Stem cells and cancer: treatment resistance and novel therapeutic targets Rob Clarke

1. Stem cells and cancer: treatment resistance and novel therapeutic targets Rob Clarke Manchester, UK Q-CROC, Montreal, 6th November, 2010 Cancer

2. Potential conflict of interests • Paid consultant for Epistem, AstraZeneca, Vertex and Pfizer • PhD studentship part-sponsored by Vertex Pharmaceuticals

3. Outline • Identification of cancer stem cells (CSCs) • CSCs and resistance to current therapies • Potential new therapies for targetting CSCs • Are cancer stem cells ready for the clinic?

4. CSC inhibitors Tumour models Current model Cancer stem cell model CSC THERAPY Tumour recurrence No tumour recurrence

5. Proliferation Self-renewal Stem cell Sphere colonies grow in vitro from cancer stem-like cells • Cancer stem-like cells • (CSCs or tumour-initiating cells) • ‘Gold standard’ is growth of human tumours in immune-deficient mice from cancer stem cell-enriched population isolated using flow cytometry with antibodies against cell surface CD proteins • Human breast cancer: CD44+/CD24lo • Al-Hajj et al., PNAS, 2003 • Human brain & colon cancer: CD133+ • Singh et al., Nature, 2004 • Ricci-Vitiani et al., Nature, 2007 • O’Brien et al., Nature, 2007

6. Primary human breast cells:Mammosphere culture • Analogous to neurospheres that enrich for brain stem cells • Undifferentiated cells survive anoikis (apoptosis), self-renew and form mammospheres (Dontu et al., 2003)

7. Hannah Harrison Ciara O’Brien Gillian Farnie

8. CD44+/CD24-/low cells (P1) are enriched for Mammosphere Forming Units (MFU) P1 = CD44+/CD24-/low P2-4 = CD44- or CD24+ Harrison et al, 2010, Cancer Res, 70, 709–18

9. Stem cell summary Breast cancer stem cell activity can be measured using: • Proportion of CD44+ CD24-/low cells • Mammosphere colonies in vitro • Tumour formation in vivo

10. Hormone Receptor Positive (ER &/or PR+ve) 70-80% HER2 +ve (eligible for Herceptin) 10-15% Triple Negative (ER/PR/HER2-ve) 10-15% Breast Cancer Subtype EARLY DISEASE 5 year DFS* 87% 75% 64% ADVANCED DISEASE Response rate 33% 60% 37% Overall Survival 34 mo 31 mo 24 mo Endocrine +/- chemotherapy Herceptin + chemotherapy TREATMENT Chemotherapy Breast tumour resistance: Opportunities to improve therapy

11. Question Are breast cancer stem-like cells responsible for resistance to therapy? Radiotherapy Chemotherapy Endocrine therapy

12. Question Are breast cancer stem-like cells responsible for resistance to therapy? Radiotherapy Chemotherapy Endocrine therapy

13. Mammosphere-initiating cells preferentially survive 6Gy irradiation Pre-invasive treatment naive cancer Advanced invasive cancer 100 80 % Mammosphere survival after 6Gy 60 40 20 0 All cells All cells Mammosphere -forming cells Mammosphere -forming cells Gillian Farnie

14. Question Are breast cancer stem-like cells responsible for resistance to therapy? Radiotherapy Chemotherapy Endocrine therapy

15. Cancer stem cells are relatively chemo-resistant in human tumours in vivo • Human breast cancer biopsies assayed after neoadjuvant chemotherapy (docetaxel or doxorubicin and cyclophosphamide) No. of MS/10,000 cells CD44+/CD24-low P <0.001 P <0.001 Initial Week 3 Week 12 Initial Week 3 Week 12 Li et al, JNCI, 2008

16. Question Are breast cancer stem-like cells responsible for resistance to therapy? Radiotherapy Chemotherapy Endocrine therapy

17. Breast cancer stem cells (CSC) and endocrine resistance CSCs in ER+ BC may respond indirectly to or function independently of estrogen. ER- breast CSCs represent a novel mechanism of resistance to endocrine therapy. ER - ER - ER+ ER+ ER+ ER + ER + ER + Cancer stem cell (CSC) ER+ breast cancer Cancer stem cell (CSC) TAMOXIFEN

18. Experimental overview HARVEST TUMOUR FOR: • Mammosphere assay • CD44/CD24/ESA cell sorting • Limiting dilution secondary transplants In vivo assay of stem cell activity in primary breast cancer xenografts after 14 days tamoxifen or vehicle control treatment 14 days Treatment Tumour growth Day 1 Day 104 Day 90 Estradiol and tumour cell implant Ciara O’Brien

19. Tamoxifen treatment of ER+ primary breast cancer xenografts enriches for CSC activity BB7 BB9 p = 0.0049 p = 0.015 * * 0.5 1 Mammosphere Formation (%) Mammosphere Formation (%) 0 0 tamoxifen placebo placebo tamoxifen 14 days treatment of tumour xenograft in vivo Ciara O’Brien

20. Current therapy CSC re-grows tumour Loss of tumour bulk Relapse What are the treatment options for targeting cancer stem cells? Fibroblast Cancer Stem Cell Endothelium Collagen Fibres Differentiated Tumour Cell Cancer stem cells are enriched for by radio-, chemo- and endocrine therapies • Potential resistance mechanisms: • Radio and chemo: Efficient DNA damage repair • Chemo: Drug efflux pumps • Endocrine: Lack of ER in stem cells

21. Targeting CSC resistance alongside current therapy Loss of CSC and differentiated cancer cells Tumour shrinkage with current therapy Cure Fibroblast Cancer Stem Cell Endothelium Collagen Fibres Differentiated Tumour Cell 1) Targeting cancer stem cell resistance • Potential stem cell resistance pathways: • DNA damage response enzymes, ie. Chk1/2, DNA-PK, PARP • p53/p63 checkpoint proteins • Drug efflux pumps

22. Targeting CSC self-renewal alongside current therapy Tumour shrinkage with current therapy Differentiation of CSC Cure Fibroblast Cancer Stem Cell Endothelium Collagen Fibres Differentiated Tumour Cell 2) Targeting cancer stem cell self-renewal • Potential stem cell self-renewal pathways: • Notch receptor • Hedgehog • Wnt • CD44

23. Self-Renewal & Survival Stem Cell Stem cell signalling pathway • First described nearly 100 years • ago as a wing mutant in Drosophila • Common integration site for mouse • mammary tumour virus (MMTV) • Viral integration produces a • truncated form of Notch, which leads • to mammary cancer Notch

24. ADAM10 NOTCH DLL/JAG Notch Receptor Signalling Pathway Notch or DLL antibodies NICD Hes Hey CoR MAML RBPJk -Secretase Cell 1 Cell 2 -secretase inhibitors DAPT or DBZ • 5 ligands: Jagged1/2 and Delta-like (DLL) 1/3/4 • 4 receptors: Notch1-4

25. Notch activation (NICD) protects normal breast cells from chemotherapy MCF10A (Notch Intra-Cellular Domain) Stylianou et al, 2006, Cancer Res

26. Melphelan Notch inhibition using the -secretase inhibitor DAPT sensitises breast cancer cells to chemotherapy -secretase inhibitor Meurette et al, 2009, Cancer Res

27. Notch inactivation using -secretase inhibitor (DAPT) reduces mammosphere formation PE – pleural effusion IDC – invasive ductal carcinoma Harrison et al, Cancer Res, 2010

28. Notch 4 activation is highest in the breast CSC-enriched population (P1) P1 = CD44+/CD24lo = breast CSC-enriched Harrison et al, 2010, Cancer Res

29. Notch4 but not Notch1 inhibition prevents tumour initiation in nude mice Notch 1 shRNA Notch 4 shRNA X Harrison et al, Cancer Res, 2010

30. Model of Notch signalling in breast cancer Harrison et al, Cancer Res, 2010

31. Are cancer stem cells ready for the clinic?

32. Is the clinic ready for breast cancer stem cells? Standard clinical trial end-points: • Tumour volume, metastases and survival CSC-related clinical end-points: • Relapse after treatment and minimal residual disease CSC-focused end points: • Tumourigenic activity and presence of CSC surface markers

33. Clinical biomarker endpoints for novel cancer stem cell (CSC) therapies Neoadjuvant trial CSC Function CSC Expression Microarray RT-PCR 2D Colony formation In vitro: Tissue Biopsies 3D Gene profiling Identify CSC population Tumour formation CSC markers In vivo: Blood Samples Serial transplantation IHC FACS Advanced cancer trial

34. Summary • Cancer stem cells may be the root cause of resistance • Potential for targeting stem cell pathways such as Notch • Clinical trial endpoints must include stem cell biomarkers in order to measure efficacy of CSC therapies

35. Thanks to: BREAST BIOLOGY GROUP Kath Spence Ciara O’Brien Matt Ablett Jagdeep Singh André Vieira Angelica Gomez-Santiago CANCER STEM CELL RESEARCH Gillian Farnie Pam Willans MOLECULAR PATHOLOGY Hannah Harrison MEDICAL ONCOLOGY Sacha Howell LIFE SCIENCES Keith Brennan SURGERY Nigel Bundred UNIVERSITY OF OXFORD Adrian Harris