, Maria A. Voronkova 1,$, James H. Fugett 1,$, Chen-Chung Lin 1, Nathaniel Linville 1, Brandon Trinh 1, Ryan H. Livengood 2, Matthew Smolkin 2, Michael Schaller 1, J. Michael Ruppert 1, Elena N. Pugacheva 1, Chad J. Creighton 3, and Alexey V. Ivanov 1# 1 WVU Cancer Institute and Department of Biochemistry, 2 Department of Pathology, West Virginia University, Morgantown, WV, 26506; 3 Department of Medicine and Dan L. Duncan Cancer Center Division of Biostatistics, Baylor College of Medicine, Houston, Texas, TX Several master regulator transcription factors (TFs) can activate the epithelial-to-mesenchymal transition (EMT), including members of the SNAI, ZEB, TWIST and TCF families. However, their individual and combinatorial contribution to EMT in breast cancer is not well defined. Here, we show that overexpression of individual SNAI1/2, ZEB1/2 or TCF3/4 in epithelial HMLE cells upregulated endogenous SNAI2, ZEB1/2, TCF4 and TWIST1/2 as a result of positive feedback. This secondary upregulation of endogenous ZEB1/2, TCF4 and SNAI2 was mediated at least in part by suppression of their negative regulator micrornas mir-200s/203/205. We identified TCF4 is a potential new target of mir200c. Expression of ZEB1/2 and TCF4, and to a lesser extent TWIST1/2 and SNAI2, correlated with the mesenchymal phenotype in human breast cancer cell lines and with lower expression of core epithelial genes in human breast tumors, indicating that the EMT program is tightly coordinated by several EMT TFs. In addition, human mammary stem-like CD24- /CD44+ cells overexpressed ZEB1/2 and TWIST1/2 but not SNAI1/2 or TCF3/4. Remarkably, ZEB1/2 and TCF4 were upregulated in super-metastatic MDA231 sublines. Knockdown of individual EMT TFs in mesenchymal MDA231LN and BT549 breast cancer cells confirmed the key role of ZEB1 and the functional cooperation of ZEB1 with other EMT TFs in the maintenance of the mesenchymal state. Furthermore, lung metastasis of MDA231LN cells in an orthotopic mouse xenograft model was blocked by inducible ZEB1+2 knockdown, even if ZEB1+2 were depleted one week after tumor cell inoculation, confirming the critical role of ZEB1/2 and EMT in dissemination from primary site. As expected, knockdown of ZEB1+2 in cells prior to tail vein injection inhibited lung colonization, supporting a key role of ZEB1/2 and EMT in extravasation. However, ZEB1+2 depletion one week post intravenous injection did not inhibit lung colonization, suggesting that ZEB1/2 and EMT are not essential for macrometastatic outgrowth. These results provide strong evidence that EMT is orchestrated by coordinated expression of several EMT TFs and establish the ZEB genes, and specifically ZEB1, as key master regulators of EMT and metastasis in breast cancer. 87

89 Abstract #70 DDR1 regulates the construction of a dormancy-supportive niche by coordinating the assembly of a pro-quiescence ECM proteome Di Martino J 1, Norbe R 2,3, Farias E 1, Mondal C 1, Fertig E 4, Naba A 5, Aguirre-Ghiso J 2, Bravo- Cordero JJ 1 1 Department of Medicine, Division of Hematology and Oncology, The Tisch Cancer Institute, Icahn School of Medicine at Mount Sinai, New York, USA 2 Division of Hematology and Oncology, Department of Medicine, Department of Otolaryngology, Department of Oncological Sciences, Tisch Cancer Institute, Black Family Stem Cell Institute, Mount Sinai School of Medicine, New York, USA. 3 Abel Salazar School of Biomedicine, Porto University, PT. 4 Department of Oncology, Sidney Kimmel Comprehensive Cancer Center, Johns Hopkins University, Baltimore, MD, USA 5 Department of Physiology & Biophysics, University of Illinois at Chicago, Chicago, USA Metastasis develops from disseminated tumor cells (DTCs) that leave the primary tumor and seed distant organs, where they can form secondary lesions. In those organs, DTCs undergo a period of dormancy during which cancer cells remain occult, in a non-proliferative state, before (macro)metastases become detectable. At the metastatic sites, DTCs encounter new microenvironments composed of blood vessels, stromal and immune cells and importantly, varying extracellular matrices (ECM). Many studies showed that certain ECM molecules like fibronectin or periostin could promote metastasis. However, how ECM sensing and remodeling can induce and sustain dormancy of DTCs is unclear. Further, whether DTCs assemble dormancysupportive ECM niches to sustain their phenotype is also an unanswered question. By using squamous carcinoma models, we have found that dormant tumor cells express a specific set of ECM and ECM remodeling genes, "the matrisome", that is lost upon break from dormancy. Further, we found that collagen receptors, such as DDR1, are upregulated upon dormancy onset and that DDR1 expression is required to maintain tumor dormancy through the regulation of key dormancy nodes (p38/erk). Depletion of DDR1 in DTCs interrupts dormancy leading to reactivation and multi-organ metastasis (lung, liver). The dormancy-to-reactivation transition is accompanied by changes in collagen-i alignment architecture measured by intravital imaging and second harmonic generation microscopy. ECM proteomic analysis further reveals significant changes in the ECM proteome of reactivated dormant tumors after DDR1 depletion, when compared with controls. Our data reveal a novel barrier to metastasis through a mechanism by which DTCs depend on DDR1 upregulation and assembly of a pro-quiescence ECM proteome to establish a niche that sustains dormancy. 88

90 POSTER SESSION II (TRAINEES ) ABSTRACTS Abstract # Abstract Title Author Y1 Characterization of Novel Drivers of Invasion and Metastasis in Lung Adenocarcinoma: Inositol Monophosphatase 3 and KDEL Receptor 2 Bajaj, Rakhee Y2 Y3 Y4 Y5 Y6 Y7 Y8 Y9 Y11 Y12 Y13 Y14 Inhibition of LCMR1 and ATG12 by demethylation-activated mir-570-3p is involved in the anti-metastasis effects of metformin on human osteosarcoma Mitochondrial genetics appear to alter immune cell development/ trafficking Copper oxide nanoparticles inhibit pancreatic tumor growth by targeting tumor initiating cells Intravital Microscopy at Single Cell Resolution Reveals the Mechanism of Cancer Cell Dissemination and Metastasis Expressions of metalloproteinases associated with p-erk in ovarian cancer tumors Bisphenol A induces focal contacts formation through a FAK, Src and GPER-dependent pathway in MDAMB-231 breast cancer cells An activated, pro-tumor lung microenvironment promotes the outgrowth of breast cancer lung metastases through lung-specific expression of the androgen receptor Elucidating skeletal muscle-derived factors that suppress breast cancer metastasis Protein Tyrosine Phosphatase Alpha (PTPα) is critical for invadopodiamediated cancer cell invasion in an MMP-dependent manner Limitrin (DICAM): a new prognostic biomarker and therapeutic target for breast cancer brain metastasis Tumor evasion from NK cell surveillance in metastatic breast cancer Extracellular matrix stiffness activates a novel mechanotransduction kinase cascade to promote EMT and tumor metastasis Bao, Xing Beadnell, Thomas Benguigui, Madeleine Borriello, Lucia Castillo, Rocio Castillo, Rocio Christenson, Jessica Crist, Sarah Decotret, Lisa Denoyer, Delphine Ducimetiere, Laura Fattet, Laurent Y15 RON kinase: a therapeutic target for cancer-induced bone destruction Fornetti Jaime 89

91 Abstract # Abstract Title Author Y16 Immediate early gene Ier2 stimulates migration and invasion of metastatic melanoma through inducing a senescence-associated secretory phenotype (SASP) Foss, Amelia Y17 Y18 Y19 Y20 Y21 Y23 Y24 Y25 Y26 Y27 Y28 Y29 Y30 Y31 Roles for Tumor-derived Matrix Metalloproteinase 3 (MMP-3) in Prostate Cancer Growth in Bone MicroRNA 520b mediated regulation of ATF5 contributes to homeostasis and reduced malignant phenotype in breast cancer cells In vitro Efficacy in Treating Metastatic Triple Negative Breast Cancer in Bone via a Targeted Calcium Phosphosilicate Nanoparticle (CPSNPs) Encapsulating Modified 5-Fluro-Uracil Microglia subset revealed by single cell analyses promotes brain metastasis outgrowth through fractalkine receptor-dependent interferon response Inhibition of Hedgehog signaling reprograms the dysfunctional immune microenvironment in breast cancer S100A4 elevation empowers expression of metastasis effector molecules in human breast cancer The Metastasis Suppressor NME1 (Nm23) Binds and Promotes the Oligomerization of Dynamin 2 to Regulate Tumor Cell Endocytosis and Motility Small molecule inhibitors of biomarker driven motility and metastasis Tumor Educated Osteoblasts Modify Osteoclasts in the Bone Microenvironment Quantifying epithelial-mesenchymal plasticity and its association with patient survival FGFR1/FGFR1 isoforms in prostate cancer progression to metastasis The role of hedgehog signaling in double-strand break repair pathway in TNBC p21-activated kinases (PAKs) regulate invadopodia activity in breast cancer cells Fascin Control Non-Small Cell Lung Cancer Metastatic expansion Through Regulating Metabolism by Remodeling Mitochondrial Actin Filaments Frieling, Jeremy Gaither, Kari Gigliotti, Christopher Guldner, Ian H. Hanna, Ann Ismail, Thamir Khan, Imran Kobelt, Dennis Kolb, Alexus Kumar Jolly, Mohit Labanca, Estefania Lama-Sherpa, Tshering Lesjak, Michaela Lin, Shengchen 90

92 Abstract # Abstract Title Author Y32 Structure-function study of POSTN: A Matricellular Protein associated with Oncogenesis and the Wnt/β-catenin Signaling Pathway Ibarra, Juan Y33 Y34 Y35 Y36 Y37 Y39 Y40 Y41 Y42 Y44 Y46 Y47 Y48 Purinergic receptor p2x7 in the maintenance of cancer stem cells and neuroblastoma metastasis Regulation of Tumor Metastasis by Allosteric regulation of E-cadherin Activation A dynamic equilibrium of differentially localized active ERK5 and its isoforms facilitates metastatic progression Deficiency of tumor suppressor Merlin facilitates metabolic adaptation by co-operative engagement of SMAD-Hippo signaling in breast cancer IL-4 receptor mediated metabolic and epigenetic changes leading to metastatic progression in triple negative breast cancer A molecular crosstalk between lung fibroblasts and highly metastatic breast cancer cells fuels metastatic colonization Recruitment and activation of hepatic stellate cells by uveal melanoma cells in a xenograft mouse model Investigating the Mechanism of CEBPD Gene Regulation by Histone Deacetylase Inhibitors in Inflammatory Breast Cancer Cells Expression of caveola-forming proteins caveolin-1 and PTRF and invasive potential of GBM The postpartum liver metastatic niche supports tumor cell survival: a role for liver lipids? Osteocytic Connexin Hemichannels in Suppression of Breast Cancer Bone Metastasis and Potential Therapeutic Application Selection of telomere homeostatic program controls metastatic behavior in breast cancer ONECUT2 is a Targetable Master Regulator of Lethal Variants of Prostate Cancer that Suppresses the Androgen Axis Martins, Poliana Mendonsa, Alisha Miranda, Mariska Mota, Mateus Williams, Demond Pein, Maren Piquet, Leo Poria, Dipak Kumar Pu, Wenjun Quackenbush, Alexandra Riquelme, Manuel Robinson, Nathaniel Rotinen, Mirja Y49 The Role of AIB1-D4 as a Driver of Breast Cancer Metastasis Sharif, Ghada Y50 Y51 The PERK/JAK2/STAT3 axis induces CEBPD expression in response to endoplasmic reticulum stress to promote cancer cell survival Crosstalk between osteoblasts and breast cancer cells alters breast cancer proliferation through multiple mechanisms Sheshadri, Namratha Shupp, Alison 91

93 Abstract # Abstract Title Author Y52 Y53 Melanoma phenotype switching and metastatic growth after dissemination. PAPP-A promotes pregnancy-associated breast cancer metastasis through collagen remodeling and a DDR2/Snail signaling axis Simmons, Jacinta Slocum, Elizabeth Y54 Y55 Y56 Y57 Y58 Y59 Y61 Y62 The Immune System Controls Dormant Metastasis in the Lungs Calpain-2 mediated amoeboid reprogramming and dissemination triggered by hypoxia Vimentin as a potential regulator of EMT-induced Tissue Factor during metastatic progression DNA Damage response drives chemo-resistance and supports metastasis in triple negative breast cancer Understanding the Role of Wnt Signaling-driven Nucleolar Dynamics in Breast Cancer Progression Nuclear Aurora-A Kinase: a novel driver of metastasis The young microenvironment selectively enhances brain metastasis in experimental models of metastatic triple-negative breast cancer Inhibition of brain metastasis by blocking MAPK12 driver kinase functions Tallon De Lara, Paulino Te Boekhorst, Veronika Vanwynsberghe, Aline Ward, Ambber Weeks, Shannon Whately, Kristina Wu, Alexus Akosua Badu- Nkansah Y63 SOX2 and the regulation of disseminated tumor cell (DTC) fate Carlini, Maria J. Y66 Y68 Y69 Y70 Y72 Y73 Inflammatory cytokines in breast tumor microenvironment lead to changes in the extracellular matrix conducive to metastasis Gain-of-function in vivo kinome screen reveals PCTK1/CDK16 as a driver of breast cancer brain metastasis Anti-androgens regulate amino acid-mtorc1 signalling and extracellular vesicle secretion to modulate prostate cancer progression WNT5A signaling: A promising target for anti-metastatic therapy MicroRNA-1205 regulation of FRYL in metastatic castration-resistant prostate cancer Establishment and characterization of highly metastatic breast cancer cell lines by orthotopic transplantation Dinca, Simion Jiang, Wayne McCormick, Kristie Mohapatra, Purusottam Naidoo, Michelle Nakayama, Jun 92

94 Abstract # Abstract Title Author Y74 Drawing a Genomic Atlas of Cancer Metastasis: Insights from Prospective Clinical Sequencing of 25,000 Patients Sánchez-Vega, Francisco Y76 Y77 Y78 Y79 Y80 Y81 Y82 Multiphase pseudo-continuous arterial spin labeling MRI for quantitative cerebral blood flow measurements in a rat model of breast cancer brain metastasis Matrix Remodeling by Semaphorin 7a promotes tumor invasion and cell survival Pleiotriphin and midkine promote metastasis in pre-clinical models of breast cancer Delineating Key Autophagic Markers in Breast Cancer Cell Lines Associated with an Aggressive Phenotype and Autophagic Inhibitor Sensitivity Loss of Tank-binding kinase 1 (Tbk1) in pancreatic cancer reduces tumor epithelial plasticity Genome-wide cooperation of the EMT inducer ZEB1 with YAP and AP-1 factors KISS1R signaling in triple negative breast cancer metastasis and metabolism Simard, Manon Tarullo, Sarah Ganguly, Debolina Giuliani, Charlett Arner, Emily Brabletz, Simone Nguyen, Mai- Uyen 93

95 POSTER SESSION II (TRAINEES ) Abstract #Y1 ABSTRACTS Characterization of Novel Drivers of Invasion and Metastasis in Lung Adenocarcinoma: Inositol Monophosphatase 3 and KDEL Receptor 2 Bajaj R 1, Kundu 1, Grzeskowiak C 2, Fradette J 1, Creighton C 2, Scott K 2, Don L Gibbons 1 1 UT-MD Anderson Cancer Center, Thoracic Head & Neck Medical Oncology, Houston, TX, 2 Baylor College of Medicine, Houston, TX Non-Small Cell Lung Cancer is the leading cause of cancer-associated deaths primarily due to its propensity to metastasize. Hence, therapeutically targeting metastasis could be a potential treatment approach for lung cancer. However, factors governing this complex process are poorly understood. To identify novel drivers of metastasis, we performed an in vitro screen by using 217 candidate driver genes. These candidates were selected based on increased gene expression in metastases versus primary tumors in the lung cancer GEMMS and in metastatic versus nonmetastatic lung cancer cell lines. This was then cross-referenced with human NSCLC data from TCGA. The invasive potential of the candidate genes was tested by performing assays on individual gain-of-function (GOF) non-metastatic murine lung cancer cell lines in 96-well invasion chambers. Driver hits were ranked based on their increase in invasion relative to the mcherry control. We identified many known drivers (e.g. MYC and SNAI2) and novel drivers (e.g. IMPAD1 and KDELR2) through our screen. We have determined that IMPAD1 and KDELR2 are necessary and sufficient for promoting invasion by using various mouse and human NSCLC cell lines. We established two distinct mechanisms of action for driving metastasis for both the hits: IMPAD1 activates AMPK, the energy sensor of the cell that promotes autophagy upon starvation, and KDELR2 increases secretion of ECM-degrading proteases. However, the study for their role in metastasis is ongoing. This exposes the multi-faceted and complex nature of metastasis and its regulation. Although EMT is a hypothesized model for metastasis initiation, downstream cellular mechanisms that connect EMT to metastasis remain unclear. We have identified IMPAD1 as a possible mir-200 target that is upregulated during EMT to promote metastasis. Overall, discovering such novel drivers and their mechanism for promoting lung cancer invasion and metastasis will help us develop new therapeutic targets for lung cancer patients. Abstract #Y2 Inhibition of LCMR1 and ATG12 by demethylation-activated mir-570-3p is involved in the antimetastasis effects of metformin on human osteosarcoma Xing B, Libo Z, Han G,Feng L. 94

96 Epidemiological studies have demonstrated that metformin could mitigate the progression of several tumors. Although it has been proved thatmetformin could cause demethylation of DNA and lead to up-regulation of some encoding genes and non-coding RNAs, there is little data about the effects of metformin on metastasis, and the interaction between metastasis and autophagy in human osteosarcoma cells. Here, we found mir-570-3p was significantly down-regulated in human metastatic osteosarcoma tissues but not in non-metastatic osteosarcoma tissues. Metformin attenuates the metastasis and autophagy in osteosarcoma. Interestingly, this autophagy favors osteosarcoma cells invasion. Moreover, reduction of metformin-induced inhibition of autophagy could reverse the invasion suppression in osteosarcoma. Mechanistically, metformin increases mir-570-3p by the demethylation of DNA, and the upregulation of mir-570-3p repressed the translation of its target, LCMR1 and ATG12. Our results, for the first time, presents evidence that the mir-570-3p-mediated suppression of LCMR1 and ATG12 is involved in the metformin-induced inhibition of metastasis in osteosarcoma cells. Abstract #Y3 Mitochondrial genetics appear to alter immune cell development/trafficking Beadnell T, Fain C, Brinker A, Vivian C, Welch D. Metastatic burden is the leading cause of cancer deaths; however, it remains unclear why some patients are more susceptible to metastatic disease. While the nuclear genome s role in tumor progression and metastasis is known, the role of mitochondrial DNA (mtdna) polymorphisms (SNP) has only recently been explored. Using mitochondrial nuclear exchange (MNX) mice, we showed that mtdna strongly influences mammary carcinoma progression and metastasis both intrinsically and via non-cell autonomous mechanisms. We hypothesized that mtdna SNP alter immune cell development/trafficking which, in turn, could influence metastasis efficiency. Peritoneal exudate and splenocytes were collected from male and female wild-type C57BL/6J (CC) and C3H/HeN (HH), and MNX mice - C57BL/6- mtmnx(c3h/hen) (CH) and C3H/HeNmtMNX(C57BL/6J) (HC) mice [first letter=nuclear; second letter=mitochondrial]. Flow cytometry identified significant and selective changes. B-cell populations in the spleen and peritoneum were primarily nuclear-driven as evident by comparison of the CC (61%;18%) and CH (61%;14%) backgrounds to the HH (55%;22%) and HC (53%;21%) backgrounds, respectively (p < 0.005). These differences remain consistent with aging [CC, 61%; CH, 59%, HH, 44%; HC, 50%]. Peritoneal macrophage numbers (F4/80 + ; CD11b +, CD11c - ) were higher in HH (61%) mice in comparison to CC (29%) mice (p < 0.001). C57mtDNA (HC) reduced the percentage of monocytes/macrophages (38%) compared to wild-type (HH) (p < 0.001). Lastly, analysis of lung metastases derived from histocompatible (i.e. ndna matched) tumor cell injection into wild-type or MNX mice, identified a 1.5-fold increase in CD8 + tumor-infiltrating lymphocytes in CH mice in comparison to wild-type CC mice (p = 0.07). Our data support the hypothesis that mitochondrial SNP modulate immune cell development and/or trafficking. Providing a plausible explanation for how metastatic potentials of syngeneic tumor cells injected into MNX mice are altered - i.e., mtdna manipulation of immune function 95

97 promotes/inhibits metastasis - which may provide insight as to why some immune therapies succeed/fail. Support: DOD BCRP BC (TCB); Susan G. Komen for the Cure SAC110037; National Foundation for Cancer Research and NIH CA (to DRW). Abstract #Y4 Copper oxide nanoparticles inhibit pancreatic tumor growth by targeting tumor initiating cells Benguigui M 1, Weitz S. I 2, Timaner M 1, Kan T 1, Shechter D 1, Sivan S 2, Raviv Z 1, Azhari H 3, and Shaked Y 1 Cancer stem cells, also termed tumor initiating cells (TIC) are a rare population of cells within the tumor mass which initiate tumor growth and metastasis. TICs have been identified in a number of cancer types and they have been shown to resist a spectrum of anti-cancer drugs. In pancreatic cancer, among other cancers, TICs significantly contribute to tumor re-growth after therapy, therefore focuses on identifying treatment modalities which can specifically eradicate TICs. Metal oxide nanoparticles (NPs) and in particular copper oxide (CuO) NPs, were shown to generate cytotoxic activity in cancer cells, in part by inducing apoptosis. Yet, the effect of CuO-NPs on TICs has not been demonstrated. Using in vitro TIC-enriched PANC1 human pancreatic adenocarcinoma culture we demonstrated that they are more susceptible to CuO-NPs cytotoxicity than standard PANC1 cultures. Specifically, TIC-enriched PANC1 cells exposed to CuO-NPs exhibited decreased viability and increased apoptosis rates compared to non-tics counterparts. Such effects are associated with increased G2 cell cycle arrest resulted in increased sub-g0 apoptotic cells. Furthermore, CuO-NPs contributed to reduced mitochondrial membrane potential and increased reactive oxygen species (ROS) levels in TIC-enriched cultures compared to standard PANC1 culture. Using an in vivo pancreatic cancer model in mice, we demonstrated that the administration of CuO-NPs significantly decreased tumor growth (by over 50%) when compared to the control untreated mice, albeit with increased toxicity. The CuO-NP-treated tumors contained apoptotic TICs, confirming a substantial CuO-NP anti-tumor activity by killing TICs. Overall, our results suggest a possible new therapeutic modality for pancreatic cancer which mainly targets TICs, hence extending survival by delaying drug resistance mediated by TICs. Abstract #Y5 Intravital Microscopy at Single Cell Resolution Reveals the Mechanism of Cancer Cell Dissemination and Metastasis Borriello L 1,2, Coste A 3, Wang Y 1,2,4, Oktay M 1,2,4,5, Entenberg D 1,2,3,4 and Condeelis J 1,2,3,4 Over the past decades, metastasis has been speculated to be an inefficient process, as the majority of disseminated tumor cells (TC) are not believed to complete all the steps of the metastatic cascade. This conclusion has been reached by studies which quantify metastatic foci 96

98 in the lung weeks after an intravenous injection of TC in mice, process called experimental metastasis model (EMM). Here, using a new technology, the Window for High Resolution Imaging of the Lung 1, we quantitatively assess, for the first time, multiple steps of the metastatic cascade: TC retention in the lung vasculature, extravasation, macrophage interaction, survival, dormancy, and growth to micro-metastases, all at single-cell resolution, longitudinally, in the same animal, and in both EMM and in mice which host a primary tumor, called the spontaneous metastasis model (SMM). Using this technology, we performed a comparative analysis which demonstrated that disseminated TC in SMM, a relevant clinical model, have drastically increased metastatic efficiencies compared to an EMM. We observed that TC in a SMM metastasize very efficiently, resulting in 62% of TC retained in the lung. In contrast, TC in an EMM are 10-fold less efficient, resulting in only 6% of TC retained in the lung. Furthermore, we observed that TC in a SMM extravasated in the lung very rapidly in contrast to the TC in an EMM (9hrs vs. 24hrs). Thus, these findings indicate that metastasis could be a more efficient process than previously thought, and the primary tumor microenvironment plays a critical role in educating TC and preparing the premetastatic niche. In conclusion, this study provides new insight into the contribution of individual steps of the metastatic cascade and identifies extravasation as the most appropriate step to target developing new strategies to prevent metastatic dissemination and death. (1). Entenberg D, Voiculescu S, Guo P, Borriello L, Wang Y, Karagiannis GS, Jones J, Baccay F, Oktay M, Condeelis J. (2017). A permanent window for the murine lung enables high-resolution imaging of cancer metastasis. Nat Methods. 15(1): PMID: / PMCID: PMC Abstract #Y6 Expressions of metalloproteinases associated with p-erk in ovarian cancer tumors Castillo Sánchez Rocío 1, Gómora María José 1, Morales-Vásquez Flavia 2, Pedernera Enrique 1, Hernández Martínez Azucena 3, Méndez Herrera María del Carmen 1. The ovarian cancer is a leading cause of death of gynecologic malignancies. The 90% of the cases correspond to epithelial ovarian cancer (EOC), which is presented in five histological subtypes: high-grade serous carcinoma, low grade serous, endometrioid, mucinous and clear cell. Extracellular signal-regulated protein kinases 1 and 2 (ERK1/2) are members of the super family MAPKs their activation carries substrate phosphorylation, including cytoskeletal proteins and transcription factors, stimulate the growth and the spread of tumor cells. There is evidence that the treatment with mitogen-activated protein kinases (MAPKs) inhibitors suppress significantly the migration and invasion, as well as the expression of matrix metalloproteinases (MMPs) in ovarian cancer cells. The aim of this study was to evaluate association of the phosphorylated 97

99 proteins ERK1/2 and MMP-2 and 9 in ovarian carcinoma. The expression of ERK1/2 and MMP-2 and 9, was evaluated in 82 surgical pieces formaldehyde- fixed and embedded in paraffin usingimmunohistochemistry technique. The results showed that ERK1/2 phosphorylated are strongly associated with MMP-2 expression in epithelium and stroma (p<0.001, p=0.014), respectively. The association of MMP-9 with ERK1/2 phosphorylated was not observed. The analysis of EOC by histological subtypes, showed p-erk1/2 and MMP-2 are associated in the endometrioid (p=0.025) and mucinous (p= 0.015) tumors; also a negative association between p- ERK1/2 and MMP-9 was observed in the stroma of borderline serous tumors (p=0.02). In conclusion, there is a clear association between activation of MAPKs signaling pathway evidenced through the phosphorylated ERK1/2 with the expression MMP-2, but absent for MMP9, this observation is differentially observed in EOC histological subtypes. Acknowledgments: Financial support of the Dirección General de Asuntos del Personal Académico, DGAPA-UNAM, PAPIIT IN224116, PAPIIT IN224617, INCan 008/034/OMI and HMEMyN SEDENA X RCS is a beneficiary of a post-doctoral grant of the DGAPA-UNAM. We are grateful to Histotecnologa Matiana Mil for their technical assistance. Abstract #Y7 Bisphenol A induces focal contacts formation through a FAK, Src and GPER-dependent pathway in MDA-MB-231 breast cancer cells. Rocío Castillo-Sánchez, Pedro Cortes-Reynosa 1, Alejandra Ordoñez-Moreno 1, Argelia Calvillo- Robledo and Eduardo Pérez-Salazar 1 Breast cancer is the most common cancer among women. Risk factors can be genetic, but some lifestyle factors and the exposure to environmental pollutants make it more likely to happen. BPA, an industrial synthetic chemical, has been related with the development of several diseases including breast cancer. In breast cancer, G-protein-coupled receptor coupled GPER expression has been associated positively with tumor size, Her-2 expression and metastasis and its activation is associated with EGFR transactivation. However, the signal transduction pathways mediated by BPA and its role as a promoter of migration and invasion in breast cancer cell that remain to be investigated. The aim in this study was to evaluate the mechanism of action through BPA promotes cell migration in breast cancer cells. Cultures MDA-MB-231 were transfected with sirna specific for GPER and later were treated with 1 µm of BPA. The activation of the proteins was evaluated using inmunoblot, cell migration was measure with scratch-wound method, the focal adhesion were visualized by immunofluorescence confocal microscopy and the role of EGFR was described using an inhibitor specific for EGFR. Our results showed that BPA promotes migration and an increase in the number of focal contacts in MDA-MB-231 breast cancer cells through a GPER dependent pathway. Moreover, BPA also induces activation of FAK, Src, and ERK2 through this receptor with the collaboration of EGFR receptor. In conclusion, our findings demonstrate that BPA induces the activation of signal transduction pathways, which mediate migration in MDA-MB-231 breast cancer cells through of a transmembrane intracellular receptor 98

100 GPER and via transactivation of the EGFR. These results suggest that BPA promotes breast cancer progression and the further support the hypothesis that BPA is a risk factor in breast cancer cases. Acknowledgments: This work was partly supported by a grant from ICYTDF (224/2012). R. C-S. was supported by a CONACYT predoctoral training grant. We are grateful to Nora Ruiz for their technical assistance. Abstract #Y8 An activated, pro-tumor lung microenvironment promotes the outgrowth of breast cancer lung metastases through lung-specific expression of the androgen receptor Christenson J, Spoelstra N, and Richer J The vast majority of breast cancer (BC) related deaths are due to metastatic disease, and the treatment options for metastatic breast cancer remain extremely limited, especially for patients with triple-negative breast cancer (TNBC). In order to improve the survival and quality of life of patients with metastatic BC, it is critical that we understand how the microenvironment supports metastatic growth and survival. We hypothesize that the lung microenvironment, in particular, responds to growing metastases by initiating wound repair, resulting in an activated microenvironment that supports the expansion and persistence of TNBC metastases within the lung. In this study, the MMTV-PyMT (mouse mammary tumor virus-polyoma middle T antigen) transgenic mouse model and late-stage metastasis models were used to study metastatic progression. In the lung surrounding large versus small metastases we measured, by immunohistochemistry, an increase in the percentage of fibroblasts and percentage/activation of type II alveolar epithelial (AEII) lung cells. This was accompanied by an increase in metastasisinfiltrating, pro-tumor M2 macrophages as well as activation of recruited neutrophils, as measured by degranulation of the activation marker S100A9. Inflammatory cytokine levels measured by cytokine array were altered in lungs with high versus low metastatic burden. Interestingly, compared to non-adjacent tumor-free MMTV-PyMT lung tissue, there was an increased percentage of androgen receptor (AR)-positive cells in the lung adjacent to metastases, and this effect was most significant within 50µm surrounding lung metastases, indicating a defined metastatic field effect. Initial co-immunofluorescence experiments demonstrated that approximately 25% of these lung-specific AR+ cells are AEII cells. Overall my data suggest that by the time patients present with established/detectable metastases, the metastatic microenvironment has already undergone fundamental alterations that render it a supportive tumor microenvironment. Consequently, in order to effectively treat metastatic disease we need to understand how to target this metastatic microenvironment. Abstract #Y9 Elucidating skeletal muscle-derived factors that suppress breast cancer metastasis Crist S 1,2, Nemkov T 3, Coleman I 1, Nelson P 1, Tapscott S 1, Hansen K 3, and Ghajar C. 1 99

101 Skeletal muscle is a striking example of a tissue that resists metastasis: only 0.2% of malignant cancer cases involve skeletal muscle metastases. Upon careful autopsy, however, microscopic lesions in skeletal muscle are detected almost 100-times more frequently. Taken together, these data suggest that disseminated tumor cells (DTCs) successfully reach skeletal muscle and survive within this tissue, but rarely colonize it. Although skeletal muscle has been recognized as an inhospitable site for metastases for decades, the anti-metastatic nature of skeletal muscle has eluded thorough molecular description. To address this biological mystery, I employed mouse models and organotypic cultures to identify a niche for DTCs within skeletal muscle, and factors within this tissue microenvironment that inhibit outgrowth of breast cancer cells. Specifically, my experiments incorporated three parallel approaches: 1) High-dimensional proteomics and metabolomics of the skeletal muscle niche; 2) an exploration of the dependence of skeletal muscle-mediated metastasis suppression on the skeletal muscle transcription factor MyoD; and 3) generation and transcriptional analyses of skeletal muscle-metastatic mammary cancer sublines. All three approaches provided insight into the molecular mechanisms required to keep disseminated breast tumor cells under growth control and pointed specifically to oxidative metabolism playing a significant role in the suppression of DTCs within skeletal muscle. Understanding microenvironments of tissues where metastases rarely, if ever, emerge could prove incredibly insightful. Our hope is that thoroughly characterizing the molecular mechanisms associated with oxidative metabolism within skeletal muscle will inspire targeted therapies to convert fertile soils into infertile ones in an enduring manner. Abstract #Y11 Protein Tyrosine Phosphatase Alpha (PTPα) is critical for invadopodia-mediated cancer cell invasion in an MMP-dependent manner Decotret, L. 1,2,4, Li, L. 2,4, Lim, C.J. 2,3, Bennewith, K. 1,4, and Pallen, C.J. 2,3,4 Metastasis has been estimated to account for over 90% of cancer-related deaths. Despite great efforts, there has been limited improvements made over the past few decades outlining a critical need for early detection and intervention. Some cancer cells have acquired the ability to move throughout the body through the formation of specialized structures called invadopodia ("invasive feet"). Invadopodia are classified as dynamic Src-regulated, actin-based protrusions of the plasma membrane that secrete matrix metalloproteases (MMPs) to degrade the surrounding extracellular matrix (ECM). Protein tyrosine phosphatase alpha (PTPa), a widely expressed transmembrane protein, acts in normal cells to promote cell migration via the formation of similar structures called focal adhesions. However, little is known about the role of PTPa in cancer cell motility. We hypothesize that PTPα regulates invadopodia formation and function to promote the invasive motility of malignant cells. PTPa knockdown MDA-MB-231 cells showed reduced migration and invasion compared to the control, which was rescued upon reintroduction of PTP suggesting PTP promotes tumour cell migration and invasion. Matrix degradation assays revealed that PTPα-depleted cells are 100

102 impaired in their ability to degrade ECM compared to the control. Interestingly, control and PTPαdepleted cells formed equivalent numbers of invadopodia, and PTPα co-localized with cortactin, actin, and MT1-MMP to punctate invadopodia-like structures. Together, these findings indicate that PTPα present within invadopodial structures, positively regulates invadopodia-mediated ECM degradation while not affecting invadopodia formation. Furthermore, PTP depletion decreased MMP9 expression and activity as well as cell surface expression of MT1-MMP (MMP14) outlining a possible reason for the dysfunctional invadopodia. These results have shown that PTPa is present within invadopodial structures and positively regulates invadopodiamediated tumour cell motility. Future research may reveal new mechanistic targets for therapeutic intervention to prevent cancer metastasis. Abstract #Y12 Limitrin (DICAM): a new prognostic biomarker and therapeutic target for breast cancer brain metastasis Denoyer D 1, Kim SH 2, Redvers RP 1, Nagpal A 1, Fuentes M 1, Anderson R 1 and Pouliot N 1. Brain metastases are associated with an extremely poor prognosis and are incurable. The mechanisms by which breast tumour cells home to and colonise the brain remain poorly understood. Accordingly, identification of predictive biomarkers or therapeutic targets is urgently needed. We used gene array profiling to identify novel brain metastasis genes differentially expressed between brain-metastatic 4T1Br4 and parental 4T1 mouse mammary tumours. These analyses revealed 17 genes significantly upregulated in brain-metastatic 4T1Br4 tumours. Limitrin (DICAM), a cell adhesion molecule never previously investigated in the context of cancer, was found to be significantly increased (~12-fold) in 4T1Br4 tumours. We validated these observations at the mrna and protein level by in silico analyses, immunoblotting and immunohistochemistry (IHC) in a panel of mouse and human cell lines or primary tumours and in a large cohort of tumours from breast cancer patients. These analyses revealed a strong prognostic association with TNBC and HER2 breast cancer, two subtypes of breast cancer associated with a high propensity to spread to the brain. Highest levels of limitrin were observed in brain-metastatic cells/tumours. In normal epithelial cells, limitrin interacts with, and modulates the function of αvβ3 integrin, a receptor previously implicated in breast cancer brain metastasis. Its expression is also elevated in brain-infiltrating lymphocytes associated with inflammatory pathologies. Consistent with these observations, we found that limitrin promotes attachment and transmigration of tumour cells across a monolayer of brain-derived endothelial cells in vitro. Further, exogenous expression of αvβ3 in limitrin-expressing TNBC cells increased the formation of intra-parenchymal brain lesions compared to cells expressing limitrin alone which formed predominantly intra-vascular lesions. 101

103 Collectively, these results indicate that limitrin has prognostic significance for brain-metastatic breast cancer and contributes to tumour cell crossing of the blood-brain barrier. Experiments evaluating the impact of limitrin suppression on TNBC brain metastasis are ongoing and will be presented. Abstract #Y13 Tumor evasion from NK cell surveillance in metastatic breast cancer Ducimetière L. 1, Tugues S. 1,2, Becher B. 1,2 Immunoediting is a process in which the immune system sculpts the highly heterogeneous tumor landscape by eliminating the most immunogenic cancer cells, thereby selecting clones that will be able to evade from it. So far, evidence in support of cancer immunoediting arises mainly from studies performed in the context of primary tumors but little is known when it comes to metastasis. Metastatic cells are exposed to further immune challenge than their primary tumor counterparts as they migrate to distant organs, where additional mechanisms are expected to take place in order for them to successfully evade the immune system. In particular, the innate lymphocytes Natural Killer (NK) cells could play a key role in this process due to their capacity to control the metastatic burden. Evading NK cell immunity could thus presumably be a requirement for tumor cells to form metastases. We used the 4T1 breast cancer model to study tumor cell recognition by NK cells in pulmonary metastasis. We found that metastatic cells arising in NK-depleted mice are functionally different from the ones growing in immune competent wild-type (WT) mice, as they could form lung metastases less efficiently upon isolation and re-injection into immune competent animals. To further understand the possible causes of this effect, we searched for phenotypical changes between tumor cells from lungs of WT and NK cell-depleted mice and observed a differential expression of various ligands reported to trigger a NK cell response. Following transcriptome analysis of these cells by Next Generation Sequencing (NGS) and epigenetic profiling by ATAC sequencing, we now aim at identifying mechanisms and pathways through which metastatic cells can escape from NK cells during metastatic disease. Abstract #Y14 Extracellular matrix stiffness activates a novel mechanotransduction kinase cascade to promote EMT and tumor metastasis Fattet L, Aubol B, Adams J, Pasquale E, Yang J. Mechanical forces exerted by extracellular matrix were recently recognized as potent regulatory signals in various cellular behaviors. Breast tumors are often detected due to their apparent 102

104 hardness compared to normal tissues, and increasing matrix stiffness correlates with distant metastasis and poor survival in breast cancer patients. These observations raise the question of how mechanical forces generated by the rigid tumor extracellular matrix (ECM) impact tumor progression and metastasis. In a polyacrylamide-based (PA) 3D Matrigel overlay culture system that recapitulates the range of physiological stiffness from normal mammary glands to breast tumors, mammary epithelial cells form normal ductal acini with intact adherens junctions and underlying basement membrane in the compliant ECM stiffness mimicking normal mammary glands, whereas they weaken their junctions and invade through basement membrane in the rigid stiffness similar to breast tumors. These cell morphological changes in response to increasing matrix rigidity resemble the Epithelial-Mesenchymal Transition (EMT) program. We previously reported that the transcription factor Twist1 is an essential mechano-mediator that promotes EMT and metastasis in response to ECM stiffness. High matrix stiffness promotes Twist1 nuclear localization through phosphorylation-dependent disruption of its interaction with cytoplasmic anchor G3BP2. Using radiometric kinase screening and mass spectrometry, we now unravel a novel mechanoresponsive kinase cascade including an ephrin receptor and a Src-family kinase that phosphorylates Twist1 to promote its nuclear translocation in response to ECM stiffness. Pharmacological or shrna-mediated inhibition of this mechanotransduction pathway rescues the Twist1-G3BP2 interaction, blocks ECM stiffness-induced EMT and invasion in 3D PA Matrigel cultures in vitro, and inhibits primary tumor invasiveness and lung metastases in a human DCIS xenograft model in vivo. Our ongoing study aims to uncover the molecular mechanism governing this novel mechanotransduction kinase cascade that senses and transmits the biomechanical signals from the tumor microenvironment to drive EMT and tumor metastasis. Abstract #Y15 RON kinase: a therapeutic target for cancer-induced bone destruction Fornetti J, Welm AL Breast cancer most commonly metastasizes to bone, where it causes bone destruction and significant problems for patients. Despite current therapies, many patients bone disease progresses, highlighting the need for additional treatments. We previously demonstrated that the macrophage stimulating protein (MSP)/RON tyrosine kinase signaling pathway is elevated in breast cancers and is associated with increased bone metastasis. In mice, MSP expression by mammary tumors causes spontaneous bone metastasis and bone destruction through activation of host RON, which is expressed by bone-resorbing osteoclasts. Based on these data, we hypothesize that MSP-expressing tumors promote bone destruction through activation of osteoclast RON, and that MSP/RON signaling may be a targetable pathway in breast cancer bone metastasis. To test this hypothesis, RON activity was depleted in osteoclasts and the effect on tumor-associated osteolysis was assessed using a PyMT intratibial tumor model. RON expression by osteoclasts was also characterized using a mcherry reporter. Data to date confirm RON 103

105 expression in mature osteoclasts and support a role for osteoclast RON as a mediator of osteolytic bone disease. MSP/RON signaling was also evaluated as a therapeutic target for bone metastasis in humans. Of 13 bone reaming samples obtained from breast cancer bone metastasis patients, 6 contained tumor by EpCAM expression and 4 of these expressed MSP. Additionally, RON inhibitor treatment in cancer patients (n=21) was associated with a decrease in the bone resorption marker CTX and increase in the bone formation marker BSAP. Notably, the best responses were in women (CTX=9 of 11, BSAP=10 of 11) leading us to speculate that the response to RON inhibitor treatment may be associated with an effect on age-related bone loss. Altogether, our clinical and preclinical data indicate that RON inhibitors may be effective against osteolytic bone metastasis and provide rationale for the continued investigation of RON inhibitors for use in patients. Abstract #Y16 Immediate early gene Ier2 stimulates migration and invasion of metastatic melanoma through inducing a senescence-associated secretory phenotype (SASP) Foss A 1, Kyjacova L 1, Rönsch K 1,2, Wallbaum S 2, Grau N 2, Utikal J 3,4, and Sleeman JP 1,2* Among the many factors contributing to tumor progression and metastasis, research indicates that premature senescence and the resulting senescence-associated secretory phenotype (SASP) induce chronic inflammation and may facilitate tumor cell dissemination. Ier2 is a member of the immediate early response family, a set of genes induced under proliferative and migratory stimuli in quiescent cells. Our analysis shows that high Ier2 expression is associated with poor survival in melanoma patients. To further evaluate the role of Ier2 in melanoma, we have utilized the RheoSwitch system to develop B16-F10 cell lines inducible for Ier2. Under Ier2 induction in vitro, B16-F10 cells demonstrated a p53/p21-dependent senescent-like phenotype, accompanied by a characteristic SASP, as well as enhanced mobility in monolayer migration assays and 3-D transwell invasion assays. In a spontaneous metastasis model, mice injected with B16-F10 Ier2- inducible melanoma cells demonstrated senescent-like characteristics within the primary tumor and developed lung metastases under induction of Ier2. Pursuing an investigation of an Ier2- mediated SASP on metastatic characteristics, we generated a B16-F10 cell line inducible for mutant Ier2 containing a mutation in the nuclear localization signal (NLS). In these mutants, the development of a senescent-like phenotype, SASP, and enhanced migration under Ier2 induction was abrogated. Among the SASP proteins downregulated in the Ier2-NLS mutant secretome was osteopontin (OPN), a matrix extracellular phosphoglycoprotein associated with metastasis and poor prognosis in melanoma patients. Importantly, functional assays using Ier2-inducible B16- F10 cells with CRISPR/Cas9 knockdown of OPN indicate that OPN in the SASP promotes the motility of non-senescent melanoma cells. In summary, this research encourages further investigation into the role of Ier2 and SASP within the tumor microenvironment and as potential targets for the prevention of metastasis in melanoma and other forms of cancer. 104

106 Abstract #Y17 Roles for Tumor-derived Matrix Metalloproteinase 3 (MMP-3) in Prostate Cancer Growth in Bone Frieling JS, Cook LM, Tauro M, Lynch CC. MMPs are integral mediators of bone metastasis due to their combined ability to degrade extracellular matrix and proteolytically regulate growth factor and cytokine bioactivity. Depending on tissue context, MMPs may either promote or inhibit tumorigenesis, therefore, it is essential to study individual MMPs in specific cancers to support the design and application of selective MMP inhibitors. Our lab has previously identified specific roles for MMP-2, -7, and -9 in bone metastatic prostate cancer, but despite elevated expression, roles for MMP-3 in the prostate cancer-bone microenvironment have not been defined. Using PCR and immunohistochemistry analyses, we observed strong MMP-3 expression in prostate cancer cell lines (PC3M, C4-2B, PaIII) and human prostate to bone metastasis biopsies. To interrogate the effects of MMP-3 on tumor growth, tumor-derived MMP-3 was ablated by shrna (PaIII). Loss of MMP-3 significantly mitigated prostate cancer cell growth in vitro, and using an in vivo intratibial model, MMP-3 ablation significantly decreased prostate cancer growth in bone as determined by bioluminescence and phospho-histone H3 staining (p<0.05). Ex vivo X-ray and microct analyses showed no significant changes in cancer-associated bone disease in this model. Taking a candidate approach, cytokine arrays were used to compare conditioned media of MMP-3 ablated cells to control. We noted a 60% increase of insulin growth factor binding protein 3 (IGFBP3), an established MMP-3 substrate, in MMP-3 ablated cells. The increased levels of IGFBP3 corresponded with a reduction in IGF receptor 1 (IGF1R) phosphorylation. Further, we observed diminished ERK and AKT phosphorylation downstream of IGF1R, which may explain the decreased growth observed following MMP-3 ablation. Here we demonstrate that tumor-derived MMP-3 significantly contributes to prostate cancer growth in bone. These data indicate that selective inhibition of MMP-3 and/or targeting of MMP generated neo-epitopes could be efficacious for the treatment of prostate to bone metastases. Abstract #Y18 MicroRNA 520b mediated regulation of ATF5 contributes to homeostasis and reduced malignant phenotype in breast cancer cells Gaither K, Madarampalli B, Liu D Breast cancer patients face a significant risk of metastasis, with 20-30% of all early diagnoses resulting in metastatic disease. According to the American Cancer Society, the overall 5-year survival for diagnosis at distant sites is just 27%. Cancer cells at distant sites face a variety of stresses and evidence is mounting that adaptation to these stressors may drive cancer metastasis. ATF5 is a widely expressed transcription factor that modulates survival, proliferation, 105

107 and differentiation and is upregulated under diverse cellular stresses such as amino acid deprivation, oxidative stress, and endoplasmic reticulum stress. We hypothesize that ATF5 is upregulated in invasive breast cancer in response to cellular stress, and that microrna (mirna) play a role in regulating the expression of ATF5. In silico analysis was used to identify potential mirna candidates. Luciferase assays were performed and transfections of precursor mirna were carried out in human MCF10A breast epithelial, MCF7 mammary epithelial, and MDA-MB-231 breast adenocarcinoma cell lines, and expression levels of ATF5 were measured under varying physiological conditions via Western Blot analysis. We demonstrate that mirna 520b reverses the upregulation of ATF5 under diverse stress conditions in MCF7 and MDA-MB-231 cell lines. Preliminary data indicate that ATF5 is upregulated during transformation of MCF10A cells with inducible Src and suppression diminishes transformation, while ATF5 is overexpressed in invasive MDA-MB-231 cells and suppression by mirna 520b reduces migratory capacity. In silico analysis of patient survival using publically available data sets in SurvExpress and SurvMicro shows that ATF5 upregulation is significantly correlated with reduced metastasis-free survival and reduction of mirna 520b is significantly correlated with reduced distant relapse-free survival in breast cancer patients, demonstrating clinical relevance. Our findings reveal a mechanism of ATF5 regulation in invasive breast cancer highlighting cellular stress, and may lead to novel therapeutic interventions for metastatic breast cancer patients. Abstract #Y19 In vitro Efficacy in Treating Metastatic Triple Negative Breast Cancer in Bone via a Targeted Calcium Phosphosilicate Nanoparticle (CPSNPs) Encapsulating Modified 5-Fluro-Uracil Gigliotti, C 1, Adair, B 2, Snyder, J 3, Gigliotti, N 2, Loc, W 4, Liu, Z-K 2, Adair, J 1,2, and Mastro, A 3 Targeted delivery of chemotherapeutics encapsulated in our CPSNPs have potential to treat human cancers such as metastatic breast cancer in bone which has no accepted protocol for treatment. The hypothesis for the current study was that nanoparticles encapsulating chemotherapeutics with surface bioconjugation of an anti-sense target molecule on the nanoparticles would promote cell death in the breast cancer without infiltrating the bone cells. The target molecule, a-cd71, for receptors associated with the triple negative human breast cancer cell membrane, MDA-MB-231, was combined with encapsulated Rhodamine WT (Red) fluorophore and a novel chemotherapeutic, phosphorylated 5-Fluoro-Uracil (FdUMP), in CPSNPs for fluorescent microscopy evaluation, DAPI staining, and MTS bioassays. The metastatic breast cancer cells in bone cell cultures within 48 hours after introduction of the CPSNP formulations at 400nM FdUMP demonstrated a fascinating range of behavior captured in fluorescent photomicrographs. There was wide spread evidence that the red fluorescing a-cd71-fdump- RhWT-CPSNPs associated with breast cancer cell membranes and, where red blossoming occurred, the small (20nm) CPSNPs dissolved and RhWT and FdUMP infiltrated the breast cancer cell cytosol. The onset of apoptosis was indicated by unraveling of many cell membranes of the breast cancer that decomposed into smaller spherical bodies, the so-called blebs associated with apoptosis. DAPI staining of the viable nucleic acid material in the cell nuclei indicated that only bone cells maintained robust viability relative to the apoptotic breast cancer cells up to 96 hours 106

108 after introduction of the nanomedical formulation. In contrast, 200uM free FdUMP was required to achieve similar effects validating the utility of the targeting of the nanoparticle formulations. MTS assays were more ambiguous in the metastatic cell cultures. Detailed approaches and experimental results to separate the effect of the mixed cell cultures on the measurements of mitochondrial activity provided by the MTS assay will be presented. Abstract #Y20 Microglia subset revealed by single cell analyses promotes brain metastasis outgrowth through fractalkine receptor-dependent interferon response Guldner I 1,4, Wang Q 1,4, Yang L 2,4, Chen D 2,4, Li J 3,4, Zhang S. 1,4 Breast cancer brain metastasis is an incurable malignancy that results from the colonization and outgrowth of disseminated tumor cells in an immunologically unique brain microenvironment. Possessing highly heterogeneous ontogenies, myeloid cells predominate the brain immune landscape. Microglia, self-sustaining brain-resident myeloid cells, and infiltrating bone marrowderived myeloid cells (BMDMs) cooperatively regulate brain homeostasis and disease. Yet, their functional roles in regulating brain metastasis outgrowth have not been fully revealed. Here, combining single cell analyses with transgenic mouse models, we elucidated the functional relevance of the myeloid heterogeneity in promoting brain metastasis outgrowth. First, genetic depletion of all myeloid cells significantly reduced brain metastasis incidence, implying a prometastatic role for myeloid cells. High-dimensional mass cytometry analysis showed that the brain metastasis myeloid landscape consisted of both microglia and BMDMs. Unexpectedly, inhibiting BMDM infiltration to metastases did not alter brain metastasis incidence or survival, suggesting that microglia are the primary myeloid effectors during brain metastasis. Furthermore, despite transcriptional heterogeneity among metastasis-associated myeloid cells (MAMs) as identified by single cell RNA-seq, all MAMs downregulated myeloid homeostatic genes, particularly Cx3cr1, compared to naïve myeloid cells. Knocking out Cx3cr1 in MAMs resulted in increased brain metastasis incidence and reduced survival. Mechanistically, ablation of Cx3cr1 led to a MAM transcriptome signature enriched in interferon signaling pathways and an upregulation of interferon-induced protein Cxcl10. Significantly, ectopic co-injection of recombinant Cxcl10 with tumor cells increased metastatic outgrowth. Therapeutically, treating mice with minocycline, an antibiotic with anti-neuroinflammatory properties, reduced MAM Cxcl10 expression and brain metastasis incidence, implicating minocycline as a potential antibrain metastasis therapy. Collectively, our results point toward a potent microglia population that drives brain metastasis through a Cx3cr1-Cxcl10 signaling axis. More broadly, our study identifies a unique myeloid state not yet characterized in other brain pathologies, which may have important implications in brain metastasis treatment. 107

109 Abstract #Y21 Inhibition of Hedgehog signaling reprograms the dysfunctional immune microenvironment in breast cancer Hanna A, Chen D, Samant RS, and Shevde LA In the tumor microenvironment, cancer cells participate in crosstalk with the surrounding stroma. This tumor-stromal interaction forms a balance dictating tumor-suppressing or tumor-promoting responses. Macrophages in the tumor microenvironment are plastic and can mediate several functions depending on their activation states. Tumor-associated macrophages co-exist as two major phenotypes: anti-tumorigenic, immune-eliciting classically activated M1 and tumorpromoting, immune-suppressive alternatively activated M2 macrophages. M2 macrophages specifically associate with more aggressive stages and poor clinical outcomes in breast cancer patients as they suppress the tumoricidal properties of the immune system, thus facilitating tumor proliferation and dissemination. Hedgehog (Hh) signaling is a critical developmental pathway; its activation modulates vital cellular processes like cell proliferation, differentiation, limb development, and angiogenesis. While the Hh pathway is normally tightly controlled, it can be deregulated, fostering tumorigenesis and tumor progression. Aberrant Hh signaling is particularly implicated in breast cancer progression and metastasis. In this study, we investigate the role of Hh signaling in polarizing breast cancer-associated macrophages toward the detrimental M2 subtype. We report that targeting different steps of the Hh signaling cascade attenuates the cytokine profile associated with M2 macrophages. We demonstrate that Hh signaling enables the molecular mechanism responsible for alternative macrophage polarization. We furthermore identify significant shifts in immune cell populations infiltrating the primary tumor upon the administration of an FDA-approved Hh inhibitor to an in vivo mammary tumor model. This altered immune profile is dynamically characterized by a reduction in immune-suppressive cells concomitant with enhanced antigen presentation properties and cytotoxic immune cell infiltration, overall culminating in reduced metastasis. Thus, we describe a novel role for Hh signaling in enabling pro-tumorigenic immunity by alternatively polarizing breast cancer-associated macrophages. This study identifies a novel strategy, with potential clinical implications to treat breast cancer through targeting aberrant tumor cell Hh activation and eliciting a robust anti-tumorigenic immune response. Abstract #Y23 S100A4 elevation empowers expression of metastasis effector molecules in human breast cancer Ismail T.M., Bennett D., Platt-Higgins A., Al-Medhity M., Barraclough R., and Rudland P. 108

110 One of the proteins of the S100 family, S100A4, is expressed at elevated levels in many forms of human cancer and has the ability to induce invasion and metastasis in benign mammary rodent cells. We demonstrate that Drosophila larvae expressing S100A4 protein, as determined by Western blot, increase invasion of the ventral nerve cord (VNC) to 75% and metastasis to distant organs in Drosophila strains expressing oncogenic Ras Val12 targeted to the optic lobes by the ey(3.5)uas enhancer-promoter combination. Drosophila strains expressing only targeted Ras Val12 and biologically inactive S100A4 produce tumours with only 22-24% invasion or metastases, while those producing S100A4 alone fail to induce any tumours or metastasis at all. Activation of JNK and production of MMP1 is increased in flies overexpressing the Ras Val12, S100A4 wild type genotype over those expressing Ras Val12 alone. These increases in activation of JNK and production of MMP1 in larvae expressing the Ras Val12 /S100A4 mutant genotype are largely lost. The inhibitory gene Basket (Bsk DN ) which suppresses the activity of c-jun N-terminal kinase (JNK) and inhibitory chemicals of JNK and MMP reduce the fraction of disseminating tumours by 2.7, 4.6 and 11.2 folds, respectively. In human breast cancer high levels of immunohistochemical staining for mammalian MMP2, 9 and 13 are independently associated with staining for S100A4 and with patient demise from metastatic disease. These results demonstrate that S100A4 can specifically induce invasion and metastasis in a non-mammalian system, via a novel pathway which in human breast cancer can terminated in metastatic dissemination. Since chemical inhibitors added directly to the medium of the Drosophila larvae can suppress metastasis within 7 days, this model represents a rapid, convenient and cheap method of screening in vivo for anti-metastatic drugs directed against specific components of this pathway. Abstract #Y24 The Metastasis Suppressor NME1 (Nm23) Binds and Promotes the Oligomerization of Dynamin 2 to Regulate Tumor Cell Endocytosis and Motility Khan I, Gril B, Steeg PS NME (NM23) functions as a metastasis suppressor gene- it inhibits the motility and migration of cancer cells in vitro and suppresses metastasis in multiple in vivo model systems. The NME homolog AWD controls Drosophila development from imaginal discs; recent studies have suggested an association of tumor cell endocytosis and SHI/dynamin function with AWD function. Dynamin is a key mediator of endocytosis; in its oligomerized form it clips off endocytic vesicles from the cell surface. We hypothesize that NME-mediated motility suppression in breast cancer metastasis is due to Dynamin-dependent increased endocytosis of cell surface receptors, needed for cellular motility. Overexpression of NMEs in two cancer cell lines increased perinuclear accumulation of transferrin receptor (TfR) and EGF receptor (EGFR) concurrent with motility and migration suppression; internalization of TfR and EGFR costained with Rab5, depleted AP2 from the cell surface, and exhibited increased Rab5-GTP levels, consistent with increased endocytosis. The dynamin inhibitors Iminodyne-22 and Dynole-34-2, or shrna-mediated downregulation of DNM2, impaired NME1 ability to augment endocytosis or suppress tumor cell motility. Using EGF- 109

111 mediated signaling through EGFR as a model in MDA-MB-231T breast cancer cells, NME1 altered pegfr and pakt expression in a DNM2-dependent manner, indicating the relevance of this interaction to downstream signaling. NME1/DNM2 interaction was confirmed in two-way coimmunoprecipitations. A novel mechanism of action for NME and DNM2 interaction was uncovered: Addition of NME1 to DNM2 in vitro facilitated DNM2 oligomerization and increased GTPase activity, both required for increased endocytosis and motility suppression. We report a novel mechanism of NME1 mediated motility/metastasis suppression by increasing DNM2- GTPase activity leading to increased endocytosis. Abstract #Y25 Small molecule inhibitors of biomarker driven motility and metastasis Kobelt D (1,2), Zincke F (1,2), Juneja M (1), Gohlke BO (2,3), Liebeskind J (1), Dahlmann M (1,2), von Kries JP (4), Lewis J (5), Gunkel N (2,6), Preissner R (2,3), Stein U (1,2) Purpose: MACC1 is a driver and prognostic biomarker for cancer progression and metastasis in a large variety of solid tumor types, particularly colorectal cancer. Combined analysis of S100A4 and MACC1 improves prognosis of high risk CRC patients. For S100A4, first small molecule inhibitors are described. Here we present first inhibitors targeting MACC1 expression for monoand combinatorial treatment. To screen for MACC1 expression inhibitors we isolated and described the MACC1 core promoter. We used the MACC1 core promoter in a reporter construct to screen different compound libraries for possible MACC1 expression inhibitors. Methods: We aimed to target MACC1 expression using two independent luciferase reporterbased high throughput screenings (HTS) comprising over compounds of four different compound libraries (ChemBioNet, Prestwick, NIH, Microsource). Most promising compounds were characterized in vitro using qrt PCR, Western Blot, EMSA, cell viability and motility assays. Possible drug target docking was shown in silico. We demonstrated the in vivo drug effects by BLI of xenografted mice. Combinatorial treatment targeting S100A4 and MACC1 in parallel was analyzed in vitro with live cell imaging and in vivo using BLI. Results: The first HTS revealed lovastatin as most potent MACC1 transcriptional inhibitor. It remarkably inhibited MACC1 promoter activity and expression resulting in reduced cell motility. Lovastatin impaired the binding of the transcription factors c-jun and Sp1 to the MACC1 promoter. In CRC-xenografted mice, it restricted MACC1 expression and liver metastasis. In the second HTS, again a statin, fluvastatin, emerged as most promising compound. Therefore, we investigated the effect of several statins on MACC1 expression and associated functional relevance. The combination of MACC1 and S100A4 inhibition is superior to the monotreatment in inhibition of motility and metastasis. Conclusions: This is the first identification of inhibitors restricting cancer progression and metastasis via the novel target MACC1. This drug repositioning might be of therapeutic value for CRC patients. 110

112 Abstract #Y26 Tumor Educated Osteoblasts Modify Osteoclasts in the Bone Microenvironment Kolb A and Bussard, K. Breast cancer cells disrupt the normal balance between osteoblasts and osteoclasts. Bone resorption exceeds bone deposition, forming osteolytic lesions. Our work reveals that osteoblasts are transformed into tumor educated osteoblasts (TEO) in the presence of disseminated breast cancer cells and have modified functions that may affect other cell types. We wanted to determine if TEOs alter osteoclast formation in bone. We hypothesized after contact with bone disseminated breast cancer cells, TEOs upregulate factors to stimulate preosteoclasts to form mature, multi-nucleated osteoclasts. We investigated whether TEOs or TEOderived factors facilitate osteoclastogenesis. Differentiated osteoblasts were treated with breast cancer conditioned media to produce TEOs, and subjected to qpcr analysis. TEOs exhibited increased mrna production of receptor activator of nuclear factor kappa-β ligand (RANKL), osteoprotegerin (OPG), and tumor necrosis factor alpha (TNFα) compared to uneducated osteoblast control. We then took a different in vitro approach to observe differences in osteoclast formation via tartrate resistance acid phosphatase (TRAP) stain. For this approach, TEOs and primary bone marrow monocytes or RAW cells (used as a surrogate for preosteoclasts) were co-cultured in the presence or absence of RANKL. Co-cultures were fixed and stained with TRAP to assess for multi-nucleated osteoclast formation. In the presence of RANKL, co-cultures formed multi-nucleated osteoclasts that were large and robustly stained for TRAP, whereas co-cultures in the absence of RANKL resulted in reduced numbers of multi-nucleated osteoclasts that were smaller in size and faintly stained compared to control. Overall, we can conclude that TEOs produce increased amounts of osteoclast-initiating factors compared to control osteoblasts yet are inefficient at forming osteoclasts in co-culture in the absence of RANKL, suggesting RANKL as the driving force behind mature osteoclast formation. Our future plans include assessing the resorptive properties of osteoclasts produced by TEO cells. Abstract #Y27 Quantifying epithelial-mesenchymal plasticity and its association with patient survival Jolly MK, George JT, Jia D, Tripathi SC, Hanash SM, Levine H Epithelial-Mesenchymal Transition (EMT) and its reverse Mesenchymal-Epithelial Transition (MET) often play crucial roles in cancer metastasis and drug/therapy resistance. Recent reports highlight that EMT and MET are not all-or-none processes; instead cells can attain a hybrid epithelial/mesenchymal (E/M) phenotype(s). But, a hybrid E/M phenotype(s) has (have) been tacitly assumed to be metastable that can be attained only transiently en route to EMT/MET, and thus remain poorly characterized. Rapid progress in mapping the regulatory networks for EMT/MET has enabled developing computational systems biology models to characterize a hybrid E/M phenotype(s). Here, using mechanism-based mathematical modeling, we identify a set of phenotypic stability factors 111

113 (PSFs) OVOL2 and GRHL2 that can help maintain cells in a hybrid E/M state. Next, we identify H1975 cells as stably maintaining a hybrid E/M state over multiple passages, and validate the role of these PSFs experimentally. We show that the knockdown of these PSFs that act as a brake on full EMT drives cells to a fully mesenchymal phenotype. Finally, we devise a statistical model built upon gene expression profiles that can quantitatively predict where a given sample lies on a scale of 0 (fully epithelial) to 2 (fully mesenchymal). Intriguingly, GRHL2 and OVOL2 were identified among the top predictors that could resolve a hybrid E/M phenotype, through an unsupervised screening, thereby reinforcing their suggested roles as PSFs. This model can recapitulate the experimentally observed behavior for multiple scenarios such as EMT induction, and unravels the association of a hybrid E/M phenotype with poor clinical outcomes across multiple tumor types. Collectively, our integrated theoretical-experimental approach enables a quantitative understanding of the role of a hybrid E/M state(s) in tumor progression, and reinforces the emerging notion that cells in a hybrid E/M state(s) may be more aggressive than those locked in a full EMT state. Abstract #Y28 FGFR1/FGFR1 isoforms in prostate cancer progression to metastasis Labanca E, Yang J, Shepherd P, Roberts J, Starbuck M, Broom B, Iyer M, Logothetis C, Chinnaiyan A, Navone N. Bone metastases typically develop in patients with advanced prostate cancer (PCa). We have previously reported that the fibroblast growth factor (FGF) axis is implicated in the pathogenesis of PCa bone growth, and that FGFR blockade has clinical activity in advanced PCa and bone metastases (PMID: ). In an RNA sequencing study of 183 human PCas we found that different samples express different FGFR1 transcripts. We then mined the TCGA PCa database to determine the expression profile associated with two well characterized FGFR1 splice variants, alpha and beta, which represent the most abundant protein coding transcripts found in PCa. We discovered that each isoform is associated with expression of different genes. Also, in gene set enrichment analysis, we found that FGFR1 beta (but not alpha) is associated with many pathways. In particular, FGFR1 beta is significantly associated with MAPK signaling cascade, signaling by FGFR in disease, and pathways in cancer, among others. In vitro studies of FGF signaling activation in PCa cells expressing FGFR1 isoforms alpha, beta or empty vector (EV), confirmed these results. Therefore, these results suggest that FGFR1 alpha and beta induce different genes. Importantly, when compared to PCa cells expressing EV, PCa cells expressing FGFR1 isoforms produce significantly more metastasis and reduced survival of mice injected intracardially with the cells. Furthermore, we found a significant increase of bone metastases in the group of mice injected with PC3 FGFR1 alpha and beta compared to PC3 EV. These results suggest that FGFR1 accelerates the metastatic phenotype of PCa cells. 112

114 In summary, our studies suggest that FGFR1 alpha and beta activate different genes and pathways in PCa cells thus conferring different phenotypes. We further propose that FGFR1 expression in PCa cells favors its metastatic dissemination to bone and this may be mediated at least partially by activating a PCa cells-bone cells interaction. Abstract #Y29 The role of hedgehog signaling in double-strand break repair pathway in TNBC Lama Sherpa T 1, 2, Lin V 3, and Shevde-Samant L. 4, 5, Ph.D. Triple-negative breast cancer (TNBC) patients have lower five-year survival rates than the other subtypes of breast cancer. Since hormone and HER2-targeted therapies are not effective for TNBC, there is a dire need for novel therapies. Treatment options for TNBC include genotoxic agents, like conventional chemotherapy and radiotherapy that induce DNA damage, including double-strand breaks (DSBs). DSBs are mainly repaired through homologous recombination (HR) and non-homologous end joining (NHEJ). HR results in slower, high-fidelity repair of DSBs, whereas NHEJ is a rapid, blunt-end ligation that is more error-prone. Ionizing radiation (IR)- induced DSBs can lead to cell death; however, an enhanced DSB repair pathway can allow resistance to radiotherapy. Hedgehog (Hh) signaling pathway, which is involved in normal embryonic development, is known to confer resistance to therapy in breast cancer. Hh signaling is aberrantly activated in breast cancer and its activation promotes proliferation and a stem celllike phenotype, allowing cancer cells to become resistant to microenvironment-imposed barriers. Importantly, RAD50 and MRE11 have previously been reported to interact with GLI1, a transcription factor that is a terminal effector of the Hh pathway. RAD50 and MRE11, along with NBS1, comprise the MRN complex, which binds and regulates the recruitment of DNA repair proteins to DSBs. Therefore, we hypothesized that Hh signaling promotes repair of IR-induced DSBs in TNBC. Our results indicate that Hh signaling is activated in TNBC cell lines after IR, and its inhibition delays DSB repair in TNBC due to higher DNA damage accumulation. Specifically, we were able to assign a role for Hh signaling in impacting the NHEJ repair pathway. Ongoing investigations are designed to further evaluate the mechanistic role of Hh signaling in NHEJ DNA repair and the functional benefits of combining Hh inhibition with radiotherapy in TNBC. This study will be crucial to develop novel therapies and improve the outcomes of TNBC patients. This work is supported by NIH grant R01CA Abstract #Y30 p21-activated kinases (PAKs) regulate invadopodia activity in breast cancer cells Lesjak MS, Wells CM p21-activated kinases (PAKs) are serine/threonine kinases best known as downstream effectors of the small GTPases Cdc42 and Rac1. PAKs have been implicated in the regulation of various 113

115 cellular processes including actin cytoskeletal dynamics, cell motility, survival, and oncogenic transformation. Overexpression of PAKs found in several cancer types correlates with poor prognosis, tumour aggressiveness and metastasis. During invasion, cancer cells form proteasesecreting protrusions, termed invadopodia, which are rich in actin and facilitate matrix degradation. These invadopodia enable cancer cells to invade the surrounding extracellular matrix and have been shown to be important for tumour cell dissemination. PAK1 and PAK4 expression is significantly increased in high-grade breast cancer patient samples. This prompted us to investigate the influence of the PAK family on the ability of breast cancer cells to form invadopodia. sirna-mediated knockdowns in MDA-MB-231 cells showed that both PAK1 and PAK4 are required for invadopodia activity. Moreover, these findings could be confirmed by isoform-specific pharmacological inhibition. In agreement with previous results gained in melanoma cells, the two isoforms have distinct functions during the invadopodia life cycle; PAK1 is needed for nascent invadopodia formation, while PAK4 drives invadopodia maturation. Importantly, injecting stable PAK4-depleted MDA-MB-231 cells into the tail vein of nude mice resulted in reduced metastasis to the lung compared to control cells. Furthermore, we could identify a protein interaction between the atypical Rho GTPase RhoU and PAK4. Interestingly, knockdown of RhoU in MDA-MB-231 cells and subsequent tail vein injection into nude mice also reduced metastasis to the lung. Therefore, we are currently investigating a potential role of RhoU for invadopodia formation. Overall, our results support a crucial role for PAKs in cancer dissemination across tissue types and identify these proteins as promising therapeutic targets. Abstract #Y31 Fascin Control Non-Small Cell Lung Cancer Metastatic expansion Through Regulating Metabolism by Remodeling Mitochondrial Actin Filaments Lin S, Huang C, Gunda V, Chellappan SP, Li Z, Izumi V, Fang B, Koomen J, Singh PK, Hao J, Yang S Although the actin cytoskeleton dysregulation is known to promote cancer cell migration and invasion, the post-dissemination role of the actin cytoskeleton in cancer metastasis is not clear.. Fascin is a pro-metastasis actin bundling protein required for the maximal crosslinking of F-actin into straight and rigid bundles. Fascin overexpression invariably correlates with aggressive clinical course, metastatic progression and shorter survival across different types of carcinoma. It is generally believed that fascin plays a mechanical role in driving tumor cell migration and invasion by facilitating membrane protrusions. However, there is also increasing evidence implicating fascin in oncogenesis, metastatic colonization, anoikis resistance, chemoresistance and cancer cell stemness. Molecular mechanisms underlying these non-canonical functions of fascin are not clear. Here we report that fascin, a pro-metastasis actin bundling protein, regulates mitochondrial metabolism and metastatic expansion in non-small cell lung cancer by remodeling mitochondrial actin filaments (mtf-actin). Mechanistically, fascin and mtf-actin controls mitochondrial oxidative phosphorylation (OXPHOS) and the biogenesis of respiratory complex through the homeostasis of mtdna nucleoids. By augmenting mitochondrial OXPHOS, fascin promotes resistance to energy stress-induced cell death. Ablation of fascin in post-extravasation 114

116 cells using inducible CRISPR/Cas9 remarkably inhibited metastatic expansion of disseminated Lewis lung cancer cells. Our data indicate that the actin cytoskeleton dysregulation in cancer cells may promote metastatic expansion by rewiring mitochondrial metabolism. Abstract #Y32 Structure-function study of POSTN: A Matricellular Protein associated with Oncogenesis and the Wnt/β-catenin Signaling Pathway Ibarra J, Cadigan K. Periostin (POSTN) is a 90-kDa matrix protein, originally known as osteoblast-specific factor-2 (OSF-2) whose elevated expression in mammalian tissues has been identified upon injury and neoplastic events. Notably, POSTN overexpression has been found in several cancers in human including breast, ovarian and colorectal cancers, as well as hepatocellular carcinomas, where it has been associated with oncogenesis, tumor progression and poor prognosis. In its role as a ligand for αvβ3 and αvβ5 integrins, POSTN has shown to support cell survival, motility, invasion and metastasis through activation of the PI3K-Akt and focal adhesion kinase (FAK) signaling. Interestingly, through an integrin-independent function it has been suggested that POSTN binds to Wnt ligands, leading to potentiation of Wnt/β-catenin signaling in cancer stem cells (CSCs). While association between POSTN and integrins and/or Wnt signaling are plausible mechanisms to explain POSTN s oncogenic activity, the functional role by which POSTN promotes tumorigenesis remains poorly understood. Recently, the Cadigan lab has found that human POSTN can mimic the action of the matrix protein Tiggrin in the hematopoietic system of Drosophila. This Tiggrin function is thought to be independent of integrin signaling. In order to gain further insights on POSTN s function in mammalian systems, I will perform a structurefunction analysis of human POSTN. I will alter a region predicted to be required for its interaction to αvβ3 and αvβ5 integrins as well as several regions of sequence similarity between POSTN and Tiggrin. POSTN function will be tested in several assays for cell motility and migration in several human cell lines. The role of POSTN in potentiating Wnt/β-catenin signaling will be analyzed through using Wnt reporter genes. These approaches will determine whether the integrin and Wnt signaling functions of POSTN are separable by mutation. These reagents will ultimately be used to determine the contributions of specific POSTN functions to oncogenesis. Abstract #Y33 Purinergic receptor p2x7 in the maintenance of cancer stem cells and neuroblastoma metastasis 1 Martins P C de M, 1 Seeger R L, 2 Lima W J R, 3 Liberti E A, 1 Ulrich H, 1 Lameu C. INTRODUCTION: Neuroblastoma is a neuroendocrine tumor of the nervous system that represents 10% of pediatric cancer mortality with 50% of cases in metastasis at diagnosis. The theory of cancer stem cells (CSC) assumes that stem-like cells in the tumor mass are resistant to 115

117 current therapies and therefore responsible for relapse and metastasis. Studies indicate the involvement of the purinergic system in tumor progression. The P2X7 receptor is well described as a purinergic mediator in cell differentiation and cell proliferation. AIM: To study purinergic signaling in the formation of CSC and neuroblastoma metastasis. MATERIAL AND METHODS: Culture of tumorspheres in the presence of ATP or ATP plus P2X7 antagonist, A Determination of pluripotency marker (SOX-2) and actin filaments by confocal microscopy. The types I and III of collagen fibers were evaluated using Picrosirius red staining under polarized light. Long-term murine model of xenotransplantation injected with human neuroblastoma cells and chronically treated with P2X7 receptor antagonist, Brilliant Blue G (BBG). RESULTS: Tumorspheres treated with ATP or ATP plus A exhibited higher expression of SOX2 and actin. In vivo, the P2X7 receptor antagonist significantly decreased tumor size and the spread of neuroblastoma cells to the metastatic niches. In addition, tumors from the BBG treated animals showed a network of type I collagen fibers in the tumor interface, suggesting a barrier to tumor invasion. CONCLUSION: The data evidence the importance of P2X7 receptor in the maintenance of CSC and metastasis. This suggests that the activation of P2X7 receptor leads to the transformation of neuroblastoma cells into a state of greater malignancy. Therefore, the use of P2X7 antagonists in therapeutic alternatives may represent a possibility of therapy for neuroblastoma. Supported by FAPESP. Abstract #Y34 Regulation of Tumor Metastasis by Allosteric regulation of E-cadherin Activation Mendonsa A, Gumbiner B. Metastasis is responsible for most of the morbidity and mortality associated with cancer. Therefore, a better understanding of the mechanisms that govern metastatic transformation of tumor cells will aid identification of novel targets for treatment. Loss of E-cadherin, the epithelial adhesion molecule, by virtue of its ability to hold tumor cells together and prevent migration has been shown to facilitate metastasis. However, many metastases still express E-cadherin and EMT is not always necessary. Additionally, mutations in E-cadherin arecausative for hereditary diffuse gastric cancer (HDGC). Even though some mutations result in protein truncation or loss of adhesive ability, many still express E-cadherin and retain adhesive function. We have developed activating antibodies that can enhance E-cadherin adhesion and activity which results in decreased p120 phosphorylation. Further, we have shown that E-cadherin activation results in contact inhibition of proliferation and induction of the Hippo signaling pathway. I thus hypothesized that alterations in E-cadherin activity due to regulation of its activity at the cell surface, p120 phosphorylation status or E-cadherin mutations allosterically effect E-cadherin mediated regulation and facilitate tumor growth and metastasis through altered downstream signaling. To test this hypothesis, I have developed tumor cell lines that have altered E-cadherin levels, p120 phosphorylation status or express HDGC E-cadherin mutations. Using these cell lines, I have evaluated cancer related phenotypes in-vitro and in-vivo. Enhanced E-cadherin activity in tumor cells led to a decrease in migration and invasion in-vitro and in-vivo using the mammary fat pad model of tumor metastasis. Further experiments to assess the use of E-cadherin 116

118 activating antibodies and whether cancer associated E-cadherin mutations alter the activation of the Hippo pathway and influence breast cancer progression and metastasis are underway. Our findings imply that homophilic binding of E-cadherin is allosterically regulated and can effect tumor progression and metastasis in multiple ways. Abstract #Y35 A dynamic equilibrium of differentially localized active ERK5 and its isoforms facilitates metastatic progression Miranda M, Saunus J, Akgül S, Kutasovic J, Simpson P, Lakhani S and Al-Ejeh F. Introduction: Triple negative breast cancer (TNBC) is a subtype of breast cancer known for its aggressive behavior and poor prognosis. We previously reported that ERK5 is an attractive target in TNBC. ERK5 activation has been implicated in proliferation, metastasis and drug resistance in several cancers. ERK5 is a convergent effector that acts downstream of multiple different cell surface receptors and is an emerging therapeutic target in oncology. ERK5 regulates processes involved in tumor growth, differentiation and microenvironmental adaptation, including adhesion, cytoskeleton organization, epithelial-to-mesenchymal transition (EMT), cytokine signaling and cancer-associated inflammation. So far, it has been unclear how ERK5 achieves specific outcomes with such multifaceted roles as a convergent signal transducer. Methods and Results: Using in vivo models of metastasis, we found that expression of ERK5 is essential for metastatic colonization in mouse models of breast cancer, and that its subcellular localization shifts during progression. Comprehensive bioinformatic and functional analyses revealed that this is underpinned by the ratio of expressed splice isoforms, which determines how the pool of signaling-active ERK5 protein (perk5) is distributed between nuclear and extranuclear compartments. Clinically, immunohistochemical analysis of activated ERK5 expression in patient cohorts revealed that primary breast tumors with extranuclear perk5 exhibit correlates of poor clinical outcome, including mesenchymal phenotype, earlier distant relapse and tamoxifen resistance, whereas nuclear perk5 is associated with epithelial differentiation and longer overall survival. Consistent with its pivotal role in transducing exogenous signals from the cell surface, isoform-specific expression of ERK5 was associated with survival for every cancer type we investigated in silico. Conclusion: Our studies reveal for the first time that ERK5 isoforms may play an important role in orchestrating metastatic progression through its dynamic localization in cancer cells and suggest that ERK5 therapeutic targeting strategies should account for its context-dependent biological functions. 117

119 Abstract #Y36 Deficiency of tumor suppressor Merlin facilitates metabolic adaptation by co-operative engagement of SMAD-Hippo signaling in breast cancer Mota M 1,2, Jackson W 1,2, Bailey S 1, Samant R 1,3, Shevde-Samant L 1,3 Merlin tumor suppressor is encoded by the NF2 gene. Merlin restrains cell proliferation through inhibiting contact-dependent growth. In breast cancer, transcripts of NF2 do not show relevant mutation; however, our laboratory has shown that Merlin protein levels are decreased in breast cancer patient samples as disease progresses to metastasis. In the current investigation, we determined that loss of Merlin in breast cancer tissues is concordant with the loss of the inhibitory SMAD, SMAD7, of the TGF-β pathway. In order to simulate Merlin deficiency, we stably knocked down NF2 in breast cancer cells, which confirmed dysregulated activation of TGF-β signaling and revealed a cooperative engagement with effectors of the Hippo pathway (YAP/TAZ/TEAD). As a consequence, the loss of Merlin in breast cancer resulted in a significant metabolic and bioenergetic adaptation of cells characterized by increased aerobic glycolysis and decreased oxygen consumption. Mechanistically, we determined that the concomitant activity of the Hippo and TGF-β transcription effectors caused upregulation of the long non-coding RNA Urothelial CancerAssociated 1 (UCA1) that disengaged Merlin s check on STAT3 activity. The consequent upregulation of Hexokinase 2 (HK2) enabled a metabolic shift towards aerobic glycolysis. In fact, Merlin deficiency engendered cellular dependence on this metabolic adaptation, endorsing a critical role for Merlin in regulating cellular metabolism. This is the first report of Merlin functioning as a molecular restraint on cellular metabolism. Thus, breast cancer patients whose tumors demonstrate concordant loss of Merlin and SMAD7 may benefit from an approach of incorporating STAT3 inhibitors. Abstract #Y37 IL-4 receptor mediated metabolic and epigenetic changes leading to metastatic progression in triple negative breast cancer Williams, D and Fingleton, B. Department of Pharmacology, Vanderbilt University, Nashville, TN, USA Introduction: Interleukin 4 (IL-4) is a Th2 cytokine that has well-defined functions in immune cells. IL-4 can increase the proliferation and survival of lymphocytes, as well as cause polarization of macrophages to the pro tumor M2 phenotype. IL-4 receptors that are distinct from the version on lymphocytes are found on the surface of other cell types, including many tumor cells. This type II IL-4 receptor is a heterodimer comprised of the IL-4 ligand binding protein IL4Rα, and a co-receptor IL-13Rα1. Previous work from our lab showed that tumor cell expression of IL4Rα significantly contributes to the metastatic phenotype in experimental metastasis models in mice. Additionally, binding of IL-4 alters metabolism of murine breast cancer cell lines. Thus, we now test the hypothesis that in human triple negative breast cancer, IL-4 alters metabolic pathways which leads to enhanced metastatic behavior. Since altered metabolic pathways can regulate 118

120 epigenetic enzymes, we are pursuing histone modifications as a mechanism for the metastatic phenotypes observed. Methods: MDA-MB-231 and BT549 triple negative breast cancer cell lines were treated with IL-4 for 2, 4, 6, and 8 days. Samples of medium were collected and assayed for glutamine, and glucose consumption, as well as lactate and glutamate secretion. Whole cell lysates were analyzed by western blotting for expression of the glucose transporter Glut1, glutamine transporter ASCT2, and the IL-4 downstream effector phospho-stat6 (pstat6). Nuclear fractions of cells with and without IL4 treatment were assessed for histone acetyltransferase (HAT) activity, and the presence of acetylated histones was determined by immuno-blotting. Clonogenic survival assays were performed in the presence of IL4 with or without HAT inhibitors. Results: We found that IL-4 increased the consumption of both glucose and glutamine, as well as increased the secretion of glutamate, and lactate in the human triple negative breast cancer lines. Enhanced expression of transporters, as well as membrane localization may be responsible for this enhanced uptake. In addition, IL-4 led to significant increases in histone acetyltransferase activity as well as increased acetylation marks on histone H3. Conclusions: The data suggests that IL-4 alters the metabolic phenotype of triple negative breast cancer cell lines, while also altering the activity of epigenetic enzymes. Together these activities appear to result in enhanced survival and outgrowth of metastatic lesions. We believe that IL4 signaling mediated through the type II IL4 receptor could be an important therapeutic target to thwart metastasis in aggressive triple negative breast cancers. Abstract #Y39 A molecular crosstalk between lung fibroblasts and highly metastatic breast cancer cells fuels metastatic colonization Pein M, Insua-Rodríguez J, Meier J, Wiedmann L, Hongu T, Sinn H-P, Spaich S, Sütterlin M, Schneeweis A, Oskarsson T Metastasis accounts for most cancer-related deaths and is still essentially incurable. In breast cancer, the lungs represent one of the main sites of metastasis. It is recognized that not only inherent attributes of cancer cells determine their ability to metastasize, but also components of the microenvironment. Activated fibroblasts are key components of the tumor microenvironment in many cancers, including breast cancer. However, it is unclear how fibroblasts evolve during metastatic colonization of the lungs to establish a metastatic niche. Isolation of fibroblasts from healthy and metastatic tissues has been challenging due to the lack of a specific fibroblast marker. To characterize fibroblasts at different stages of metastatic colonization in an unbiased manner, we established a xenograft mouse model of breast cancer metastasis to the lungs and used a combination of positive and negative markers for FACSassisted fibroblast isolation. Transcriptomic analysis revealed that fibroblasts undergo dynamic changes during metastatic colonization, encompassing enhanced proliferation, contractility, 119

121 extracellular matrix production and inflammatory signaling. Interestingly, highly metastatic cancer cells induced fibroblast transition more efficiently than less metastatic cells. Using mouse models and in vitro assays, we found that highly metastatic breast cancer cells secrete IL-1α/β that induce CXCL9/10 in lung fibroblasts in an NF-κB-dependent manner. Fibroblast-secreted CXCL9/10 in turn enhanced metastatic growth via binding to CXCR3, which is expressed by a subset of highly metastatic cells. Intriguingly, CXCR3-positive cancer cells expressed higher levels of IL-1α/β, fueling the pro-metastatic CXCL9/10-CXCR3 crosstalk. Thus, the metastatic ability of breast cancer cells can be partly attributed to their ability to engage with lung fibroblasts. Disrupting the IL-1α/β-CXCL9/10-CXCR3 axis significantly reduced metastatic colonization in xenograft and syngeneic mouse models. Therefore, targeting this specific cancer cell-fibroblast crosstalk may represent a promising strategy against metastatic breast cancer. Overall, our study provides novel insights into the dynamic evolution of fibroblasts during metastasis. Abstract #Y40 Recruitment and activation of hepatic stellate cells by uveal melanoma cells in a xenograft mouse model Piquet L,(1-3), Gerges P,(2-3), Pelletier W, (2-3), Bérubé J,(2-3), Mouriaux F(1, 4), de la Fouchardière A,(5), Landreville S,(1-3) Purpose: Uveal melanoma (UM) spreads to the liver in half of the cases, and the survival rate following the report of metastasis is less than 10% at 2 years. Activated hepatic stellate cells (HSCs) play pivotal roles in the metastatic progression of colorectal and pancreatic cancers. We thus tested the hypothesis that HSCs would provide a suitable microenvironment that will enhance the UM cell invasion using a xenograft mouse model of metastatic UM. Methods: Fluorescent metastatic UM cell lines H79, MU2F, TJU-UM001 and OMM2.3 were inoculated alone or with human HSCs in the spleen of immunodeficient mice to generate microand macrometastases in the liver in 3-6 weeks. The growth of the hepatic lesions was imaged with an in vivo imaging system. Histological analyses were performed on formalin-fixed paraffinembedded (FFPE) tissue sections using hematoxylin & eosin and Masson s trichrome stains, and antibodies against MART-1, α-sma, Ki67 and CD31 (imaging software : NDP.view 2). We compared our findings with stainings in UM liver metastases from patients treated by partial hepatectomy (N=12). Results: All UM cell lines, inoculated alone or with human HSCs, spread to the liver as micro- or macrometastases. Furthermore, UM cell lines exhibited various invasive capabilities. Histological analyses revealed multiple metastasis foci, a significant recruitment of both host and injected human HSCs near the lesions, and an increase in extracellular matrix production. Interestingly, activated HSCs surrounding micro- and macrometastasis, as well as desmoplastic areas were also found in patient hepatectomy samples. Conclusion: The results in both our xenograft model and a cohort of patients are consistent with our hypothesis that HSCs are recruited and activated by UM lesions, thus providing a permissive 120

122 microenvironment to invasion. Further characterization of the paracrine signaling of activated HSCs and their desmoplastic stroma will be needed to understand how to antagonize these prometastatic contributors. Abstract #Y41 Investigating the Mechanism of CEBPD Gene Regulation by Histone Deacetylase Inhibitors in Inflammatory Breast Cancer Cells Poria D 1, Balamurugan K 1, Krishnamurthy S 2, Sharan S 1, Kim S 3,Baek S 3, Fujiwara S 3, Hager GL 3, Ueno NT 2, and Sterneck E 1 Inflammatory breast cancer (IBC) is a highly metastatic subtype of breast cancer. Skin invasions and formation of emboli within the lymphovascular space are hallmarks and causation for the clinical diagnosis of IBC. The transcription factor C/EBPdelta (CEBPD) promotes metastasis, hypoxia adaption and inflammatory signaling (Balamurugan et al., EMBO, 2010; Nat. Com. 2013) and supports the generation and/or maintenance of breast cancer stem cells (CSCs), which are implicated in treatment resistance and metastasis (Balamurugan et al, submitted). Investigating a potential role of CEBPD in IBC, we found that CEBPD was expressed in a subset of IBC tissues including emboli. In IBC cell lines, CEBPD not only promoted stemness (sphere formation) but also emboli formation in culture. Inhibitors of histone deacetylases (HDACs) including Romidepsin, which has shown efficacy against IBC cell lines in pre-clinical models (Robertson et al., J Exp Ther Oncol, 2013), rapidly downregulated CEBPD expression at the transcriptional level in breast cancer cell lines including IBC cell lines. We are investigating the molecular mechanism by which HDACi inhibit CEBPD transcription to determine the molecules/pathways that drive CEBPD expression in breast cancer cells, and to eventually identify more specific agents to inhibit CEBPD expression. As an unbiased approach, we used RNA-Seq together with ATAC-seq (Assay for Transposase-Accessible Chromatin using Sequencing) to assess the gene expression changes linked with chromosomal accessibility upon Romidepsin treatment in SUM149 mammospheres. Modulated signaling pathways are interrogated to address their role in the regulation of CEBPD transcription and the global cellular response to HDACi therapy. Deeper insight into the mechanisms of CEBPD gene regulation in cancer cells may lead to more specific approaches for pharmacological inhibition of CEBPD and IBC metastasis. Furthermore, inhibition of potential survival pathways activated by HDACi may increase efficacy of HDACi therapy. Abstract #Y42 Expression of caveola-forming proteins caveolin-1 and PTRF and invasive potential of GBM 1 Pu W, 2 Nassar Z, 1 Khabbazi S, 1 Xie N, 1 Peters AA, 3 McMahon KA, 4 Harris J, 1 Parat MO 121

123 Glioblastoma (GBM) is the most malignant brain tumour. The short survival time and poor prognosis for GBM patients is due to high invasiveness. Caveolae are plasma membrane subdomains with a key role in several biological functions. Two major proteins, caveolin-1 and polymerase I and transcript release factor (PTRF), are required for caveola formation. Cancer type- and cancer stage-dependent overexpression of caveola-forming proteins has been reported. We hypothesized that high expression of caveolin-1 or PTRF in GBM promotes invasiveness via upregulation of matrix-degrading enzymes. The mrna expression of caveolaforming proteins and matrix proteases in GBM samples, and survival after stratifying patients according to caveolin-1 or PTRF expression, were analyzed from the TCGA and REMBRANDT databases. Expression of both caveolin-1 and PTRF (i) was increased in GBM compared to normal samples and (ii) correlated with expression of urokinase plasminogen activator (upa) and MMP9. In addition, overexpression of caveolin-1 and PTRF correlated with a worse prognosis of GBM. The proteolytic profile among cultured GBM cell lines expressing or devoid of caveola-forming proteins was investigated using zymography and real-time qpcr; in vitro invasion through basement membrane was quantified. GBM cell lines that formed caveolae produced more upa and gelatinases, and were more invasive than GBM cells devoid of caveolae. Caveolae may modulate GBM cell invasion by regulating upa and gelatinase expression. Abstract #Y44 The postpartum liver metastatic niche supports tumor cell survival: a role for liver lipids? Quackenbush A, Schedin P Breast cancer patients within 10 years of childbirth are ~three times more likely to develop liver metastases compared to their never-pregnant (nulliparous) peers, even after controlling for patient age, year of diagnosis, breast cancer subtype, and tumor size. We predict that this elevated risk for liver metastasis is due to establishment of a pre-metastatic niche in the postpartum liver, driven by the physiologic event of weaning-induced liver involution. We have shown that after weaning, the pregnancy-enlarged rodent liver returns to a pre-pregnant size via hepatocyte death, extracellular matrix remodeling, and myeloid cell infiltration. Furthermore, when mice are injected with mammary tumor cells via the portal vein, involution group mice develop ~3x more liver lesions than nulliparous hosts. Here we tested whether the involuting liver environment preferentially supports tumor growth. At ~6 weeks post tumor cell injection, we find higher mammary tumor burden in the involution group, but no differences in lesion size, tumor cell proliferation or death between groups. Surprisingly, at 90 minutes, 1 day, and 3 days post-tumor cell injection, there are more tumor cells present in the nulliparous host liver. Since more tumor cells advance to overt tumors in involution mice, these early time-point data suggest a tumor cell survival advantage in the involuting liver that becomes manifest after tumor cell extravasation. One possible mediator of differential tumor cell survival may be lipid based, as others demonstrate lipid impacts on tumor growth and macrophages and immature monocytes, immune cells that contribute to the metastatic niche. We find a transient increase in liver lipid 122

124 droplets during the first week of weaning-induced liver involution. Further the composition of these lipids is distinct from that found in the nulliparous or lactation stage mouse livers. To evaluate if involution-specific changes to liver lipids contribute to liver metastasis, lipid intervention studies are ongoing. Abstract #Y46 Osteocytic Connexin Hemichannels in Suppression of Breast Cancer Bone Metastasis and Potential Therapeutic Application Riquelme M, Gu S, and Jiang JX Bone is a fertile place for metastasized breast cancer cell (BCC), however, mechanical loading is reported to inhibit BCC growth. Osteocytes representing over 95% of bone cells are mechanosensitive; they coordinate bone remodeling and the extracellular environment. Mechanical loading activates osteocityc connexin43 (Cx43) hemichannels, which mediates communication between the intracellular and extracellular space with message of molecules below 1 kda. We have shown that ATP released by osteocytic hemichannels through the activation of BCC P2X7 receptor, inhibits BCC growth and migration. Here, we evaluate if Cx43 hemichannels are a therapeutic target for treating breast cancer bone metastasis and understand the underlying inhibitory mechanism. Interestingly, transgenic mice with osteocyte-specific overexpression of Cx43R76W, a dominant negative mutation block gap junction channels without affecting Cx43 hemichannels, reduced BCC growth compared with WT mice. However, mechanical loading increased tumor growth in R76W compared with WT mice, which was reverted with an inhibitor that blocked prostaglandin synthesis. We develop a monoclonal antibody, which binds to extracellular domain of Cx43 and increases the activity of osteocytic hemichannels (MHC2). This antibody was immune-reactive in HeLa cells expressing Cx43, osteocytes cell line MLO-Y4 and mouse osteocytes in vivo. MHC2 enhanced Cx43 hemichannels activity in cells and in vivo osteocytes. The weekly injection of MHC2 reduced the BCC growth in the tibia of WT mice, but not in specific osteocyte Cx43 Knockout mice. A P2X7 specific agonist, reduced in vitro BCC migration, increase cytoplasmic free Ca 2+ and induced formation of tubulin bundles, indicator of cytoskeleton reorganization. The effect of the ATP agonist required functional CamKII. Our results suggest that Cx43 hemichannels are likely a new therapeutic target in treating breast cancer bone metastasis, probably through the enhancement of the extracellular ATP levels and the activation of purinergic/camkii axis to inhibit BCC growth and metastasis. Abstract #Y47 Selection of telomere homeostatic program controls metastatic behavior in breast cancer Robinson NJ, Canfield DR, Taylor DJ, Schiemann WP 123

125 Breast cancer (BC) is the most commonly diagnosed cancer in women globally. Importantly, 90% of BC-associated deaths are attributable to metastasis. However, the cell-intrinsic determinants of metastasis remain unclear. In the present study, we employ a forward genetic screen to delineate genetic regulators of BC metastasis. In doing so, we find the balance between two prevailing telomere maintenance mechanisms (TMMs), telomerase and alternative lengthening of telomeres (ALT), is central in dictating the propensity of BC cells to metastasize. Nontumorigenic disseminated cancer cells (DCCs) harboring ectopic telomerase exhibit increased outgrowth in vitro and pulmonary tumor formation in vivo, suggesting that telomerase promotes metastatic progression in a clinically relevant BC niche. Conversely, inert DCCs display evidence of ALT. Mechanistically, telomerase-driven DCCs rely on the non-canonical activity of telomerase reverse transcriptase (TERT) as a central regulator of a diverse transcriptional network that includes vascular endothelial growth factor (VEGF) and Wnt/β-catenin. Specifically, TERT promotes BC cell growth through its recruitment to the VEGF promoter. In addition, Wnt and TERT constitute a feed-forward loop that mediates the tumorigenic effects of Wnt signaling. CRISPR/Cas9-mediated loss of TERT abrogates these transcriptomic changes and reverses acquired metastatic features via institution of p21-mediated senescence. Analyses of patientderived xenograft (PDX) and central nervous system (CNS) metastasis samples, as well as BC patient cohorts, reveal that TMM selection is predictive of patient outcome in a subtypedependent manner. Similarly, small molecules that selectively target each TMM or their downstream pathways are capable of eradicating BC cells at different stages of metastatic progression. This study demonstrates that elucidating the consequences of telomere dysfunction within the metastatic niche may provide a novel therapeutic approach for patients with metastatic BC. Abstract #Y48 ONECUT2 is a Targetable Master Regulator of Lethal Variants of Prostate Cancer that Suppresses the Androgen Axis Rotinen M, You S, Yang J, Coetzee S, Reis-Sobreiro M, Huang W-C, Huang F, Pan X, Yáñez A, Steadman K, Hazelett D, Chu C-Y, Chung L-W.K, Freedland SJ, Di Vizio D, Garraway I, Murali R, Knudsen BS and Freeman MR. BACKGROUND: Treatment of prostate cancer by hormone suppression leads to the appearance of aggressive variants of metastatic castration-resistant prostate cancer (mcrpc) with variable or no dependence on the androgen receptor (AR). Here we identify the transcription factor ONECUT2 as a negative regulator of the AR axis, that emerges in aggressive PC variants to control transcriptional networks linked to CRPC and neuroendocrine (NE) differentiation. We further demonstrate that ONECUT2 can be targeted with a small molecule that inhibits metastasis in mice. METHODS: ONECUT2 was confirmed as a mcrpc-relevant protein and to be targetable by computational modeling, enforced expression, silencing, microarray, ChIP-Seq, immunohistochemistry, functional assays, in vivo experiments and surface plasmon resonance. 124

126 RESULTS: We have performed a master regulator analysis using 260 mcrpc transcriptome profiles and developed a model transcription factor network that associates ONECUT2 with metastatic progression. Gene expression profiling of ONECUT2-engineered PC cell lines, has allowed us to generate a ONECUT2 activity signature that reveals high positive correlation with pro-neural and aggressive PC signatures, and a negative correlation with AR activation pathways. We find that ONECUT2 is a negative regulator of AR expression and a repressor of its transcriptional program through direct binding to AR target genes. We also find that ONECUT2 is significantly increased in human NEPC and that confers NE properties to CRPC through direct down-regulation of the NEPC inhibitor FOXA1 and direct up-regulation of the NEPC driver PEG10. Finally, we show that ONECUT2 is required for cell growth and survival and that it can be targeted with a small-molecule that, by binding to the ONECUT2 DNA binding domain, inhibits metastasis in mice. CONCLUSIONS: ONECUT2 is a targetable master regulator of lethal mcrpc variants that drives AR-dependent adenocarcinoma toward NEPC differentiation by blocking AR/FOXA1-activity and inducing PEG10. Patients with OC2 active tumors may benefit from ONECUT2 inhibitor therapy. Abstract #Y49 The Role of AIB1- Sharif GM, Nasir A, Wellstein A and Riegel AT It has been challenging to determine what drives Ductal Carcinoma In Situ (DCIS) progression to invasive ductal carcinoma as both disease stages share a similar gene expression profile. High levels of Amplified in Breast Cancer 1 (AIB1) in breast cancer have been correlated with highgrade tumors and poor prognosis. Our lab has identified a splice variant of AIB1 missing exon 4 (AIB1-. The resulting protein lacks the N-terminal 224 amino acids, which comprise an inhibitory domain previously shown to interact with the tumor suppressor ANCO1. Due to this truncation, AIB1- loses negative regulation allowing it to act as a more potent transcription coactivator. In mice studies, AIB1- expression significantly increases as DCIS xenografts transition to invasive lesions. AIB1- expression levels were higher in breast cancer cell lines that metastasize to the lung, brain and bone. Our analysis of RNA-Seq data from breast cancer patients shows higher expression of AIB1- in late stage cancer compared to early stage or locally advanced disease. To determine how AIB1- contributes to the invasive phenotype in breast cancer, we engineered clones from MCF10A and MCF10DCIS.com cell lines to predominantly express AIB1- using CRISPR technology. ChIP-seq of histone marks revealed a different set of engaged and activated genes after expression of AIB1- neomorphic shift in genes involved in luminal to basal as well as epithelial to mesenchymal transitions. RNA-seq data also confirmed that the identified gene sets are enriched by AIB1- expression. Consistent with a contributory role for AIB1- in progression, MCF10Ashow spindle-like morphology and disorganized 3D spheres. DCIS- cell motility and transendothelial invasion. In vivo, DCIS- 4 injected into zebrafish embryos showed 125

127 higher extravasation from the tail vasculature compared to the parental line. In conclusion, these data suggest a model where the increased splicing of AIB1 to the 4 variant alters transcription at specific target genes and contributes to the metastatic phenotype. Abstract #Y50 The PERK/JAK2/STAT3 axis induces CEBPD expression in response to endoplasmic reticulum stress to promote cancer cell survival Sheshadri N, Poria D, Sharan S and Sterneck E Cancer cells experience endoplasmic reticulum (ER) stress due to activated oncogenes and microenvironmental conditions of nutrient deprivation and hypoxia. To support adaptation to this stress, cells activate the unfolded protein response (UPR) mediated by ATF6, IRE1/XBP1s and PERK/ATF4 pathways. While the UPR in cancer cells contributes to survival and metastasis, the elevated ER stress renders cells hypersensitive to stress-inducing agents like Thapsigargin, which is currently in clinical trial as anti-cancer agent. A peptide interactome screen suggested that the C/EBPdelta (CEBPD) transcription factor can heterodimerize with the ER stress effectors ATF4 and CHOP. We recently found that silencing CEBPD compromised tumor growth in xenograft mouse models (see poster by Balamurugan et al. submitted). Therefore, we hypothesized that CEBPD may have a role in the ER stress response. Using a panel of cancer cell lines, we show that CEBPD is transcriptionally induced in response to various chemical and physiological inducers of ER stress. Through pharmacological inhibition and RNAi, we found that the PERK pathway induced CEBPD transcription, in part through JAK2/STAT3 activation. Global transcriptional profiling by RNAseq revealed that CEBPD specifically regulates genes involved in ER stress adaptation such as chaperones, components of the ER associated degradation pathway, and inhibitors of apoptosis, e.g. BIRC3/cIAP2. CRISPR-based knockout of CEBPD in cancer cells caused mild basal ER stress, which provided protection from low doses of Thapsigargin, but caused prolonged Thapsigargin-induced UPR hypersensitivity to higher doses. Furthermore, we found that metastasis-derived variants of prostate and breast cancer cell lines have elevated basal levels of CEBPD and/or persistent expression in response to ER stress-inducing agents. Taken together, our data show that CEBPD is a novel proximal ER stress response gene in cancer cells which confers a survival advantage by promoting adaptive responses. Ongoing studies are assessing how targeting of this pathway may affect tumor metastasis. Abstract #Y51 Crosstalk between osteoblasts and breast cancer cells alters breast cancer proliferation through multiple mechanisms Shupp A, Kolb A, Mukhopadhyay D, Bussard K 126

128 Breast cancer frequently travels to bone, where cancer cells can survive in a dormant state for years before patients discover that their cancer has metastasized and they have relapsed. When breast cancer cells (BCCs) disseminate to bone they interact with a variety of stromal cells, including osteoblasts. Osteoblasts have been shown to regulate cancer cell proliferation, and may contribute to cancer dormancy. We have discovered that crosstalk between osteoblasts and BCCs educates osteoblasts to produce factors that may regulate progression of bone-disseminated breast cancer by altering BCC proliferation. Therefore, we hypothesize that tumor-educated osteoblasts (TEOs) utilize multiple mechanisms of intercellular communication to regulate proliferation of BCCs. TEOs have a unique secretory protein profile compared to uneducated osteoblasts, and exhibit altered production of cytokines, growth factors, and extracellular matrix proteins. We found that treating BCCs with TEO conditioned media was capable of decreasing BCC proliferation, while uneducated osteoblast conditioned media did not affect BCC proliferation. We found this effect to be mediated in part through alterations in TEO production of decorin and nephroblastoma overexpressed (NOV). We also identified TEO conditioned media as a rich source of exosomes (exo). We confirmed the presence of an exo population via iodixanol density gradient separation and western blotting for specific exosomal protein markers. We found that TEO-derived exo, but not uneducated osteoblast-derived exo, decreased proliferation of both ER-positive and triple negative BCCs, in a concentration dependent manner, while maintaining BCC viability. Moreover, we labeled TEO exo with RFP-conjugated CD63, which is a characteristic exosomal protein marker. This allowed us to visually confirm exo transfer from TEO cells to BCCs using confocal microscopy. Our data suggest that TEOs use multiple mechanisms of intercellular communication to regulate BCC proliferation in the bone microenvironment. Abstract #Y52 Melanoma phenotype switching and metastatic growth after dissemination Simmons JL, Pierce CJ, Al-Ejeh F and Boyle GM Metastatic melanoma is inherently aggressive and rapidly develops resistance to all currently available targeted therapies. The highly heterogeneous nature of melanoma and plasticity between cell populations of distinct phenotypes (known as phenotype switching) contribute to the aggressive nature of the disease. Specifically; proliferative cells in the primary tumour switch to an invasive state during dissemination then back to the proliferative phenotype to initiate growth of a metastasis. The transcriptional programs responsible for phenotype switching have been widely studied with Microphthalmia-associated Transcription Factor (MITF) expression often associated with the proliferative phenotype. Similarly the POU domain transcription factor BRN2 (POU3F2) is present in the invasive population. These two transcription factors are heterogeneously expressed in metastatic melanoma and are mutually exclusive in tumours. Therefore switching between a proliferative MITF expressing 127

129 population and the invasive BRN2 expressing population has been suggested to contribute to progression of metastatic melanoma. To further understand the functional significance of MITF and BRN2 expression in melanoma growth and metastasis we developed of system of inducible depletion via shrna. Cell cycle progression was slowed and in vitro invasion perturbed in cells depleted of MITF, as were primary tumour growth and experimental metastasis. Interestingly, cells depleted of BRN2 displayed no disruptions to growth or invasion in vitro and primary xenograft tumour growth was unchanged. However, growth after metastatic dissemination was significantly reduced by BRN2 depletion, indicating the presence of both MITF and BRN2 expressing populations in melanoma tumours may be important for metastatic growth. We have now extended our research to understanding the function of these two transcription factors in resistance to targeted therapy. Preliminary data indicates a rare population of MITF and BRN2 negative melanoma cells found in patient tumours are extremely resistant to BRAF targeted inhibition. Abstract #Y53 PAPP-A promotes pregnancy-associated breast cancer metastasis through collagen remodeling and a DDR2/Snail signaling axis Slocum E, Germain D Pregnancy-associated breast cancer (PABC) is currently empirically defined as breast cancers diagnosed 1-2 years following pregnancy. However, epidemiological studies show that the risk of breast cancer remains higher for up to 6 years post-pregnancy. Therefore, what factors drive the extended susceptibility to breast cancer following pregnancy remains a fundamental question. In addition, PABC is typically more aggressive than other breast cancers and have an increased rate of metastasis, but the underlying mechanisms remain unknown. We previously reported that the secreted protease PAPP-A is an involution dependent oncogene that promotes PABC through its ability to degrade the IGF inhibitor, IGFBP5. Involution is a transient process that occurs after lactation is complete and uses a complex coordination of cell signaling, selected cell death, and matrix remodeling to reset the breast architecture back to that seen prior to pregnancy. A hallmark of involution is an increase in collagen deposition and remodeling to a TACS3, a highly aggressive and invasive collagen signature. This unique collagen phenotype during involution has been implicated in the development of PABC. While a TACS3 phenotype is transient and specific to the involution period, here we show that PAPP-A alters collagen in late post-partum mammary glands to pro-tumorigenic TACS3 collagen. Further, we show that PAPP-A activates the collagen receptor DDR2, promoting the stabilization of Snail and cell invasion. This PAPP-A/DDR2/Snail axis in conjunction with TACS3 maintenance promotes invasion, and the inhibition of DDR2 by CRISPR abolishes the growth and metastasis of PAPP-A-driven PABC. Collectively, these findings suggest that PAPP-A, in the collagen-rich environment of a post-partum breast, contributes to the metastasis of PABC through sustaining 128

130 a TACS3 orientation and promoting cell invasion through DDR2/Snail activation. Furthermore, targeting DDR2 may be a potential therapeutic approach against the aggressive nature of PABC. Abstract #Y54 The Immune System Controls Dormant Metastasis in the Lungs Tallón de Lara P, Vermeer M, Núñez N, Hiltbrunner S, Cecconi V, Movahedian Attar F, Glarner I, B Becher, van den Broek M Metastasis requires that disseminated tumor cells evade immune surveillance and adapt to an ectopic environment in the metastatic organ. Although metastatic disease can be present already at diagnosis, in some patients metastasis appears months or years after resection of the primary tumor. This is thought to be due to the ability of some tumor cells to remain dormant after metastatic seeding. It is not known how dormancy is controlled although there is evidence for tumor cell-intrinsic and -extrinsic mechanisms including immune defense. To investigate whether and how immune defense controls metastasis to the lungs, we used two breast cancer cell lines derived from the same spontaneous tumor in immunocompetent BALB/c mice. We found that orthotopic 4T1 tumors gave rise to progressive metastatic disease, whereas orthotopic 4T07 tumors did not. However, both tumors gave rise to circulating and disseminated tumor cells. Thus, disseminated 4T07 cells displayed a dormant behavior. We next investigated whether 4T07 cells were dormant in immunodeficient NOD.Cg-Prkdc scid Il2rg tm1 (NSG) mice that lack T, B and NK cells and found that 4T07 tumors induced progressive lung metastasis, suggesting that the immune system essentially controls dormancy. In further experiments we deeply characterize the role of different immune populations in the control of dormant disseminated tumor cells. Loss of 4T07 dormancy was also observed in BALB/c mice bearing a contralateral 4T1 tumor, suggesting that 4T1 tumors promote the escape from dormancy of 4T07 cells. We propose that 4T1 tumors compromise immune surveillance escaping from dormancy by a mechanism that is yet to be unraveled. Abstract #Y55 Calpain-2 mediated amoeboid reprogramming and dissemination triggered by hypoxia te Boekhorst V 1, Jiang L 1, Yang Y 3, Chen X 4, Durst F 1, Zuidema A 2, Levine H 3, Shenoy V 4, Friedl P 1,2 Cancer invasion and metastatic dissemination occurs through single cell and collective strategies. Tumor hypoxia, by elevating hypoxia-inducible factors (HIFs), is an established inducer of metabolic and invasive reprogramming driving cancer metastasis. We recently identified hypoxia as an inducer of amoeboid-like individualization and migration in otherwise collectively invading epithelial cancer cells. However, the mechanisms underlying reprogramming to this primordial, amoeboid strategy remain unclear. 129

131 Using epithelial carcinoma tumoroids invading into 3D fibrillar collagen, we here classify amoeboid dissemination modes and their molecular and mechanical adaptations in response to hypoxic stress or pharmacological HIF-stabilization. As central regulator of amoeboid switching after HIF induction, the cysteine protease calpain-2 became upregulated and activated, mediated cleavage of the focal adhesion adapter talin-1 and thereby diminished β1 integrin function and cell elongation, whereas migration speed remained unperturbed. In silico modeling of cell shape identified reduction of cell-substrate adhesion as mechanism for cell rounding, and further predicts calpain-2 as molecular converter of filamentous-to-cortical actin transition, which stabilizes the round amoeboid state. Consistently, calpain activity was elevated in scattered cells of roundish shape in human carcinoma xenografts and required for amoeboid migration after HIF-stabilization in vivo monitored by intravital microscopy. Molecular targeting of calpain-2 presents as promising adjuvant strategy to limit adaptive amoeboid escape and plasticity of cancer invasion, as interference with calpain expression or function prevented talin-1 cleavage, restored β1 integrin function and induced mesenchymal-like phenotypes in vitro and in vivo. The dominance of amoeboid cells in vitro/silico/vivo after induction of HIF signaling reveals a microenvironmentally regulated amoeboid reprogramming of cancer cells with calpain-2 as central regulator of phenotypic conversion. HIF-induced integrin shutdown may thus support mechanochemical plasticity and migratory escape from metabolically perturbed tissue regions. Abstract #Y56 Vimentin as a potential regulator of EMT-induced Tissue Factor during metastatic progression Vanwynsberghe A M 1 & Francart M-E 1, Lambert J 1, Gilles C 1 The contribution of epithelial-to-mesenchymal transitions (EMTs) to the biology of Circulating Tumor Cells is today well recognized and supported by abundant literature associating EMT with higher survival, invasive and metastatic properties. The presence of CTCs with EMT traits has further been extensively reported in cancer patients. Aiming at understanding the mechanisms by which EMT could provide CTCs with enhanced metastatic competence, previous work of the laboratory demonstrated the existence of a link between EMT and coagulation. EMT pathways were indeed shown to induce the expression of Tissue Factor (TF) (a cell-associated activator of the coagulation cascade) and to provide tumor cells with enhanced coagulant properties and early metastatic properties when injected intravenously in mice. Such an activation could generate a protective fibrin-rich cocoon around EMTpositive CTCs, thereby enhancing their survival and metastatic potential. We recently identified vimentin as a potential regulator of TF and therefore examined the implication of vimentin in this EMT/TF/coagulation axis. Silencing vimentin by sirna transfection decreased TF protein expression and in vitro coagulant properties. In addition, intravenous injection of vimentin sirna transfected cells diminished lung metastases after 24h in mice. In the light of literature data demonstrating a contribution of 130

132 vimentin in RNA stabilization, we explored here the possibility that vimentin could interact with TF mrna and stabilize it. Performing RNA immunoprecipitation, we observed an enrichment of TF mrna in the vimentin immunoprecipitate. Vimentin silencing was also found to accelerate TF mrna decay after actinomycin D treatment, supporting a stabilization of TF mrna by vimentin. We further identified a regulatory mechanism by which vimentin could interfere with a mir 520- dependent dowregulation of TF mrna. Taken together, our results thus bolster a functional implication of vimentin in the stabilization of TF mrna, thereby favoring coagulation-dependent early metastasis. Abstract #Y57 DNA Damage response drives chemo-resistance and supports metastasis in triple negative breast cancer Ward A, Kelly G, Simmons J, van Oosterhout R, Lee J, and Wiegmans A. Triple negative breast cancer (TNBC) accounts for up to 15-20% of all breast cancers and is defined by the absence estrogen receptor (ER), progesterone receptor (PR) and human epidermal growth factor receptor 2 (HER2) expression. TNBC is the most aggressive of the subtypes with the poorest prognosis and highest rates of recurrence within 5 years of diagnosis. The standard of care treatment for TNBC is systemic chemotherapy. There are currently no targeted therapies available for TNBC patients and few biomarkers to guide treatment. The development of chemotherapy resistance is a common problem, with relapse rates up to 38% with less than 6 months median survival. Relapse is often associated with aggressive metastatic disease that no longer responds to chemotherapy. To mimic this clinical scenario we derived TNBC cell lines that are resistant to the frontline chemotherapy combination; Doxorubicin and Docetaxel. A known mechanism by which cancer cells acquire chemo-resistance is by deregulation of the DNA damage response (DDR). This can involve both down and upregulation of normal DNA repair pathways. We have previously published that RAD51; a key protein in the homologous recombination DNA repair pathway has increased expression in distant metastases compared to matched primary breast tumours and that knockdown of RAD51 inhibits metastatic growth and tumour progression. Here we characterize the DDR of chemotherapy resistant TNBC cell lines and show that enhanced expression of RAD51 support chemoresistance. Further we show that RAD51-driven resistance correlates with changes in capability of cells to migrate in the presence of chemotherapy. We find that targeting RAD51 with a small molecule inhibitor (Recombinib) resensitizes chemo-resistant cells with potential to inhibit metastases as observed with RAD51 knockdown. Future work will utilize a TNBC xenograft murine model to test the efficacy of treatment with Doxorubicin and Docetaxel in combination with Recombinib in vivo. We propose the use of a specific DDR inhibitor can overcome acquired resistance to effectively treat the spectrum of TNBC cancers that present to the clinic. 131

133 Abstract #Y58 Understanding the Role of Wnt Signaling-driven Nucleolar Dynamics in Breast Cancer Progression Weeks SE 1, Chen D 2,3, Shevde LA 1,3, Samant RS 1,3 The nucleolus was one of the first recognized diagnostic pathological features of a cancerous cell. Until recently, abnormal nucleoli were noted as an indicator of cancerous lesion, and were used as one of the pathologic readouts of neoplasia. More recently, nucleolar hypertrophy has been implicated in increased mortality rates in breast cancer. Although various functions of the nucleolus have been investigated, it remains widely understudied in the context of diseases such as cancer. The nucleolus is a sub-nuclear compartment that was initially thought to be solely the site of ribosome biogenesis. However, nucleolar proteome sequencing, and studies done by the Human Protein Atlas revealed nearly 1,300 proteins localize to the nucleolus. More than half of the proteins found in the nucleolus were associated with cell cycle regulation, stress response, and other biological functions. Thus it is evident that the nucleolus plays major roles in stress response, and cellular aspects that influence cancer progression. The nucleolus is one of the first responders in cellular metabolic changes, and its altered morphology is a readout for metabolic stress, both of which are important cellular determinants for malignant progression of cancer cells. One key signaling pathway known to regulate tumor growth, metabolic adaptation, and cellular stress response is Wnt signaling. However, the effects of Wnt/β-catenin signaling on the nucleolus and the resultant impact on cancer development and progression remain unclear. Our recent work on nucleolar proteomics of multiple breast cancer cell lines uncovered exciting evidence that several key protein members of Wnt signaling such as GSK3β, TLE1, and β-catenin reside in the nucleolus. Additionally, we found that multiple breast cancer cell lines exhibited a notable reduction in the number of nucleoli per nucleus in response to Wnt/β-catenin signaling inhibition. We will present our work that elaborates mechanistic impacts of Wnt modulation on nuclear functionality. Abstract #Y59 Nuclear Aurora-A Kinase: a novel driver of metastasis Marinak-Whately K 1,5, Loskutov Y*, Smolkin M 4, Margaryan M,1,2, and Pugacheva E 1,2,5. Aurora-A Kinase (AURKA) is a serine/threonine kinase that is critical for mitosis. AURKA is overexpressed in 96% of human cancers, including breast cancer. It has been previously shown by our group that AURKA localizes to the nucleus in breast cancer metastases and especially in metastases of Triple Negative Breast Cancer (TNBC). TNBC is currently lacking efficient treatment strategies and often metastatic. Our objective is to define the role differential AURKA localization 132

134 plays in metastatic dissemination and colonization of distant organs, so that novel treatment strategies could be developed based on this knowledge. To achieve this objective we created TNBC cell lines with CRISPR/Cas9 based deletion of endogenous AURKA and rescue with exogenous AURKA specifically targeted to the nucleus by addition of a nuclear localization signal (NLS) or cytoplasm via addition of a nuclear exclusion signal (NES), respectively. These cells were used in orthotopic xenograft study. Here we report that TNBC cells expressing NLS-AURKA show significant increase in proliferation, dissemination and colonization potential of multiple organs when compared to control, WT-AURKA, or AURKA-NES group. Interestingly, cytoplasmic AURKAexpressing cells had decreased colonization potential when compared to WT or control mice. RNA sequencing analysis of these cell lines shows distinct changes in gene expression profile in the AURKA-NLS group associated with cell cycle, angiogenesis and invasion supporting the aggressive metastatic phenotype observed in vivo. Further research and validation of these genes will unveil the mechanisms executed by nuclear AURKA to promote metastasis. Overall, our results indicate that nuclear AURKA is a novel driver of metastatic outgrowth in breast cancer. Thus targeting of these genes or inhibition of AURKA via targeted nuclear delivery of inhibitor is critical for the development of new, less toxic treatment strategies for control and eradication of metastatic disease. Abstract #Y61 The young microenvironment selectively enhances brain metastasis in experimental models of metastatic triple-negative breast cancer Wu A, Gossa S, Dolan E, Robinson C, Difilippantonio S, Gril B, McGavern D, Wakefield L, Steeg, PS Young women (< 40 years old) with breast cancer often have a poorer outcome compared to older women with breast cancer despite receiving more aggressive treatment. Multivariate analyses have shown that even after accounting for differences in tumor characteristics such as tumor grade, nodal status, and molecular subtype, young age is still an independent predictor of poorer outcome. For this reason, we hypothesize that young host age promotes breast cancer progression and metastasis. To test this hypothesis, brain-seeking human MDA-MB-231-BR triple negative breast cancer cells were injected into the left cardiac ventricle of young and old athymic nude mice. Young mice developed two-fold more brain metastases (p <0.05) compared to old mice. Similar results were also observed in young and old immunocompetent BALB/c mice injected with brain-seeking syngeneic 4T1-BR breast cancer cells. This effect of age was not observed in multiple hematogenous and orthotopic models of metastatic breast cancer (parental MDA-MB-231, 6DT1, MVT1, E0771 and HRM1) where the primary metastatic site is the lungs. We next sought to identify the mechanism responsible for this age effect in the brain. Fluorescent immunohistochemical analysis of MDA-MB-231-BR brain metastases for Ki67 and Glia Fibrillary Acid Protein revealed no significant difference in level of tumor cell proliferation or reactive astrocytes between the two age cohorts. However, flow cytometry-based immunophenotyping of brains from young and old athymic nude mice, with and without MDA- MB-231-BR brain metastases, showed that the aged brain had reduced numbers of inflammatory 133

135 Ly6C + monocyte/macrophages. Future work will focus on how these changes in inflammatory cells may alter the capacity of breast cancer cells to form brain metastases. Abstract #Y62 Inhibition of brain metastasis by blocking MAPK12 driver kinase functions Badu-Nkansah A The frequency of breast cancer metastasis to the brain is 10-16% and half of this cohort will die within 3-27 months. Additionally, as therapies that target the primary cancer improve, this prolongs the life of patients and, in turn, allows enough time for metastatic brain tumors to form. To treat brain metastases, multiple strategies are used including surgery, chemotherapy, targeted therapies, whole-brain radiotherapy and stereostatic radiosurgery. These strategies on their own or in combination only slow the progression of the metastasis by a few months; death is a common outcome for individuals suffering from this disease. A comprehensive understanding of mechanisms facilitating brain metastasis, resulting tumor formation and resistance to current treatments will empower the development of new therapeutic strategies to directly target, treat and potentially prevent brain metastasis. As a means to determine mechanisms promoting brain metastasis our lab conducted an in vivo gain of function kinase screen. Briefly, we expressed a 354-kinase library (divided into 17 pools of ~21 kinases each) in the MDA-MB-231 (231) human breast cancer cell line and injected the resulting cells into the carotid artery of nude mice to induce experimental brain metastases. Several pools of these kinases led to dramatically increased brain metastasis-specific mortality compared to 231 vector control cells. Next generation sequencing identified that these aggressive brain metastases were enriched with certain kinases. Among these, MAPK12 was not previously known to promote brain metastasis. Importantly, MAPK12 is overexpressed in highly aggressive human breast cancers, and patients with MAPK12 high-expressing breast cancers have higher incidences of brain metastasis at a later stage. Thus, we established MAPK12- overexpressing breast cancer sublines and injected them into the carotid artery of mice to test their brain metastasis potential. The data validated that MAPK12-overexpressing cells indeed significantly increased brain metastases formation compared to their vector control cells. Therefore, we hypothesize that activation and/or overexpression of MAPK12 coordinates signaling pathways in breast cancer cells to promote brain metastasis. The focus of my project is to investigate novel mechanisms of MAPK12-mediated breast cancer brain metastasis. Abstract #Y63 SOX2 and the regulation of disseminated tumor cell (DTC) fate Carlini M, Sosa M 134

136 Metastasis remains the main cause of human cancer deaths. After treatment of the primary tumors, disseminated tumor cells (DTCs) may enter a quiescence phase called dormancy and may become the source of future metastasis. The nuclear retinoic responsive receptor NR2F1 was shown to be responsible for the dormancy phase of DTCs in a head and neck squamous carcinoma model. Also, NR2F1 upregulated pluripotency genes like NANOG, SOX2 and to a lesser extend OCT4. Here we show that SOX2 knock-down induces reactivation of dormant BM-HEp3 cells, as assessed in vivo using the chick choriallantoic membrane assay. In accordance, SOX2 knockdown was accompanied by a decrease in the protein levels of the cell cycle inhibitor p27 in BM-HEp3 cells. Also, we took advantage of an in vivo mouse dormancy model to further demonstrate the role of SOX2 during dormancy phase of DTCs. After surgery, we observed loco regional recurrences in lymph nodes with 66% incidence in DOX-treated mice (SOX2-knockdown DTCs) compared with 33% in vehicle mice. Interestingly, the time to recurrence was shorter when SOX2 was knockdown. Regarding distant relapses in lungs, we measured total burden of DTCs by alu- QPCR method in the right lung, observing an increase in the median of total DTCs in the SOX2- knockdown group. Moreover, when examining the digital images of H&E lung staining we found a higher number and size of micrometastasis in SOX2 depleted group. Overall, these results argue that SOX2 is required to maintain the dormancy phase of DTCs. We are exploring the underlying mechanisms in current studies. Abstract #Y66 Inflammatory cytokines in breast tumor microenvironment lead to changes in the extracellular matrix conducive to metastasis Dinca S 1,2, Greiner D 1, Jorcyk C 1,2 Recent studies suggest that the tumor microenvironment (TME) plays an important role in the invasive and metastatic potential of breast cancer. Specifically, the ability to cross-link and align the collagen fibers of the extracellular matrix (ECM) provides tracks on which tumor cells can metastasize. Enzymatically active lysyl oxidases (LOX, LOXL1, 2, 3, and 4) are responsible for priming collagen fibers to be cross-linked by reacting with peptidyl lysine residues on collagen molecules. In this study, we investigate the effects of proinflammatory cytokines of the interleukin-6 (IL-6) family on ECM-modifying proteins of the lysyl oxidase family. Using MCF7, MDA-MB-468, and MDA-MB-231 human breast cancer cells, our studies demonstrate that lysyl oxidase-like 2 (LOXL2) is induced by inflammatory cytokines via qrt-pcr and immunostaining. Specifically, we found that oncostatin M (OSM), but not other IL-6-related cytokines, is responsible for a significant increase in protein expression of the glycosylated, enzymatically active LOXL2 (~105kD). These studies are supported by Kaplan-Meier plots demonstrating that patients with high OSM/ high LOXL2 have significantly worse survival than those with low or normal levels of each. To assess the activity of LOXL2 on collagen cross-linking, we employed collagen I gels in which MCF7, MDA-MB-468, and MDA-231 cells were cultured. OSM treatment leads to significant increases in collagen I cross-linking, alignment, and density as well as 135

137 increased mesenchymal phenotype. Inhibition of LOXL2 leads to a reversal of collagen I remodeling in ECM. These outcomes demonstrate that OSM causes drastic changes to the TME that allows for easier invasion and metastasis of breast cancer tumor cells through LOXL2 induction. Collectively, our results, supported by bioinformatics data, suggest that LOXL2 plays a critical role in OSM-induced invasion and metastasis. This is significant as the relationship between OSM and LOXL2 can be exploited as a potential therapeutic target. The project described was supported by NIH grants #YP20GM103408, #YP20GM109095, and #YR25GM123927, the METAvivor Quinn Davis Northwest Arkansas METSquerade Fund, the Smylie Family Cancer Fund, and Biomolecular Research Center Travel Award. Abstract #Y68 Gain-of-function in vivo kinome screen reveals PCTK1/CDK16 as a driver of breast cancer brain metastasis Lowery FJ*, Jiang WY*, Zhang PC, Yu D In spite of the advance in managing primary breast cancer, the incidence and mortality of breast cancer brain metastases (BCBM) have been increasing continuously. To tackle the challenge of understanding the biology of BCBM and pave the way for therapeutic development, a gain-offunction in vivo kinome screen was performed on MDA-MB-231 (231) model to find out potential driver kinases of BCBM. A human kinome library was retrovirally introduced into 231 cells, which were subsequently pooled and delivered into nude mice through intracarotid injection. Nextgeneration sequencing was utilized to reveal enriched kinases in resected brain metastatic lesions compared to the pre-injected control cells. Among the hits, PCTK1 has been validated to promote BCBM and cause worse survival in vivo on both 231 and HCC-1954Br models. TGCA analysis has shown that PCTK1 is positively correlated with multiple genes involved in glutamine metabolism. With an in vitro system that mimics brain nutrient condition, PCTK1 has been shown to promote cell growth by utilizing glutamine as carbon and nitrogen sources. Metabolomics analysis showed that PCTK1 enables 231 cells to utilize glutamine to synthesize serine, which is not abundantly available in brain parenchymal environment. PCTK1 upregulates PHGDH, the rate-limiting enzyme in de novo serine synthesis, at transcriptional level. Given that serine in blood circulation cannot readily pass blood-brain barrier, PCTK1-driven de novo serine synthesis allows breast cancer cells to overcome brain-specific metabolic obstacle during brain tropism. Targeting PCTK1 has the potential to limit BCBM outgrowth by abolishing specific metabolic adaptation to brain nutrient environment. Abstract #Y69 Anti-androgens regulate amino acid-mtorc1 signalling and extracellular vesicle secretion to modulate prostate cancer progression 136

138 McCormick K 1, Fan S-J 1, Stevens D 1, Turley H 2, Harris, AL 2, Verrill C 1, Bryant RJ 3, Hamdy FC 3, Goberdhan DCI1 Metastatic prostate cancer (mpca) is currently incurable, and despite recent improvements in next-generation androgen deprivation therapy (ADT) and chemotherapy most patients develop treatment resistance within a small number of years. A better understanding of the mechanisms underlying treatment resistance is necessary to develop novel therapies. Extracellular vesicles (EVs) are nanosized, membrane-bound carriers of proteins, lipids and nucleic acids, which mediate intercellular communication and have been increasingly implicated in numerous processes involved in PCa progression. The ways in which anti-cancer treatments such as ADT affect EVs released from PCa cells, and whether these EVs influence treatment resistance, remains poorly understood. We have characterised the effects of the non-steroidal anti-androgen bicalutamide on EV secretion from PCa cells. We find that bicalutamide promotes secretion of EVs carrying the androgen receptor (AR) via a mechanism involving inhibition of the mtorc1 microenvironmental sensor. Our data suggests that anti-androgen treatment inhibits mtorc1 activity by downregulating expression of SLC36A4 (PAT4), a glutamine-sensitive amino acid transporter. Depletion of the PAT4 substrate, glutamine, promotes secretion of AR-containing EVs. Furthermore, we explore the functional effects of these two EV sub-populations with respect to PCa progression and drug resistance. These findings led us to hypothesise that ADT may select for more aggressive PCa tumours that express high levels of PAT4. In support of this theory we further report that mrna expression and copy number changes in PAT4 are associated with disease progression in PCa patients. Overall, we propose that anti-androgen therapies promote PCa progression via mechanisms involving altered EV signalling and modulation of the PAT4-mTORC1 regulatory axis. This work highlights novel future therapeutic strategies and defines EV protein signatures that could be used to monitor treatment responsiveness. Abstract #Y70 WNT5A signaling: A promising target for anti-metastatic therapy Mohapatra P, Prasad CP, Linnskog R, Moradi F, Södergren K, Andersson T WNT5A signaling has been associated with tumor progression in melanoma, while WNT5A loss led to early relapse and overall reduced survival in breast cancer. The WNT5A ligand mediates its effects via interaction with different receptor or co-receptors complexes i.e. tyrosine-kinase, Frizzled, LRP5/6 etc. It is very difficult to target or mimic all these receptors with a single inhibitor to either potentiate or block the effect of WNT5A-triggered signaling in different cancer. In the last few years, we closely studied the mechanistic aspects of WNT5A inhibition or WNT5A supplementation on the metastatic behavior of melanoma and breast cancers. In melanoma models, we investigated the effect of the combined inhibition of WNT5A and IL-6 signaling on the 137

139 invasive migration of melanoma cells. Our results showed that the combined knockdown of WNT5A and IL-6 or the simultaneous inhibition of WNT5A and IL-6 signaling efficiently inhibited melanoma cell migration and invasion. In the context of breast cancer, we have used two WNT5A deficient metastatic breast cancer cell lines i.e. MDA-MB-468 and MDA-MB-231 and stably transfected them with WNT5A plasmid. Evaluation of glycolytic markers in these WNT5A expressing cell lines showed selective downregulation in Platelet type-phosphofructokinase (PFKP) expression and lactate secretion, without affecting cell proliferation. Interestingly, we found that the down-regulation of PKFP and lactate was mediated through WNT5A inhibition of β-catenin signaling. Functionally, we showed that specific downregulation of either β-catenin or PFKP resulted in reduced lactate production, accompanied by impaired breast cancer cell migration and invasion. Our therapeutic approach clearly demonstrates that targeting WNT5A signaling can be beneficial in treating metastatic melanoma and breast cancer. Abstract #Y72 MicroRNA-1205 regulation of FRYL in metastatic castration-resistant prostate cancer Naidoo M, Levine F, Gillot T, Ali T, Krampis K, Orunmuyi A, Ogun G, Adebayo S, Olapade-Olaopa E, Ogunwobi O High mortality rates from prostate cancer (PCa) are associated with metastatic castrationresistant prostate cancer (mcrpc) due to maintenance of androgen receptor (AR) signaling in PCa cells despite androgen ablation therapy. The 8q24 chromosomal locus is a highly susceptible PCa region that has amplifications of the PVT1 gene. PVT1 is a non-protein coding gene that encodes microrna-1205 (mir-1205). We examined mir-1205 mrna expression in normal prostate (n=22), benign prostatic hyperplasia (n=42), and PCa (n=26) tissues obtained from human prostatectomy or transrectal biopsies. A Tukey post hoc test revealed decreased mir expression in benign prostatic hyperplasia (4.61 ± 7.5) and PCa (3.39 ± 3.53) when compared to normal prostate tissues (6.55 ± 9.5), indicating that mir-1205 may have tumor suppressive functions. In vitro studies revealed lower mir-1205 expression in mcrpc cells (C4-2B, 22RV1 and PC-3) when compared to non-crpc cells (MDA-PCa-2b or LNCaP), suggesting a role for mir-1205 in mcrpc. Furthermore, we observed significant inhibition of tumor volume in NOD/SCID-gamma mice implanted with C4-2B cells that were administered our novel synthetic analog of mir-1205 (patent pending) when compared to mice treated with a scramble oligonucleotide. We identified Fry-like (FRYL) as a putative target of mir-1205 using a misvr algorithm and subsequently observed FRYL and AR overexpression in prostate tumors compared to normal tissue from fourteen PCa patients. Moreover, FRYL mrna was overexpressed in mcrpc cells compared to non-crpc cells, suggesting oncogenic functions in mcrpc. A luciferase assay confirmed direct binding of mir-1205 to FRYL when C4-2B cells were transfected with mir-1205 and a luciferase vector containing the 3 UTR of FRYL. These observations strongly suggest that mir-1205 acts as a tumor suppressor by directly targeting the 3 UTR of FRYL in PCa cells. Understanding the role of mir-1205 regulation of FRYL and AR signaling may provide novel insights into the molecular mechanisms of mcrpc. 138

140 Abstract #Y73 Establishment and characterization of highly metastatic breast cancer cell lines by orthotopic transplantation Nakayama J 1,2, Fujimoto J 1,3, Semba K 1,4 Metastasis signature genes (MSGs) have been identified form signature genes in highly metastatic cells, most of which had been established by intra-circulation or subcutaneous transplantation. Metastasis is multi-step phenomena which regulated by metastasis promoting genes. We thought that orthotopic model could mimic more spontaneous and early steps of metastasis than intra-circulation model in vivo. We thus established highly metastatic MDA-MB- 231-luc2 cell lines which metastasized to lung by the two animal transplantation models: the orthotopic transplantation method, which mimics all steps from primary tumor to metastatic sites, or tail vein injection (TVI) method, one of the methods for intra-circulation transplantation. Orthotopic lung metastatic cell lines had high metastatic activities and tumorigenicity in only orthotopic metastasis assay. However, results of tail vein injection metastasis assay using orthotopic cell lines showed that these did not promote extravasation and metastatic colonization in lung. To characterize the molecular expression profiles of early stage in metastasis, we performed comparative analysis of transcriptome in established lung metastatic cells using DNA microarray. Interestingly, lung MSGs were distinct between orthotopic and TVI methods although metastatic lines were derived from an identical cell line. 189 genes included known metastasis promoting genes, for example MMP1, IL8 and IL13RA2, were upregulated and 286 genes downregulated in the signature of orthotopic lung. 91 upregulated genes were common in TVI MSGs, while 98 genes were identified specifically in orthotopic MSGs (Nakayama J et al., 2017). In this research, we suggested that characterization of orthotopic MSGs will be helpful in understanding of the multi-step mechanisms of metastasis. Signature genes in orthotopically metastatic cells may also have the potential for novel metastasis promoting genes and prognosis markers. Abstract #Y74 Drawing a Genomic Atlas of Cancer Metastasis: Insights from Prospective Clinical Sequencing of 25,000 Patients Sánchez-Vega F, Fong CJ, Chatila WK, Ganesh K, DiNatale RG, Penson A, Camacho N, Jonsson P, Reznik E, Gao J, Yaeger R, Berger MF, Taylor BS and Schultz N. Since January 2014, tumors from more than 25,000 patients at Memorial Sloan Kettering Cancer Center have been sequenced using MSK-IMPACT, a targeted sequencing assay that identifies mutations, copy number changes and structural rearrangements in 468 genes using tumor and matched normal DNA. Using text mining techniques on our institutional database of electronic medical records, we were able to identify the list of anatomic locations affected by metastatic 139

141 disease in each individual patient. We used this data, covering >50 cancer types and >20 metastatic sites, to draw a comprehensive map of metastatic transitions and we then investigated statistical associations between genomic alterations and specific patterns of metastatic dissemination. We quantified and compared the heterogeneity in affected metastatic sites per patient across different cancer types and relevant molecular subtypes. We identified site-specific enrichment of somatic alterations for tumors from the same cancer type, with a focus on therapeutically relevant targets. Lung metastases of esophagogastric cancer were enriched in HER2 amplified tumors (9/18, 50% vs. 24/175, 14% in other metastatic sites; p<0.001). Brain metastases of lung adenocarcinoma (n=34/468) exhibited increased aneuploidy (median fraction of genome altered 0.55 vs. 0.44; p<0.001) and adrenal gland metastases (n=21/468) exhibited increased mutational burden (13.3 mut/mb vs. 6.1 mut/mb; p<0.001). MITF/ETV1 amplifications were enriched in melanoma patients with brain metastases (13/63, 21% vs. 10/234, 4%; p<0.001) and 20q chromosomal amplifications were enriched in oligometastatic colorectal patients (158/223, 71% vs. 28/58, 48%; p=0.002). Ongoing work includes the development of probabilistic models to estimate the risk of future metastasis based on the genomic profile of early stage tumors, which has the potential to directly influence patient care by optimizing metastatic surveillance protocols and guiding personalized treatment. Abstract #Y76 Multiphase pseudo-continuous arterial spin labeling MRI for quantitative cerebral blood flow measurements in a rat model of breast cancer brain metastasis Simard MA 1, Larkin JR 1, Chappell MA 2, Sibson NR 1. Arterial spin labelling (ASL) MRI has numerous potential applications in the clinic as a noninvasive method to measure perfusion in the brain. We have previously determined optimised parameters for ASL in rats by using a multiphase pseudo-continuous ASL (pcasl). Here, our aim was to use our optimised pcasl approach to provide accurate measures of tumour perfusion in a rat model of brain metastasis. Female Berlin-Druckrey IX (BD-IX) rats were injected in the left striatum with 1000 ENU1564 cells in 1 µl PBS. MRI experiments were performed on isoflurane anaesthetised rats at weeks 2, 3, and 4 post-injection using a 9.4 T MRI spectrometer (Agilent). Metastatic tumours were visible on T2-weighted images of metastasis-bearing rats from 2 weeks post-injection onward. Blood-brain barrier breakdown occurred between weeks 2 and 3 postinjection, as demonstrated by hyper-intense regions on post-gadolinium T1-weighted images. At this time, multiphase pcasl showed a significant decrease in cerebral blood flow (CBF) within the tumour in ENU-injected rats. The tumour core showed the greatest decrease in CBF and histologically was found to be hypoxic (anti-pimonidazole staining) in regions distant to blood vessels (anti-cd31 staining). The significant decrease in tumour perfusion is indicative of dysregulated blood flow within the tumour. Additionally, the elevated presence of pimonidazole staining in areas further from CD31 stained vessels within brain metastases provides evidence of hypoxia within these tumours, a 140

142 consequence of reduced blood flow in these areas. The presence of hypoxia in the tumours suggests that these would be poor responders to radiation therapy. With our multiphase pcasl methodology, we obtained reproducible CBF values in ENU1564 injected rats. The metastatic tumours proved to be hypoxic and this correlated with reduced CBF observed using multiphase pcasl. We propose, therefore, that multiphase pcasl may provide a useful tool for treatment planning in brain metastasis. Abstract #Y77 Matrix Remodeling by Semaphorin 7a promotes tumor invasion and cell survival Tarullo S, Hill R, Elder A, Hansen K, Nelson A, Behbod F, and Lyons T Breast cancer (BC) remains the second leading cause of cancer related deaths in women in the US -- mainly due to metastatic disease. Thus, understanding how BC progresses is of the utmost importance to developing new strategies to block metastasis. Young women diagnosed with BC generally have poor prognosis due to increased rates of metastasis. Additionally, women who are within 5 years of most recent child-birth at diagnosis are ~3 times more likely to develop metastasis and die from BC than age and stage matched nulliparous women. We define these cases as postpartum breast cancers (PPBC) and propose that the unique biology of the postpartum breast drives tumor progression and metastasis. Semaphorin 7a (SEMA7A) is a unique member of the Semaphorin family as it is the only GPI-anchored member that can be shed into the extracellular environment. Our published results show that SEMA7A expression is associated with decreased overall survival in BC patient cohorts and revealed roles for SEMA7A in breast tumor cell growth, motility, invasion, and tumor associated-lymphangiogenesis all of which are also increased in PPBC. However, whether SEMA7A is a driver PPBC and how SEMA7A promotes BC progression remains largely unexplored. To test the role of SEMA7A in PPBC we implanted MCF10DCIS breast cancer cells stably expressing scrambled or SEMA7A shrnas into postpartum hosts and nulliparous controls. Our results show loss of SEMA7A is sufficient to decrease postpartum tumor growth. Further, using silencing and overexpression systems model we show that SEMA7A effects multiple pro-metastatic tumor attributes such as survival in detached conditions, tumor associated COX-2 expression, fibronectin deposition, and fibroblastmediated collagen deposition. These studies suggest SEMA7A as a key mediator of BC progression and targeting SEMA7A alone or in combination may open avenues to novel therapeutic strategies to prevent BC progression to metastasis. Abstract #Y78 Pleiotriphin and midkine promote metastasis in pre-clinical models of breast cancer Sorrelle N, Dominguez A. Dominguez, Ganguly D, Huang H, Berens Eric*, Toombs Jason, Poczobutt J**, Rosenfield S*, Du W, Nemenoff R**, Wellstein A* and Brekken R. 141

143 Introduction: Pleiotrophin (PTN) and midkine (MDK) are present in serum and primary tumors of breast cancer patients where their expression correlates with worse outcome. Previous studies indicate these heparin-binding cytokines contribute to angiogenesis, hematopoiesis, and inflammation. However, the functional significance of PTN and MDK in breast cancer remains to be elucidated. Methods: The effect of anti-ptn therapy (mab; 3B10) on breast tumor progression and pulmonary metastasis was evaluated in four preclinical models of breast cancer: 4T1, E0771, Met-1, and the MMTV-PyMT transgenic model of breast cancer. Tissues from these experiments were analyzed for changes in angiogenesis, hematopoiesis, macrophage infiltration, and cytokine/chemokine expression. The effect of anti-mdk therapy (mab; IP10) was evaluated in the 4T1 breast cancer model and tissues were analyzed by immunohistochemistry. Results: 3B10 reduced primary tumor growth only in the poorly metastatic Met-1 model. However, 3B10 reduced pulmonary metastasis in the 4T1, E0771, and MMTV-PyMT models of breast cancer. Additionally, 3B10 therapy had no effect in PTN-null mice, suggesting that the antibody is specific and that host stromal cells, rather than cancer cells, are the main source of PTN in these models. Further, 3B10 therapy resulted reduction in CXCL1/4/5, MIP3α, and MCP1 in breast tumors. PTN blockade also decreased neutrophil infiltration and macrophage/metastatic aggregates at in lungs of mice bearing 4T tumors. In vitro, we observed that PTN induced RAW macrophages to produce MIP3α, CXCL4 (PF4), and CXCL5. PTN increased haptotaxis of primary macrophages, but not breast cancer cell lines. Anti MDK therapy (IP10) reduced pulmonary metastasis in 4T1 model. Furthermore, IP10-treated tumors showed a significant increase in E-cadherin and decrease in Vimentin, suggesting that MDK may participate in tumor cell plasticity. Conclusion: Stromal-derived PTN and MDK promote breast cancer progression in preclinical models of breast cancer. We hypothesize that this is mediated, in part, by promoting tumorassociated neutrophil and macrophage infiltration and metastasis-associated cytokine/chemokine expression by myeloid cells. Abstract #Y79 Delineating Key Autophagic Markers in Breast Cancer Cell Lines Associated with an Aggressive Phenotype and Autophagic Inhibitor Sensitivity Giuliani C, Price JT Victoria University, Western Centre for Health Research and Education, Sunshine Hospital, Victoria, Australia Autophagy is a highly conserved catabolic process resulting in the degradation of organelles and proteins into recyclable nutrients in response to cellular stress to maintain homeostasis. However, its role in the context of cancer has been somewhat paradoxical in that it has been shown to prevent tumorigenesis via protein aggregate degradation yet paradoxically it can also facilitate the survival and maintenance of the metastatic phenotype. The role of autophagy and 142

144 the mechanisms that underlie its activity, within the varying contexts of cancer, are still not well understood and require elucidation. We have examined a breast cancer cell line panel for key autophagy markers which were found to be increased in expression in breast cancer subtypes with a more aggressive phenotype. This increase was supported by functional studies of autophagic activity, where a high autophagic flux was observed at basal conditions. Interestingly, activation of autophagy above baseline resulted in a dysregulation of its activity, indicating that the basal level of autophagic activity in aggressive breast cancer cells had reached maximal autophagic capacity. The requirement for autophagy in these cells was confirmed by treatment of the cell lines with specific autophagic inhibitors resulting in cell death. No cell death was observed in the immortalised breast epithelial cell line, MCF10A. A key modulator of autophagy, MAP1LC3, was investigated in regards to isoform expression in patient data, significantly, expression of two of the isoforms associated with increased overall patient survival while one correlated with decreased patient survival. Consistent with this, in vitro investigation of the MAP1LC3 isoforms in the breast cancer panel demonstrated isoform expression at basal conditions and in response to stress that could delineate the aggressive phenotype of the cell line. We propose that variations in MAP1LC3 isoform expression may lend themselves as future biomarkers for targeted autophagic therapeutics. Abstract #Y80 Loss of Tank-binding kinase 1 (Tbk1) in pancreatic cancer reduces tumor epithelial plasticity Cruz VH, Arner EN, Bremauntz AE, Hwang TH, Brekken RA. Pancreatic ductal adenocarcinoma (PDA) is the third leading cause of cancer-related deaths in the United States and eighth worldwide. PDA is commonly characterized by an activating mutation in KRAS, yet direct inhibition of KRAS through pharmacological means remains a challenge, indicating a need for effective therapies. Higher levels of TANK-binding kinase 1 (TBK1) mrna, a critical downstream effector of mutant active KRAS in lung cancer, are associated with poorer overall survival in human PDA patients. Given the high mutation frequency of KRAS in human pancreatic cancer and the association of high TBK1 expression with poor outcome in PDA, we hypothesize that TBK1 is a critical mediator of KRAS in PDA. Here we report that TBK1 is expressed and more active in human PDA cell lines relative to immortalized pancreatic epithelial lines (HPNE). Additionally, KRAS mutant PDA cell lines are selectively sensitive to a small molecule inhibitor of TBK1 in the low micromolar range. In a KRAS driven genetic mouse model of PDA, Tbk1 supports the growth of spontaneous pancreatic tumors as evidenced by longer survival and smaller tumors in Tbk1Δ/Δ: PDA mice relative to Tbk1+/+: PDA mice. We found that tumors from Tbk1Δ/Δ: PDA mice are more epithelial in gene expression and show significantly less collagen deposition compared to Tbk1+/+: PDA tumors. Furthermore, cell lines isolated from Tbk1Δ/Δ: PDA tumors are more epithelial in morphology and less able to migrate and invade relative to Tbk1+/+: PDA cell lines in vitro and in vivo, as evidenced by scratch, transwell migration, and lung colonization assays. Current work is focused on mechanistic understanding of the epithelial 143

145 phenotype in Tbk1Δ/Δ: PDA tumors as well as the contribution of TBK1 to metastasis. These results will further our understanding of KRas signaling in pancreatic cancer and are critical for exploring new avenues of targeted therapy. Abstract #Y81 Genome-wide cooperation of the EMT inducer ZEB1 with YAP and AP-1 factors Feldker N 1, Lukassen S², Ferrazzi F², Widholz S 1, Kleemann J 1, Mock K 1, Stemmler MP 1, Brabletz S 1, Brabletz T 1. 1 Department of Experimental Medicine I, Nikolaus-Fiebiger-Center for Molecular Medicine, University of Erlangen/Nürnberg, Erlangen 2 Human Genetics, University Clinic, Erlangen Metastasis and therapy resistance have been established as the leading cause of cancerassociated deaths. In the past years, we could identify the EMT-inducing transcription factor ZEB1 as a crucial stimulator of these processes. While ZEB1 used to be considered as a transcriptional repressor, we and others have recently demonstrated that it is also capable of acting as a transcriptional activator. We showed that activating abilities of ZEB1 involve direct interaction with the Hippo pathway effector YAP. However, the precise mechanisms by which ZEB1 transforms from a repressor to an activator remain elusive. Our results suggest that the ZEB1 activator complex not only comprises YAP, but also the AP-1 family members Fra-1 and Jun. Analysis of ChIP-seq datasets revealed a strong overlap between genomic regions bound by ZEB1, YAP, or JUN. Moreover, integration with gene expression data showed that promoters co-bound by all three factors preferentially induce target gene transcription. Our findings further support a direct interaction between ZEB1 and Fra-1 or Jun and a cooperativity of all four factors in inducing target gene transcription. In summary, our results provide evidence for a direct link between ZEB1 and two other major signalling pathways crucial for cancer progression. Elucidating this link further may enable us to develop new treatment strategies directly targeting essential traits of aggressive cancers. Abstract #Y82 KISS1R signaling in triple negative breast cancer metastasis and metabolism Nguyen M 1,3, Guzman S 1,3, Dragan M 1,3, Su X 1,3,4, Wondisford F 1, Brackstone M 5, Tuck A 6, Babwah A 2,3, Bhattacharya M 1,3,4 Kisspeptins (KPs), peptide products of the KISS1 gene are endogenous ligands for the kisspeptin receptor (KISS1R), a G protein-coupled receptor. In numerous cancers (e.g. melanoma, ovarian, bladder) KISS1R signaling plays anti-metastatic roles. However, in triple negative breast cancer 144

146 (TNBC), KISS1R signaling stimulates cell invasion by crosstalking with the epidermal growth factor receptor (EGFR), a biomarker associated with this subtype of breast cancer. TNBC comprise a heterogeneous group of basal-like tumors lacking estrogen receptor (ER ), progesterone receptor (PR) and HER2 (ErbB2). TNBC occurs frequently in women under 50 years of age and these patients have poor prognosis compared to other breast cancer subtypes since tumors are high grade and develop metastasis. Unfortunately, these patients lack targeted therapy. The mechanisms regulating metastasis remain poorly understood. We found that the ER status of breast epithelia critically dictates the ability of KISS1R to activate EGFR and promote invasiveness. KISS1/KISS1R mrna and protein are upregulated in TNBC patient tumors, compared to healthy breast tissue. Using orthotopic and experimental xenograft metastasis models, we demonstrate for the first time that human KISS1R signaling promotes vascularization of primary tumors and metastasis. Mechanistically, KISS1R signaling induces epithelial to mesenchymal transition and the activation of key invadopodia proteins as well as matrix metalloproteases (MT1-MMP, MMP-9). Lastly, we found that tumor expression of KISS1R is associated with altered metabolic activity. Taken together, our results indicate that targeting KISS1R might have therapeutic potential for the treatment of metastatic TNBC patients. 145

147 Alexander Hall Princeton University Acknowledgement Congress Organizing Staff: Jennifer Lopez Metastasis Research Society Marianna Bogucki Princeton University Kate Bankaitis Metastasis Research Society Special Thanks to: Irene Shih Wayne Y. Jiang Akosua Badu-Nkansah UT MD Anderson Cancer Center 146