1. Stem cells and cancer: treatment resistance and novel therapeutic targets Rob Clarke Manchester, UK Q-CROC, Montreal, 6 th 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. Tumour recurrence Current modelCancer stem cell model No tumour recurrence CSC CSC inhibitors THERAPY Tumour models

5. 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+/CD24 lo 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 Self-renewal Proliferation Stem cell Sphere colonies grow in vitro from cancer stem-like cells

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

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 Harrison et al, 2010, Cancer Res, 70, 709–18 P2-4 = CD44- or CD24+

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

10. Breast tumour resistance: Opportunities to improve therapy Hormone Receptor Positive (ER &/or PR+ve) 70-80% HER2 +ve (eligible for Herceptin) 10-15% Triple Negative (ER/PR/HER2-ve) 10-15% 5 year DFS*87%75%64% Overall Survival34 mo 60%Response rate EARLY DISEASE ADVANCED DISEASE 33%37% 24 mo31 mo Breast Cancer Subtype TREATMENT Endocrine +/- chemotherapy Herceptin + chemotherapy Chemotherapy

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

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

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

14. Question Are breast cancer stem-like cells responsible for resistance to therapy? i.Radiotherapy ii.Chemotherapy iii.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) CD44+/CD24 -low No. of MS/10,000 cells Initial Week 3Week 12 Initial Week 3Week 12 Li et al, JNCI, 2008 P <0.001

16. Question Are breast cancer stem-like cells responsible for resistance to therapy? i.Radiotherapy ii.Chemotherapy iii.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. Cancer stem cell (CSC) ER - ER+ ER+ breast cancer TAMOXIFEN Cancer stem cell (CSC)

18. Experimental overview Day 1 Estradiol and tumour cell implant Tumour growth Day 90 14 days Treatment Day 104 In vivo assay of stem cell activity in primary breast cancer xenografts after 14 days tamoxifen or vehicle control treatment Ciara O’Brien 1.Mammosphere assay 2.CD44/CD24/ESA cell sorting 3.Limiting dilution secondary transplants HARVEST TUMOUR FOR:

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

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

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

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

23. Notch 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

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

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

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

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

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