Stem Cells and Novel Targeted Therapeutics in Cancer

Transcription

1 Stem Cells and Novel Targeted Therapeutics in Cancer David M. Jablons, M.D. Professor and Chief Thoracic Surgery Ada Distinguished Professor of Thoracic Oncology Helen Diller Family Comprehensive Cancer Center University of California at San Francisco

2 Definition Understanding Development WHY IS THE CONCEPT OF CANCER STEM CELLS (CSC) IMPORTANT? Self renewal and differentiation pathways of CSCs Mechanisms by which CSCs escape conventional therapies Early detection Prevention Novel targeted therapies overcoming resistance to cytotoxics and recurrence

3 PLURIPOTENT STEM CELL THEORY IS OLD Increasing evidence for pluripotent stem cell theory Oscar Auerbach, MD

4 THE STEM CELL HYPOTHESIS Tumor tissue architecture: Features of normal structures with a cellular hierarchy that regulates the balance between cell renewal and cell differentiation Interactions between cancer and the microenvironment rely on deregulated physiological feedback mechanisms normally responsible for tissue maintenance E.g. INFLAMMATION + paracrine actions of Wnt, Hedgehog and NOTCH Normal stem cells: 1- Self-renewal throughout life after injury etc: Homeostasis 2- Strict regulation of stem cell numbers 3- Capacity to differentiate to clonally repopulate functional cells in an organ There is a variation in the intrinsic ability to do the above Gutova et al. PLoS ONE 2007

5 THE CANCER STEM CELL HYPOTHESIS A subset of cells within the tumor behave like stem cells using stem cell signaling pathways to their advantage This subset of cells (rather than the remainder of the cells in the tumors or the progenitor cells ) initiate tumor formation, progression and metastasis through aberrant self-renewal and differentiation The stem cell niche or microenvironment (ue) is an important component regulating the interaction of stem cells and their neighbouring cells both during homeostasis and during carcinogenesis INFLAMMATION ACTIVE WNT B E A +/- C REPAIR D Reya et al. Nature 2001; Clarke et al. Cell 2006; Beltrán et al. Springer, 2009

6 CONVENTIONAL THERAPIES MAY SHRINK TUMORS BY KILLING MAINLY CELLS WITH LIMITED PROLIFERATIVE POTENTIAL

7 EVIDENCE FOR PULMONARY NICHES IN MICE Regionally distinctive lesions following a proximal-to-distal tracheo-pulmonary distribution SCC in trachea centrally SCLC, adenocarcinomas and brochioloalveolar cancers distally Transgenic mouse models Maintenance of specific cascades + permanent mutations needed to induce & maintain tumors. Stem cell expansion is not enough Finite and distinct pulmonary microenvironment with cells capable of initiating, promoting and maintaining tumorigenesis NEB Niche: Bronchiolar SCLC BADJ Niche: Broncoalveolar cell carcinoma BASCs: Jackson et al. and Kim et al. Cell 2005 Berns et al. Genes Dev 2005; Meuwissen et al. Oncogene 2001; Jackson et al. Genes Dev 2001; Kim et al. Cell 2005

8 STEM CELL NICHES IN THE HUMAN BRONCHOPULMONARY TREE? TRACHEA BRONCHI BRONCHIOLES ALVEOLI NEB BADJ - Certain lung tumors contain specific subpopulations of cells resistant to chemoradiotherapy - Regionally distinctive but mixed lesions following a proximal-to-distal tracheopulmonary distribution (similar to mouse studies) - Increasing evidence that in humans either Clara or AT2 cells > Precursors of adenocarcinoma: A human BADJ niche Beltrán et al. Lung Cancer Stem Cells Stem Cells and Cancer, Springer, NYC, Adapted from Giangrecco et al. Am J Physiol Lung Cell Mol Physiol 2004

9 CONTROL CONTROL A549 CD45 - CD44 + CD133 + CD45 - CD34 + ckit + CD44 Beltrán, A et al ;

10 FLOW SORTING OF NSCLC A549 CD44/CD133 Beltrán, A; Jablons, DM 2008

11 PROSPECTIVE FRESH FACS + PARAFFIN HUMAN TISSUE COLLECTION Surgical Resection Lab within 1 hour of resection Prospective: FACS: Live single cell suspension for sorting Retrospective: Formalin/paraffin, OCT and Protein studies

12 FACS INFORMATION AVAILABLE ON EVERY SINGLE PATIENT Beltrán and Jablons: Current 15 Adenoca. 13 UnTx 2 Tx 23 NSCLC 4 SCC Esophageal 4 Other 26 Mesothelioma

13 CD133 + /CD45 - CELLS ARE DETECTED BY IMMUNOFLUORESCENCE AND BY FLOW CYTOMETRY IN FRESHLY RESECTED NSCLC A B CD133 is expressed in a small percentage of primary NSCLC Xu, Z et al. 2007

14 CD133 + /CD45 - CELLS ARE DETECTED BY FACS IN FRESHLY RESECTED NSCLC IN DIFFERING PERCENTAGES: WHY? CLINICAL SIGNIFICANCE? PATIENT 1071 PATIENT 1074 PATIENT 1075 Blue: Green: Red: Negative control for tumor Normal lung Tumor Beltrán, A et al. 2007

15 FROM NSCLC PRIMARY CULTURE (PATIENT 1099) CONTROL Beltrán, A & Xu, Z 2007

16 FLOW SORTING OF HUMAN 1RY CULTURE CD44/CD133

17 SUCCESSFUL DEVELOPMENT OF LUNG CANCER 3D- ORGANOIDS Mechanisms regulating malignant conversion. Paracrine interactions between cells not feasibly studied in vivo. Debnath et al. Nature 2005.

18 LONG TERM IN VITRO CULTURE OF CANCER STEM CELL SPHERES Surgical Resection: 11/08/2007 resected 10/02/2008 Re-cultured into 3-D Spheres

19 IN VITRO, EX VIVO AND IN VIVO CANCER STEM CELL STUDIES IN NSCLC Culture/Expansion mrna profiles (Wnt + Hh receptors) 3-D organoids from putative stem cells Functional Studies 3-D organoids from putative stem cells Animal Functional Studies Functional Studies Novel targeted agents against CSC (e.g. Wnt + Hh inhibitors)

20 LUNG ADENOCARCINOMA TUMOR MOUSE XENOGRAFT

21 CD133 IHC STAINING OF SINGLE CELLS ISOLATED FROM PRIMARY LUNG TUMOR XENOGRAFTS Control CD133+ Staining

22 CONCLUSION CANCER STEM CELLS AS A FOUNDATION FOR DISCOVERY Library of cancer stem cells - Drug screening - Gene expression profiling - Signaling pathway discovary - Proteomic profiling - Cancer vaccine development Novel cancer stem cell surface antigen-antibody discovery - Cancer stem cell antigens - Inflammatory antigens Cancer Stem Cells Lung cancer xenograft In vivo injection Cells

23 NSC Niche for epithelial stem cells NPC NPC Genetic mutations Epigenetic changes Signaling pathway alterations Redifferentiation to stem-like cell CSC Solid tumor formation Mutated NPC CC Cancer

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25 Human Mesothelioma Contains a High Percentage of Macrophages IHC Representative IHC of mesothelioma showing macrophages stained in brown.

26 % of CD45 + cells Macrophage Infiltration is Significantly Higher in Mesothelioma Compared to Other Thoracic Malignancies Normal NSCLC Lung N=23 * Normal Tumor Esophagus N=10 Normal N=4 MesoT N=26 Pleura Other CD13 + CD14 + CD3 + CD4 - CD8 - CD3 + CD8 + CD3 + CD4 + CD19 + Magnus Johannson, Adam Yagui-Beltrán, Nikita Kolhatkar

27 Tumour Differential Expression of Wnt-1 and Gli-3 Expression Wnt-1 Tumour Merge a b Human mesothelioma co-stained with immunofluorescent Wnt-1 and Gli-3. Gli-3 is expressed in a nuclear punctuate manner in tumour cells (yellow arrow). However stromal cells including inflammatory leukocytes (white arrows), do not express Gli-3 but they stain positively for Wnt-1. Beltrán

28 CO-CULTURE OF 3D-ORGANOIDS WITH INFLAMMATORY CELLS INDUCES AN INVASIVE PHENOTYPE Day 3 Day 5 Day 7 Day 10 Day 3 Day 5 Day 7 Day 10 Non-invasive phenotype Invasive phenotype Proliferation: E-cadherin/pRb/DAPI Johansson, M & Beltrán, A 2007

29 Deregulated Pathways Leading Stem Cells to Cancer

30 Hedgehog and Wnt Signaling Pathways

31 SHH PATHWAY THE LAMB & THE FLOWER Wild corn lily 1960 s: Lynn James and Richard Keeler 1996: Philip Beachy (Stanford University) Blueprint for new cancer drugs

32 Cancer Therapeutics Targeting Components of the Hh Pathway Scales S. & de Sauvage F. Trends in Pharmacological Sciences, 2009

33 N ENGL J MED VOLUME 361(12): SEPTEMBER 17, 2009

34 GDC-0449 ACTIVITY IN PATIENTS WITH LOCALLY ADVANCED BASAL-CELL CARCINOMA

35 PHARMACOKINETIC ANALYSIS AND MOLECULAR CORRELATES OF GDC-0449 ADMINISTRATION

36 Targeting the Gli Transcription Activation Domain by Small Molecules in Human Cancer (Gli: glioma-associated oncogene homology)

37 Hot Show = Hot Target

38 Therapeutic Significance of Gli Inhibition Hh/SMO Gli inhibitors AKT RAS TGFß Gli Tumor development Alternative pathways

39 8xGli-BS Luc activity (%) Compound Screening: Transcription Activity Assay 100% 75% 50% 25% 0% Compounds

40 JABLONS UNPUBLISHED DATA ON SHH & LUNG CANCER First generation library screening of small molecule inhibitors of Hh show promising in vitro and in vivo effects: Functional optimization ongoing DOSE RESPONSE OF SMALL MOLECULE FN1-8 IN LUNG CANCER CELLS H358 A549

41 A490 A490 EFFECT OF SMALL MOLECULE FN1-8 ON PROLIFERATION OF LUNG CANCER CELLS A549 MTS Assay H358 MTS Assay DMSO FN um FN um 1.6 DMSO FN um FN um Days Days

42 DMSO FN1-5 FN1-8 DMSO FN1-5 FN1-8 The Hit Compound Suppress Canonical Wnt signaling in Lung Cancer cells H460 A549 Dvl-3 Cytosolic beta-catenin Survivin Actin

43 Tumor volume (mm3) Hit Compound Suppresses Tumor Growth in vivo DMSO FN Time (days)

44 Toxicity Analysis of the Hit Compound in Mice DMSO Treatment Kidney Lung Spleen Liver Small intestine Colon FN1-8 Treatment (30mg/kg for 2-week)

45 Conclusions Stem cell hypothesis gaining traction Chronic inflammation (COPD,GERD) to stem cell escape of niche autonomy Chronic carcinogen exposure to acquired mutation, then niche autonomy to cancer Stem cell developmental pathways, Wnt, Hedghog, Notch now proven in cancer Novel therapeutics to these pathways are here and more to come..stay tuned!!

46 Acknowledgement Thoracic Oncology Laboratory David Jablons, MD Liang You, PhD Zhidong Xu, PhD Genevieve Clement, PhD Adam Beltran, MD Johannes Kratz, MD Dawn Bravo, PhD Tomomi Hirata, MD, PhD Fengfeng Xu, PhD Roshni Ray, BS Eric Hung, MD Zhao Chen, BS Iwao Mikami, MD Junichi Okamoto, MD, PhD Eric Lin, MD Yihui Shi, PhD Collaborators Magnus Johansson, PhD Lisa Coussens, PhD (Pathology Dept. UCSF) Naoaki Fujii, PhD (St. Jude Children s Hospital)