UNRAVELING THE SIGNALING NETWORKS IN HUMAN CELL TRANSFORMATION. By ROCKY CIPRIANO

Size: px
Start display at page:

Download "UNRAVELING THE SIGNALING NETWORKS IN HUMAN CELL TRANSFORMATION. By ROCKY CIPRIANO"

Transcription

1 UNRAVELING THE SIGNALING NETWORKS IN HUMAN CELL TRANSFORMATION By ROCKY CIPRIANO Submitted in partial fulfillment of the requirements For the degree of Doctor of Philosophy Thesis Advisor: George R. Stark Department of Pathology CASE WESTERN RESERVE UNIVERSITY May, 2010

2 CASE WESTERN RESERVE UNIVERSITY SCHOOL OF GRADUATE STUDIES We hereby approve the thesis/dissertation of Rocky Cipriano Ph.D. candidate for the degree *. Mark Jackson, Ph.D. (signed) (chair of the committee) George Stark, Ph.D. David Danielpour, Ph.D. Edward Stavnezer, Ph.D. Charis Eng, M.D., Ph.D. Clive Hamlin, Ph.D. 03/31/2010 (date) *We also certify that written approval has been obtained for any proprietary material contained therein.

3 TABLE OF CONTENTS List of Figures List of Abbreviations Acknowledgements Abstract Chapter I: Introduction A. Overview B. Current Statistics C. Human Breast Architecture D. Breast Cancer Subtypes E. Breast Cancer Treatments F. Forward genetics in gene discovery G. Validation Based Insertional Mutagenesis (VBIM) H. Tumor Suppressors p PTEN p RB p16 and p14arf I. Oncogenes c-myc and RAS

4 J. Cellular Transformation K. Acquisition of persistent growth signals L. TGF-β Signaling M. Human Mammary Epithelial Cells N. Summary and Research Aims Chapter II: Inactivation of p53 signaling by p73 or PTEN ablation results in a transformed phenotype that remains susceptible to Nutlin-3 mediated apoptosis A. Abstract B. Introduction C. Materials and Methods D. Results E. Discussion Chapter III: Suppression of TGF-β signaling overcomes RAS-induced senescence in HMECs leading to neoplastic transformation A. Abstract B. Introduction C. Materials and Methods

5 D. Results E. Discussion Chapter IV: Discovery of FAM83B as a novel oncogene using a validation-based forward genetic strategy A. Abstract B. Report C. Materials and Methods Chapter V: Supplemental Data Section A. Discovery of a novel family of oncogenes Chapter VI: Discussion and Future Directions A. Summary B. Discussion: Chapter II C. Future Directions: Chapter II D. Discussion: Chapter III E. Future Directions: Chapter III F. Discussion: Chapter IV & V G. Future Directions: Chapter IV & V H. Conclusions

6 References

7 LIST OF FIGURES Chapter I Figure I-1: Morphological architecture of the mammary gland in vivo. 22 Figure I-2: Forward genetics paradigm Figure I-3: VBIM lentiviral construction and use Figure I-4: Use of the VBIM technology Figure I-5: Acquired Capabilities of Cancer Figure I-6: Normal Human Cell Transformation Figure I-7: Schematic of receptor tyrosine kinase (RTK) and RAS Signaling Figure I-8: Generation of Epithelial Cell Cultures from Organoids. 50 Chapter II Figure II-1: Transformation of normal human diploid fibroblast by ablating p53, p73, or PTEN Figure II-2: Nutlin-3 treatment of parental ERT derivates and ERT derivates recovered from soft agar Figure II-3: p53 is efficiently stabilized in the absence of p73 and PTEN. 65 Figure II-4: Nutlin-3 induces a caspase-mediated apoptosis in ERT derivatives, dependent on functional p Figure II-5: Transformation by BCL2 results in cells sensitive to Nutlin

8 Chapter III Figure III-1: HMECs expressing a shrna targeting p53 are resistant to Nutlin Figure III-2: RAS-mediated OIS is independent of DDR proteins, p53, and p Figure III-3: Multiple RAS effectors are required for RAS-mediated OIS in HMECs Figure III-4: TGF-β signaling is required for RAS-mediated OIS.. 85 Figure III-5: Inhibition of TGF-β signaling in shp53/ras-g12v- HMECs Figure III-6: Western analysis of HMECs expressing c-myc.. 88 Figure III-7: Treatment of shp53-hmecs with exogenous TGF-β. 89 Figure III-8: RAS-resistant (RAS-R) HMECs are untransformed.. 91 Figure III-9: RAS-resistant (RAS-R) HMECs western analysis.. 93 Figure III-10: Neoplastic transformation of HMECs requires inhibition of TGF-β signaling Figure III-11: Neoplastic transformation of primary HMECs.. 96 Figure III-12: c-myc and RAS-G12V expression does not confer resistance to Nutlin Figure III-13: Elevated HDMX expression overcomes TGF-β suppression enhancing transformation Chapter IV 6

9 Figure IV-1: A forward genetic screen identifies FAM83B as a driver of anchorage-independent growth in human mammary epithelial cells Figure IV-2: Validation of cell libraries SD3-1 and SD Figure IV-3: Schematic diagram of the VBIM integration site in the FAM83B gene Figure IV-4: Inhibition of FAM83B suppresses the growth of breast cancer cells Figure IV-5: Validation of FAM83B shrna constructs Figure IV-6: Detection of endogenous FAM83B protein in various cell lines Figure IV-7: FAM83B and transformation Figure IV-8: The DUF1669 drives AIG Figure IV-9: Comparison of FAM83B expression in mutant cells Figure IV-10: PLD assay of mutant FAM83B expressing HMECs Figure IV-11: FAM83B is required for RAS mediated transformation and overexpressed in several cancer subtypes Figure IV-12: FAM83B expression in cancer Figure IV-13: FAM83 member expression in cancer Chapter V Figure V-1: Additional FAM83 members drive HMEC transformation and are overexpressed in cancer

10 Figure V-2: Alignment of FAM83 members demonstrating the conservation in the DUF Figure V-3: Mass Spectrometry analysis of FAM83 members Figure V-4: Western analysis of FAM83 members expressed in immortalized HMECs Figure V-5: FAM83 members can substitute for the loss of FAM83B. 152 Figure V-6: Expression analysis of FAM83 members in normal tissues. 153 Figure V-7: Expression analysis of FAM83 members in cancer

11 LIST OF ABBREVIATIONS σ 48R Stratifin 48RS cells, HMECs obtained from Martha Stampfer 4E-BP1 Eukaryotic translation initiation factor 4E-binding protein 1 AIG AKT AML ARF ATM Anchorage-Independent Growth v-akt murine thymoma viral oncogene homolog Acute Myeloid Leukemia Cyclin-Dependent Kinase inhibitor 2A ataxia telangiectasia mutated B99 G-2 and S-phase Expressed 1 BAX BCL2-Associated X protein BCL-2 B-cell CLL/Lymphoma 2 B-CLL BRAF B-Chronic Lymphocytic Leukemia v-raf murine sarcoma viral oncogene homolog B1 CA CDK cdna CHK2 CMV Constitutively-Active Cyclin Dependent Kinase complementary DNA CHK2 checkpoint homolog (S. pombe) cytomegalovirus 9

12 c-myc CO 2 v-myc myelocytomatosis viral oncogene homolog (avian) Carbon Dioxide D1/CDK DCIS DMEM DN DNA Constitutively-Active Cyclin D1/CDK fusion protein Ductal Carcinoma In Situ Dulbecco's Modified Eagle Medium Dominant Negative Deoxyribonucleic acid DUF 1669 Domain of Unknown Function 1669 E2F ER ERBB2 ERK ESR1 E2F Transcription Factors Estrogen Receptor Human Epidermal Growth Factor Receptor 2 (also HER2) Mitogen-Activated Protein Kinase (also MAPK) Estrogen Receptor FAM83 Family with sequence similarity 83 FAS TNF receptor superfamily, member 6 G1 G2 GADD45 GAPDH Gap 1 phase, cell cycle Gap 2 phase, cell cycle Growth arrest and DNA-damage-inducible Glyceraldehyde 3-phosphate dehydrogenase 10

13 GFP Green Fluorescent Protein h HDM2 HDMX HER2 HIV HME1 HMEC htert Hours Mdm2 p53 binding protein homolog (mouse) Mdm4 p53 binding protein homolog (mouse) Human Epidermal Growth Factor Receptor 2 (also ERBB2) Human immunodeficiency virus htert Immortalized Human Mammary Epithelial Cells Human Mammary Epithelial Cells Human Telomerase Reverse Transcriptase INK4A/ARF L LT LTR Cyclin-dependent kinase inhibitor 2A Liter Large T Antigen Long Terminal Repeat M-phase M MAP MAPK MCDB170 MCL1 MEK Mitosis Phase, cell cycle Molar Mitogen-Activated Protein Mitogen-Activated Protein Kinase (also ERK) Mammary Epithelial Basal Medium Myeloid cell leukemia sequence 1 (BCL2-related) Mitogen-Activated Protein Kinase Kinase 11

14 mirna mrna ml MLV mmol mol mtor Micro RNAs Messenger RNA Milliliters murine leukemia virus Millimole Moles mammalian target of rapamycin NF-kB nm nuclear factor kappa-light-chain-enhancer of activated B cells nanometer NOXA Phorbol-12-myristate-13-acetate-induced protein 1 OIS Oncogene Induced Senescence P p14 ARF p16 INK4a p21 p27 p53 p73 PCR PI3K Phosphorylated [protein] Cyclin-Dependent Kinase Inhibitor 2A Cyclin-Dependent Kinase Inhibitor 2A Cyclin-Dependent Kinase Inhibitor 1A Cyclin-Dependent Kinase Inhibitor 1B Tumor protein p53 Tumor protein p73 Polymerase Chain Reaction Phosphoinositide-3-Kinase 12

15 PIGs PLD PP2A PR PTPases PTEN p53 Inducible Genes Phospholipase D protein phosphotase 2A Progesterone Receptor protein tyrosine phosphatases phosphatase and tensin homolog PUMA BCL2 Binding Component 3 RAF RAL RAL-GEFs RAS RB RNA RTKs v-raf-1 murine leukemia viral oncogene homolog v-ral simian leukemia viral oncogene homolog Ral Guanine Exchange Factors Rat Sarcoma Viral Oncogene homologs Retinoblastoma Ribonucleic Acid Receptor Tyrosine Kinases S Synthesis Phase, cell cycle S6K1 p70 S6 Kinase 1 SCID SERM SFS shchk2 shp53 Severe Combined Immunodeficiency Selective Estrogen Receptor Modulators Serum-Free Supplements shrna to CHK2 shrna to p53 13

16 shrna SIN SMADs SOS st Short-Hairpin Ribonucleic Acid self inactivating LTR Mothers Against DPP homologs (Drosophila) Son of sevenless small t antigen SV40 Simian Virus 40 SV40-ER Simian Virus 40 Early Region TAp73 TDLUs TGFβ TGFβRII Transactivating p73 Terminal Ductal Lobular Units Transforming Growth Factor β Transforming Growth Factor β Receptor II TSC1 tuberous sclerosis 1 TSC2 tuberous sclerosis 2 µg Microgram µl Microliter µm Micromolar µmol Micromole VBIM VEGF VSV-G Validation Based Insertional Mutagenesis Vascular Endothelial Growth Factor vesicular stomatitis virus G 14

17 ACKNOWLEDGEMENTS There are many people I need to acknowledge that have contributed to my development as a graduate student. I want to thank my advisor, George Stark, PhD, for all his guidance over the years. I truly appreciate that he has always taken time out of his busy schedule to meet with me about my research. My co-mentor, Mark Jackson, PhD, deserves an enormous amount of credit for his contributions to my scientific development and his leadership directing this research program. I would also like to recognize my thesis committee members, Charis Eng, MD, PhD, David Danielpour, PhD, Edward Stavnezer, PhD, and Clive Hamlin, PhD, for all their time and effort. My time as a graduate student would not have been as pleasant or intellectually stimulating without the wonderful members of the Stark and Jackson laboratories: Charlene Kan, PhD, Sarmishtha De, PhD, Gretchen Larusch, John Patton, James Graham, and Damien Junk, PhD. Thank you, to all those on the Wolstein Research Building 3 rd floor for various help throughout the years. Last, but not least, I want to thank my family and especially my wife Linnea for all her support throughout the years. 15

18 Unraveling the Signaling Networks in Human Cell Transformation Abstract By ROCKY CIPRIANO The use of cellular transformation models has aided our understanding of tumorigenesis. The continued refinement of these cellular models is critical to expanding our understanding of the signaling pathways dysregulated in cancer and linking specific genetic alterations with the therapeutic efficiencies of new treatment options. In studies described here ablation of p73, or PTEN, or overexpression of BCL-2 resulted in a transformed phenotype which retained wild-type p53. Using this model of cellular transformation has allowed the dissection of genetic alterations that confer a sensitive or resistant phenotype to Nutlin-3, an HDM2 antagonist currently in clinical trials. Our results suggest that treating tumors harboring inactive PTEN, p73, or with elevated BCL- 2 expression with Nutlin-3 could re-engage p53 activity and induce apoptosis, arguing that therapies aimed at reactivating p53 could prove efficient in tumors harboring these genetic alterations. Such studies are critical in light of findings demonstrating HDMX overexpression protects cells from Nutlin-3 toxicity. As cell culture models of human cell transformation evolve, a more detailed analysis of the pathways commonly altered during cancer development can begin to be defined. Using normal human mammary epithelial cells (HMECs) obtained from reduction mammoplasties, we established a cellular model of the basal or triple negative subtype of breast cancer. This progressive model of breast 16

19 cancer initiation uncovered a novel TGF-β mediated suppressive barrier engaged by dysregulated oncogenic signaling. Unlike fibroblasts, we report here that HMECs undergo a TGF-β-mediated OIS in response to oncogenic RAS, which does not require p53, p16, ATM or CHK2. Finally, my studies led to the discovery and characterization of a novel oncogene, FAM83B, identified by combining a phenotypic based forward genetic screen with a HMEC transformation model. FAM83B is a previously uncharacterized protein, identified here as a driver of HMEC transformation that functions by hyperactivating the MAPK and mtor signaling cascades. We further demonstrate that FAM83B is a member of an eight protein family which contains a conserved N-terminal DUF 1669, that is necessary and sufficient for a transformed phenotype. The results presented here have (1) linked transforming events with the therapeutic efficiency of Nutlin-3, (2) identified a novel growth arrest pathway responsible for suppressing breast cancer initiation and (3) identified a novel oncogene family which may result in the development of novel therapeutics. 17

20 Chapter I: Introduction A. Overview Designing strategies for efficient diagnosis and effective treatment for cancer patients remains one of the biggest challenges cancer researchers face. Our understanding of the complex interactions between tumor suppressor and oncogene signaling pathways continues to evolve as components of these pathways are identified. The development of human cell transformation models has led to the identification of novel regulators in human cancer development and linked specific genetic alterations with therapeutic efficiency 1-3. In this dissertation I will discuss the use of human cell transformation models to (1) link specific genetic alterations with the therapeutic efficiency of Nutlin-3, (2) uncover a novel component of the tumor suppressive OIS engaged in response to activated oncogenes in mammary epithelial cells, and (3) discover a novel oncogene family. I first examined how ablation of p73, PTEN, or overexpression of BCL-2 promoted transformation in primary human fibroblasts which retain wild-type p53 and evaluated whether p53 could be efficiently activated by Nutlin-3. Since Nutlin-3 is a compound currently in clinical trials for the treatment of tumors with wild-type p53, my findings provide insight into which tumors may respond to this promising treatment. My studies next examined the signaling networks that must be disrupted early in the progression towards breast cancer. Breast cancers can be stratified into several subtypes based on their expression profiles. The breast cancer model that will be discussed in this thesis mirrors the basal-like or triple-negative breast cancer subtype. This subtype is very aggressive and has proven difficult to treat. Therefore, a better 18

21 understanding of how basal-like breast cancers develop may allow for the development of more effective treatment options. Since the majority of human breast cancers arise from epithelial cells, normal human mammary epithelial cells (HMECs) obtained from reduction mammoplasties were utilized in my studies. The use of normal HMECs allowed an in depth analysis of the genetic alterations that are required for neoplastic transformation. A novel tumor suppressive pathway to OIS was uncovered in response to oncogenic RAS signaling. Overcoming this suppressive barrier is a key step in transforming normal HMECs to malignant variants. Understanding the mechanisms that prevent OIS will provide the foundation for future therapies aimed at reengaging these hidden limits to proliferation as a cancer therapy. Finally, I will describe the successful use of a powerful genetic technique that permits identification of novel factors involved in positively or negatively regulating signaling pathways. This method was applied to the discovery of genetic changes that can substitute for oncogenic RAS to promote the transformation of immortalized HMECs. I will describe the discovery of a previously uncharacterized protein, name Family with Sequence Similarity 83, member B (FAM83B), which drives HMEC transformation and is required for RAS mediated effector activation. Interestingly, FAM83B is one of eight members of a protein family (FAM83) characterized by a highly conserved domain of unknown function 1669 (DUF 1669), which is required for the transforming ability of FAM83 members. This significant discovery of a novel family of oncogenes, and their critical role in growth signaling provides new targets in the pursuit for novel therapeutics. B. Current Statistics 19

22 Cancer is the result of numerous genetic and epigenetic changes that breakdown suppressive checkpoints and drive uncontrolled cell proliferation. Genetic changes encompass point mutations, amplifications, translocations, and deletions throughout the genome, while epigenetic changes encompass gene silencing through promoter methylation or gene activation by promoter demethylation. Mutations can occur through a variety of causes including exposure to radiation or mutagenic chemicals 4. The most important risk factor for developing cancer is age, as 77% of all cancers are diagnosed in people over the age of As we age, genetic and epigenetic changes accumulate within our cells due to increased exposure to carcinogens. In addition to acquiring genetic changes throughout one s life, there is also a subset of people who inherit mutations which predispose them to developing cancer. Despite the many advances made in cancer treatment, cancer remains a leading causes of mortality in the United States, resulting in about one in four deaths 6. Over 1.4 million new cases of invasive cancer are predicted to be diagnosed each year 5. In women, breast cancer is the mostly commonly diagnosed cancer, accounting for about 26% of all new cancer cases 6 and remains the leading cause of cancer death, despite billions of dollars spent each year 7. Further research aimed at understanding the mechanisms underlying breast cancer development will allow for more effective treatments that can link specific genetic alterations with therapeutic efficiency. C. Human Breast Architecture Each breast contains about 15 to 20 lobes composed of several lobules and terminal ducts that make up the terminal ductal lobular units (TDLUs) 8. The parenchyma of the adult breast ducts and lobules are mainly composed of luminal epithelial cells, 20

23 lining the ducts, and basal or myoepithelial cells which make up the outer layer 8. The luminal epithelial cells form a continuous layer while the basal cells lie in a single, discontinuous layer below the luminal cells 8 (Figure I-1). During menstruation, the secretion of hormones, such as estrogen and progesterone, stimulates proliferation leading to an increase in the height of alveolar epithelium and an increase in milk producing cells. As hormonal stimulation decreases, regression through apoptosis of alveolar epithelium is observed 8. Although there is apoptosis and involution following each menstrual cycle, there is an overall increase in total epithelial cell number and the TDLUs become more complex with each subsequent cycle 9. The increase in hormonal exposure during menstrual cycles has been proposed to explain the increased risk of breast cancer in women who do not have children or become pregnant late in life, since pregnancy reduces the total number of menstrual cycles a woman has in a lifetime 10. D. Breast Cancer Subtypes In recent years, breast cancer has increasingly been recognized as a collection of separate diseases defined by distinct genetic alterations 11. About 75% of breast cancers are estrogen receptor (ER) and progesterone receptor (PR) positive 12, two markers clinically used to classify breast cancers. Another marker used in classifying breast cancer is human epidermal growth factor receptor 2 (HER2), which has been found to drive the growth of about 15-30% of breast cancers 13. This has allowed breast cancers to be classified as ER+ or ER- and HER2+ or HER2-. The stratification of breast cancer has begun to link genetic alterations with specific therapies. For example, patients with ER/PR+ tumors often respond positively to compounds that inhibit estrogen activity or production

24 Figure I-1: Morphological architecture of the mammary gland in vivo. Schematic of a lobule from human mammary gland; mammary epithelium possesses a polarized architecture surrounding a hollow lumen, which is surrounded by an inner layer of luminal epithelial cells and an outer layer of myoepithelial and basal epithelial cells 15 22

25 In recent years researchers have started classifying breast cancers using their gene expression profiles in combination with ER, PR, and HER2 status. Each subtype of breast cancer has a unique gene expression signature and these signatures can be used to predict prognosis and treatment outcomes. Hierarchical clustering of gene expression signatures has stratified breast cancers into five distinct types, including normal breast-like, luminal A (ER/PR+; HER2-; low grade), luminal B (ER/PR+; HER2-; high grade), basal-like (ER/PR-; HER2-), and HER2 positive (ER/PR- or +; HER2+) sub-types 16, 17. The most significant difference in long term survival was observed between patients with a luminal A subtype, which had the longest survival time, and patients with a basal-like or HER2+ subtype, which had the shortest survival time 18, 19. The basal-like or triple-negative (ER/PR-; HER2-) subtype has the worst disease free survival indicative of a faster rate of recurrence 20. Also, patients with ER/PR-; HER2+ tumors have survival rates most similar to triple-negative tumors 20. Although both luminal A and luminal B subtypes express the estrogen receptor, the overall survival differs between the two, with the luminal B subtype having a shorter overall survival rate and a greater rate of relapse 18. Stratifying breast cancers has proven advantageous due to its ability to link efficiency of treatment options with specific subtypes. For example, low grade luminal A tumors are sensitive to anti-estrogens while higher grade luminal B and HER2+; ER+ tumors are less sensitive 17. Patients with luminal B subtype tumors have not benefited from the use of selective estrogen receptor modulators (SERMs), likely due to lower levels of ER expression 18. Also, basal-like tumors which are typically more aggressive have increased sensitivity to preoperative chemotherapy when compared to luminal tumors

26 E. Breast Cancer Treatments Currently the treatment of breast cancer includes a combination of several modalities which encompass surgery, radiation, chemotherapy, targeted therapy and hormonal therapy 22. Surgical options can range from a full mastectomy, where the breast is removed, to a lumpectomy combined with radiation therapy, which aims to remove tumor without removing large amounts of normal breast tissues. Radiation therapy is often used following surgery to eradicate subclinical residual disease and minimize local recurrence. Chemotherapy is also used in the treatment of breast cancer. However, since chemotherapies are systemic therapies, they also affect normal cells in the body, limiting the doses that can be given to patients. This limitation has resulted in a push for more targeted therapies that work by inhibiting a particular oncoprotein that drives the tumor s growth. Such therapies represent a more personalized treatment since they are only effective if the patients harbor a particular mutation or overexpress a protein that drives the growth of their malignancy. Targeted therapies can range from small molecules which inhibit an enzymatic activity that is elevated in cancer cells, often due to amplification or activating mutations, to monoclonal antibodies that work by blocking receptor activation and ultimately block tumor growth. For example, Avastin (bevacizumab) is a monoclonal antibody currently being used in breast cancer treatment, which binds to vascular endothelial growth factor (VEGF) and prevents it from interacting with its receptors, resulting in decreased angiogenesis 23. Another monoclonal antibody currently used in breast cancer treatment is Herceptin, which blocks the growth 24

27 factor receptor HER2 and is currently used in patients who have HER2+ breast cancers 23, 24. As described earlier, hormonal therapy is the current standard of care for the treatment of breast cancers that depend on estrogen and progesterone to proliferate. Many of these breast cancers have been successfully treated by blocking the activity of the estrogen receptor. Treatment with SERMs blocks the effects of estrogen at certain target organs, such as the breast, and can be used in both pre- and post-menopausal women. Tamoxifen, a commonly used SERM is a competitive inhibitor of estrogen, that binds to ER inhibiting ER activity 25. The use of aromatase inhibitors such as Arimidex can be used in post-menopausal women to inhibit estrogen production 14. Post-menopausal woman no longer have secretion of estrogen from their ovaries; however, the estrogen is replaced by the conversion of androstenedione to estrone and testosterone to estradiol. Thus, inhibiting aromatase with drugs such as Arimidex, lowers circulating estrogen levels and prevents estrogen dependent breast cancers from growing 26. In this thesis we evaluate the directed therapeutic agent Nutlin-3 and its efficacy on various genetic backgrounds with wild-type p53 status. The Nutlin compounds are excellent candidates for breast cancer treatment since seventy five percent of breast cancers have wild-type p53 status 27. The Nutlins stabilize the tumor suppressor p53 by competing for binding to its negative regulator HDM2, preventing the two proteins from interacting and resulting in the stabilization of wild-type p53. Nutlin-3 has been shown in tumor cell lines and xenographs in nude mice to lead to toxicity and prevent tumor growth 28. Although reactivation of p53 by Nutlin-3 holds great therapeutic promise, several laboratories have demonstrated that normal and transformed cells overexpressing 25

28 HDMX, a protein with significant homology to HDM2, are resistant to Nutlin-3 2, 29, 30. The failure of Nutlin-3 to affect HDMX-p53 interactions demonstrates the importance of being able to determine the genetic contexts where its clinical use may be most effective. F. Forward genetics in gene discover Forward genetics in mammalian cells is a powerful tool, used to delineate novel functions for known and unknown proteins alike 31. Typically, a selection for a given phenotype is established whereby rare cells obtain a growth or survival advantage due to a genetic alteration induced by chemical mutagens, cdna or shrna libraries, or insertional mutagens (Figure I-2). Each of these approaches creates distinct genetic changes and poses specific challenges. We have focused on promoter insertion, delivered by retroviruses, to modify cellular gene expression. Promoter insertion can induce dominant changes in the expression of genomic sequences neighboring the insertion sites, including high-level expression of a full-length or truncated protein. Since truncated proteins may have constitutive or dominant-negative functions, genes that facilitate or inhibit the phenotype of interest can be identified. Typically, insertional mutagenesis studies have been performed using murine leukemia virus (MLV)-based vectors. However, MLV integrase preferentially targets promoter regions, while HIV integrations occur throughout coding regions with an avoidance of CpG islands found in and around promoters 32, 33. This pattern of HIV 26

29 Figure I-2: Forward genetics paradigm. A diverse cell library is produced such that each cell has a unique genetic alteration. The cell library is placed into selection for a rare phenotype, and the genetic alteration driving the mutant phenotype is identified. Introduce Genetic alteration Selection Identify Genetic alteration Parental Cell library Rare Mutants 27

30 integration should increase the probability of generating truncated proteins and antisense transcripts, since insertion would statistically occur more frequently downstream of the initiation codon, with equal chances of integrating in either orientation. Both products increase the likelihood of identifying genes with growth arrest or apoptosis-inducing functions, thereby expanding the use of insertional mutagenesis to identify tumor suppressors. G. Validation-Based Insertional Mutagenesis (VBIM) Retroviruses have been used as insertional mutagens for decades. We sought to improve their utility in forward genetics screen by modifying the lentiviral vector backbone in a number of ways, the most significant being the placement of the mutationinducing CMV promoter so that it drives transcription through the 3 LTR 34. This could only be accomplished if we first mutated the polyadenylation signals in the 5 and 3 LTRs to permit transcriptional read-through into the genomic sequences near the viral insertion site. In addition to the mutant poly(a) signals, a deletion to eliminate 3 LTR promoter activity was introduced (termed self-inactivating (SIN) LTRs) and a LoxP site was included in the 3 LTR to permit excision of all but 238 nucleotides of inert proviral LTR sequence upon expression of Cre recombinase (Figure I-3A and B) 34, 35. Using this vector backbone, we added a highly active CMV promoter that can be regulated by an upstream tetracycline operator sequence, if desired. A GFP reporter gene is under the control of the CMV promoter, and a downstream internal ribosome entry site (IRES) allows translation of the cellular protein encoded by the genomic DNA downstream of the insertion site (Figure I-3C) 34. The cellular protein will have a virallyencoded FLAG tag on its amino-terminus. A splice donor sequence is provided in the 28

31 Figure I-3: VBIM lentiviral construction and use. (A) Illustration of the Validation- Based Insertional Mutagenesis vector backbone, with deletion of the poly(a) signals in the 5 and 3 LTRs (* and shown in the gray box), and addition of the promoter insertion components. (B) Illustration of the provirus following integration, including splicing into the neighboring exon. (C) Transcription from the CMV promoter would produce a single mrna encoding GFP and a FLAG/cellular fusion protein, provided splicing is correct. Abbreviations: tetracycline operator (TO), woodchuck hepatitis virus post-transcriptional regulatory element (WPRE), splice acceptor site (SA), splice donor site (SD), internal ribosome entry sequence (IRES), long terminal repeat (LTR), self-inactivating LTR (SIN), green fluorescent protein (GFP), cytomegalovirus promoter (CMV), central polypurine tract (cppt), site for cre-mediated recombination (LoxP). A B TAR U G G G C A C G A G U U U G C U C A G A C wild-type HIV R region E mutant HIV R region Δ Poly(A) G = E mutation Δ G = Adenovirus Klasens et. al NAR 1998 FLAG Splice donor 3 LTR * LTR TO CMV GFP WPRE IRES SIN * polya LoxP * U C U G SIN C G A G C A U U A U A G C G U LoxP poly(a) U A C G U G C G U A C U G G A C C A G C U U A G A C U C A U A U C G U A C G U C G G C A U A U C U A U A C G G U U A C G A U C G TAR U G G G A C C G A G U U U A G G C U C A U C G C G A G C A U U A U A G C G U U A C G G G U C U TO CMV U C C U G G deleted poly(a) A A C C poly(a) U A U A C G G C U A C G A U C G GFP WPRE IRES * SIN LoxP exon C mrna: GFP WPRE IRES exon SIN Proteins: GFP FLAG 29

32 vector, with three different reading frames of FLAG-SD (splice donor) present in three separate constructs, so that each splice acceptor sequence in the genome can be targeted to produce a FLAG-tagged fusion protein. The VBIM viruses have general features that are extremely useful and convenient. The lentiviral backbone allows infection of most cell types, and even growth arrested cells can be infected. The viral particles are pseudotyped with VSV-G protein, so that any cell can be infected with high efficiency and virus stocks are quite stable when stored long term at C. Importantly, the dominant mutations expected following promoter insertion include elevated expression of (1) a full-length protein, (2) a truncated protein, or (3) production of an anti-sense mrna. Since truncated proteins may have constitutive or dominant-negative functions, we can identify genes that either facilitate or inhibit the phenotype of interest. Experimental evidence proving the functionality of the VBIM vectors currently exists for different biological systems including multiple mutants with acquired chemotherapy resistance and inhibitors of NFkB 34, 36. A screen for genes that can substitute for RAS in an HMEC transformation model is presented in this thesis. Once a mutant has been created via VBIM-mediated promoter insertion, the goal is to remove the promoter by Cre-mediated excision or silence the promoter using TR- KRAB-mediated repression and determine whether the mutant phenotype reverts to wildtype (Figure I-4). Reverse transcription and integration of the VBIM viruses will yield a provirus having identical 5 and 3 SIN LTRs with the CMV promoter in between (Figure I-3B). Since each LTR contains a LoxP site (the recognition site for the Cre recombinase enzyme), expression of Cre recombinase will excise the majority of the proviral DNA, leaving behind one 238 nucleotide LTR lacking promoter activity and poly(a) signals. 30

33 Figure I-4: Use of the VBIM technology. (1) The creation of a cell library and selection for a rare mutant phenotype; (2) the validation that the mutant phenotype is promoterdependent; (3) the identification of the gene responsible for the mutant phenotype. Selection Vec Cre TR KRAB Selection Validated Promoter dependent Identify mutation VBIM infected Cell library Mutants Vec Cre TR KRAB NON Validated Promoter independent Discard 31

34 In most cases, integration will occur within introns (since they comprise the majority of each gene), therefore, we hypothesize that a 238 base pair SIN LTR with a deleted poly(a) signal will not usually influence appropriate expression from the endogenous promoter. Another option is reversion by promoter silencing. Using a fusion protein consisting of the Kruppel-associated box (KRAB) domain of the human Kox 1 zinc finger protein to the teto DNA binding domain from the prokaryotic tetracycline transrepressor (ttr) gives rise to a protein that represses promoters in the vicinity of a nearby teto sequence (ttr-krab) 37, 38. Expression ttr-krab in cells with integrated VBIM virus can strongly repress even a full-length CMV promoter. Additionally, the repressive activity of ttr-krab can be inhibited by adding tetracycline or doxycycline to the medium, thereby reversing the reversion and giving rise to the initially selected mutant phenotype (Figure I-4). Once a mutant has passed the validation testing, one can be confident that the VBIM promoter insertion is responsible for the mutant phenotype. The next step in the process is to identify the VBIM insertion site. The VBIM insertion sites can be identified using inverse PCR or ligation mediated PCR methods 34. After integration site identification, the mutant phenotype must be validated in naïve cells. This can be accomplished by either knockdown of a gene product such as in the case of a promoter being inserted in the antisense orientation relative to the endogenous gene. Or one can over express either a full-length or truncated gene product that corresponds to a forward orientated promoter, relative to the endogenous gene. Since truncated proteins may have constitutive or dominant-negative functions, we can identify genes that either facilitate or 32

35 inhibit the phenotype of interest. H. Tumor suppressors In this thesis, I will delineate the role of critical tumor suppressors in human cell transformation. I will link the loss of tumor suppressors to the therapeutic efficiency of Nutlin-3. This thesis will also discuss the role that tumor suppressors play in response to dysregulated oncogenic signaling in normal HMECs and how these tumor suppressive pathways must be perturbed for neoplastic transformation. p53 The p53 protein has been found to be mutated in about 50% of human cancers and is functionally inhibited in most cancers 39, 40. p53 is a transcription factor that regulates the expression of genes involved in cell cycle arrest, DNA repair, apoptosis and functions as a tumor suppressive barrier to cancer 41, 42. The levels and activity of p53 can be suppressed by interactions with its negative regulators HDM2 and HDMX, which bind to and prevent p53 from initiating transcription. HDM2 ubiquitinates p53 and leads to its proteosomal degradation, while HDMX does not degrade p53 but rather inhibits p53 transcriptional activity The functional consequences of p53 activation can lead to cell cycle arrest, senescence, or apoptosis. Cell cycle arrest occurs via upregulation of the cyclin-dependent kinase (CDKs) inhibitor p21 and other genes such as σ, Reprimo, GADD45, and B99 46, 47. Apoptosis is induced by p53 responsive genes such as BAX, NOXA, PUMA, PIGs, and FAS 48. The genetic autosomal dominant hereditary disorder, Li-Fraumeni syndrome, which is caused by germline mutations in p53, is associated with an increased risk of breast cancer 49. Also, analysis of breast cancers shows about 20% harbor a mutation in 33

36 the p53 gene 50. Inhibition of p53 can also occur by amplification or overexpression of the p53 negative regulators HDMX 51 or HDM2 52. HDMX expression is observed in about 20% of breast cancers 51. Also, mutation or suppression of upstream activators or downstream target genes can lead to a suppression of p53 function 53, 54. Inactivation of p53 in breast cancer has also been linked to the loss or methylation of the HOXA5 transcription factor, which is responsible for p53 gene transcription 55, 56. PTEN A common event in human cancers is alterations of the tumor suppressor Phosphatase and tensin homolog deleted on chromosome 10 (PTEN), which has been documented in primary cancers, such as brain, prostate and breast cancers 57, 58. The autosomal dominant multineoplasia syndrome, Cowden disease, is associated with germline PTEN mutations and an increased incidence of breast cancer PTEN function has been implicated in a diverse range of biological processes, such as cell cycle arrest, apoptosis, and inhibition of cell migration 63. PTEN is a protein and lipid phosphatase involved in cell cycle control, apoptosis, cell migration and cellular proliferation A major function of PTEN is the dephosphorylation of phosphatidylinositol-3,4,5-triphosphate (PIP3), which is a lipid second messenger and a product of the proto-oncogene phosphatidylinositol 3-kinase (PI3K) 65. PI3K is activated by a number of tyrosine kinase growth factor receptors and integrins 67, 68. The accumulation of PIP3 at the membrane allows recruitment of proteins containing a pleckstrin homology (PH) domain. PIP3 recruits the downstream effector AKT/PKB to the plasma membrane through its PH domain. AKT is a serine threonine protein kinase that, once activated, acts to inhibit apoptosis by phosphorylation of several 34

37 key substrates 69. It has been reported that PTEN overexpressing cells exhibited lower levels of AKT phosphorylation and also that PTEN mediated repression of gene expression was rescued by activated AKT but not activated PI3K 70. The regulation of AKT by PTEN is mediated by its ability to reduce PIP3 levels, thus preventing activation of AKT 71. This regulation of AKT activity by PTEN is pertinent in the regulation of cell survival and cell proliferation. Most point mutations identified in PTEN during tumorigenesis lead to either complete loss or reduced catalytic activity and mutations are clustered within residues important for the secondary structure of PTEN 72. This suggests that the loss of PTEN phosphatase activity is a required step for tumorigenesis. Also, p53 protein stability has been shown to be dependent on PTEN through phosphatase dependent and independent mechanisms 66, 73. The p53-pten interaction has been demonstrated to enhance p53 binding to p53 target genes, including the p21 and PTEN promoters 73, 74. Also, it was demonstrated that PTEN enhances acetylation by p300 on p53 independent of its catalytic activity and this results in the tetramerization of p These reports outline a mechanism of tumor suppression for PTEN by modulating p53 function. p73 The p53 family member p73 is structurally similar to p53 and can transactivate p53 responsive genes in response to various stimuli. The p73 gene is located at chromosome 1p36, a region frequently deleted in neuroblastoma, colon cancer, melanoma, and breast cancer 76, 77. The p73 gene leads to several gene products and numerous isoforms based on cell type and alternative splicing. The two major classes of isofroms include the TA isoforms, which express a tranactivation domain that has 35

38 significant homologue to p53, while the ΔN isoforms lack this transactivation domain and functions in a dominant-negative fashion towards TAp73 and p53, blocking transactivation of p53-responsive genes 78. The TA isoforms of p73 have similar properties to wild type p53 and have the ability to transactivate p53 responsive genes. Therefore, TAp73 isoforms are suspected to play a tumor suppressor role in human tumors 79. The ΔNp73 isoforms are thought to play a role in blocking the transactivation of both, p53 and TAp73 target genes, such as p21, Bax, HDM2 or σ, and are considered to be a dominant-negative transcriptional inhibitor of p53 family members 80, 81. The ΔNp73 isoforms have been shown to be elevated in various human cancers 82. Also, it has been shown that inactivation of p73 occurs at the transcriptional level and is associated with promoter hypermethylation in oligodendroglial tumors 83. Mouse models have also been used to examine the tumor suppressive functions of p73. It has reported that p73+/- mice develop lung adenocarcinomas, thymic lymphomas, and hemangiosarcomas by two years of age 84. In combination with the observation of loss of heterozygosity in p73+/- tumors, this report supports the tumor suppressive functions of p73 and its prominent role in suppressing tumorigenesis 84. RB One of the first tumor suppressors to be discovered was the Retinoblastoma protein (RB) 85. Defects in RB led to its discovery as a tumor suppressor and the cause of retinoblastoma 86, 87. RB belongs to a family of proteins that includes p107 and p130 and is phosphorylated by cyclin-dependent kinases (CDKs) 88, 89. RB regulates the cell cycle by binding to members of the E2 transcription factor family (E2Fs) when 36

39 hypophosphorylated, inhibiting their transcriptional activity. The phosphorylation of RB by CDKs disrupts the interaction with E2Fs and allows for E2F induced transcription to occur 90. E2F1 induces the transcription of a number of genes needed for DNA replication and cell cycle progression 91. Disruption of the normal functions of RB can lead to tumor development, through unregulated cell cycle control and E2F1 transcription 92. p16 and p14arf The INK4A/ARF locus encodes two vital tumor suppressors through alternative reading frames, cyclin-dependent kinase inhibitor 2A, p16 INK4a (p16), and the p53 activator p14 ARF (ARF). Although these two proteins are encoded by the same gene they share no amino acid homology and have separate molecular functions 93. p16 is a negative regulator of the CDKs and can induce a G1 arrest by ultimately dephosphorylating and activating RB family members. The p14arf tumor suppressor functions by regulating p53 stability by inactivating its negative regulator HDM2, leading to the stabilization of p53 protein, which can result in cell cycle arrest or apoptosis 94. I. Oncogenes c-myc and RAS Two oncogenes discussed in this thesis are avian myelocytomatosis viral oncogene homolog (c-myc) and rat sarcoma viral oncogene homologs (RAS). Both oncogenes been used in transformation models to provide cells with constitutive proliferative signals Many cancers have elevated levels of c-myc caused by amplification, translocation or mutation, altering the normal regulation of its functions. c- MYC is a transcription factor that can increase proliferation and growth but can also induce apoptosis or G2 arrest and also has effects on metabolism 98. Overexpression of c- MYC can result in the loss of cell contact inhibition and the requirement for cell matrix 37

40 attachment 99, 100. Also, elevated c-myc can result in genomic instability, promote angiogenesis, and promote the development of cancer 99, 100. Gene expression and chromatin immunoprecipitation analysis has led to the estimation that c-myc regulates the expression of roughly 15% of the genome 101. It has yet to be demonstrated that one single c-myc regulated gene can recapitulate the cell transforming activity of c-myc. Therefore, the cooperative activity of several c-myc target genes are required to transform cells 100. Approximately 70% of human breast cancers have elevated c-myc expression 102. Many studies examining MYC expression using immunohistochemistry demonstrated that % of breast cancer cases had increased protein levels of c-myc 103. RAS is a powerful oncogene that normally acts as an intermediate between growth factor receptor tyrosine kinases and downstream serine/threonine kinases, such as mitogen-activated protein (MAP) kinases 104. MAP kinases phosphorylate and regulate the activity of nuclear transcription factors. When RAS activity becomes dysregulated, cells experience mitogenic signals in the absence of growth factors, leading to the oncogenic activity of RAS 105. In normal cells, the introduction of constitutive activation of RAS signaling leads to cellular senescence, usually through the actions of p53 and p This proliferative arrest engaged in response to persistent oncogene activation has been termed oncogene-induced senescence (OIS). However, cells which have disrupted p53 and/or p16-rb tumor suppressive pathways can tolerate and become transformed by activated RAS expression 97, 107. RAS is commonly used in transformation models to provide cells with a constitutive proliferative signal, following inactivation of the RB and p53 tumor 38

41 suppressor pathways 95, 97, 108, 109. While activating point mutations of RAS are representative of many cancer types, with 50% of colon and thyroid cancers, and 90% of pancreatic cancers harboring such mutations, they are infrequent in breast cancer (less than 5%) 104, 110. The reason for the differences between cancers that tolerate RAS mutations and those that do not remains unclear. However, breast carcinoma cells still acquire alterations in the RAS-signaling pathway, most notably through amplification of RAS 104. There is also extensive support that deregulated RAS signaling, such as overexpression of wild-type RAS or downstream proteins, can contribute to the transformation of mammary epithelial cells J. Cellular Transformation Cellular transformation models have provided important insights regarding the specific genetic changes that permit the malignant transformation of human cells. The ability to transform normal human cells in vitro allows us to understand the impact of specific genetic alterations on tumor development. It also allows one to determine what tumor suppressive mechanisms must be overcome to drive transformation and also allow for the development of novel tissue-specific therapeutics. Cancer cells typically acquire a number of capabilities that can be categorized as cell autonomous hallmarks and tumor expansion hallmarks (Figure I-5) 114. Cell autonomous hallmarks allow the formation of the tumor and include self-sufficiency in growth signals, insensitivity to growth-inhibitory signals, evasion of apoptosis, and limitless replicative potential (immortalization). Tumor expansion hallmarks include tissue invasion, metastasis, and sustained angiogenesis. To circumvent anti-growth and 39

42 Figure I-5: Acquired Capabilities of Cancer. We [Hanahan and Weinberg] suggest that most if not all cancers have acquired the same set of functional capabilities during their development, albeit through various mechanistic strategies

43 pro-apoptotic signals, the RB and p53 tumor suppressor pathways are commonly inactivated in cancer. The transformation of normal human cells has proven to be more complex than the transformation of normal rodent cells. Expression of either adenoviral E1A, c-myc, polyoma Large T, or mutant p53, in combination with activated-ras lead to the transformation of normal rodent cells 96, 115, 116. However, expression of these combinations of events in human cells, such as fibroblasts or lymphocytes, did not result in a transformed phenotype 117, 118. This indicates that murine cells have fewer suppressive barriers to transformation compared to normal human cells 95. The first successful transformation of human cells incorporated the expression of htert in combination with the simian virus 40 (SV40) early region (SV40-ER) and activated-ras in embryonic kidney cells and normal human fibroblasts 97. Further studies later demonstrated that the SV40-ER encoded two protein, Large T antigen (LT) and small t antigen (st), and that both were required for transformation (Figure I-6) 119, 120. The SV40 LT antigen has been shown to inactivate both the p53 and RB tumor suppressor pathways 121. Transient expression of SV40 st in mammalian cells inhibits protein phosphotase 2A (PP2A), which leads to the activation of growth factor-stimulated signaling through activation of Phosphoinositide 3-kinase (PI3K) 120, 122, 123 and mitogenactivated protein kinase kinase (MAPK) 124, 125. Also, in a fibroblast model a shrna directed at PTEN was able to substitute for the SV40 st antigen 95. Transformation of normal human fibroblasts without the use of viral proteins was finally achieved by expression of htert, dominant-negative p53, shrnas directed at the tumor suppressors RB and PTEN, and overexpression of MYC and RAS

44 Figure I-6. Normal Human Cell Transformation. Normal human cells can be transformed by using a multistep protocol. (1) Immortalization through htert expression. (2) Inhibiting oncogene induced senescence (OIS) through p53 and RB inhibition. (3) Providing a persistent growth signal. 42

45 K. Acquisition of persistent growth signals The acquisition of persistent growth signaling by cancer cells is a hallmark of transformation. One of the studies described in this thesis describes a novel protein involved in driving aberrant growth signaling by RAS and receptor tyrosine kinases (RTKs). A number of important proteins can drive aberrant growth signaling when mutated or overexpressed. Often, the aberrant activation of RTKs results in a cascade of events that change the cells transcriptional and translational programs (Figure I-7). RTK activation recruits adaptor proteins, such as Grb2 or Shc, which in turn recruit son of sevenless (SOS) and RAS to the receptor, resulting in RAS activation 126. Following its activation, RAS initiates a series of signaling changes that result in the recruitment of RAF to the membrane where RAF becomes activated by phosphorylation, and subsequently phosphorylates MEK1 and MEK2 which activate ERK1 and ERK The ERK proteins then activate transcription factors responsible for regulating growth and proliferation. RAS activation also activates PI3-Kinase, by binding to a conserved Ras binding domain (RBD) in the p110 catalytic subunit 128. Activation of PI3K results in the phosphorylation of phosphatidylinositol (4,5) bisphosphate (PIP2) to form PIP3, which activates Phosphoinositide-dependent protein kinase (PDK-1), and recruits AKT to the membrane for activation. AKT phosphorylates and activates mammalian target of rapamycin (mtor), which regulates numerous cell division and growth pathways by modulating transcription, translation and protein degradation. My studies will describe a role for mtotr activation in HMEC transformation. In addition, AKT phosphorylates TSC2, which activates Rheb, a GTPase 43

46 Figure I-7. Schematic of receptor tyrosine kinase (RTK) and RAS signaling. RAF P P MEK P ERK PA Transcription RTK RAS RAL PLD PI3K PDK1 P AKT P TSC1/2 Rheb P PA mtor + + Raptor Rictor P S6K P P 4E BP1 PKC P AKT Translation Proliferation 44

47 that promotes an interaction between mtor and Raptor (regulatory associated protein of mtor), to create mtor complex 1 (mtorc1). mtorc1 phosphorylates S6K and 4E- BP1, which activates S6K and inhibits eif-4e binding protein (4E-BP1), resulting in increased translation by eif-4e 129. Collectively, these processes all promote cell growth and survival in response to RAS-PI3K-AKT activation. A second mtor complex is formed when mtor interacts with Rictor (rapamycin-insensitive companion of mtor) to create mtorc2. The function of TORC2 is not as well characterized as mtorc1, but it is clear that mtorc1 and mtorc2 phosphorylate distinct targets. mtorc2 does not phosphorylate S6K or 4E-BP1, but rather phosphorylates AKT, SGK1, and PKC 130. Importantly, the mtorc1-specific component Raptor has been shown to have an inhibitory effect on mtorc2, while the mtorc2-specific component Rictor inhibits mtorc Collectively, these signaling cascades have become the subject of intense research aimed at identifying pharmacological inhibitors that will suppress growth signaling and prevent cancer cell proliferation. Targeted therapies aimed at disrupting RTKs (including antibodies and small molecule inhibitors targeting EGFR, HER2 and VEGF), RAS (Farnesyltransferase Inhibitors Tipifarnib and lonafarnib), RAF (Sorafenib, RAF265 and PLX4032), MEK (PD , AZD6244, ARRY and ARRY ), AKT (VDQ-002) and mtor (Rapamycin, CCI-779, RAD001, and AP-23573), have been developed and are currently being evaluated in a number of clinical trials 129, 132. However, the complexity of signaling interactions continues to limit the effectiveness of these therapies, since resistance is easily obtained due to the plasticity of cancer cells. It is 45

48 therefore important that novel proteins involved in these signaling cascades be identified to provide new opportunities for drug development and expansion of therapeutic options. In addition to RAF-MEK-ERK and canonical PI3K-AKT-mTOR signaling, RAS activation also stimulates RalA and RalB, small GTPases that facilitate intracellular signal transduction. RAL proteins are required for RAS-mediated tumorigenesis and tumor survival 133, 134, due to their stimulation of phospholipase D (PLD) activity, which hydrolyzes phosphatidylcholine into phosphatidic acid (PA) and choline 135, 136. PA is an important signaling lipid involved in recruiting cytosolic proteins to the membrane where they can be activated to potentiate growth signaling. Two important signaling molecules that require PA for their activation are RAF and mtor 137. In fact, the activation of PLD- 1 has been proven to be critical for the transforming activity of RAS. Inhibition of PLD activity prevented the anchorage-independent growth and tumorigenicity of both H-RAS and K-RAS-expressing cells. This effect was specific for PA, since transformation could be restored simply by providing the cells exogenous PA 138. Given that many tumors have elevated PLD activity, considerations for the role of PA in RAS-mediated signaling pathways will be important for devising future therapies 139. Studies of RAF activation have defined an important regulatory process mediated by the direct binding of PA to a site in the C-terminus of RAF 140. Upon growth factor receptor activation, PLD-mediated PA production induces the translocation of RAF to the plasma membrane. Mutation of key basic residues located in the putative PA binding region (PA-BR) of RAF efficiently prevents RAF translocation and activation Moreover, the production of PA is also required to couple ERK activation in cell membranes 143. In addition to regulating RAF activation, the role of PA in activating 46

49 mtor is a developing research area with important implications for cancer therapeutics, since PA and rapamycin (a therapy that targets mtor) compete for the same binding domain on mtor 137. Therefore, the overall PLD activity within a cancer cell influences its sensitivity to rapamycin. Recent studies have shown that by reducing the overall PLD activity in cancer cells, the sensitivity of mtor complexes to inhibition by rapamycin could be significantly increased 144. Importantly, therapeutic doses of rapamycin that were unable to affect mtorc2 in the presence of elevated PLD activity could be rendered inhibitory simply by suppressing PA levels 145. This has important implications for the use of rapamycin and its derivatives in the treatment of breast cancers, since recent studies have demonstrated that breast cancers have significant elevation of overall PLD activity relative to normal adjacent tissue 146. L. TGF-β Signaling Transforming growth factor beta (TGF-β) is a cytokine that is involved in a variety of cellular processes and has both tumor suppressing and tumor promoting activities 147, 148. Activation of the TGF-β receptor leads to initiation of downstream signaling through the phosphorylation of SMAD (mothers against DPP homologs (Drosophila)) proteins, a family of transcription factors 149. Growth inhibition occurs via the induction of CDK inhibitor proteins, such as p21, and repression of c-myc expression 150, 151. The ability of TGFβ to inhibit tumor growth is often associated with lower tumor stage and its growth promotion properties is associated with advanced tumor stage and metastasis 152, 153. Tumors which have disabled the growth inhibitory arm of TGF-β signaling, have enhanced cell invasion, migration, and evade the immune system, thereby promoting tumor growth

50 M. Human Mammary Epithelial Cells (HMECs) The majority of human cancers arise from epithelial cells and develop by overcoming cell cycle checkpoints, acquiring persistent growth signals, and immortality. Cell culture models of human cell transformation have begun to elucidate the pathways commonly altered during this process. However, these models commonly use fibroblasts rather than epithelial cells. It was previously observed that inhibition of the RB and p53 pathways by SV40 LT antigen, along with activation of PI3K signaling, is sufficient to transform immortalized HMECs 155. While the use of viral proteins has greatly aided in the dissection of pathways necessary for transformation, they have also complicated the interpretation of these results because viral proteins have multiple intracellular targets. Therefore to examine human breast cancer, the ability to begin with normal HMECs allows us to examine the suppressive barriers in transformation. We obtained commercially available, htert-immortalized HMECs (HME1) which maintain a normal phenotype and karyotype 155. The expression of htert, which encodes the catalytic subunit of telomerase, has proven important for conferring limitless replication in human cells 97. Indeed, studies show that at least 80-95% of human breast cancers possess telomerase activity 156. HME1 cells can be transformed through the use of SV40 LT and activated RAS 155. It was also discovered that late-passage immortalized HMECs (HME1), commonly upregulate endogenous c-myc expression and lack p16 expression due to methylation 155. Therefore, HME1 cells require htert, c-myc, and RAS expression to exhibit a transformed phenotype. We have also collaborated with Dr. Martha Stampfer at the Lawrence Berkeley National Laboratory who has developed a method to obtain cultures of human mammary 48

51 epithelial cells from reduction mammoplasties. The reduction mammoplasty specimens are grossly dissected to obtain the epithelial containing tissue which is then minced and digested with collagenase and hyaluronidase. Upon completion of the digestion step, microscopic examination shows organoids, clumps of cells, with ductal, alveolar or ductal-alveolar structure (Figure I-8A). These organoids are then plated onto tissue culture dishes and an outgrowth of cells will develop (Figure I-8B). A process of differential trypsinization allows for an epithelial subculture to be obtained (Figure I- 8C) 157. HMECs obtained through this process will encounter a proliferation barrier and senesce after about 10 to 15 population doublings, however, a subpopulation of epithelial cells will grow out 158. These HMECs (48R) were found to have methylated p16 allowing them to overcome the proliferation barrier which is a stress-induced growth arrest (stasis) mediated by p p16 inactivation due to promoter methylation is observed in histologically normal mammary epithelium, and is now believed to be an important early event in breast carcinogenesis resulting in diminished RB responsiveness 159. N. Summary and Research Aims Advances in our understanding of the complex interactions between tumor suppressor and oncogene signaling pathways will lead to the discovery of novel therapeutic options. The development of human cell transformation models has led to the identification of novel regulators in human cancer development and linked specific genetic alterations with therapeutic efficiency 1-3. Therefore, in this dissertation we expand on our understanding of cellular transformation by, linking specific genetic alterations with Nutlin-3 efficiency, uncovering a novel suppressive barrier to persistent oncogenic signaling, and discovering a new family of oncogenes involved in persistent growth 49

52 Figure I-8. Generation of Epithelial Cell Cultures from Organoids. (A) Ductalalveolar organoid derived from enzymatic digestion of reduction mammoplasty tissue. (B) Morphology of normal human mammary epithelial cells grown in MEGM. Growth of the primary culture 5 days after being seeded. The remnant of the organoid is in the center, with actively dividing cells on the outside of the patch (38X). (C) Primary mammary epithelial cell culture growing as a monolayer culture (Adapted from 157 ). 50

53 signaling. The studies presented in this dissertation provide us with novel therapeutic targets for the treatment of cancer. Also, they expand our understanding of the interplay between tumor suppressor and oncogene signaling pathways, which may be exploited in the pursuit for novel therapeutic strategies. In Chapter II, the effects of p73 or PTEN ablation are examined in a model of transformation that requires inhibition of p53 function. The effectiveness of Nutlin-3 is assessed in these transformed cells and demonstrates the possibility of reengaging p53 as a therapeutic agent. Also, the study links genetic alterations, such as loss of PTEN, p73, or overexpression of BCL-2 to Nutlin-3 efficiency. The use of Nutlin-3 to reengage p53 is an attractive therapeutic strategy for the treatment of breast cancer, since p53 remains wild-type in a significant number of patients. Therefore, an understanding of which genetic alterations render a tumor susceptible to Nutlin-3 treatment is vital to the success of this therapeutic strategy. Advances in breast cancer treatment and prevention will come as novel tumor suppressor and oncogene interactions in the genesis of breast cancer are identified and characterized. The studies presented in Chapter III, utilize cell culture models, starting with non-transformed cells and use non-viral proteins to model the various stages of transformation. The use of a model based on normal HMECs and nonviral proteins is novel and allows for the study of intact tumor suppressive pathways. In Chapter III, HMECs were used to dissect the role of tumor suppressors and oncogenes in transformation, these studies identified a TGF-b mediated OIS, engaged by activated RAS signaling, which must be overcome to permit the expansion of malignant breast epithelial cells. This tumor suppressive barrier could be overcome by supressing TGF-β 51

54 signaling, and did not involve DDR proteins as described in studies using murine and human fibroblast models. This study was key to understanding the multistep protocol that leads to the malignant conversion of normal HMECs. This work demonstrates the importance of using normal cells to study the tumor suppressive pathways that are dismantled during the tumorigenic process, as these novel pathways can aid in the discovery of novel treatment targets. Also, the breast cancer model discussed in this thesis mirrors the basal-like or triple-negative breast cancer subtype, which has proven very aggressive and difficult to treat. Finally, I will discuss the use of a genetic technique (VBIM) that permits identification of novel factors involved in positively or negatively regulating signaling pathways. VBIM was used to identify genetic changes that can substitute for oncogenic RAS to promote the transformation of immortalized HMECs. We describe in Chapter IV the discovery of a previously uncharacterized protein, name Family with Sequence Similarity 83, member B (FAM83B), which behaves like RAS in our HMEC transformation model, and is required for RAS-signaling and transformation. Interestingly, FAM83B is one of eight members of a protein family (FAM83) characterized by a highly conserved domain of unknown function 1669 (DUF 1669), which is required for the transforming ability of FAM83 members. Understanding the mechanism of FAM83B signaling will provide the foundation for future therapies aimed at targeting its function. 52

55 Chapter II: Inactivation of p53 signaling by p73 or PTEN ablation results in a transformed phenotype that remains susceptible to Nutlin-3 mediated apoptosis. This work was published in the journal of Cell Cycle Cell Cycle 2010 April 1; 9(7) Authors: Rocky Cipriano John T. Patton Lindsey D. Mayo Mark W. Jackson A. Abstract The p53 signaling pathway is frequently disrupted in carcinogenesis. However, roughly 50% of all cancers express wild-type p53 and have alterations in accessory signaling components required for p53 activity. Using the well described E1A/RAS transformation model, in which p53 activity must be suppressed for transformation, we show here that p53 is inactive and unable to suppress transformation following ablation of p73 or PTEN. However, despite the transformed phenotype conferred by p53 inactivation following p73 or PTEN loss, p53 could be fully activated by Nutlin-3, resulting in efficient caspasemediated apoptosis. Our novel and unexpected finding provides important information 53

56 regarding the efficacy of Nutlin-3 and indicates that patients with tumors deficient in p53 function due to p73 or PTEN loss may benefit from Nutlin-3 treatment. B. Introduction Cancer cells have acquired genetic alterations that disrupt cellular checkpoints established to limit abnormal proliferation. In many cancers, these checkpoints are simply suppressed rather than absent, leaving the option to reengage these hidden limits to proliferation and survival as a cancer therapy. The TP53 gene is mutated in approximately 50% of all human cancers, with some tissues having low frequencies of p53 mutations (leukemia, liver, breast and prostate), and others having high mutation frequencies (lung and skin) 27. Cancers retaining wildtype p53 are thought to have inadequate p53 function due to other aberrations in the p53 signaling pathway. These include (1) inactivation of upstream effectors responsible for activating p53 (such as ATM, CHK2, p14arf), (2) overexpression of proteins that directly bind p53 to suppress its function (such as HDM2, HDMX, COP1), and (3) overexpression of proteins that suppress the downstream effectors of p53 (including Cyclin overexpression to inactivate p21/waf1 and BCL2/BCL-XL overexpression to inactivate pro-apoptotic proteins induced by p53-dependent transactivation) 160. A novel approach that has gained significant ground over the last few years has been the use of small molecules to stabilize or activate wild-type p53, with the goal of regressing tumors due to p53-mediated apoptosis or senescence. The Nutlin compounds are a group of low molecular weight HDM2-antagonists that stabilize p53 by displacing p53 from its 54

57 negative regulator HDM2, leading to p53-dependent toxicity in tumor cell lines and xenograft tumors in mice 28. While Nutlins provide a directed therapeutic approach to the potential treatment of wild-type p53 tumors, several reports have shown that normal and transformed cells overexpressing HDMX, a protein with significant homology to HDM2, are resistant to Nutlin-3 mediated toxicity 2, 29, 30. We and others have recently shown the failure of Nutlin-3 to effect cells overexpressing HDMX, raising concerns about which genetic events confer susceptibility to p53 reactivation. The identification of genetic elements capable of cooperating to transform human cells has permitted us to examine the contribution of specific genes to the transformation process and evaluate the response of these isogenic cells to specific therapeutic agents. In this report, we expand upon a model of human cell transformation that is dependent upon p53 inactivation and which allows one to identify genetic events responsible for inactivating p53 function. Our model system consists of normal human fibroblasts transduced with adenoviral E1A, oncogenic Ras mutant (RasV12), and the htert subunit of telomerase. As previously described by us and others, when p53 expression is inhibited by a short hairpin RNA (shrna), or the function of p53 is inhibited due to overexpression of key negative regulators HDM2 or HDMX, cells become transformed and grow anchorage independently 2, 108. Two proteins that have been shown to interact with p53 to promote apoptosis are PTEN and p73. PTEN loss leads to hyperactivation of AKT which in turn phosphorylates HDM2 and causes its nuclear accumulation, resulting in decreased p53 activity 161. In addition, PTEN physically associates with endogenous p53 and regulates the transcriptional activity of p53 by modulating its DNA binding, independent of its 55

58 phosphatase activity 74. PTEN is required for the maintenance of p53 acetylation, which is also required for gene transcription 75. Similarly, p73 is required for p53-dependent apoptosis in response to DNA damage 162. In this report, we combined a human fibroblast transformation model that requires p53 inactivation with targeted ablation of p73 or PTEN. The resulting cells grew anchorage-independently, indicating that p53 requires p73 and PTEN for its transformation suppressing function. Surprisingly, we found that Nutlin-3 efficiently induced p53-dependent transcription, caspase activation and apoptosis, in cells lacking p53 function due to p73 or PTEN ablation. While the transactivation of p53 target genes remained intact, additional studies implicate a transcription-independent function for p53 stabilized by Nutlin-3. Understanding which alterations in the p53 pathway lead to a transformed phenotype, and also permit p53-dependent apoptosis is important to the future clinical use of Nutlin-3, a promising new therapeutic approach. C. Materials and Methods Cell Lines and Culture Conditions. Normal diploid BJ and IMR90 fibroblasts were grown in a humidified atmosphere containing 5% CO 2 in DMEM (with glucose and L- glutamine; Life Technologies) with 10% fetal bovine serum and 50 units/ml of penicillin and 50 µg/ml of streptomycin sulfate (U.S. Biochemical Corp.). Cells were plated 24 hours before Nutlin-3 was added to fresh media at the indicated concentrations. For survival and growth assays, medium containing Nutlin-3 was removed after 24 hours, replaced with fresh medium, and cells were grown an additional 5 days to 2 weeks before 56

59 staining with methylene blue in 50% methanol. The stain was quantified following extraction with 0.5 mol/l HCl by reading the absorbance at 595 nm. Two independent experiments were performed in triplicate. Retroviral Constructs. pbabe-bleo H-Ras-V12 (Bleo-RAS), pbabepuro htert, LNCX2-E1a and LVTHM-shp53 and shgfp were described previously 2. Primers (5 CGCGTCCCCGCGTATACAGGAACAATATTGTTCAAGAGACAATATTGTTCC TGTATACGCTTTTTGGAAAT 3 ) and (5 CGATTTCCAAA AAGCGTATACAGGAACAATATTGTCTCTTGAACAATATTGTTCCTGTATACGC GGGGA 3 ) were annealed and ligated into LVTHM to create LVTHM-shPTEN. Two shrna constructs targeting p73 were constructed by annealing primers (5 CGCGTCCCCACGTCCATGCTGGAATCCGTTCAAGAGACGGATTCCAGCATG GACGTTTTTTGGAAAT 3 ) and (5 CGATTT CCAAAAAACGTCCATGCTGGAATCCGTCTCTTGAACGGATTCCAGCATGGAC GTGGGGA 3 ) or (5 CGCGTCCCCGGCCATGCCTGTTTACAAGTTCAAGAGACTTGTAAACAGGCA TG GCCTTTTTGGAAAT 3 ) and (5 CGATTTCCAAAAAGGCCATGCCTGTTTACAAGTCTCTTGAACTTGTAAACA GGCATGGCCGGGGA 3 ) and ligated into LVTHM to create LVTHM-shp73 (1) and (2). ERT cells expressing HdmX have been described previously 2. The BCL-2 cdna was provided in pcdna3.1 by Shigemi Matsuyama (Case Western Reserve University, Cleveland, OH), and sub-cloned into pwzl-neo. Virus Production and Infection. Retroviruses were produced as described 163. Briefly, retroviral vectors were transfected into Phoenix-Ampho cells together with a packaging 57

60 plasmid encoding the MLV-gag-pol and env genes. Viruses encoding shrnas were packaged in 293T cells using the second-generation packaging constructs pcmv-dr8.74 and pmd2g, kind gifts from Didier Trono (University of Geneva, Switzerland). Supernatant media containing virus, collected at h, were supplemented with 4 ug/ml polybrene before being frozen in aliquots or used to infect cells for 6-24 hours. Western Analysis. Whole cell extracts were prepared by incubating cell pellets in lysis buffer containing 50 mmol/l of Tris (ph 8.0), 150 mmol/l of NaCl, 1.0% NP40, 10 µg/ml of aprotinin, 100 µg/ml of phenylmethane sulfonyl fluoride, 5 µg/ml of leupeptin, 5 µg/ml of pepstatin, and 1 mmol/l of NaVO 4. Cell extracts containing equal quantities of proteins, determined by the Bradford method, were separated by SDS-PAGE (8 12.5% acrylamide) and transferred to polyvinylidene difluoride membranes (Millipore). Antibodies included: p53 (DO-1 and C19 goat polyclonal, Santa Cruz Biotechnology); p21 Waf1 (C19, Santa Cruz Biotechnology); PTEN (A2B1, Santa Cruz Biotechnology; HDM2 (2A10; Oncogene Research Products, La Jolla, CA); HDMX (BL1258; Bethyl laboratories, Montgomery, TX); Glyceraldehyde-3-phosphate dehydrogenase (Calbiochem); p73 (Bethyl Laboratories); STAT3 (Cell Signaling); PARP, BCL-2, and Tubulin (Santa Cruz Biotechnology). Primary antibodies were detected with goat anti-mouse or goat anti-rabbit conjugated to horseradish peroxidase (Hoffman-La Roche), using enhanced chemiluminescence (Perkin-Elmer). Soft Agar Assays. Cells (2 x 10 5 ) were suspended in 0.6% type VII agarose (Sigma) and plated onto a bottom layer of 1.2% agar in a 60mm plate in triplicate as described 1. The medium was changed every 3 days until cells were analyzed after 3 weeks. To quantify colonies, each plate was scanned using an automated multi-panel scanning microscope, 58

61 and the digital images analyzed using MetaMorph image quantification software. Two independent experiments were performed in triplicate. Cell staining and fluorescence-activated cell sorting analysis. Cells (1 x 10 6 per 10 cm dish) were treated with Nutlin-3 for 24 hours and then harvested with trypsin (including nonadherent cells) were fixed in 70% ethanol and washed with washing buffer (PBS). Cells were then resuspended in propidium iodide (10 µg/ml) in PBS and analyzed by flow cytometry. For each sample, 20,000 cells were analyzed in triplicate in two independent experiments. RNA isolation and reverse transcription-pcr. Total RNA was isolated using Qiagen RNeasy miniprep kit (Qiagen, Valencia CA). RNA was analyzed for HDM2 and p21 expression using an Applied Biosystems GeneAMP PCR System 9700 and Applied Biosystems assays Hs _m1 (p21) and Hs _m1 (HDM2) at the Gene Expression Array Core Facility of the Comprehensive Cancer Center of Case Western Reserve University and University Hospitals of Cleveland. Relative HDM2 and p21 expression was normalized to beta-actin expression. Experiments were performed in triplicate. D. Results Transformation of normal human diploid fibroblasts by ablation of p53, p73, or PTEN expression. 59

62 Identification of genetic elements capable of cooperating to transform normal human cells has permitted the analysis of a specific genes contribution to the transformation process and its involvement in therapeutic responses 1, 2. Since p73 and PTEN have been implicated as key co-factors required for p53-dependent transactivation, we hypothesized that ablation of p73 and PTEN would inhibit the ability of p53 to suppress cellular transformation. Human fibroblasts expressing E1A, RAS and htert (called ERT cells) require ablation of p53 function, either by p53-shrna, or HDM2 or HDMX overexpression to become fully transformed 2. Therefore, to test whether p73 or PTEN serve as key co-factors required for p53-dependent transformation suppression, we expressed shrnas targeting TAp73 or PTEN and examined their transforming effect relative to ablation of p53 itself. Knockdown of either p73 or PTEN resulted in a significant increase in colony formation compared to control shgfp cells, although not as robust, in either colony number or size, when compared to the derivatives expressing shp53 (Figure II-1A and II-1B). Western analysis confirmed the knockdown of each target protein and thus we concluded that inhibiting p73 or PTEN expression in ERT cells can substitute for p53 ablation, resulting in a transformed phenotype. ERT cells transformed by p73 or PTEN ablation remain susceptible to Nutlin-3 mediated apoptosis. We hypothesized that cells which were unable to efficiently activate p53 in response to plating in anchorage-independent conditions, would also be insensitive to targeted therapies which disrupt the p53-mdm2 interactions, such as Nutlin-3. Our hypothesis was that even though p53 was free of its negative regulator, the absence of p73 or PTEN would prevent the efficient activation of p53-dependent target genes, and 60

63 Figure II-1: Transformation of normal human diploid fibroblast by ablating p53, p73, or PTEN. Normal human diploid fibroblasts expressing E1A, RAS and htert, together with the indicated lentiviral shrnas targeting GFP, p53 (2 targets), p73 (2 targets), or PTEN, were plated into soft agar to assess anchorage-independent growth. The results represent 2 independent experiments that were done in triplicate. Phase contrast images of the colonies are shown in (A). Quantification of colony number was determined three weeks after plating, using an automated multi-panel scanning microscope and MetaMorph image quantification software (B). 61

64 inefficient cell cycle arrest or apoptosis. To test this hypothesis, ERT derivatives were treated with 10 µmol/l of Nutlin-3 for 24 hours, then Nutlin-3 was removed and the cells were grown an additional 5 days before quantification of cell number. As previously demonstrated, treatment of control ERT cells expressing a shrna targeting GFP with Nutlin-3 efficiently decreased cell number, whereas cells expressing shrna targeting p53 showed no signs of Nutlin-3 induced growth inhibition or toxicity 2. Surprisingly, ERT cells expressing shrnas targeting p73 or PTEN remained sensitive to Nutlin-3, with comparable decreases in cell number relative to control cells (Figure II-2A). In addition to testing the ERT derivatives prior to plating them into agar, we recovered the cells that formed colonies in agar and reestablished a monolayer culture to investigate whether successful anchorage-independent growth resulted in an increased resistance to Nutlin-3. Selection in agar often enriches for the most robustly transformed cells within the population, and therefore we expected to see an increased resistance to Nutlin-3 from the recovered shp73 and shpten derivatives. However, p73 and PTEN ablation, coupled with selection in agar, was still unable to confer any increase in resistance to Nutlin-3 treatment (Figure II-2B). We conclude that ablation of p73 and PTEN, while sufficient to prevent the transformation-suppressing function of p53, does not prevent p53-mediated growth inhibition or toxicity in response to Nutlin-3. p53 transcriptional activity remains intact in response to Nutlin-3 treatment. To assess the levels of p53 protein and p53-dependent transcription in ERT derivatives expressing shp73 and shpten following Nutlin-3 treatment (10 µmol/l for 24hours), we performed western analysis and real-time PCR analysis on p53 and its 62

65 Figure II-2: Nutlin-3 treatment of parental ERT derivates and ERT derivates recovered from soft agar. (A) ERT cells expressing shrnas targeting GFP, p53, p73, or PTEN were treated with 10 µmol/l of Nutlin-3 for 24 hours, Nutlin-3 was removed the cells were grown an additional 5 days prior to quantification of relative survival, as determined by methylene blue staining. (B) ERT derivatives recovered from soft agar and reestablished as monolayer cultures were treated, and quantified as described. The results represent 2 independent experiments that were done in triplicate. 63

66 transcriptional target genes, p21 and Hdm2. In both the shp73 and shpten expressing cells, including those recovered from agar, Nutlin-3 induced the efficient stabilization of the p53 protein, and induced p21 protein expression (Figure II-3A and II-3B). ERTshGFP cells had an increase in p73 protein levels after exposure to Nutlin-3; however, ablation of p73 did not prevent the growth inhibition or toxicity observed in response to Nutlin-3. This result suggests that p73 induction is likely of little importance in the suppression of growth or survival following Nutlin-3 treatment. In addition, Western analysis confirmed the knockdown of p53, p73, and PTEN in the ERT derivatives (Figure II-3A, II-3B, and II-3C). Furthermore, real-time PCR confirmed the results obtained by Western analysis. As expected, control cells treated with Nutlin-3 induced p21 and HDM2 transcription efficiently, whereas ERT-shp53 cells showed no induction of either gene (Figure II-3D). ERT-shp73 and ERT-shPTEN cells showed similar inductions in p21 and HDM2 mrna levels relative to control cells, again confirming that p53 is fully functional when treated with Nutlin-3. Taken together, our results indicate that the disruption of p53 that occurs during the transformation process may differ significantly from that which may be exploited therapeutically. Nutlin-3 induces p53-dependent apoptosis mediated by caspase activation. To assess whether the p53-dependent decrease in cell number observed following Nutlin-3 treatment is due to cell-cycle arrest or apoptosis, we performed propidium iodide staining and DNA content determination using flow cytometry, and the cell cycle distribution of each population was examined after exposure to Nutlin-3 (10 µmol/l for 24 hours). Control ERT-shGFP cells showed a clear dose dependent increase in the sub- G1 DNA content, indicative of apoptosis. In agreement with this, the accumulation of the 64

67 Figure II-3: p53 is efficiently stabilized in the absence of p73 and PTEN. (A and B) Western blot analysis was performed on ERT derivatives (A) and cells recovered from agar after anchorage-independent growth (B). The shgfp cell extract was included on each Western in order to compare p53 levels and the p53-dependent expression of p21. The efficiency of p53 and p73 knockdown is also confirmed. (C) Western blot analysis of ERT shgfp and shpten cells was performed to confirm the knockdown of PTEN expression in shpten cells. (D) ERT derivatives were treated with 10 µmol/l of Nutlin- 3 for 24 hours, RNA was isolated, and real-time PCR was performed to examine p21 and HDM2 mrna levels in response to Nutlin-3. The results represent 2 independent experiments that were done in triplicate. 65

68 sub-g1 peak could be significantly blocked by the addition of Z-VAD-FMK, a pan caspase inhibitor that irreversibly binds and inhibits the induction of apoptosis (Figure II- 4). In contrast, there was no accumulation of sub-g1 DNA in ERT-shp53 cells, demonstrating that p53 is required for the Nutlin-3 mediated apoptosis observed. Accumulation of sub-g1 DNA was observed in ERT-shp73 and ERT-shPTEN cells, which again could be blocked by the addition of Z-VAD-FMK (Figure II-4). We conclude that Nutlin-3 induces p53-dependent apoptosis, even in cells lacking p73 or PTEN, making it a potentially useful therapy for tumors harboring deficiencies in either of these tumor suppressors. Recent studies have determined that a transcription-independent function of p53 is activated following Nutlin-3 treatment, in which p53 translocates to the mitochondria to induce apoptosis in some tumor cells. To further examine whether p53-dependent transcription was responsible for inducing apoptosis or whether transcription-independent functions of p53 could also be responsible for Nutlin-3 mediated cell death, ERT cells transformed by BCL-2 were examined. BCL-2 expression conferred a transformed phenotype, similar to that previously observed with HDMX (Figure II-5A). Overexpression of BCL-2 and HDMX in ERT cells was confirmed by western analysis (Figure II-5A). Expression of BCL-2 did not impair the induction of p53 by Nutlin-3, or the p53-mediated induction of p21 or HDM2 (Figure II-5B). In both the short term and long term assays, BCL-2 provided only a marginal protection from Nutlin-3 treatment, similar to our results made following p73 or PTEN ablation. Vector control cells and BCL-2 expressing cells showed a significant increase in the proportion of cells with sub- G1 DNA content, indicating that the cells were undergoing apoptosis (Figure II-5C). In 66

69 Figure II-4: Nutlin-3 induces a caspase-mediated apoptosis in ERT derivatives, dependent on functional p53. ERT derivatives were treated with increasing doses of Nutlin-3 (5 and 10 µmol/l) for 24 hours and analyzed by FACS to monitor the percentage of cells with a sub-g1 DNA content, which was quantified and graphed. The caspase inhibitor Z-VAD-FMK was added together with Nutlin-3 to confirm the involvement of caspase activity in Nutlin-3-induced apoptosis. Graphs of DNA content (A) and quantification of sub-g1 content (B) are presented. The results represent 2 independent experiments that were done in triplicate. 67

70 Figure II-5: Transformation by BCL2 results in cells sensitive to Nutlin-3. ERT cells expressing HdmX or BCL-2 were plated into soft agar to assess anchorage-independent growth. Phase contrast images of the colonies are shown in the top panel, Western analysis for HDMX and BCL-2 expression are shown in the bottom panel. (B) The ERT derivatives were treated with increasing doses of Nutlin-3 (5 and 10 µmol/l) and subjected to western analysis to examine p53, Hdm2 and p21. (C) The ERT derivatives were treated with increasing doses of Nutlin-3 (5 and 10 µmol/l) for 24 hours and analyzed by FACS to monitor the percentage of cells having sub-g1 DNA content. (D) Following 24 hours of Nutlin-3 treatment (5 and 10 µmol/l), ERT derivatives were given fresh media and grown an additional 5 days prior to quantification of survival by Methylene Blue staining. The results represent 2 independent experiments that were done in triplicate. (E) Nuclear and cytoplasmic extracts were prepared from ERT cells treated with Nultin-3 (10 µmol/l) to determine the relative localization of p53. Long exposure of the p53 blot shows the ubiquitinated forms of p53 concentrated in the cytoplasm. 68

71 contrast, HDMX-expressing cells, which we previously showed were resistant to Nutlin-3 treatment, showed very little apoptosis following Nutlin-3 treatment 2. This observation is consistent with the diminished long term survival observed for the Vector control cells and BCL-2 expressing cells relative to HDMX-expressing cells (Figure II-5D). Finally, we examined the cellular localization and ubiquitination of p53 following Nutlin-3 treatment. It was recently shown that p53 is still efficiently ubiquitinated following Nutlin-3 treatment, and that the ubiquitinated p53 resides in the cytoplasm. Importantly, this ubiquitinated p53 can promote mitochondrial depolarization independently of its transcriptional activity, resulting in efficient apoptosis in certain cancer cells 164. Indeed, we observed a significant accumulation of ubiquitinated p53 in the cytoplasm of ERT cells following Nutlin-3 treatment (Figure II-5E). Together with our observation that BCL-2 overexpression cannot prevent Nutlin-3 mediated apoptosis, our results indicate that p53 can induce apoptosis independently of transcription in the ERT transformation model. This is consistent with previous observations that mitochondrial p53 binds to the proapototic protein BAK, disrupting it from preexisting inhibitory complexes with the anti-apoptotic MCL-1 protein and leads to p53-mediated apoptosis 165. E. Discussion To study how tumors respond to a specific therapy, researchers commonly use a limited set of genetically undefined tumor cell lines. Such studies can provide only a correlative analysis, at best, to link known genetic alterations with therapeutic mechanism and efficacy. For example, the apoptotic responses observed in human cancer cell lines 69

72 treated with Nutlin-3 are highly variable, even when all lines examined express wild-type p These findings exemplify the difficulty in using genetically undefined cancer cell lines to delineate genetic components responsible for the sensitivity or resistance to a therapeutic agent. The use of isogenic cell lines differing in a single genetic element permits more precise interrogation of a specific proteins involvement in the resistance or sensitivity to a given treatment. Here, we have used a well-described E1A/RAS transformation model, in which p53 activity must be suppressed for transformation. Our data demonstrate that p53 is not active at suppressing transformation in the absence of p73 and PTEN, confirming and extending observations made using mouse models, which demonstrate that p73 and PTEN are required components for p53-dependent apoptosis 74, 162. The novel and unexpected finding from our studies is that while p53 requires p73 and PTEN to suppress transformation, it does not require p73 or PTEN for the observed toxicity following Nutlin-3 treatment. We hypothesized that cells would be transformed by p73 or PTEN ablation and also be insensitive to p53 reactivation by Nutlin-3 due to studies showing (1) p53 is unable to promote apoptosis in the absence of p and (2) a direct interaction between PTEN and p53 is necessary for efficient p53-dependent promoter binding and transactivation 74. However, we observed striking differences in p53-dependent responses to anchorage-independent growth assays and Nutlin-3 treatment, with a fully competent p53 response to Nutlin-3 treatment. Our study provides important observations into the differences underlying distinct stressors and delineates which components of the p53 signaling cascades are critical for tumor suppression versus Nutlin-3 mediated therapeutic efficacy. Previous studies have shown that p73 can be activated by Nutlin-3 in cells 70

73 lacking p53 or expressing mutant p53. However, these studies used considerably higher doses of Nutlin-3 compared to our studies, up to 50 micromolar. Upon closer examination, these studies are in full agreement with ours, whereby 10 micromolar Nutlin-3 induces insignificant levels of apoptosis in the absence of p53 or in the presence of mutant p53 74, 162. We conclude that cancers that harbor mutations or loss of p73 or PTEN, and retain wild-type p53, would have a favorable prognosis if treated with an HDM2 antagonist, such as Nutlin-3. Our studies using cells transformed by the anti-apoptotic protein BCL-2 implicate a transcription-independent function for at least part of the toxicity observed following Nutlin-3 treatment. Although p53-dependent transactivation of target genes was not altered following p73 and PTEN ablation, the inability of BCL-2 to prevent Nutlin-3 induced apoptosis indicates that transcription of pro-apoptotic target genes is responsible for little of the observed Nutlin-3 induced toxicity. Previous studies have shown that ubiquitinated p53 readily translocates to the mitochondria, where it can liberate the proapoptotic protein BAK from MCL-1, resulting in efficient mitochondrial depolarization and apoptosis 165. In agreement with this, we observed significant amounts of ubiquitinated p53 in the cytoplasm of E1A/RAS-expressing cells following Nutlin-3 treatment. Using the same E1A/RAS model of transformation, we previously showed that HDMX-expressing cells were refractory to Nutlin-3 treatment because Nutlin-3 was unable to prevent HDMX-p53 interactions 2. Interestingly, we noted that HDMX bound a significant amount of ubiquitinated p53. This may indicate that HDMX suppresses the transcription-independent function of p53 by preventing its translocation to the mitochondria following Nutlin-3 treatment. 71

74 Delineating the divergent genetic programs that (1) promote tumorigenesis and (2) dictate the response to specific therapies is an important first step to personalized treatment regimes. We have recently identified additional genetic alterations that drive transformation by inhibiting p53 signaling, but result in distinct responses to Nutlin-3 treatment, including HDMX and CyclinD1 1, 2. While Nutlin-3 and other HDM2/HDMX antagonists continue to excite the cancer research community due to their potential, defining how efficiently small molecule inhibitors aimed at re-awakening wild-type p53 will work is a critical first step to advancing this approach. Our results suggest that tumors harboring inactive PTEN, p73 and BCL-2 will respond well to Nutlin-3 based therapies. 72

75 Chapter III: Suppression of TGF-β signaling overcomes RAS-induced senescence in HMECs leading to neoplastic transformation. Authors: Rocky Cipriano Charlene E. Kan James Graham David Danielpour Martha R. Stampfer Mark W. Jackson A. Abstract Oncogene-induced senescence (OIS), the proliferative arrest engaged in response to persistent oncogene activation may serve as a tumor suppressive barrier in preneoplastic lesions. We show here that HMECs undergo a p16- and p53-independent OIS in response to oncogenic RAS that requires TGF-β signaling. Suppression of TGF-β signaling by expressing a dominant negative receptor, use of a TGF-β receptor I inhibitor, or elevated expression of c-myc permitted continued proliferation upon RAS expression. Ablation of ATM or CHK2 was unable to prevent RAS-mediated OIS in HMECs, arguing that the DNA damage response is not required. Ablation of the cytostatic effects of TGF-β by a number of mechanisms not only allowed HMECs to tolerate persistent growth signaling, but also resulted in neoplastic transformation. Thus, TGF-β mediated OIS in preneoplastic breast lesions provides an early barrier to malignant progression. We propose that understanding the mechanisms that prevent OIS 73

76 will provide the foundation for future therapies aimed at reengaging these hidden limits to proliferation as a cancer therapy. B. Introduction Cancer cells typically acquire a number of capabilities including, among others: (1) a loss of sensitivity to anti-growth and/or pro-apoptotic signals, (2) persistent growth signals and (3) limitless replication potential 114. The idea that these capabilities could be imparted to normal cells by using a limited set of genetic manipulations was confirmed in mouse cells nearly 25 years ago by combining either c-myc, polyoma large-t antigen, mutant p53, or adenoviral E1A with a hyperactive RAS gene 96, 115, 116. It took an additional 16 years before the same goals were accomplished with human cells, indicating that more stringent tumor suppressive pathways exist in human cells than in mouse cells. For instance, human cells undergo a growth arrest in response to telomere depletion and the activation of protein phosphatase 2A (PP2A), suppressive pathways that do not exist in rodent cells. In addition, multiple RAS effectors are required for human cell transformation, while a single RAS effector transforms rodent cells 134. The development of human cell transformation models have led to the identification of complex interactions between the tumor suppressor and oncogene signaling pathways involved in human cancer development and linked specific genetic alterations with therapeutic efficiency 1-3. The observation that dysregulated oncogenic signaling could promote OIS, a permanent growth arrest, rather than promoting proliferation, implicates OIS as an important tumor suppressive barrier. Research examining OIS in human fibroblasts has 74

77 identified genes encoding the cyclin-dependent kinase inhibitor p16, DNA-damage signaling and p53 as critical signaling components for OIS 167. In these studies, OIS could be bypassed by disabling the DNA damage response (DDR), either by eliminating ATM, CHK2 or p53 function prior to RAS, MOS or STAT5 expression Additional studies have also confirmed the existence of OIS in human tumors and mouse tumor models, providing much needed evidence that OIS is physiologically relevant in vivo 93, The differential presence of senescent cells in benign tumors, but not advanced cancers, argues that OIS is a tumor suppressive barrier that needs to be dismantled for full tumorigenic progression. Much like the research that defined differences between signaling events required for rodent and human cell transformation, such differences are also being identified between fibroblast and epithelial cells. For example, HMECs have a unique arrest pathway that is independent of p53 and the p16-rb axis following oncogenic RAF expression, indicating that the signaling networks responsible for initiating and maintaining OIS differ between fibroblast and epithelial cells 106, 179. In addition, there are significant cell-type differences in requirements for RAS effector signaling, with RAF and RAL-GEFs required for fibroblast transformation and RAF, PI3K, and RAL-GEFs required for HMEC transformation 134. Furthermore, the response of fibroblasts and epithelial cells to cytokines, such as TGF-β is markedly different, with epithelial cells undergoing a cell-cycle arrest and fibroblasts increasing proliferation and exhibiting characteristics of morphological transformation. Proteins responsible for the cell-type specific responses are beginning to emerge, but it is clear that studies aimed at 75

78 understanding the OIS signaling pathways responsible for suppressing the transformation of epithelial cells must be performed using appropriate cell models. We show here that HMECs undergo a p16 and p53-independent senescence that requires intact TGF-β signaling following activated RAS expression. In contrast to studies performed using fibroblasts, ablation of ATM or CHK2 did not prevent RASmediated OIS in HMECs, whereas suppression of TGF-β signaling by expressing a dominant negative receptor or use of a TGF-β receptor I inhibitor prevented RASinduced senescence and contributed to a transformed phenotype. Elevated c-myc expression abrogated the cytostatic effects of TGF-β, prevented RAS-mediated OIS, and together with ablation of p16 and p53, permitted the expansion of malignant breast epithelial cells. C. Materials and Methods Materials. Senescence Beta-Galactosidase Staining Kit #9860 was purchased from Cell Signaling and the standard protocol was performed. Nutlin-3 and SB were obtained from Calbiochem and TGF-β was obtained from PeproTech. Retroviral Constructs. LV-RasV12, pbabebleo-rasv12, pbabepuro-htert, LPCX- HDMX and LVTHM-shp53 and shgfp were described previously 2. pbabepuro-rasv12, pbabepuro-rasv12-y40c, pbabepuro-rasv12-t35s, pbabepuro-rasv12-e37g (Addgene plasmid 1768, 12276, 12274, and 12275) were obtained from Addgene, deposited by Dr. Robert Weinberg (Whitehead Institute, Cambridge, MA). pretrosuper-shchk2, pretrosuper-shatm, and pretrosuper-shgfp were 76

79 obtained from Dr. Yosef Shiloh (Department of Neurobiology, Tel Aviv University, Tel Aviv, Israel) 180. LNCX2-DNTGFβRII was created by subcloning DNTGFβRII from pmfg-dnr-ires-neo and recloning it into the LNCX2 vector 181. SINpuro-shp16 and SINhygro-shp16 were provided by Dr. Scott Lowe (Cold Spring Harbor Laboratory, New York). LNCX2-GFP, LNCX2-GFP-IRES-RasV12 (GiR), LNCX2-MYC-IRES-GFP (MiG), and LNCX2-MYC-IRES-RasV12 (MiR) were created by subcloning RasV12 from pbabepuro-rasv12, IRES and GFP from pires-gfp, and c-myc from pwzl- MYC (Addgene plasmid 10674). After successful cloning all 4 constructs were sequence verified. Virus Production and Infection. Retroviruses were produced as described 163. Briefly, retroviral vectors were transfected into Phoenix-Ampho cells together with a packaging plasmid encoding the MLV-gag-pol and env genes. Lentiviruses were packaged in 293T cells using the second-generation packaging constructs pcmv-dr8.74 and pmd2g, kind gifts from Didier Trono (University of Geneva, Switzerland). Supernatant media containing virus, collected at h, were supplemented with 4 ug/ml polybrene before being frozen in aliquots or used to infect cells for 6-24 hours. Cell Lines and Culture Conditions. Pre-stasis and post-selection HMECs (specimen 48R, Batch S) were obtained from Dr. Martha R. Stampfer (Lawrence Berkeley National Laboratory, Berkeley, California). Post-selection HMECs were grown in a humidified atmosphere containing 5% CO 2 in Medium 171 with mammary epithelial growth supplement (Cascade Biologics) and 50 units/ml of penicillin and 50 µg/ml of streptomycin sulfate (U.S. Biochemical Corp.) as described 182. Pre-stasis HMECs 77

80 (specimen 48R) were grown in a humidified atmosphere containing 5% CO 2 in M87A media as previously described 183. Soft Agar Assays. For HMECs (48R), 2 x 10 5 cells were suspended in 0.6% type VII agarose (Sigma) and plated onto a bottom layer of 1.2% agar in a 60mm plate in triplicate as described 1. The medium was changed every 3 days until cells were analyzed after 3 weeks. To quantify colonies, each plate was scanned using an automated multi-panel scanning microscope, and the digital images analyzed using MetaMorph image quantification software. SB (5uM and 10uM) was obtained from Calbiochem and was added to the medium during feeding for indicated experiments. TGF-β (10ng/mL) was obtained from PeproTech and was added to the medium during feedings for indicated experiments. Multiple independent experiments were performed in triplicate. Immunoblot Analysis. Whole cell extracts were prepared by incubating cell pellets in lysis buffer containing 50 mmol/l of Tris (ph 8.0), 150 mmol/l of NaCl, 1.0% NP40, 10 µg/ml of aprotinin, 100 µg/ml of phenylmethane sulfonyl fluoride, 5 µg/ml of leupeptin, 5 µg/ml of pepstatin, and 1 mmol/l of NaVO 4. Cell extracts containing equal quantities of proteins, determined by the Bradford method, were separated by SDS-PAGE (8 12.5% acrylamide) and transferred to polyvinylidene difluoride membranes (Millipore). Antibodies to c-myc (sc-40), RAS (sc-520), RB (sc-50), p21(sc-397), p53 (sc-126), p16 (sc-9968) were from Santa Cruz Biotechnology, antibodies to ß-actin (pan Ab-5) and p14arf (Ab-1) were from Neomarkers, antibodies to glyceraldehyde-3- phosphate dehydrogenase were from Calbiochem, antibodies to HDMX and ATM were from Bethyl Laboratories, antibodies to HDM2 were from Chemicon, antibodies to p-rb (Ser 249/Thr 252) were from Biosource, and antibodies for ERK1/2, P-ERK1/2 78

81 (Thr202/Tyr204), P-p53 (Ser 15), CHK2, P-SMAD2 (Ser 465/467), AKT, and P-AKT (Ser473) were from Cell Signaling. Primary antibodies were detected with goat antimouse or goat anti-rabbit conjugated to horseradish peroxidase (Hoffman-La Roche), using enhanced chemiluminescence (Perkin-Elmer). Relative Growth Assay. For HMECs (48R), cells were plated in triplicate at 20,000 cells per well and cell number was determined on a Beckman Coulter counter after 5 days of growth. SB (10uM) and Nutlin-3 (10uM) was obtained from Calbiochem and was added to the medium during feeding for indicated experiments. TGF-β (10ng/mL) was obtained from PeproTech and was added to the medium during feedings for indicated experiments. Multiple independent experiments were performed in triplicate. D. Results Previous studies have identified a p53- and p16-rb-independent growth arrest pathway in HMECs following RAF activation 179. To examine the mechanism by which aberrant oncogenic signals lead to this p53- and p16-independent growth arrest, we created HMECs that express a shrna targeting p53 and lack p16 protein expression due to promoter methylation 184, 185. The lack of p53 expression and activity was confirmed using Nutlin-3 growth assays and western analysis. HMECs expressing a control shrna (shgfp) or a shrna targeting p53 were treated with Nutlin-3, a small-molecule inhibitor that disrupts the interaction between p53 and its negative regulator, HDM2. Western analysis confirmed the knockdown of p53 protein levels, and the ablation of p53- dependent transactivation of target genes HDM2 and p21 (Figure III-1a). Furthermore, 79

82 Figure III-1: HMECs expressing a shrna targeting p53 are resistant to Nutlin-3. (a) HMECs expressing a control shrna (shgfp) or a shrna targeting p53 (shp53) were treated with Nutlin-3. Western analysis confirmed the knockdown of p53 protein levels and the ablation of p53-dependent transactivation of target genes HDM2 and p21. (b) Treatment of control HMECs with Nutlin-3 resulted in p53-mediated growth arrest, while cells expressing a shrna targeting p53 remained unaffected. 80

83 treatment of control HMECs with Nutlin-3 resulted in p53-mediated growth arrest, as previously reported 1, 2, while cells expressing a shrna targeting p53 remained unaffected (Figure III-1b). Having cells lacking p53 protein and function, we proceeded to examine their response to activated RAS by infecting shp53-expressing HMECs with a lentivirus encoding RAS-G12V. Cells expressing RAS-G12V underwent a growth arrest that was characterized morphologically by an increase in cell size, cell spreading, and vacuolization typical of senescence (Figure III-2a) 186. RAS-G12V-expressing cells also stained positive for the presence of senescence-associated β-galactosidase activity, and were strongly growth inhibited, as determined by cell counts 5 days post infection (Figure III-2a and III-2b). Western analysis confirmed the expression of RAS-G12V, as well as the induction of the cyclin-dependent kinase inhibitor p21, and a consequent decrease in phosphorylated RB protein levels (Figure III-2c). We next examined whether various RAS-G12V point mutants capable of activating specific effector pathways including RAF (T35S), RAL-GEF (E37G), or PI3K (Y40C) also resulted in a senescent phenotype 134. Each of these point mutations abolished the ability of RAS-G12V to induce p21 expression and suppress the growth of shp53-expressing HMECs, arguing that multiple RAS-signaling effectors are required to engage the p53- and p16-independent OIS (Figure III-3). Previous reports using fibroblasts have demonstrated a requirement for DNA damage-responsive proteins, including ATM and CHK2, in RAS-G12V-mediated senescence 106, 167, 187. To determine whether the p53/p16-independent senescence observed in HMECs was dependent on ATM or CHK2, we ablated ATM and CHK2 in 81

84 Figure III-2: RAS-mediated OIS is independent of DDR proteins, p53, and p16. (a) Representative images of shp53 expressing HMECs infected with a retrovirus encoding RAS-V12G or a control retrovirus (Vector). Shp53/RAS-G12V-HMECs stained positive for the presence of senescence-associated β-galactosidase activity. (b) Shp53/RAS- G12V-HMECs were strongly growth inhibited compared to control HMECS as determined by cell counts 5 days post-infection. (c) Western analysis of shp53/ras- G12V-HMECs (R) confirmed the expression of RAS-G12V, as well as the induction of the cyclin-dependent kinase inhibitor p21 and a decrease in phosphorylated RB protein levels compared to shp53/vector-hmecs (V). (d) Shp53-HMECs were infected with retroviruses encoding a shrna targeting ATM, CHK2, or GFP. The knock-down of ATM and CHK2 was confirmed by Western analysis. (e) The shp53-hmecs with knockdown of either ATM, CHK2, or GFP were infected with a retrovirus encoding RAS- G12V or a control retrovirus (Vec). Five days after RAS-G12V infection, cell number was quantified and plotted as relative growth compared to vector infected cells. 82

85 Figure III-3: Multiple RAS effectors are required for RAS-mediated OIS in HMECs. Various RAS-G12V point mutants capable of activating only one specific effector pathways including RAF (T35S), RAL-GEF (E37G), or PI3K (Y40C) were expressed in shp53-hmecs. Each of these point mutations abolished the ability of RAS- G12V to induce p21 expression and suppress the growth of shp53-expressing HMECs, as determined by western analysis and cell number was quantified 5 days post infection. 83

86 shp53-expressing HMECs. The knock-down of ATM and CHK2 was confirmed by western analysis, and the ATM- and CHK2-expressing HMECs were infected with a retrovirus encoding RAS-G12V (Figure III-2d). Five days after RAS-G12V infection, cell number was quantified and plotted as relative growth compared to vector-infected cells (Figure III-2e). The results demonstrate that ATM and CHK2 are dispensable for the p53/p16-independent senescence induced by RAS-G12V. We conclude that HMECs do not require p16, p53, ATM or CHK2 to mount a tumor suppressive arrest in response to aberrant oncogene activation. This response is distinct from the response observed with human fibroblasts, and may be indicative of cell-type specific oncogenic sensing mechanisms. The expression of v-ras in murine keratinocytes resulted in p19arf, p53, p15 and p16 induction, elevated secretion of TGF-β, dephosphorylation of RB, and an OIS phenotype 188. While this study did not address the role of p53 and p16 in the RASinduced senescence, abrogation of TGF-β signaling suppressed the OIS phenotype 188. To examine the role of TGF-β signaling in the p53/p16-independent senescence observed in HMECs in response to RAS-G12V, we treated shp53-expressing HMECs with SB431542, a TGF-β receptor I antagonist prior to RAS-G12V infection 189. Cells treated with SB showed a significant increase in cell number 5 days after RAS-G12V expression (Figure III-4a). However, this phenotypic rescue was not permanent since removal of the inhibitor led to a reduction of proliferation and a growth arrested phenotype (Figure III-4b). 84

87 Figure III-4: TGF-β signaling is required for RAS-mediated OIS. (a) Shp53-HMECs were treated with SB431542, a TGF-β receptor I antagonist, prior to RAS-G12V infection. Shp53-HMECs treated with SB showed a significant increase in cell number 5 days after RAS-G12V expression. (b) Shp53/RAS-G12V-HMECs grown in SB were plated plus or minus inhibitor and relative growth was measured after 5 days of inhibitor withdrawal. (c) Shp53-expressing HMECs were infected with a retrovirus encoding a dominant-negative TGF-β type II receptor (DN-TGFβRII) or an empty vector and subsequently infected with a retrovirus encoding RAS-G12V. Relative growth was determined 5 days post RAS-G12V infection. (d) Shp53-expressing HMECs were infected with a retrovirus encoding c-myc or an empty vector and subsequently infected with a retrovirus encoding RAS-G12V. Relative growth was determined 5 days post RAS-G12V infection. (e) Shp53-HMECs expressing either Vector, c-myc, or DN- TGFbRII were plated plus or minus exogenous TGF-β and relative growth determined after 5 days of continuous treatment. 85

88 Since pharmacological inhibition of TGF-β signaling was able to overcome the RAS-mediated growth arrest, we next examined whether a dominant-negative TGFβ type II receptor (DN-TGFβRII) would also rescue cells from the growth arrest. Shp53- expressing HMECs were infected with a retrovirus encoding DN-TGFβRII or an empty vector and subsequently infected with a retrovirus encoding RAS-G12V. Control shp53/vector-hmecs underwent RAS-mediated OIS while shp53/dn-tgfβrii- HMECs continued proliferating in the presence of RAS-G12V (Figure III-4c). In addition, shp53/dn-tgfβrii-hmecs appeared morphologically distinct from control cells, were refractory to contact inhibition, and resembled many cancer-derived cell lines (Figure III-5). Importantly, these data suggest that this previously uncharacterized p53/p16-independent OIS in HMECs is dependent on functional TGF- β signaling. Previous reports have implicated the overexpression of c-myc, an oncoprotein frequently overexpressed in breast cancer, in suppressing the cytostatic effects of TGFβ 102, 103, 190, 191. We next examined whether c-myc could rescue HMECs from RASmediated OIS. Shp53-expressing HMECs were infected with a retrovirus encoding c- MYC or an empty vector and subsequently infected with a retrovirus encoding RAS- G12V (Figure III-6). Control shp53/vector-hmecs underwent RAS-mediated senescence, while shp53/c-myc-hmecs continued proliferating in the presence of RAS-G12V (Figure III-4d). TGF-β induces a growth arrest in shp53-expressing HMECs that is associated with SMAD phosphorylation, p21 induction and hypophosphorylation of RB, as previously shown following RAS-G12V expression (Figure III-7). As expected, HMECs expressing 86

89 Figure III-5: Inhibition of TGF-β signaling in shp53/ras-g12v-hmecs. Representative images of shp53/ras-g12v-hmecs expressing either an empty vector or DN-TGFβRII. Shp53/DN-TGFβRII-HMECs appeared morphologically distinct from control cells, were refractory to contact inhibition, and resembled many cancer-derived cell lines. 87

90 Figure III-6: Western analysis of HMECs expressing c-myc. Western blot analysis of shp53-hmecs infected with a retrovirus encoding c-myc or a control retrovirus (Vector). 88

91 Figure III-7: Treatment of shp53-hmecs with exogenous TGF-β. Shp53-HMECs were plated plus and minus exogenous TGF-β (2 hour exposure) and western analysis was performed for p21 induction, phosphorylation of SMAD2, and hypophosphorylation of RB. 89

92 either c-myc or DN-TGFβRII were resistant to the growth inhibitory properties of exogenous TGF-β treatment (Figure III-4e). Our results suggest that RAS-mediated OIS requires a functional TGF-β receptor and downstream signaling, which can be circumvented by receptor inactivation or c-myc expression. We hypothesized that cells lacking three prominent tumor suppressor pathways, including p53, p16 and TGF-β signaling, would be transformed following expression of RAS-G12V. To test this we examined whether shp53/dn-tgfβrii expressing HMECs were transformed following RAS-G12V expression. The cells were plated into soft agar and anchorage-independent growth (AIG) was assessed three weeks later using an automated multi-panel scanning microscope and MetaMorph image quantification software. Control shp53/dn-tgfβrii expressing HMECs failed to form colonies, while shp53/dn-tgfβrii/ras expressing HMECs efficiently formed anchorage-independent colonies (Data not shown). Interestingly, these data demonstrate that the transformation of HMECs can occur independently of htert expression. However, despite their transformed phenotype, the transformed variants eventually stop dividing, owing to telomere depletion, further arguing that telomerase activation is a key rate-limiting step to advanced tumorigenicity (manuscript in preparation). In addition, a population of shp53-expressing HMECs emerged from the RAS- G12V-induced senescent cultures, which we termed RAS-resistant (RAS-R) HMECs (Figure III-8a and III-8b). Based on their ability to grow in the presence of RAS-G12V, we predicted they would be resistant to TGF-β signaling. However, the control and RAS- R cells showed equivalent sensitivities to exogenous TGF-β treatment (data not shown), 90

93 Figure III-8: RAS-resistant (RAS-R) HMECs are untransformed. (a) Representative images of shp53-hmecs infected with a retrovirus encoding RAS-G12V or a control retrovirus at 4, 12, and 6 days post infection. (b) Growth analysis of shp53-hmecs expressing Vector or RAS-G12V 4, 8, 12, 16, and 20 days post infection. (c) A population of shp53-expressing HMECs emerged from the RAS-G12V-induced senescent cultures, which we termed RAS-resistant (RAS-R) HMECs. RAS-R cells were unable to efficiently form anchorage-independent colonies compared to control HMECs. Treatment of RAS-R cells with SB significantly enhanced anchorage-independent growth in a dose-dependent manner compared to control HMECs. 91

94 and were unable to efficiently form anchorage-independent colonies (Figure III-8c). However, the RAS-R cells showed a significant decrease in RAS-G12V expression and decreased expression of p21 after prolonged passage in culture (Figure III-9). Surprisingly, treatment of RAS-R cells with SB significantly enhanced anchorage-independent growth in a dose-dependent manner, arguing that TGF-β signaling remained at least partially intact or was induced in anchorage-independent conditions (Figure III-8c). We next confirmed that elevated c-myc expression, which suppressed RAS- G12V-mediated OIS by circumventing TGF-β signaling, also cooperated with RAS- G12V to drive HMEC transformation. For this, shp53-expressing HMECs were infected with retroviruses encoding c-myc and RAS-G12V, either alone or in combination, and assessed for anchorage-independent growth. Cells expressing shp53, c-myc and RAS- G12V efficiently formed colonies in soft agar, whereas shp53-hmecs expressing c- MYC alone or RAS-G12V alone did not form colonies efficiently (Figure III-10a). Previous studies have correlated RAS-G12V expression with an increase in anchorageindependent growth 192. We increased the level of RAS-G12V expression using different retroviral vectors in shp53/c-myc-hmecs and c-myc-hmecs retaining wild-type p53 expression. Increasing RAS levels increased colony formation of shp53-hmecs until a critical threshold was surpassed, which then resulted in growth inhibition (Figure III- 10b). Little AIG was observed in the presence of p53. These data argue that the suppression of OIS by exogenous c-myc can be overcome by increasing RAS-G12V expression, and the resulting increase in TGF-β signaling. Interestingly, in cells 92

95 Figure III-9: RAS-resistant (RAS-R) HMECs western analysis. RAS-R cells showed a significant decrease in RAS-G12V expression and decreased expression of p21 after prolonged passage in culture by western analysis. 93

96 Figure III-10: Neoplastic transformation of HMECs requires inhibition of TGF-β signaling. (a) Shp53-HMECs were infected with retroviruses encoding c-myc and RAS-G12V, either alone or in combination, and assessed for anchorage-independent growth (AIG). (b) Four different retroviral vectors encoding RAS-G12V were used to increase the level of RAS-G12V expression in shp53/c-myc-hmecs and c-myc- HMECs retaining wild-type p53 expression. These HMEC derivatives were examined for AIG. (c) Western analysis of RAS-G12Vexpression in the HMEC derivatives infected with 4 different retroviral vectors encoding RAS-G12V. (d) Western analysis of HMECs and shp53-hmecs expressing GFP, RAS-G12V, c-myc, or RAS-G12V and c-myc together. (e) C-MYC/RAS-G12V-HMECs were infected with a retrovirus either encoding a shrna targeted at GFP, p53, ATM, or CHK2 and AIG was examined. 94

97 recovered from soft agar, it was clear that each recovered population expressed an optimal level of RAS-G12V that was significantly reduced from the initial population (Figure III-10c). These findings argue that the balance between RAS and c-myc expression is critical for neoplastic transformation, since increasing levels of RAS signaling would engage the cytostatic functions of TGF-β. We have confirmed that primary HMEC cultures have a nearly identical transformed phenotype when p16 is silenced using shrna, rather than by promoter methylation (Figure III-11). We have also confirmed that p53 remained functional in HMECs expressing c-myc and RAS-G12V using Nutlin-3 assays, as described earlier. As expected, p53 suppressed the growth of c-myc/ras-g12v-hmecs following treatment with Nutlin-3. In contrast, the growth of shp53/c-myc/ras-g12v-hmecs was not inhibited at the same dose of Nutlin-3, confirming that p53 was fully inactive (Figure III-12). Western analysis of each cell population determined that p53 was phosphorylated and stabilized by c-myc expression, but actually decreased in the RAS- G12V expressing cells, likely due to elevated HDM2 levels, as described previously (Figure III-10d) 193. In addition, CHK2 was phosphorylated and p14arf was induced in c-myc expressing cells, but not in RAS-G12V expressing cells (Figure III-10d). Despite the elevated levels of p14arf, p53 and DNA damage responsive signaling, c-myc expression in parental HMECs or shp53-expressing HMECs does not alter their growth (data not shown). In addition, suppression of ATM or CHK2 does not substitute for p53 loss in our transformation model, indicating that DNA damage signaling to p53 via these proteins is not a significant suppressor of transformation (Figure III-10e). 95

98 Figure III-11: Neoplastic transformation of primary HMECs. Shp16-HMECs and shp16/shp53-hmecs were infected with retroviruses encoding c-myc and RAS-G12V, either alone or in combination, and assessed for anchorage-independent growth (AIG). Western analysis of shp16-hmecs and shp16/shp53-hmecs expressing GFP, RAS- G12V, c-myc, or RAS-G12V and c-myc together. 96

99 Figure III-12: c-myc and RAS-G12V expression does not confer resistance to Nutlin-3. HMECs and shp53-hmecs expressing c-myc and RAS-G12V were treated with Nutlin-3 and relative growth was assessed after 5 days of continuous treatment. 97

100 If DNA damage does not prime the tumor suppressive activities of p53, then what does? In a murine TGF-β1 knock-out model, the lack of TGF-β signaling suppresses p53 activation 194. Therefore, we examined whether p53 function was linked to TGF-β signaling by treating HMECs expressing c-myc and RAS-G12V with SB following their plating into soft agar. As expected, cells retaining functional p53 did not form soft agar colonies efficiently; however, treatment with SB resulted in a significant enhancement of anchorage-independent growth (Figure III-13a). Moreover, SB treatment also enhanced colony formation in cells lacking functional p53, indicating that TGF-β signaling suppresses transformation via p53-dependent and p53- independent mechanisms. Additional studies of genes that contribute to a transformed phenotype in HMECs led us to the observation that HDMX, a well-characterized suppressor of p53, was significantly more efficient at transforming c-myc/ras-g12v- HMECs than p53 shrna (Figure III-13b). Surprisingly, expression of HDMX also increased the anchorage-independent growth of shp53-expressing c-myc/ras-g12v- HMECs, suggesting that HDMX may also suppress transformation via p53-dependent and p53 independent mechanisms. Previous studies have shown that HDMX can directly bind to SMAD2 to prevent SMAD-mediated transcriptional activation 195, 196. These data suggest that elevated HDMX expression may simultaneously suppress two tumor suppressive pathways necessary for neoplastic transformation of HMECs. 98

101 Figure III-13: Elevated HDMX expression overcomes TGF-β suppression, enhancing transformation. (a) HMECs and shp53-hmecs expressing c-myc and RAS-G12V were plated in soft agar plus and minus continuous exposure to SB and AIG was assessed. (b) C-MYC-HMECs, c-myc/ras-g12v-hmecs were infected with a retrovirus encoding HDMX or a control retrovirus and assessed for AIG. Also, shp53/c-myc-hmecs, shp53/c-myc/ras-g12v-hmecs were infected with a retrovirus encoding HDMX or a control retrovirus and assessed for AIG. Western analysis of the HMEC derivatives expressing HDMX was performed. 99

102 E. Discussion It is clear from recent studies that OIS is a critical tumor suppressive barrier in vivo, since senescent cells are commonly identified in early hyperplastic lesions and naevi A number of genetic and epigenetic events can result in the generation of dysregulated proliferative signals 132, which triggers OIS in normal cells. The eventual breakdown of OIS signaling allows pre-cancerous cells to tolerate the dysregulated proliferative signals and continue towards neoplastic transformation. To date, much of the work delineating OIS has been performed using human fibroblasts, identifying p16, RB, DDR proteins and p53 as signaling components necessary for OIS. Ablation of p53, ATM, CHK2 or p19arf allows human and murine fibroblasts to tolerate activated RAS, MOS or STAT5 expression and continue dividing rather than undergoing senescence , 197. Together with the observation that tumor cells have persistent DNA damage and often lose DDR signaling responsible for activating p53, the current hypothesis poses an integral role for DDR signaling in OIS. Unlike fibroblasts, HMECs have a uncharacterized OIS response that functions independently of p53 and p16 following oncogenic RAF or RAS expression 106, 179. We report here that HMECs undergo a TGF-β-mediated OIS in response to oncogenic RAS, which does not require p53, p16, ATM or CHK2. In our studies, suppression of TGF-β signaling by expressing a dominant negative receptor, use of a TGF-β receptor I inhibitor, or elevated expression of c-myc prevented RAS-induced senescence. Nearly all epithelial tumors, including breast cancer, acquire a resistance to the cytostatic effects of TGF-β, although the mechanisms for this resistance in early hyperplasia remain poorly defined. TGF-β receptors and the SMAD proteins responsible for generating TGF-β- 100

103 mediated growth inhibitory signals are not often mutated in breast cancer 198. Rather, genetic changes that prevent the growth inhibitory signaling, which involves the induction of cyclin-dependent kinase inhibitors such as p15 and p21, are frequently observed 198. These include the elevated expression of cyclins (D1 and E), c-myc, MDM Several reports have shown that expression of RAS or its downstream effectors leads to the secretion of several cytokines, including TGF- β 188, 199. Primary mouse keratinocytes transduced with RAS undergo a TGF-β dependent G1 growth arrest accompanied by an increase in secreted TGF- β leading to senescence. However, this arrest is still mediated via p53 and p16, and is quite different from the RAS-induced p53/p16-independent senescence we observe in HMECs 188. The response of fibroblasts and epithelial cells to TGF-β is markedly different, with fibroblasts increasing proliferation and exhibiting characteristics of morphological transformation, while epithelial cells undergo a cell-cycle arrest. The use of HMECs has uncovered a role for TGF-β signaling in RAS-mediated OIS, which has not been observed as a tumor suppressive barrier in studies of OIS that utilize human fibroblasts. Suppression of TGF-β signaling in HMECs suppressed the OIS response, allowing RAS to drive a transformed phenotype. This observation led us toward the identification of a physiologically appropriate set of four genetic events that consistently drive HMEC transformation and are commonly observed in breast cancer. These include the suppression of p16 and p53 function, acquired resistance to the cytostatic effects of TGF-β, and acquisition of persistent growth signaling. p16 inactivation due to promoter methylation, as observed in HMECs, occurs in histologically normal mammary epithelium, and is now believed to be an important early event in breast carcinogenesis 101

104 resulting in diminished RB responsiveness 200. In addition, p53 is inactivated by gene mutation, overexpression of negative regulators HDM2 and HDMX, or loss of HOXA5, a transcription factor responsible for p53 gene transcription 55. Our study has identified that the acquisition of persistent growth signaling must be accompanied an acquired resistance to the cytostatic effects of TGF-β, either by TGFβ receptor inhibition or elevated c-myc expression. c-myc is a well-known suppressor of TGF-β signaling due, in part, to its ability to repress the transcription of the TGF-β activated genes p21 and p c-myc is amplified in up to 52% of breast cancer specimens, depending on the study, and it is likely that a tumors that do not harbor a c- MYC amplification overexpress c-myc protein via additional mechanisms 102, 191, 201, 204. We propose that the suppression of TGF-β-mediated growth inhibition by deregulated c- MYC expression is a key event in inhibiting OIS when breast epithelial cells encounter inappropriate oncogenic signaling. Interestingly, HDMX has been implicated in suppression of TGF-β signaling through its interaction with SMAD proteins, preventing SMAD transactivation 195, 196. Our results showed that elevated HDMX expression increased AIG even in the absence of p53, suggesting a p53-independent role for HDMX in the transformation of HMECs. Similarly, HMECs expressing c-myc and RAS-G12V had substantially more colonies when TGF-β signaling was inhibited pharmacologically, similar to our observation with elevated HDMX expression. We suggest that HDMX suppresses both the p53 and TGF-β tumor suppressive pathways simultaneously, resulting in enhanced transformation. Consistent with our findings, HDMX has been shown to be elevated or amplified in a variety of cancer types, including breast cancer 51, and we propose that elevated HDMX expression may allow early breast lesions to bypass 102

105 OIS. Importantly, our model does not employ the expression of exogenous htert, and while HMECs expressing shrna-p53, c-myc, and RAS grew anchorage-independently, the absence of telomerase activity eventually led the cells to cease dividing and undergo crisis. This observation illustrates another important difference between our studies and previous transformation models, as every other human epithelial cell transformation model utilizes cells immortalized by exogenous htert expression. Immortality is a critical factor in the malignant progression of human cancer, and it has been proposed that acquisition of telomerase activity is the rate-limiting step in human breast cancer progression 205. Importantly, previous studies of htert-immortalized HMECs have shown that htert expression induces resistance to TGF-β signaling, allowing oncogenes become potent, growth promoting signals rather than engaging senescence 179, 206. This critical switch, provided by immortalization, permits the progression of earlystage breast hyperplasia to more aggressive, malignant phenotypes. We credit the use of non-immortalized, finite lifespan cells with allowing us to identify the critical TGF-βmediated OIS barrier responsible for suppressing transformation in response to hyperactivated RAS signaling. RAS mutations are surprisingly infrequent in breast cancer (less than 5%) relative to other cancers which frequently have RAS mutations (50% of colon and thyroid cancers, and 90% of pancreatic cancers) 207. The reason for the differences between cancers that tolerate RAS mutations and those that do not remains unclear. However, we speculate that point-mutant RAS may initiate the TGF-β-mediated OIS described here more potently than elevated RAS signaling via growth factor receptors or amplification of wild-type RAS, which are far more frequently observed in 103

106 breast cancer 104. Finally, the paradox that TGF-β signaling suppresses the growth of normal epithelial cells, and yet, is often required for the maintenance of a transformed phenotype remains puzzling. However, much like the differences in biological outcomes observed between fibroblasts and epithelial cells exposed to TGF-β, the response of normal, hyperplastic, and transformed epithelial cells can be explained by the SMADindependent signals generated from the TGF-β receptor, which include pathways involving TAK1, NFkB, JNK, MAPK, PI3K/AKT and mtor 208. The relative contribution of the SMADs and these additional signaling arms control whether cells arrest or continue dividing, and also regulate the epithelial-to-mesenchymal transition (EMT) often described following TGF-β treatment. EMT results in cells that have increased motility, invasiveness, and stem-like characteristics commonly associated with more aggressive cancer phenotypes. Hence, elevated c-myc expression suppresses the growth arrest arm of the TGF-β signaling pathway and maintains the TGF-β receptor in a functional state with respect to these additional signaling cascades. Interestingly, the HMECs used in this study exhibit characteristics consistent with EMT, including increased vimentin and decreased E-cadherin expression. We suggest that, in breast cancer, the anti-proliferative effects of TGF-β are simply suppressed rather than absent, leaving the option to reengage these hidden limits to proliferation as a cancer therapy. 104

107 Chapter IV: Discovery of FAM83B as a novel oncogene using a validation-based forward genetic strategy. Authors: Rocky Cipriano James Graham Ronald C. Bruntz Sarah A. Scott H. Alex Brown George R. Stark Mark W. Jackson A. Abstract Aberrant regulation of growth signaling is essential for the development of cancer and often occurs through the constitutive activation of growth factor receptors or their downstream effectors 114. Here, we describe the discovery of a novel oncoprotein capable of hyperactivating the Mitogen Activated Protein Kinase (MAPK) and mammalian Target of Rapamycin (mtor) signaling cascades, critical regulators of cellular growth and proliferation. Using validation-based insertional mutagenesis (VBIM) 34, we identified FAM83B (Family with Sequence Similarity 83, member B), based on its ability to substitute for RAS in the transformation of immortalized human mammary epithelial 105

108 cells (HMECs). Interestingly, FAM83B is a member of an uncharacterized protein family (FAM83) defined by a highly conserved domain of unknown function (DUF1669). The DUF1669 of FAM83B is sufficient for HMEC transformation, and site-directed mutation of lysine 230 within the DUF1669 abolishes its transforming activity. Ablation of FAM83B in tumor-derived cells or in HMECs transformed by activated RAS, inhibited their proliferation and malignant phenotypes, implicating FAM83B as a critical mediator of aberrant growth signaling. Analysis of human tumor specimens revealed that the levels of FAM83B were significantly elevated in several types of cancer, with elevated expression associated with specific cancer subtypes, increasing tumor grade, and decreased overall survival. Together these results establish FAM83B as a novel oncogene and provide a promising new target for therapeutic intervention. B. Report The unique transcriptomes of different human epithelial cell types provide opportunities for the identification of tissue-specific oncogenes and tumor suppressors using phenotypic-based forward genetic screens. We recently created an improved validation-based insertional mutagenesis (VBIM) strategy that expands the application of reversible promoter insertion to nearly any type of mammalian cell 34. The VBIM lentiviruses alter the unique transcriptome of the model system under investigation by introducing promoters into the genome that result in dominant genetic alterations that increase the expression of sequences neighboring the insertion sites. To identify genes capable of driving HMEC transformation, a VBIM forward genetic screen was performed 106

109 using immortalized human mammary epithelial (HME1) cells (Figure IV-1a). Expression of a single event, such as constitutive Cyclin D1 1 or constitutive activation of RAS, promotes the anchorage-independent growth (AIG) of HME1 cells (Figure IV-1b). The strategy involves the creation of HME1 cell libraries in which each cell in a library has a different insertion event, resulting in a unique genetic alteration. Following expansion, each library was plated into soft agar to select for rare mutants capable of AIG, a hallmark of transformed cells. Subsequently, mutant cells were recovered from agar and infected with a retrovirus encoding Cre recombinase to remove the VBIM mutagenic promoter. Those mutants that lost the ability to grow anchorage-independently were considered validated, promoter-dependent mutants worthy of further study. Those that showed no decrease in AIG following promoter excision were considered promoterindependent mutants and were not further analyzed (Figure IV-1a). The ability of cells to undergo phenotypic reversion was first examined using RAS-infected HME1 cells. RAS expression alone resulted in significant AIG, which could be reversed following the expression of Cre recombinase to excise the promoter and RAS cdna (Figure IV-1b). After establishing the feasibility of reversion by Cre recombinase, we performed a VBIM screen to identify novel genes capable of inducing AIG. Our VBIM screen consisted of six libraries for each of the three VBIM lentiviruses, representing three different splice-donor reading frames (SD1, SD2 and SD3) 34. Following infection, each of the 18 cell libraries were expanded, plated into soft agar and assessed three weeks later for AIG, using an automated multi-panel scanning microscope and MetaMorph image quantification software. Five libraries had more than twice as many colonies as the GFP control (Figure IV-1c). To test the promoter- 107

110 Figure IV-1: A forward genetic screen identifies FAM83B as a driver of anchorageindependent growth in human mammary epithelial cells. (a) Illustration of the VBIM strategy: (1) numerous cell libraries were created using VBIM lentiviruses such that each infected cell in the library contains a distinct genetic alteration. (2) The libraries were plated into soft agar to select for rare mutants capable of anchorage-independent growth (AIG), a hallmark of transformed cells. (3) Mutants recovered from agar were tested for validation by eliminating the VBIM promoter using CRE recombinase and analyzed for phenotypic reversion. Libraries that lost the ability to grow anchorage-independently were deemed to be validated. (b) Confirmation of reversible AIG. HME1 cells were infected with retroviruses encoding GFP loxp and RAS loxp and analyzed for AIG 3 weeks later, using an automated multi-panel scanning microscope and MetaMorph image quantification software. Cells were recovered from agar and infected with a retrovirus encoding CRE-recombinase (CRE, C), or a control retrovirus (Vec, V), and analyzed to confirm the reversibility of the phenotype. (c) A phenotypic-based forward genetic screen for genes that promote AIG. The screen was conducted using 10,000 cells per well and 6 wells for each of the 3 VBIM viruses, resulting in 18 distinct cell libraries. The initial 10,000 cells were expanded to 200,000 cells and analyzed for AIG. Cells expressing GFP (-) or RAS (+) alone served as the negative and positive controls, respectively. (d) Validation of mutant libraries. Five pools were identified that had twice as many colonies as the negative control (background). The colonies from these libraries were recovered from agar, infected with a retrovirus encoding CRE (C), or control vector (V) and analyzed for phenotypic reversion. (e) Identification of FAM83B in the validated SD3-5 mutant. RT-PCR was performed on RNA from SD3-5 cells using a primer targeting the 5 region of the VBIM-driven mrna and a 3 FAM83B-specific primer, confirming that the VBIM-FAM83B spliced mrna (V-FAM83B) was dependent on the sustained activity of the VBIM promoter. Primers for GAPDH served as a loading control. (f) Northern analysis of FAM83B expression in parental HME1 cells and SD3-5 cells infected with a retrovirus encoding CRE (C), or control vector (Vector, V), confirming the promoter-dependent elevation of FAM83B mrna. (g) Overexpression of FAM83B in naïve HME1 cells drives AIG. Naïve HME1 cells were infected with a retrovirus encoding full-length FAM83B, VBIM-FAM83B, or a control retrovirus (Vector) and analyzed for AIG, confirming that the phenotype of the SD3-5 mutant observed during the original screen was caused specifically by FAM83B. (h) FAM83B expression activates MAPK signaling and elevates Cyclin D1 expression. Immunoblot analysis of phospho-erk1/2, ERK1/2, phospho-akt, Cyclin D1, FLAG, and Actin in HME1 cells expressing FAM83B and VBIM-FAM83B. (i) Subcutaneous tumors formed from HME cells expressing GFP (Vector), FAM83B, or RAS-V12 constructs together with the SV40 Large T genomic region in immunocompromised mice. Error bars represent the mean +/- standard deviation for a representative experiment performed in triplicate. 108

111 Figure IV-1 109

112 dependence of these mutants, they were recovered as individual colonies or as pools of colonies and monolayer cultures were re-established. The recovered cells were expanded and infected with a retrovirus encoding Cre-Recombinase (C) or control retrovirus (V) and plated into soft agar, to test for phenotypic reversion. The successful validation of cells from libraries SD3-1 and SD3-5 is shown in Figure IV-1d (recovered pools of colonies) and Figure IV-2a (individual colonies). The additional three pools either did not revert, or failed to continue as a manageable culture, and were not further analyzed. The failure of some pools to revert highlights the superior efficiency of the validation-based approach used, since non-validated mutants were likely to be caused by an unidentified spontaneous genetic alteration that would be extremely difficult to analyze. The VBIM insertion sites were indentified from the SD3-1 and SD3-5 colonies using inverse PCR 34. The promoter insertion in the SD3-1 mutant mapped to the second intron of the gene SLC30A7 and encoded a truncated protein (Figure IV-2b). However, expression of either full-length SLC30A7 or the VBIM-driven SLC30A7 truncation in naïve HME1 cells failed to confirm that it alone could promote AIG and therefore no additional work was performed (Figure IV-2c). The promoter insertion in the SD3-5 mutant was mapped to the first intron of an uncharacterized gene named Family with Sequence Similarity 83, member B (FAM83B). Using a primer targeting the 5 region of the VBIM-driven mrna and a 3 FAM83B-specific primer, RT-PCR was performed to show the expression of the VBIM-FAM83B fusion transcript, and its loss following Cremediated promoter excision (Figure IV-1e). The VBIM-mediated expression of FAM83B was also confirmed by Northern analysis (Figure IV-1f), and the VBIM driven mrna was cloned and sequenced. Based on the orientation of the CMV promoter relative to the 110

113 Figure IV-2: Validation of cell libraries SD3-1 and SD3-5. (a) Individual soft agar colonies (A, B, and C) were recovered from pools SD3-1 and SD3-5, expanded and infected with a retrovirus encoding Cre Recombinase (C), or a control (Vector, V). Following puromycin selection, the cells were assessed for AIG. (b) The VBIM viral insertion site in the SD3-1 mutant was identified in the SLC30A7 gene by inverse PCR. RT-PCR was performed on RNA from SD3-1 cells using a primer targeting the 5 region of the VBIM-driven mrna and a 3 SLC30A7 specific primer, confirming the VBIM- SLC30A7 spliced mrna (V-SLC30A7) is dependent on the sustained activity of the VBIM promoter. Primers for GAPDH served as a loading control. (c) Naïve HME1 cells expressing full-length FAM83B, VBIM-FAM83B (cloned from SD3-5 mutant), full-length SLC30A7, and VBIM-SLC30A7 (cloned from SD3-1 mutant) were assessed for AIG. 111

114 FAM83B coding region, a truncated FAM83B protein lacking the first 148 amino acids of the 1011 amino acid protein was expected (Figure IV-3). To ensure the AIG observed in the SD3-5 mutant during the original screen was caused specifically by FAM83B, the cdnas encoding full length FAM83B and truncated VBIM-FAM83B proteins were cloned and expressed in naïve HME1 cells. Both promoted AIG, confirming the results obtained with the original mutant SD3-5 (Figure IV-1g and IV-1h). Since FAM83B was able to substitute for RAS in our genetic screen, we examined whether, the activities of RAF or PI3K, downstream effectors of RAS were altered in FAM83B-expressing cells. Indeed, the expression of either full-length FAM83B or VBIM-FAM83B resulted in elevated ERK1 and ERK2 phosphorylation relative to control cells, while AKT phosphorylation was unaltered (Figure IV-1h). The level of Cyclin D1 protein, which is transcriptionally elevated by MAPK signaling, was also elevated following ectopic FAM83B expression (Figure IV-1h) 209. Elevated expression of Cyclin D1, a critical cell cycle regulator, is already known to drive HMEC transformation 1, 210. Finally, FAM83B expressing cells formed tumors in immunodeficient animals, confirming its role as a novel putative oncogene (Figure IV-1i). Consistent with the ability of FAM83B to drive HMEC transformation, public microarray data confirmed a significant elevation of FAM83B mrna expression in breast cancer specimens (Figure IV-4a) 211, 212. Furthermore, in two independent NCI-60 microarray data sets, MCF7 breast carcinoma cells had the highest level of FAM83B mrna of the cell lines examined. Northern analysis confirmed that FAM83B levels are similar in MCF7 and SD3-5 mutant cells and both are significantly higher than in normal HME1 cells (Figure IV-4b). To further substantiate that FAM83B expression is a critical 112

115 Figure IV-3: Schematic diagram of the VBIM integration site in the FAM83B gene. (a) The number of nucleotides comprising each of the 5 exons of the FAM83B gene is shown in blue. The number of nucleotides comprising each intron is shown in green. The number of amino acids encoded by each exon is shown in red. The yellow arrow denotes the VBIM insertion site within intron 2. (b) Sequence analysis of the VBIM-FAM83B fusion mrna illustrates that the splice donor of the VBIM provirus properly splices into exon 2 of FAM83B. 113

116 Figure IV-4: Inhibition of FAM83B suppresses the growth of breast cancer cells. (a) FAM83B expression is upregulated in breast cancer. Microarray data was acquired from Array Express ( for two data sets, E-MEXP-882 and E-TABM-276, which examined transcriptional changes between normal (N) and cancerous (C) human breast tissues. The relative expression of FAM83B in each specimen was plotted and the statistical significance determined using a Welch s t-test. Sample distribution and p-values are shown. (b) FAM83B is overexpressed in MCF7 breast carcinoma cells. Northern analysis of FAM83B expression was performed using RNA from MCF7, HME1, and SD3-5 mutant cells, demonstrating that MCF7 cells express elevated FAM83B mrna compared to HME1 cells. The SD3-5 mutant has two distinct mrnas, a slower migrating VBIM-driven product containing the functional elements of the VBIM viruses, and a faster migrating endogenous message. (c-e) Ablation of FAM83B inhibits the proliferation of MCF7 cells. MCF7 cells were infected with lentiviruses encoding shrnas targeting GFP (G) or FAM83B (1 and 2). Northern analysis was performed to confirm the suppression of FAM83B mrna. The intensity of the FAM83B signal was captured using a Typhoon Phosphorimager, and normalized to the 18S ribosomal RNA (value shown below 28S/18S panel) (c). Colony formation assay of MCF7 cells expressing shrna towards FAM83B (1 and 2) (d). MCF7 cells expressing shrnas targeting GFP (G) or FAM83B (1 and 2) were plated, grown for 3, 5, 7 or 10 days, and the cell number was determined (e). (f) A panel of breast cancer cell lines (MCF7, MDA468, BT474, T47D, and MDA436) were infected with shrnas targeting GFP (G) or FAM83B (1 and 2) and cell number was assessed after 8 days. (g) Northern analysis for FAM83B demonstrated that the inhibitory effect of the FAM83B shrnas correlated with the initial level of FAM83B expressed in the parental cancer cell lines. (h) Northern analysis of FAM83B expression in MDA468 cells expressing shrnas towards FAM83B (1 and 2). (i and j) Ablation of FAM83B (1 and 2) by shrna inhibits AIG in MCF7 (i) and MDA468 (j) cells compared to control cells expressing a shrna targeting GFP (G). (k and l) MDA468 cells were infected with shrnas targeting GFP (G) or FAM83B (2) and injected subcutaneously into immunocompromised mice to assess tumor formation. All experiments were performed in triplicate and mean +/- standard deviation is shown. 114

117 Figure IV-4 115

118 mediator of transformation, we examined whether its ablation would alter MCF7 proliferation and colony formation. Four shrnas targeting FAM83B (1-4) or a control shrna (GFP, G) were delivered to MCF7 cells by lentiviral infection (Figure IV-4c and Figure IV-5). Expression of FAM83B shrnas in MCF7 cells lead to decreased colony formation compared to control cells (Figure IV-4d). Also, cells expressing the FAM83B shrnas were plated and grown for various times, and cell number was determined (Figure IV-4e and Figure IV-5). Whereas parental MCF7 and shgfp-expressing cells grew similarly, the growth of cells expressing FAM83B-shRNAs was significantly inhibited, by 84-87% at the 10-day time point (Figure IV-4e). Additional analysis of other breast cancer cell lines identified MDA468 cells as equally dependent on FAM83B expression for efficient growth, whereas other cell lines were either moderately affected (BT474 and T47D) or unaffected by FAM83B knockdown (MDA436) (Figure IV-4f). Notably, the inhibitory effect of the FAM83B shrnas correlated with the initial level of FAM83B expressed in the parental cancer cell lines (Figure IV-4g). The ablation of FAM83B from MDA468 cells was confirmed by northern analysis (Figure IV-4h). We next examined whether FAM83B ablation would alter AIG and tumorigenesis in breast cancer cell lines that required FAM83B expression for proliferation. MCF7 and MDA468 AIG were inhibited by 70-90% when FAM83B expression was suppressed by shrna (Figure IV-4i and IV-4j). In addition, the tumorigenicity of MDA468 cells was examined following FAM83B-shRNA expression using a xenograft tumor model. Again, ablation of FAM83B resulted in a significant decrease in tumor volume (p=0.013) and tumor weight (p=0.0036) compared to control cells expressing a shrna targeting GFP (Figure IV-4k and IV-4l). These data demonstrate that cell lines derived from human cancers can have a 116

119 Figure IV-5: Validation of FAM83B shrna constructs. MCF7 cells expressing shrnas targeting GFP (G) or FAM83B (1, 2, 3, and 4) were plated, grown for 8 days, and the cell number was determined. Northern analysis for FAM83B demonstrated that the inhibitory effect of the FAM83B shrnas correlated with growth inhibition. 117

120 dependence for, or addiction to, sustained FAM83B expression to maintain their growth and tumorigenicity, providing a novel target for therapeutic intervention in a subset of breast cancers. The FAM83B gene was originally annotated as a RefSeq gene (chromosome 6 open reading frame 143), and included as a member of a hypothetical protein family (FAM83) based on the presence of a DUF1669 N-Terminal domain among FAM83 members. FAM83B protein expression has been confirmed in various human cell lines following immunoprecipitation using phospho-tyrosine, phospho-serine or phosphothreonine antibodies, and mass spectrometry (Figure IV-6). The Domain of Unknown Function (DUF1669) contains a putative phospholipase D (PLD) motif and is the only predicted functional domain within FAM83B (Figure IV-7a). To determine whether the DUF1669, which comprises amino acids 1-283, is the critical functional domain within FAM83B responsible for its transforming activity, we tested whether deletion mutants comprising amino acids (284) or (482) promoted AIG in HME1 cells (Figure IV-7a and Figure IV-8). Combined with our results using the truncated VBIM- FAM83B protein, which lacks the first 148 amino acids, these data implicate the region between amino acids 148 and 284, as sufficient for the transforming activity of FAM83B (Figure IV-7a and Figure IV-8). To examine whether FAM83B-expressing cells have elevated PLD activity, a cell-based mass spectrometric PLD assay was performed 213. HME1 cells expressing GFP or FAM83B were left untreated or stimulated with serum in the presence of 1-butanol-d 10 to trap the PLD product as phosphatidylbutanol-d 9 (PtdBuOH), which was analyzed by mass spectrometry. FAM83B-expressing cells had elevated PLD activity in both basal 118

121 Figure IV-6: Detection of endogenous FAM83B protein in various cell lines. The FAM83B protein has been detected by mass spectrometry following immunoprecipitation with phospho-tyr/ser/thr antibodies. The human FAM83B peptides recovered are shown alongside the corresponding mouse sequence. The references provided by Phosphosite ( describe the identified peptides and the conditions under which they were identified. 119

122 Figure IV-7: FAM83B and transformation. (a) Schematic of the FAM83B protein denoting the Domain of Unknown Function 1669 (DUF1669), the putative PLD motif compared to a consensus PLD motif, and the deletion mutants analyzed here. Summary of the FAM83B constructs that drive HMEC anchorage-independent growth (AIG). (b) Analysis for PLD activity. HME1 cells expressing GFP or FAM83B were left untreated or stimulated with serum in the presence of 1-butanol-d 10 to trap the PLD product as phosphatidylbutanol-d 9 (PtdBuOH), which was detected by mass spectrometric analysis. Data is plotted as a ratio of PtdBuOH to internal standard. (c) Inhibition of FAM83Bmediated transformation by 1-butanol, but not tert-butanol. FAM83B-expressing HME1 cells (or GFP-expressing control cells) were plated into soft agar and left untreated (-), or treated with 0.5% or 1.0% tert-butanol (as a control) or 1-butanol and AIG was assessed. (d) Site-directed mutagenesis of the PLD motif abolishes FAM83B-mediated AIG. HME1 cells expressing wild-type (WT) FAM83B, H224/K226A double point mutant, K230A single point mutant, or deletion of the DUF1669 (1-283 amino acids) were analyzed for AIG. (e) HME1 cells expressing GFP, FAM83B or FAM83B-K230A were grown as 3-dimensional (3D) cultures in laminin-rich basement membrane (lrbm) for 10 days. Pictures were taken and colony size determined using MetaMorph image quantitation software. (f) FAM83B expression activates MAPK and mtor signaling. Immunoblot analysis of phospho-erk1/2, phospho-s6k, phospho-akt, phospho- 4EBP1 and total protein in HME1 cells expressing FAM83B or FAM83B-K230A (B- K230A). (g) FAM83B-mediated AIG requires MEK and mtor activity. FAM83Bexpressing HME1 cells were plated into soft agar and treated with MEK inhibitor U0126 (1.5uM), or mtor inhibitor Rapamycin (11nM). All experiments were performed in triplicate and mean +/- standard deviation are shown. 120

123 Figure IV-7 121

124 Figure IV-8: The DUF1669 drives AIG. (a) HME1 cells expressing full-length (FL) FAM83B or FAM83B deletion mutants encoding amino acids (482) or (284) were assessed for AIG. (b) Immunoblot analysis of FLAG and GAPDH in HME1 cells expressing Vector, FAM83B-284, FAM83B-482, the deletion of the DUF1669 (1-283 amino acids), and wild-type (WT) FAM83B. 122

125 and serum-stimulated conditions relative to control GFP-expressing cells (Figure IV-7b). Furthermore, treatment of FAM83B-expressing cells with 1-butanol, a competitive nucleophile that prevents phosphatidic acid production, strongly inhibited FAM83Bmediated AIG. Tert-butanol, which is unable to serve as a competitive nucleophile in the transphosphatidylation reaction was used as a negative control (Figure IV-7c). Together, these data support a model in which FAM83B mediates elevated PLD activity resulting in increased PA production that drives transformation. We next examined whether disruption of histidine and lysine residues (H224, K224, and K230) in the putative PLD motif shown in Figure IV-7a, played a role in the elevated PLD activity observed in FAM83B-expressing cells and whether this motif was required for transformation. To determine whether these residues are important for the transforming activity of FAM83B, we created three mutations, one in which the aminoterminal 283 amino acids of FAM83B were deleted (Δ DUF) and two site-directed mutations targeting the histidine and lysine residues (H224/K226A and K230A). Interestingly, each of these mutations prevented FAM83B from promoting AIG, further implicating this region in FAM83B-mediated transformation (Figure IV-7d and Figure IV-9). In contrast to cells expressing wild-type FAM83B protein, cells expressing the FAM83B-K230A mutant showed no increase in PLD activity (Figure IV-10). However, FAM83B immunoprecipitates do not display PLD activity using exogenous assays capable of detecting PLD activity from PLD1 immunoprecipitates (data not shown). PLD activity results in the production of phosphatidic acid (PA), which is required for the activation of RAF and mtor 137, 214, 215, two signaling pathways commonly altered in cancer. These observations provide the first indication that it may be beneficial to 123

126 Figure IV-9: Comparison of FAM83B expression in mutant cells. Immunoblot analysis of FLAG and GAPDH in HME1 cells expressing wild-type (WT) FAM83B, H224/K226A double point mutant, and K230A single point mutant. 124

127 Figure IV-10: PLD assay of mutant FAM83B expressing HMECs. HME1 cells expressing GFP, FAM83B, or FAM83B-K230A were left untreated or stimulated with serum in the presence of 1-butanol-d 10 to trap the PLD product as phosphatidylbutanol-d 9 (PtdBuOH), which was detected by mass spectrometric analysis. Data is plotted as a ratio of PtdBuOH to internal standard. 125

128 therapeutically target FAM83B through this motif to treat FAM83B-dependent malignancies. The activation of RAF and mtor 137, 214, 215 are dependent on phosphatidic acid, therefore, we examined the activation of their downstream effectors, such as phospho-erk1/2, phospho-s6k and phospho-4e-bp1 in FAM83B-mediated transformation. HME1 cells expressing GFP, FAM83B or FAM83B-K230A were grown in laminin-rich basement membrane (lrbm), colony size was measured (Figure IV-7e) and western analysis was performed. Consistent with the results shown in Figure IV-1h, FAM83B expression resulted in elevated ERK1 and ERK2 phosphorylation relative to control cells. In contrast, the FAM83B-K230A mutant is unable to activate ERK, again demonstrating the requirement for PLD activity (Figure IV-7f). This is consistent with previous reports that the direct binding of PA to a site in the C-terminus of RAF induces the translocation of RAF to the plasma membrane for activation and that the production of PA is required to couple ERK activation at cell membranes 140, 143. Moreover, mtor activation, measured by S6K and 4E-BP1 phosphorylation, was elevated following FAM83B expression, but not following expression of the FAM83B-K230A mutant (Figure IV-7f). AKT phosphorylation was not elevated in FAM83B-expressing cells, suggesting that FAM83B activates mtor independently of the PI3K-AKT axis. Since MAPK and mtor signaling were activated following FAM83B expression, we examined whether inhibition of either pathway prevented FAM83B-mediated transformation. Treatment of FAM83B-expressing cells with an inhibitor of MEK (U0126) or mtor (rapamycin) prevented AIG (Figure IV-7g). Mechanistically, this is explained by the observation that PA and rapamycin compete for the same binding 126

129 domain on mtor 137. These data strongly support a model in which FAM83B-mediated increases in PA production initiates signaling through the MAPK and mtor pathways, both of which are required for the transformed phenotype. It has been previously reported that phosphatidic acid production by PLD1 is a critical intermediate for RAS-mediated transformation 138. We hypothesized that the increases in PLD activity conferred by FAM83B expression may also play an important role in RAS-mediated transformation. To test this hypothesis, two commonly used RASdependent transformation models were examined. The first model consisted of HME1 cells expressing a constitutively activated RAS(V12) and SV40 Large T-antigen (HME1- TRAS), which results in a transformed phenotype 3. HME1-TRAS were infected with lentiviruses encoding shrnas targeting GFP or FAM83B, and the proliferation and AIG of these cells was examined. Importantly, ablation of FAM83B resulted in a significant decrease in cell number and AIG, supporting a role for FAM83B as an important intermediary in aberrant RAS signaling (Figure IV-11a and IV-11b). In the second model, BJ fibroblasts were transformed by expressing adenoviral E1A, RAS(V12) and htert (BJ-ERT) and analyzed for their FAM83B dependency. It has been previously shown, by our laboratory and others, that ablation of p53 expression by shrna in BJ- ERT cells confers a phenotype that is capable of AIG and tumorigenicity 2, 108. Following lentiviral delivery of the FAM83B shrnas to BJ-ERT/shp53 cells, they were tested for their ability to grow anchorage-independently. AIG was inhibited in the cells expressing the FAM83B shrnas, further confirming a role for FAM83B in RAS-mediated transformation (Figure IV-11c). Furthermore, Northern analysis of FAM83A and FAM83B in BJ fibroblasts expressing htert alone (BJ-T) or together with adenoviral 127

130 Figure IV-11: FAM83B is required for RAS mediated transformation and overexpressed in several cancer subtypes. (a and b) Sustained FAM83B expression is required for RAS-mediated transformation. HME1 cells expressing SV40 Large T (HME1-T) were infected with a retrovirus encoding RAS, or a control (Vector; V). The RAS-expressing HME1-T cells (HME1-TRAS) were subsequently infected with lentiviruses encoding shrnas targeting FAM83B (1 and 2) or GFP (G). The cells were plated for 5 days, and growth was assessed (a). Northern analysis confirmed the knockdown of FAM83B mrna relative to the control shrna. HME1-T cells expressing Vector (V), RAS alone, or RAS together with shrnas targeting FAM83B (1 & 2) or GFP (G) were assessed for AIG (b). (c) FAM83B expression is required for RASmediated transformation. BJ-ERT cells were infected with a lentivirus expressing an shrna targeting p53. The BJ-ERT-shp53 cells were subsequently infected with lentiviruses encoding control (GFP, G) or FAM83B (1 & 2) shrnas and assessed for AIG. (d) Northern analysis of FAM83A and FAM83B in BJ fibroblasts expressing htert alone (BJ-T) or together with adenoviral E1A and RAS (BJ-ERT) demonstrated a specific elevation of FAM83B. (e) FAM83B is overexpressed in several cancer subtypes. Origene TissueScan Cancer Survey Panels (384-well panels covering 17 tumor types) were analyzed by Real-Time PCR for FAM83B. The relative expression of FAM83B for specimens that had a statistically significant difference between normal, unassociated (N) and cancerous (C) tissues is presented. The expression of FAM83B in each normal and cancer set was normalized to the highest normal, unassociated specimen, which was set equal to 1, and the statistical significance was determined using a Welch s t-test. The cancer subtypes with p-values reaching statistical significance (p<0.05) are shown here. (f) Ablation of FAM83B in ovarian, cervical and lung cancer cell lines inhibits their growth. SKOV3, HeLa, and H1299 cancer cell lines and normal diploid BJ fibroblasts expressing htert (BJ-T) were infected with lentiviruses encoding shrna targeting GFP (G) or FAM83B (1 and 2) and cell number was assessed 8 days after plating. Error bars represent the mean +/- standard deviation for a representative experiment performed in triplicate. 128

131 Figure IV

132 E1A and RAS (BJ-ERT) demonstrated a specific elevation of FAM83B in BJ-ERT cells (Figure IV-11d). Importantly, BJ-ERT cells require the induced expression of FAM83B by E1A and RAS for their transformed phenotype. These data support the possibility to therapeutically target malignancies that depend on constitutive RAS signaling through FAM83B inhibition. Since FAM83B was able to drive HMEC transformation and required for the transformed phenotype of tumor-derived cells and HMECs transformed by activated RAS, we next examined whether its expression was elevated in human cancers. Analysis of FAM83B in human tumors was performed using a real-time PCR cancer array containing normal and tumor samples from 17 different tissue types. FAM83B mrna expression was elevated, relative to the normal unassociated tissue, in a number of cancers including breast, lung, ovary, cervical, testis, thyroid, bladder, and lymphoid cancers (Figure IV-11e and Figure IV-12). This observation demonstrates that FAM83B is overexpressed in a sizeable fraction of human cancers. Also, ablation of FAM83B in the lung cancer (H1299), ovarian cancer (SKOV3) or cervical cancer (HeLa) cell lines inhibited their growth, as previously shown in breast cancer cells (Figure IV-11f). Delivery of the FAM83B shrnas to normal diploid BJ fibroblasts expressing htert (BJ-T), which do not express detectable levels of FAM83B mrna (Figure IV-11d), did not alter their growth (Figure IV-11f). Furthermore, analysis of tumor microarray datasets implicate elevated expression of FAM83B (1) in specific cancer subtypes, (2) with increasing tumor grade, and (3) with decreased overall survival (Figure IV-12). Importantly, increased expression of FAM83B in breast cancer is significantly associated with estrogen-receptor (ER) and progesterone-receptor (PR) negative tumors, with higher 130

133 Figure IV-12: FAM83B expression in cancer. Summary of tumor microarray datasets obtained from Oncomine ( demonstrating significant association of FAM83B expression with specific clinical or pathologic parameters. 131

134 grade and poorer outcomes. The lack of ER and PR expression correlates with the basal or triple negative phenotype. While not necessarily synonymous, these classifications describe tumors that are not responsive to hormonal therapy and tend to be less responsive to standard systemic chemotherapies. This information provides an important relevance for studies aimed at targeting FAM83B in cancer. FAM83B is one of eight members of the FAM83 protein family based solely on the presence of the DUF1669, which is essential for FAM83B-mediated transformation. Similar to FAM83B, analysis of tumor microarray datasets implicate elevated expression of various FAM83 members in specific cancer subtypes, with increasing tumor grade, and with decreased overall survival (Figure IV-13). Furthermore, in addition to our discovery of FAM83B, the laboratory of Dr. Mina Bissell has identified another FAM83 member, FAM83A, in a separate forward genetic screen for cdnas that confer resistance to EGFR inhibition in three-dimensional culture, resulting in a tumorigenic phenotype (M. Bissell, personal communication). Their results are consistent with those presented here and suggest that the regulation of FAM83A is important for appropriate EGFR signaling, epithelial cell polarity and proper tissue architecture. FAM83A has also been identified as a lung cancer biomarker, where it has been reported to be elevated in ~70% of lung adenocarcinomas 216. These observations demonstrate that FAM83 members are overexpressed in a sizeable fraction of human cancers. Our significant discovery of FAM83B as a novel oncogene and a critical mediator of growth signaling provide a new avenue in the pursuit for novel therapeutics. 132

135 Figure IV-13: FAM83 member expression in cancer. Summary of tumor microarray datasets obtained from Oncomine ( demonstrating significant association of FAM83 member expression with specific clinical or pathologic parameters. 133

136 134

137 135

138 136

139 137

140 C. Materials & Methods Retroviral Constructs. The lentiviral vectors encoding GFP-LoxP (WPXL) was kindly provided by Didier Trono (University of Geneva, Switzerland, Addgene Plasmid #12257). Ras-V12 was cloned into the BamHI site of WPXL to create RAS-LoxP. The pbabe-cre was kindly provided by K. Wagner (University of Nebraska Medical Center), LNCX2-SV40 Large T was provided by Andrei Gudkov (Roswell Park Cancer Institute), and pbabe-puro htert, LNCX2-E1a and LVTHM-shp53 were described previously 2. The FAM83B cdna (BC112275) was amplified by PCR using primers (5 CGCGGATCCATGGACTACAAGGACGACGATGACAAGGTGAGACCTCATCAAT CTTTCC 3 and 5 CCATCGATGTTACAAAGACTGTCCACAATTTTCTTT 3 ) which introduced a FLAG tag epitope and was subsequently cloned into the retroviral vector plpcx (Clontech). The VBIM-FAM83B cdna was amplified by PCR using RNA isolated from mutant SD3-5 cells and two primers (5 CGCGGATCCCCACAAGGAGACGACCTTCCGG 3 and 5 AATATTCCTGAGACGCTGAACTGAACAACC 3 ). The VBIM-FAM83B PCR fragment was cloned into LPCX-FLAG-FAM83B using BglII and XhoI. Additional FAM83 members were ultimately cloned into LPCX following PCR amplification with primers encoding a FLAG epitope. The cdna for SLC30A7 (BC064692) was PCR amplified using two primers (5 CGCGGATCCATGGACTACAAGGACGACGATGACAAGGTGTTGCCCCTGTCCA TCAAAGAC 3 and 5 CCATCGATAAAGCTGGGTACAATGTTTTGG 3 ) which introduced an N-terminal FLAG epitope tag and was subsequently cloned into plpcx. The VBIM-SLC30A7 cdna was amplified by PCR using RNA isolated from mutant 138

141 SD3-1 cells and two primers (5 CGCGGATCCCCACAAGGAGACGACCTTCCGG 3 and 5 CCATCGATAA AGCTGGGTACAATGTTTTGG 3 ) and was cloned into plpcx. All cdna plasmids were acquired from Open Biosystems and sequence verified following PCR. To create FAM83B truncation mutants, the FAM83B cdna (BC112275) was amplified by PCR using the following primers, cloned into the pcmv-flag2 vector (Sigma), and then subcloned into LPCX. LPCX-FLAG-FAM83B-284 was created using using primers (5 CGCGGATCCATGGACTACAAGGACGACGATGACAAGGTGAGACCTCATCA ATCTTTCC 3 and 5 CCATCGACTATGCTGATTCTTCCTGAGCAAA 3 ), LPCX- FLAG-FAM83B-482 was created using primers (5 CGCGGATCCATGGACTACAAGGACGACGATGACAAGGTGAGACCTCATCAAT CTTTCC 3 and 5 CCATCGATCTAGGTTGGCATTCGTTGTTGCAA 3 ). LPCX- FLAG-FAM83B-ΔDUF plasmid was created using primers (5 CGCGGATCCATGGACTACAAGGACGACGATGACAAGTCATTTGCTCAGGAAG AATCAGCA 3 and 5 CCATCGATGTTACAAAGACTGTCCACAATTTTCTTT 3 ). The LPCX-FLAG-FAM83B-H224/K226A point mutant was made by amplifying the LPCX-FLAG-FAM83B plasmid with primers (5 GCTGGCGCCATGGAACAGAAATTTTTGTTAGTT 3 and 5 GAATTTTGCCCCTGTTTTTGA 3 ) which was subsequently ligated with T4 DNA ligase and sequence verified. The LPCX-FLAG-FAM83B-K230A point mutant was made by amplifying the LPCX-FLAG-FAM83B plasmid with primers (5 139

142 GCATTTTTGTTAGTTGACTGCCAG 3 and 5 CTGTTCCATTTTTCCATGGAA 3 ) which was subsequently ligated with T4 DNA ligase and sequence verified. Virus Production and Infection. Retroviruses were produced as described 163. Briefly, retroviral vectors were transfected into Phoenix-Ampho cells together with a packaging plasmid encoding the MLV-gag-pol and env genes. Detailed information on VBIM vector construction and use has been described previously 34. Plasmids encoding shrnas targeting FAM83B or GFP in plko.1 were acquired from Open Biosystems. Viruses were packaged in 293T cells using the second-generation packaging constructs pcmvdr8.74 and pmd2g, kind gifts from Didier Trono (University of Geneva, Switzerland). Supernatant media containing virus, collected at h, were supplemented with 4 ug/ml polybrene before being frozen in aliquots or used to infect cells for 6-24 hours. Inverse PCR. Inverse PCR was performed as described 34, 217. Genomic DNA from the mutants was isolated using the Blood & Cell Culture DNA kit (Qiagen). Ten ug of genomic DNA was digested with EcoRI and MfeI overnight at 37C and 2 ug of this DNA was then ligated with T4 DNA ligase overnight. The reaction products were purified by using a Qiagen MinElute PCR purification kit and one-half of the purified reaction mixture was used for the first PCR reaction, using primer #2 (5 CCAGAGTCACACAACAGACG 3 ) and primer #3 (5 GTAAGACCACCGCACAGC 3 ) in a 50 ul reaction volume, using a Phusion PCR kit (NEB). A second PCR reaction was performed using 0.5 ul of the first PCR reaction mixture, with primer #1 (5 CCAGAGAGACCCAGTACAAGC 3 ) and primer #4 (5 GATCTTCAGACCTGGAGGAG 3 ). PCR products were separated by agarose gel electrophoresis, excised from the gel and cloned into the pcr8/gw/topota vector 140

143 (Invitrogen). DNA sequencing was performed and the insertion site was identified using bioinformatics from the NCBI web site. Cell Lines and Culture Conditions. htert-hme1 cells were purchased from (Clontech) and grown in a humidified atmosphere containing 5% CO 2 in Medium 171 with mammary epithelial growth supplement (Cascade Biologics) and 50 units/ml of penicillin and 50 µg/ml of streptomycin sulfate (U.S. Biochemical Corp.) as described 182. Cancer cell lines (MCF7, MDA468, MDA436, T47D, BT474, HeLa, H1299, and SKVO3) and normal diploid BJ fibroblasts were grown in a humidified atmosphere containing 5% CO 2 in DMEM (with glucose and L-glutamine; Life Technologies) with 5% fetal bovine serum and 50 units/ml of penicillin and 50 µg/ml of streptomycin sulfate (U.S. Biochemical Corp.). Soft Agar Assays. For htert-hme1 cells, 1 x 10 5 cells were suspended in 0.6% type VII agarose (Sigma) and plated onto a bottom layer of 1.2% agar in a 60mm plate in triplicate as described 1. The medium was changed every 3 days until cells were analyzed after 3 weeks. To quantify colonies, each plate was scanned using an automated multipanel scanning microscope, and the digital images analyzed using MetaMorph image quantification software. For MCF7 and BJ cells, 2 x 10 5 cells were plated per 60mm dish and medium was changed every 3 days until cells were analyzed after 3 weeks. AG1478 (33nM), U0126 (1.5uM), and Rapamycin (11nM) were obtained from Calbiochem and were added to the medium during feeding for indicated experiments. Tert-Butanol (0.5% & 1.0%) and 1-Butanol (0.5% & 1.0%) were obtained from Fisher Scientific and were added to the medium during feedings for indicated experiments. 141

144 Subcutaneous Tumorigenicity Assays. NOD/SCID mice were bred and maintained under defined conditions at the Athymic Animal and Xenograft Core facility at the Case Western Reserve University, Case Comprehensive Cancer Center an accredited facility that acts in compliance with NIH guidelines and provides veterinary care by several full time veterinary personnel. MDA468 (2x10 6 ) cancer cells and HMECs (4x10 6 ) were suspended in a 1:1 mixture of media and matrigel (BD Biosciences) and injected subcutaneously into mice (6-8 weeks of age) that were sublethally irradiated with 300 rad 4 hours prior to injection. Tumor volume was calculated with the formula 4/3πr 3 and student t-tests were performed. HME cells expressing GFP, RAS-V12, and FAM83B were infected with a retrovirus encoding SV40 Large T genomic to increase the efficiency of tumor take in immunocompromised mice 192. FAM83B expressing HME cells formed tumors when transplanted subcutaneously into immunocompromised mice with a latency of 12 weeks, while RAS-V12 expressing HME cells served as a positive control for tumor formation. Western Analysis. Whole cell extracts were prepared by incubating cell pellets in lysis buffer containing 50 mmol/l of Tris (ph 8.0), 150 mmol/l of NaCl, 1.0% NP40, 10 µg/ml of aprotinin, 100 µg/ml of phenylmethane sulfonyl fluoride, 5 µg/ml of leupeptin, 5 µg/ml of pepstatin, and 1 mmol/l of NaVO 4. Cell extracts containing equal quantities of proteins, determined by the Bradford method, were separated by SDS-PAGE (8 12.5% acrylamide) and transferred to polyvinylidene difluoride membranes (Millipore). Antibodies to cyclin D1 (C-20) were from Santa Cruz Biotechnology, antibodies to ß-actin (pan Ab-5) were from Neomarkers, antibodies to glyceraldehyde-3- phosphate dehydrogenase were from Calbiochem, antibodies to FLAG (M2) were from 142

145 Sigma-Aldrich, and antibodies for ERK1/2, P-ERK1/2 (Thr202/Tyr204), AKT, P-AKT (Ser473), P-S6K (Thr389), and P-4E-BP1 (Ser65) were from Cell Signaling. Primary antibodies were detected with goat anti-mouse or goat anti-rabbit conjugated to horseradish peroxidase (Hoffman-La Roche), using enhanced chemiluminescence (Perkin-Elmer). Relative Growth Assay. htert-hme1, MCF7, MDA468, BT474, T47D, MDA436, BJ, H1299, SKOV3, and HeLa cells were plated in triplicate at 20,000 cells/well in triplicate and cell number was determined on a Beckman Coulter counter. RNA Isolation and Northern Analysis. Total RNA was isolated using Qiagen RNeasy miniprep kit (Qiagen, Valencia CA). Ten to twenty micrograms of total RNA was fractionated in a 1% agarose/formaldehyde gel and transferred to a Hybond-XL membrane as described 218 (Amersham Biosciences). The RNAs were cross-linked to the membrane by using a UV cross-linker before hybridization. [ - 32 P]-dCTP (Perkin Elmer) was used to generate 32 P-labeled cdna fragments of FAM83B and FAM83A by using the Megaprime DNA labeling system (Amersham Biosciences, Piscataway, NJ). The membrane was prehybridized for 6 hours with 5x saline-sodium phosphate-edta (75 mmol/l NaCl, 50 mmol/l NaH 2 P0 4, and 4 mmol/l EDTA) plus 2% SDS and 100 µg/ml ssdna at 65 C. After the addition of specific probes, hybridization took place for 24 hours at 65 C. The membranes were washed three times with 2x saline-sodium phosphate-edta plus 0.1% SDS. Transfers were normalized for loading by comparing the intensities of the 18 S and 28 S rrna bands. 143

146 Real Time Quantitative PCR and Statistical Analysis. TissueScan Cancer Survey Panel I, comprising of 381 tissues covering 17 different cancers (Origene), was subjected to Real Time quantitative PCR analysis. RNA was analyzed for FAM83B and actin expression using an Applied Biosystems GeneAMP PCR System 9700 and Applied Biosystems assay Hs _m1. Analysis was performed at the Gene Expression Array Core Facility of the Comprehensive Cancer Center of Case Western Reserve University. The cycle threshold (Ct) of each FAM83 gene was determined in the 381 specimens within 40 cycles of PCR, or was set equal to 40 if it remained undetected by the 40 th cycle. The delta Ct was calculated by subtracting the FAM83B gene Ct from the actin Ct for each sample. The relative expression within each normal/cancer subtype was calculated by dividing each specimen s value by the value of the normal tissue with the highest expression within the subtype; such that its value became 1.0. The mean of the normal and cancer specimens was determined and the p-value calculated using an unpaired Welch s t-test. For Figure 4d, the relative expression of the normal and cancer specimens that were significantly different is plotted together with the mean for each (black bar). The p-value is included below each graph. Cell-Based Mass Spectrometric PLD Assay. Cell-based PLD activity was determined using a modified in vivo deuterated 1-butanol PLD assay 213. HME1 cells were seeded at 3.5 x 10 5 cells/well in 6-well tissue culture plates 48 hours prior to the assay in complete growth medium. At the time of the assay cells were treated with either Medium 171 alone, Medium % (v/v) 1-butanol-d 10 or Medium % (v/v) 1- butanol-d % fetal bovine serum for 60 min at

147 C. After treatment, cellular lipids were extracted as previously described and internal standard was added. The resulting lipids were dried and resuspended in MS solvent. Samples were directly injected into a Finnigan TSQ Quantum triple quadrupole MS and data were collected in negative ion mode. Data were analyzed and are represented as a ratio of major phosphatidylbutanol-d 9 lipid products and internal standard. Background signal was subtracted using cells not treated with 1-butanol-d 10 as a negative control. Samples were generated in triplicate in multiple independent experiments. 145

148 Chapter V: Supplemental Data Section A. Discovery of a novel family of oncogenes. In Chapter IV we found that FAM83B was able to drive a transformed phenotype in immortalized HMECs and required for the growth of tumor derived cell lines. Also, FAM83B expression was elevated in a number of cancer subtypes. FAM83B is one of eight members of the FAM83 protein family, based solely on the presence of the DUF1669 (Figure V-1a). An alignment of the eight family members highlights the conserved amino terminus and the location of the DUF1669 (Figure V-2) 219, 220. In addition to our discovery of FAM83B, the laboratory of Dr. Mina Bissell has identified another FAM83 member, FAM83A, in a separate forward genetic screen for cdnas that confer resistance to EGFR inhibition in three-dimensional culture, resulting in a tumorigenic phenotype (personal communication). Their results are consistent with those presented here and suggest that the regulation of FAM83A is important for appropriate EGFR signaling, epithelial cell polarity and proper tissue architecture. The expression of all FAM83 proteins, except FAM83C and FAM83E, have been confirmed following phosho-tyr/ser/thr immunoprecipitation and mass spectrometry (Figure V-3). Since all FAM83 members share a DUF1669, which is essential for FAM83B-mediated transformation, we tested the ability of FAM83 members (A-E) to transform HME1 cells. All five FAM83 members produced significantly more AIG than control cells (Vector), yet were less potent than the positive control, a constitutively activated CyclinD1-CDK2 fusion protein (Figure V-1b, V-1c, and Figure V-4) 1. Of note, lysine 230 in FAM83B is critical for the AIG phenotype, and is positionally conserved among the other family 146

149 Figure V-1: Additional FAM83 members drive HMEC transformation and are overexpressed in cancer. (a) Schematic diagram of the eight FAM83 members, illustrating the conserved Domain of Unknown Function (DUF1669), the putative PLD motif, and the number of amino acids encoding each protein. (b and c) FAM83A-E promotes HME1 AIG. The cdnas encoding each FAM83 member A-E were cloned, expressed in HME1 cells and analyzed for AIG. Representative images of soft agar growth for each family member and a CyclinD1-CDK fusion protein, used as a positive control, are shown. (d) FAM83 members are overexpressed in cancer. Origene TissueScan Cancer Survey Panels (384-well panels covering 17 tumor types) were analyzed by Real-Time PCR for FAM83 members A-E. Cancer specimens were scored as having elevated expression of a FAM83 member if their mrna levels were at least three times higher than the highest normal for each tissue type. These data are represented as the percentages of the tumor specimens having elevated expression. In addition, the statistical significance was determined for each normal and cancer set using a Welch s t-test. The cancer subtypes with p-values reaching statistical significance (p<0.05) are indicated. 147

150 Figure V-2: Alignment of FAM83 members demonstrating the conservation in the DUF1669. A Clustalw amino acid alignment was performed on the 8 FAM83 members showing the conserved N-terminal DUF1669, which is required for the transforming activity of the family. A ClustalX colour scheme (Jalview 2.3) was used. The PLD motif described in Figure 3a is indicated by the red rectangle and the conserved amino acid of each FAM83 member corresponding to K230 of FAM83B is indicated by the asterisk. 148

151 Figure V-3: Mass Spectrometry analysis of FAM83 members. Table indicating the number of FAM83 member peptides (human) identified by mass spectrometry following immunoprecipitation with phospho-tyr/ser/thr antibodies ( 149

RAS Genes. The ras superfamily of genes encodes small GTP binding proteins that are responsible for the regulation of many cellular processes.

RAS Genes. The ras superfamily of genes encodes small GTP binding proteins that are responsible for the regulation of many cellular processes. ۱ RAS Genes The ras superfamily of genes encodes small GTP binding proteins that are responsible for the regulation of many cellular processes. Oncogenic ras genes in human cells include H ras, N ras,

More information

Transformation of Normal HMECs (Human Mammary Epithelial Cells) into Metastatic Breast Cancer Cells: Introduction - The Broad Picture:

Transformation of Normal HMECs (Human Mammary Epithelial Cells) into Metastatic Breast Cancer Cells: Introduction - The Broad Picture: Transformation of Normal HMECs (Human Mammary Epithelial Cells) into Metastatic Breast Cancer Cells: Introduction - The Broad Picture: Spandana Baruah December, 2016 Cancer is defined as: «A disease caused

More information

Deregulation of signal transduction and cell cycle in Cancer

Deregulation of signal transduction and cell cycle in Cancer Deregulation of signal transduction and cell cycle in Cancer Tuangporn Suthiphongchai, Ph.D. Department of Biochemistry Faculty of Science, Mahidol University Email: tuangporn.sut@mahidol.ac.th Room Pr324

More information

Cancer. The fundamental defect is. unregulated cell division. Properties of Cancerous Cells. Causes of Cancer. Altered growth and proliferation

Cancer. The fundamental defect is. unregulated cell division. Properties of Cancerous Cells. Causes of Cancer. Altered growth and proliferation Cancer The fundamental defect is unregulated cell division. Properties of Cancerous Cells Altered growth and proliferation Loss of growth factor dependence Loss of contact inhibition Immortalization Alterated

More information

p53 and Apoptosis: Master Guardian and Executioner Part 2

p53 and Apoptosis: Master Guardian and Executioner Part 2 p53 and Apoptosis: Master Guardian and Executioner Part 2 p14arf in human cells is a antagonist of Mdm2. The expression of ARF causes a rapid increase in p53 levels, so what would you suggest?.. The enemy

More information

Cell cycle, signaling to cell cycle, and molecular basis of oncogenesis

Cell cycle, signaling to cell cycle, and molecular basis of oncogenesis Cell cycle, signaling to cell cycle, and molecular basis of oncogenesis MUDr. Jiří Vachtenheim, CSc. CELL CYCLE - SUMMARY Basic terminology: Cyclins conserved proteins with homologous regions; their cellular

More information

Introduction. Cancer Biology. Tumor-suppressor genes. Proto-oncogenes. DNA stability genes. Mechanisms of carcinogenesis.

Introduction. Cancer Biology. Tumor-suppressor genes. Proto-oncogenes. DNA stability genes. Mechanisms of carcinogenesis. Cancer Biology Chapter 18 Eric J. Hall., Amato Giaccia, Radiobiology for the Radiologist Introduction Tissue homeostasis depends on the regulated cell division and self-elimination (programmed cell death)

More information

Multistep nature of cancer development. Cancer genes

Multistep nature of cancer development. Cancer genes Multistep nature of cancer development Phenotypic progression loss of control over cell growth/death (neoplasm) invasiveness (carcinoma) distal spread (metastatic tumor) Genetic progression multiple genetic

More information

Cancer. The fundamental defect is. unregulated cell division. Properties of Cancerous Cells. Causes of Cancer. Altered growth and proliferation

Cancer. The fundamental defect is. unregulated cell division. Properties of Cancerous Cells. Causes of Cancer. Altered growth and proliferation Cancer The fundamental defect is unregulated cell division. Properties of Cancerous Cells Altered growth and proliferation Loss of growth factor dependence Loss of contact inhibition Immortalization Alterated

More information

CELL CYCLE MOLECULAR BASIS OF ONCOGENESIS

CELL CYCLE MOLECULAR BASIS OF ONCOGENESIS CELL CYCLE MOLECULAR BASIS OF ONCOGENESIS Summary of the regulation of cyclin/cdk complexes during celll cycle Cell cycle phase Cyclin-cdk complex inhibitor activation Substrate(s) G1 Cyclin D/cdk 4,6

More information

number Done by Corrected by Doctor Maha Shomaf

number Done by Corrected by Doctor Maha Shomaf number 19 Done by Waseem Abo-Obeida Corrected by Abdullah Zreiqat Doctor Maha Shomaf Carcinogenesis: the molecular basis of cancer. Non-lethal genetic damage lies at the heart of carcinogenesis and leads

More information

Introduction to Cancer Biology

Introduction to Cancer Biology Introduction to Cancer Biology Robin Hesketh Multiple choice questions (choose the one correct answer from the five choices) Which ONE of the following is a tumour suppressor? a. AKT b. APC c. BCL2 d.

More information

CANCER. Inherited Cancer Syndromes. Affects 25% of US population. Kills 19% of US population (2nd largest killer after heart disease)

CANCER. Inherited Cancer Syndromes. Affects 25% of US population. Kills 19% of US population (2nd largest killer after heart disease) CANCER Affects 25% of US population Kills 19% of US population (2nd largest killer after heart disease) NOT one disease but 200-300 different defects Etiologic Factors In Cancer: Relative contributions

More information

Tumor suppressor genes D R. S H O S S E I N I - A S L

Tumor suppressor genes D R. S H O S S E I N I - A S L Tumor suppressor genes 1 D R. S H O S S E I N I - A S L What is a Tumor Suppressor Gene? 2 A tumor suppressor gene is a type of cancer gene that is created by loss-of function mutations. In contrast to

More information

mirna Dr. S Hosseini-Asl

mirna Dr. S Hosseini-Asl mirna Dr. S Hosseini-Asl 1 2 MicroRNAs (mirnas) are small noncoding RNAs which enhance the cleavage or translational repression of specific mrna with recognition site(s) in the 3 - untranslated region

More information

7.012 Problem Set 6 Solutions

7.012 Problem Set 6 Solutions Name Section 7.012 Problem Set 6 Solutions Question 1 The viral family Orthomyxoviridae contains the influenza A, B and C viruses. These viruses have a (-)ss RNA genome surrounded by a capsid composed

More information

Determination Differentiation. determinated precursor specialized cell

Determination Differentiation. determinated precursor specialized cell Biology of Cancer -Developmental Biology: Determination and Differentiation -Cell Cycle Regulation -Tumor genes: Proto-Oncogenes, Tumor supressor genes -Tumor-Progression -Example for Tumor-Progression:

More information

TUMOR-SUPPRESSOR GENES. Molecular Oncology Michael Lea

TUMOR-SUPPRESSOR GENES. Molecular Oncology Michael Lea TUMOR-SUPPRESSOR GENES Molecular Oncology 2011 Michael Lea TUMOR-SUPPRESSOR GENES - Lecture Outline 1. Summary of tumor suppressor genes 2. P53 3. Rb 4. BRCA1 and 2 5. APC and DCC 6. PTEN and PPA2 7. LKB1

More information

Negative Regulation of c-myc Oncogenic Activity Through the Tumor Suppressor PP2A-B56α

Negative Regulation of c-myc Oncogenic Activity Through the Tumor Suppressor PP2A-B56α Negative Regulation of c-myc Oncogenic Activity Through the Tumor Suppressor PP2A-B56α Mahnaz Janghorban, PhD Dr. Rosalie Sears lab 2/8/2015 Zanjan University Content 1. Background (keywords: c-myc, PP2A,

More information

Lecture 8 Neoplasia II. Dr. Nabila Hamdi MD, PhD

Lecture 8 Neoplasia II. Dr. Nabila Hamdi MD, PhD Lecture 8 Neoplasia II Dr. Nabila Hamdi MD, PhD ILOs Understand the definition of neoplasia. List the classification of neoplasia. Describe the general characters of benign tumors. Understand the nomenclature

More information

Chapt 15: Molecular Genetics of Cell Cycle and Cancer

Chapt 15: Molecular Genetics of Cell Cycle and Cancer Chapt 15: Molecular Genetics of Cell Cycle and Cancer Student Learning Outcomes: Describe the cell cycle: steps taken by a cell to duplicate itself = cell division; Interphase (G1, S and G2), Mitosis.

More information

Oncogenes and Tumor Suppressors MCB 5068 November 12, 2013 Jason Weber

Oncogenes and Tumor Suppressors MCB 5068 November 12, 2013 Jason Weber Oncogenes and Tumor Suppressors MCB 5068 November 12, 2013 Jason Weber jweber@dom.wustl.edu Oncogenes & Cancer DNA Tumor Viruses Simian Virus 40 p300 prb p53 Large T Antigen Human Adenovirus p300 E1A

More information

Cancer and Gene Alterations - 1

Cancer and Gene Alterations - 1 Cancer and Gene Alterations - 1 Cancer and Gene Alteration As we know, cancer is a disease of unregulated cell growth. Although we looked at some of the features of cancer when we discussed mitosis checkpoints,

More information

Genetics and Cancer Ch 20

Genetics and Cancer Ch 20 Genetics and Cancer Ch 20 Cancer is genetic Hereditary cancers Predisposition genes Ex. some forms of colon cancer Sporadic cancers ~90% of cancers Descendants of cancerous cells all cancerous (clonal)

More information

MOLECULAR BASIS OF ONCOGENESIS

MOLECULAR BASIS OF ONCOGENESIS MOLECULAR BASIS OF ONCOGENESIS MUDr. Jiří Vachtenheim, CSc. 1 Cell processes which result also in cell cycle effects. Differentiation. Differentiated cells are usually in the G0 phase of the cell cycle.

More information

VIII Curso Internacional del PIRRECV. Some molecular mechanisms of cancer

VIII Curso Internacional del PIRRECV. Some molecular mechanisms of cancer VIII Curso Internacional del PIRRECV Some molecular mechanisms of cancer Laboratorio de Comunicaciones Celulares, Centro FONDAP Estudios Moleculares de la Celula (CEMC), ICBM, Facultad de Medicina, Universidad

More information

oncogenes-and- tumour-suppressor-genes)

oncogenes-and- tumour-suppressor-genes) Special topics in tumor biochemistry oncogenes-and- tumour-suppressor-genes) Speaker: Prof. Jiunn-Jye Chuu E-Mail: jjchuu@mail.stust.edu.tw Genetic Basis of Cancer Cancer-causing mutations Disease of aging

More information

Cancer Biology How a cell responds to DNA Damage

Cancer Biology How a cell responds to DNA Damage 1 Cancer Biology How a cell responds to DNA Damage Jann Sarkaria Department of Oncology Mayo Clinic 2 EDUCATIONAL GOALS How proteins can transmit signals to each other. The definition of a tumor suppressor

More information

Oncogenes and Tumor. supressors

Oncogenes and Tumor. supressors Oncogenes and Tumor supressors From history to therapeutics Serge ROCHE Neoplastic transformation TUMOR SURESSOR ONCOGENE ONCOGENES History 1911 1960 1980 2001 Transforming retrovirus RSV v-src is an oncogene

More information

Cancer Genetics. What is Cancer? Cancer Classification. Medical Genetics. Uncontrolled growth of cells. Not all tumors are cancerous

Cancer Genetics. What is Cancer? Cancer Classification. Medical Genetics. Uncontrolled growth of cells. Not all tumors are cancerous Session8 Medical Genetics Cancer Genetics J avad Jamshidi F a s a U n i v e r s i t y o f M e d i c a l S c i e n c e s, N o v e m b e r 2 0 1 7 What is Cancer? Uncontrolled growth of cells Not all tumors

More information

LESSON 3.2 WORKBOOK. How do normal cells become cancer cells? Workbook Lesson 3.2

LESSON 3.2 WORKBOOK. How do normal cells become cancer cells? Workbook Lesson 3.2 For a complete list of defined terms, see the Glossary. Transformation the process by which a cell acquires characteristics of a tumor cell. LESSON 3.2 WORKBOOK How do normal cells become cancer cells?

More information

BIO360 Fall 2013 Quiz 1

BIO360 Fall 2013 Quiz 1 BIO360 Fall 2013 Quiz 1 1. Examine the diagram below. There are two homologous copies of chromosome one and the allele of YFG carried on the light gray chromosome has undergone a loss-of-function mutation.

More information

Name Section Problem Set 6

Name Section Problem Set 6 Name Section 7.012 Problem Set 6 Question 1 The viral family Orthomyxoviridae contains the influenza A, B and C viruses. These viruses have a (-)ss RNA genome surrounded by a capsid composed of lipids

More information

Karyotype analysis reveals transloction of chromosome 22 to 9 in CML chronic myelogenous leukemia has fusion protein Bcr-Abl

Karyotype analysis reveals transloction of chromosome 22 to 9 in CML chronic myelogenous leukemia has fusion protein Bcr-Abl Chapt. 18 Cancer Molecular Biology of Cancer Student Learning Outcomes: Describe cancer diseases in which cells no longer respond Describe how cancers come from genomic mutations (inherited or somatic)

More information

Early Embryonic Development

Early Embryonic Development Early Embryonic Development Maternal effect gene products set the stage by controlling the expression of the first embryonic genes. 1. Transcription factors 2. Receptors 3. Regulatory proteins Maternal

More information

Problem Set 8 Key 1 of 8

Problem Set 8 Key 1 of 8 7.06 2003 Problem Set 8 Key 1 of 8 7.06 2003 Problem Set 8 Key 1. As a bright MD/PhD, you are interested in questions about the control of cell number in the body. Recently, you've seen three patients

More information

Oncolytic virus strategy

Oncolytic virus strategy Oncolytic viruses Oncolytic virus strategy normal tumor NO replication replication survival lysis Oncolytic virus strategy Mechanisms of tumor selectivity of several, some of them naturally, oncolytic

More information

The functional investigation of the interaction between TATA-associated factor 3 (TAF3) and p53 protein

The functional investigation of the interaction between TATA-associated factor 3 (TAF3) and p53 protein THESIS BOOK The functional investigation of the interaction between TATA-associated factor 3 (TAF3) and p53 protein Orsolya Buzás-Bereczki Supervisors: Dr. Éva Bálint Dr. Imre Miklós Boros University of

More information

Molecular Cell Biology. Prof. D. Karunagaran. Department of Biotechnology. Indian Institute of Technology Madras

Molecular Cell Biology. Prof. D. Karunagaran. Department of Biotechnology. Indian Institute of Technology Madras Molecular Cell Biology Prof. D. Karunagaran Department of Biotechnology Indian Institute of Technology Madras Module 9 Molecular Basis of Cancer, Oncogenes and Tumor Suppressor Genes Lecture 2 Genes Associated

More information

BIO360 Quiz #1. September 14, Name five of the six Hallmarks of Cancer (not emerging hallmarks or enabling characteristics ): (5 points)

BIO360 Quiz #1. September 14, Name five of the six Hallmarks of Cancer (not emerging hallmarks or enabling characteristics ): (5 points) Name: BIO360 Quiz #1 September 14, 2012 1. Name five of the six Hallmarks of Cancer (not emerging hallmarks or enabling characteristics ): (5 points) 2. The controversial hypothesis that only a small subset

More information

Genome of Hepatitis B Virus. VIRAL ONCOGENE Dr. Yahwardiah Siregar, PhD Dr. Sry Suryani Widjaja, Mkes Biochemistry Department

Genome of Hepatitis B Virus. VIRAL ONCOGENE Dr. Yahwardiah Siregar, PhD Dr. Sry Suryani Widjaja, Mkes Biochemistry Department Genome of Hepatitis B Virus VIRAL ONCOGENE Dr. Yahwardiah Siregar, PhD Dr. Sry Suryani Widjaja, Mkes Biochemistry Department Proto Oncogen and Oncogen Oncogen Proteins that possess the ability to cause

More information

What causes cancer? Physical factors (radiation, ionization) Chemical factors (carcinogens) Biological factors (virus, bacteria, parasite)

What causes cancer? Physical factors (radiation, ionization) Chemical factors (carcinogens) Biological factors (virus, bacteria, parasite) Oncogenes What causes cancer? Chemical factors (carcinogens) Physical factors (radiation, ionization) Biological factors (virus, bacteria, parasite) DNA Mutation or damage Oncogenes Tumor suppressor genes

More information

Disorders of Cell Growth & Neoplasia. Lecture 4 Molecular basis of cancer

Disorders of Cell Growth & Neoplasia. Lecture 4 Molecular basis of cancer General Pathology VPM 152 Disorders of Cell Growth & Neoplasia Lecture 4 Molecular basis of cancer Enrique Aburto Apr 2010 Skin tumor in a 10-year-old Rottweiler. Considering the external appearance and

More information

Signaling. Dr. Sujata Persad Katz Group Centre for Pharmacy & Health research

Signaling. Dr. Sujata Persad Katz Group Centre for Pharmacy & Health research Signaling Dr. Sujata Persad 3-020 Katz Group Centre for Pharmacy & Health research E-mail:sujata.persad@ualberta.ca 1 Growth Factor Receptors and Other Signaling Pathways What we will cover today: How

More information

A particular set of insults induces apoptosis (part 1), which, if inhibited, can switch to autophagy. At least in some cellular settings, autophagy se

A particular set of insults induces apoptosis (part 1), which, if inhibited, can switch to autophagy. At least in some cellular settings, autophagy se A particular set of insults induces apoptosis (part 1), which, if inhibited, can switch to autophagy. At least in some cellular settings, autophagy serves as a defence mechanism that prevents or retards

More information

609G: Concepts of Cancer Genetics and Treatments (3 credits)

609G: Concepts of Cancer Genetics and Treatments (3 credits) Master of Chemical and Life Sciences Program College of Computer, Mathematical, and Natural Sciences 609G: Concepts of Cancer Genetics and Treatments (3 credits) Text books: Principles of Cancer Genetics,

More information

Biochemistry of Carcinogenesis. Lecture # 35 Alexander N. Koval

Biochemistry of Carcinogenesis. Lecture # 35 Alexander N. Koval Biochemistry of Carcinogenesis Lecture # 35 Alexander N. Koval What is Cancer? The term "cancer" refers to a group of diseases in which cells grow and spread unrestrained throughout the body. It is difficult

More information

TARGETS OF CYCLIN D1-CDK

TARGETS OF CYCLIN D1-CDK TARGETS OF CYCLIN D1-CDK FIRST TARGET OF THE COMPLEX CYCLIN D-KINASI: prb, IS THE PRODUCT OF THE GENE CONFERRING SUSCEPTIBILITY TO RETINOBLASTOMA - ABSENT OR MUTATED IN SEVERAL HUMAN CANCERS - TRANSCRIPTIONL

More information

SSN SBPM Workshop Exam One. Short Answer Questions & Answers

SSN SBPM Workshop Exam One. Short Answer Questions & Answers SSN SBPM Workshop Exam One Short Answer Questions & Answers 1. Describe the effects of DNA damage on the cell cycle. ANS : DNA damage causes cell cycle arrest at a G2 checkpoint. This arrest allows time

More information

Breast Cancer Statistics

Breast Cancer Statistics 1 in 8 Breast Cancer Statistics Incidence Mortality Prevalence 2 Breast Cancer Incidence Breast Cancer Mortality Breast Cancer Prevalence ~$100,000 Female Breast Anatomy Breasts consist mainly of fatty

More information

BIO360 Fall 2013 Quiz 1

BIO360 Fall 2013 Quiz 1 BIO360 Fall 2013 Quiz 1 Name: Key 1. Examine the diagram below. There are two homologous copies of chromosome one and the allele of YFG carried on the light gray chromosome has undergone a loss-of-function

More information

Neoplasia 18 lecture 6. Dr Heyam Awad MD, FRCPath

Neoplasia 18 lecture 6. Dr Heyam Awad MD, FRCPath Neoplasia 18 lecture 6 Dr Heyam Awad MD, FRCPath ILOS 1. understand the role of TGF beta, contact inhibition and APC in tumorigenesis. 2. implement the above knowledge in understanding histopathology reports.

More information

Chapter 4 Cellular Oncogenes ~ 4.6 -

Chapter 4 Cellular Oncogenes ~ 4.6 - Chapter 4 Cellular Oncogenes - 4.2 ~ 4.6 - Many retroviruses carrying oncogenes have been found in chickens and mice However, attempts undertaken during the 1970s to isolate viruses from most types of

More information

The mutations that drive cancer. Paul Edwards. Department of Pathology and Cancer Research UK Cambridge Institute, University of Cambridge

The mutations that drive cancer. Paul Edwards. Department of Pathology and Cancer Research UK Cambridge Institute, University of Cambridge The mutations that drive cancer Paul Edwards Department of Pathology and Cancer Research UK Cambridge Institute, University of Cambridge Previously on Cancer... hereditary predisposition Normal Cell Slightly

More information

Cancer genetics

Cancer genetics Cancer genetics General information about tumorogenesis. Cancer induced by viruses. The role of somatic mutations in cancer production. Oncogenes and Tumor Suppressor Genes (TSG). Hereditary cancer. 1

More information

Prof. R. V. Skibbens

Prof. R. V. Skibbens Prof. R. V. Skibbens December 2, 2011 BIOS 10: BioScience in the 21 st Century Cell Cycle, Cell Division and Cancer (Part 2) Directionality The Cell Cycle clock goes in only one direction S-phase cells

More information

Regarding techniques of proteomics, there is:

Regarding techniques of proteomics, there is: Molecular الحلقة biology 14 واألخيرة To put you back in the context; the discussion was about Trancriptomics (the study of transcription). The following topic will be PROTEOMICS, which is the study of

More information

Chapter 9, Part 1: Biology of Cancer and Tumor Spread

Chapter 9, Part 1: Biology of Cancer and Tumor Spread PATHOPHYSIOLOGY Name Chapter 9, Part 1: Biology of Cancer and Tumor Spread I. Cancer Characteristics and Terminology Neoplasm new growth, involves the overgrowth of tissue to form a neoplastic mass (tumor).

More information

BIT 120. Copy of Cancer/HIV Lecture

BIT 120. Copy of Cancer/HIV Lecture BIT 120 Copy of Cancer/HIV Lecture Cancer DEFINITION Any abnormal growth of cells that has malignant potential i.e.. Leukemia Uncontrolled mitosis in WBC Genetic disease caused by an accumulation of mutations

More information

Prof. R. V. Skibbens. Cell Cycle, Cell Division and Cancer (Part 2)

Prof. R. V. Skibbens. Cell Cycle, Cell Division and Cancer (Part 2) Prof. R. V. Skibbens November 22, 2010 BIOS 10: BioScience in the 21 st Century Cell Cycle, Cell Division and Cancer (Part 2) Directionality - clocks go in only one direction G1 doesn t have replication-inducing

More information

VIRUSES AND CANCER Michael Lea

VIRUSES AND CANCER Michael Lea VIRUSES AND CANCER 2010 Michael Lea VIRAL ONCOLOGY - LECTURE OUTLINE 1. Historical Review 2. Viruses Associated with Cancer 3. RNA Tumor Viruses 4. DNA Tumor Viruses HISTORICAL REVIEW Historical Review

More information

1. Basic principles 2. 6 hallmark features 3. Abnormal cell proliferation: mechanisms 4. Carcinogens: examples. Major Principles:

1. Basic principles 2. 6 hallmark features 3. Abnormal cell proliferation: mechanisms 4. Carcinogens: examples. Major Principles: Carcinogenesis 1. Basic principles 2. 6 hallmark features 3. Abnormal cell proliferation: mechanisms 4. Carcinogens: examples Carcinogenesis Major Principles: 1. Nonlethal genetic damage is central to

More information

Biochemistry of Cancer and Tumor Markers

Biochemistry of Cancer and Tumor Markers Biochemistry of Cancer and Tumor Markers The term cancer applies to a group of diseases in which cells grow abnormally and form a malignant tumor. It is a long term multistage genetic process. The first

More information

Development of Carcinoma Pathways

Development of Carcinoma Pathways The Construction of Genetic Pathway to Colorectal Cancer Moriah Wright, MD Clinical Fellow in Colorectal Surgery Creighton University School of Medicine Management of Colon and Diseases February 23, 2019

More information

ONCOGENES. Michael Lea

ONCOGENES. Michael Lea ONCOGENES 2011 Michael Lea ONCOGENES - Lecture Outline I. Introduction 2. Identification of oncogenic genes in retroviruses 3. Homologous sequences in transformed and untransformed cells 4. Methods of

More information

Cell Signaling part 2

Cell Signaling part 2 15 Cell Signaling part 2 Functions of Cell Surface Receptors Other cell surface receptors are directly linked to intracellular enzymes. The largest family of these is the receptor protein tyrosine kinases,

More information

Emerging" hallmarks of cancer, a. Reprogramming of energy metabolism b. Evasion of the immune system, Enabling characteristics, a.

Emerging hallmarks of cancer, a. Reprogramming of energy metabolism b. Evasion of the immune system, Enabling characteristics, a. HALLMARKS OF CANCER - Together dictate the malignant phenotype. 1. Self-sufficiency in growth signals 2. Insensitivity to growth inhibitory signals 3. Evasion of cell death 4. Limitless replicative potential

More information

Phospho-AKT Sampler Kit

Phospho-AKT Sampler Kit Phospho-AKT Sampler Kit E 0 5 1 0 0 3 Kits Includes Cat. Quantity Application Reactivity Source Akt (Ab-473) Antibody E021054-1 50μg/50μl IHC, WB Human, Mouse, Rat Rabbit Akt (Phospho-Ser473) Antibody

More information

Eukaryotic Gene Regulation

Eukaryotic Gene Regulation Eukaryotic Gene Regulation Chapter 19: Control of Eukaryotic Genome The BIG Questions How are genes turned on & off in eukaryotes? How do cells with the same genes differentiate to perform completely different,

More information

Osamu Tetsu, MD, PhD Associate Professor Department of Otolaryngology-Head and Neck Surgery School of Medicine, University of California, San

Osamu Tetsu, MD, PhD Associate Professor Department of Otolaryngology-Head and Neck Surgery School of Medicine, University of California, San Osamu Tetsu, MD, PhD Associate Professor Department of Otolaryngology-Head and Neck Surgery School of Medicine, University of California, San Francisco Lung Cancer Classification Pathological Classification

More information

BioSci 145A Lecture 15 - Oncogenes and Cancer

BioSci 145A Lecture 15 - Oncogenes and Cancer BioSci 145A Lecture 15 - Oncogenes and Cancer Topics we will cover today Introduction to normal and cancer cells Characteristics of cells in culture Cancerous changes in cells Viruses can harbor transforming

More information

KEY CONCEPT QUESTIONS IN SIGNAL TRANSDUCTION

KEY CONCEPT QUESTIONS IN SIGNAL TRANSDUCTION Signal Transduction - Part 2 Key Concepts - Receptor tyrosine kinases control cell metabolism and proliferation Growth factor signaling through Ras Mutated cell signaling genes in cancer cells are called

More information

Computer Science, Biology, and Biomedical Informatics (CoSBBI) Outline. Molecular Biology of Cancer AND. Goals/Expectations. David Boone 7/1/2015

Computer Science, Biology, and Biomedical Informatics (CoSBBI) Outline. Molecular Biology of Cancer AND. Goals/Expectations. David Boone 7/1/2015 Goals/Expectations Computer Science, Biology, and Biomedical (CoSBBI) We want to excite you about the world of computer science, biology, and biomedical informatics. Experience what it is like to be a

More information

Crosstalk between Adiponectin and IGF-IR in breast cancer. Prof. Young Jin Suh Department of Surgery The Catholic University of Korea

Crosstalk between Adiponectin and IGF-IR in breast cancer. Prof. Young Jin Suh Department of Surgery The Catholic University of Korea Crosstalk between Adiponectin and IGF-IR in breast cancer Prof. Young Jin Suh Department of Surgery The Catholic University of Korea Obesity Chronic, multifactorial disorder Hypertrophy and hyperplasia

More information

BCHM3972 Human Molecular Cell Biology (Advanced) 2013 Course University of Sydney

BCHM3972 Human Molecular Cell Biology (Advanced) 2013 Course University of Sydney BCHM3972 Human Molecular Cell Biology (Advanced) 2013 Course University of Sydney Page 2: Immune Mechanisms & Molecular Biology of Host Defence (Prof Campbell) Page 45: Infection and Implications for Cell

More information

Understanding and Optimizing Treatment of Triple Negative Breast Cancer

Understanding and Optimizing Treatment of Triple Negative Breast Cancer Understanding and Optimizing Treatment of Triple Negative Breast Cancer Edith Peterson Mitchell, MD, FACP Clinical Professor of Medicine and Medical Oncology Program Leader, Gastrointestinal Oncology Department

More information

Cancer. Questions about cancer. What is cancer? What causes unregulated cell growth? What regulates cell growth? What causes DNA damage?

Cancer. Questions about cancer. What is cancer? What causes unregulated cell growth? What regulates cell growth? What causes DNA damage? Questions about cancer What is cancer? Cancer Gil McVean, Department of Statistics, Oxford What causes unregulated cell growth? What regulates cell growth? What causes DNA damage? What are the steps in

More information

Generating Mouse Models of Pancreatic Cancer

Generating Mouse Models of Pancreatic Cancer Generating Mouse Models of Pancreatic Cancer Aom Isbell http://www2.massgeneral.org/cancerresourceroom/types/gi/index.asp Spring/Summer 1, 2012 Alexandros Tzatsos, MD PhD Bardeesy Lab: Goals and Objectives

More information

Chapter 9. Cells Grow and Reproduce

Chapter 9. Cells Grow and Reproduce Chapter 9 Cells Grow and Reproduce DNA Replication DNA polymerase Addition of a nucleotide to the 3 end of a growing strand Use dntps as substrate Release of pyrophosphate Initiation of Replication Replication

More information

Neoplasia 2018 lecture 4. Dr Heyam Awad MD, FRCPath

Neoplasia 2018 lecture 4. Dr Heyam Awad MD, FRCPath Neoplasia 2018 lecture 4 Dr Heyam Awad MD, FRCPath ILOS To understand the concept of the hallmarks of cancer and that they are phenotypic changes needed in all cancer cells. To list the tumor enablers

More information

CELL BIOLOGY - CLUTCH CH CANCER.

CELL BIOLOGY - CLUTCH CH CANCER. !! www.clutchprep.com CONCEPT: OVERVIEW OF CANCER Cancer is a disease which is primarily caused from misregulated cell division, which form There are two types of tumors - Benign tumors remain confined

More information

Introduction to Genetics

Introduction to Genetics Introduction to Genetics Table of contents Chromosome DNA Protein synthesis Mutation Genetic disorder Relationship between genes and cancer Genetic testing Technical concern 2 All living organisms consist

More information

Early cell death (FGF) B No RunX transcription factor produced Yes No differentiation

Early cell death (FGF) B No RunX transcription factor produced Yes No differentiation Solution Key - Practice Questions Question 1 a) A recent publication has shown that the fat stem cells (FSC) can act as bone stem cells to repair cavities in the skull, when transplanted into immuno-compromised

More information

Enzyme-coupled Receptors. Cell-surface receptors 1. Ion-channel-coupled receptors 2. G-protein-coupled receptors 3. Enzyme-coupled receptors

Enzyme-coupled Receptors. Cell-surface receptors 1. Ion-channel-coupled receptors 2. G-protein-coupled receptors 3. Enzyme-coupled receptors Enzyme-coupled Receptors Cell-surface receptors 1. Ion-channel-coupled receptors 2. G-protein-coupled receptors 3. Enzyme-coupled receptors Cell-surface receptors allow a flow of ions across the plasma

More information

Regulators of Cell Cycle Progression

Regulators of Cell Cycle Progression Regulators of Cell Cycle Progression Studies of Cdk s and cyclins in genetically modified mice reveal a high level of plasticity, allowing different cyclins and Cdk s to compensate for the loss of one

More information

G-Protein Signaling. Introduction to intracellular signaling. Dr. SARRAY Sameh, Ph.D

G-Protein Signaling. Introduction to intracellular signaling. Dr. SARRAY Sameh, Ph.D G-Protein Signaling Introduction to intracellular signaling Dr. SARRAY Sameh, Ph.D Cell signaling Cells communicate via extracellular signaling molecules (Hormones, growth factors and neurotransmitters

More information

Regulation of the Cell Cycle

Regulation of the Cell Cycle Regulation of the Cell Cycle 21 I. OVERVIEW Quiescent differentiated cell / can be induced to re-enter the active cell cycle. urvival Cell division Apoptosis 1 Daughter cells Apoptic cell enescent cell

More information

Activation of cellular proto-oncogenes to oncogenes. How was active Ras identified?

Activation of cellular proto-oncogenes to oncogenes. How was active Ras identified? Dominant Acting Oncogenes Eugene E. Marcantonio, M.D. Ph.D. Oncogenes are altered forms of normal cellular genes called proto-oncogenes that are involved in pathways regulating cell growth, differentiation,

More information

Basic tumor nomenclature

Basic tumor nomenclature Jonas Nilsson jonas.a.nilsson@surgery.gu.se Sahlgrenska Cancer Center Bilder gjorda av Per Holmfeldt och Jonas Nilsson Benign tumor Basic tumor nomenclature Malignant tumor = cancer Metastasis Carcinoma:

More information

BL 424 Test pts name Multiple choice have one choice each and are worth 3 points.

BL 424 Test pts name Multiple choice have one choice each and are worth 3 points. BL 424 Test 3 2010 150 pts name Multiple choice have one choice each and are worth 3 points. 1. The plasma membrane functions as a a. selective barrier to the passage of molecules. b. sensor through which

More information

Problem Set 5 KEY

Problem Set 5 KEY 2006 7.012 Problem Set 5 KEY ** Due before 5 PM on THURSDAY, November 9, 2006. ** Turn answers in to the box outside of 68-120. PLEASE WRITE YOUR ANSWERS ON THIS PRINTOUT. 1. You are studying the development

More information

CHAPTER VII CONCLUDING REMARKS AND FUTURE DIRECTION. Androgen deprivation therapy is the most used treatment of de novo or recurrent

CHAPTER VII CONCLUDING REMARKS AND FUTURE DIRECTION. Androgen deprivation therapy is the most used treatment of de novo or recurrent CHAPTER VII CONCLUDING REMARKS AND FUTURE DIRECTION Stathmin in Prostate Cancer Development and Progression Androgen deprivation therapy is the most used treatment of de novo or recurrent metastatic PCa.

More information

CELL CYCLE REGULATION AND CANCER. Cellular Reproduction II

CELL CYCLE REGULATION AND CANCER. Cellular Reproduction II CELL CYCLE REGULATION AND CANCER Cellular Reproduction II THE CELL CYCLE Interphase G1- gap phase 1- cell grows and develops S- DNA synthesis phase- cell replicates each chromosome G2- gap phase 2- cell

More information

Src-INACTIVE / Src-INACTIVE

Src-INACTIVE / Src-INACTIVE Biology 169 -- Exam 1 February 2003 Answer each question, noting carefully the instructions for each. Repeat- Read the instructions for each question before answering!!! Be as specific as possible in each

More information

Diabetes Mellitus and Breast Cancer

Diabetes Mellitus and Breast Cancer Masur K, Thévenod F, Zänker KS (eds): Diabetes and Cancer. Epidemiological Evidence and Molecular Links. Front Diabetes. Basel, Karger, 2008, vol 19, pp 97 113 Diabetes Mellitus and Breast Cancer Ido Wolf

More information

C) The graph should look exactly like the graph on the left (Mut1 cells + Mating Pheromone for 3 hours at 25 degrees). The cells arrest in G1.

C) The graph should look exactly like the graph on the left (Mut1 cells + Mating Pheromone for 3 hours at 25 degrees). The cells arrest in G1. 706-2000-Exam 4 Answer Key 1) The question asks you to explain peaks A and B in the top graph. The other two graphs were there to give you hints. The question did not ask for these other two graphs to

More information

Cancer Cells. It would take another 20 years and a revolution in the techniques of biological research to answer these questions.

Cancer Cells. It would take another 20 years and a revolution in the techniques of biological research to answer these questions. Cancer Cells Cancer, then, is a disease in which a single normal body cell undergoes a genetic transformation into a cancer cell. This cell and its descendants, proliferating across many years, produce

More information

Breast Cancer Carcinogenesis: Mechanisms and Pathways in Hormone Receptor Positive Disease

Breast Cancer Carcinogenesis: Mechanisms and Pathways in Hormone Receptor Positive Disease Breast Cancer Carcinogenesis: Mechanisms and Pathways in Hormone Receptor Positive Disease 1 Nearly Two-Thirds of Metastatic Breast Cancers Express Hormone Receptors Breast cancer tumors are often classified

More information

Chapter 6: Cancer Pathways. Other Pathways. Cancer Pathways

Chapter 6: Cancer Pathways. Other Pathways. Cancer Pathways Chapter 6: Cancer Pathways Limited number of pathways control proliferation and differentiation Transmit signals from growth factors, hormones, cell-to-cell communications/interactions Pathways turn into

More information

Chapter 18- Oncogenes, tumor suppressors & Cancer

Chapter 18- Oncogenes, tumor suppressors & Cancer Chapter 18- Oncogenes, tumor suppressors & Cancer - Previously we have talked about cancer which is an uncontrolled cell proliferation and we have discussed about the definition of benign, malignant, metastasis

More information