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

Potential conflict of interests

• Paid consultant for Epistem, AstraZeneca, Vertex and Pfizer • PhD studentship part-sponsored by Vertex Pharmaceuticals

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?

Tumour models

Current model Cancer stem cell model CSC

THERAPY

Tumour recurrence CSC inhibitors No tumour recurrence

Self-renewal

Cancer stem-like cells

(CSCs or tumour-initiating cells)

Stem cell Proliferation

‘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

Sphere colonies grow in vitro from cancer stem-like cells

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)

Hannah Harrison Ciara O’Brien Gillian Farnie

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

Stem cell summary

Breast cancer stem cell activity can be measured using:

i.

Proportion of CD44

+

CD24

-/low

ii. Mammosphere colonies

in vitro

cells iii. Tumour formation

in vivo

Breast tumour resistance: Opportunities to improve therapy

Breast Cancer Subtype Hormone Receptor Positive

(ER &/or PR+ve)

70-80% HER2 +ve

(eligible for Herceptin)

10-15%

Triple Negative

(ER/PR/HER2-ve)

10-15%

EARLY DISEASE 5 year DFS* 87% 75% 64% ADVANCED DISEASE Response rate Overall Survival 33% 34 mo 60% 31 mo 37% 24 mo TREATMENT Endocrine +/ chemotherapy Herceptin + chemotherapy Chemotherapy

Question

Are breast cancer stem-like cells responsible for resistance to therapy?

i.

Radiotherapy ii. Chemotherapy iii. Endocrine therapy

Question

Are breast cancer stem-like cells responsible for resistance to therapy?

i.

Radiotherapy ii. Chemotherapy iii. Endocrine therapy

Mammosphere-initiating cells preferentially survive 6Gy irradiation 100 80 60 40 20 0 Pre-invasive treatment naive cancer Advanced invasive cancer Gillian Farnie

Question

Are breast cancer stem-like cells responsible for resistance to therapy?

i.

Radiotherapy ii. Chemotherapy iii. Endocrine therapy

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) P <0.001

P <0.001

Initial Week 3 Week 12 Initial Week 3 Week 12 Li et al, JNCI, 2008

Question

Are breast cancer stem-like cells responsible for resistance to therapy?

i.

Radiotherapy ii. Chemotherapy iii. Endocrine therapy

Breast cancer stem cells (CSC) and endocrine resistance ER Cancer stem cell (CSC) Cancer stem cell (CSC) ER+ ER + ER + ER ER+ ER+ breast cancer • 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.

TAMOXIFEN

Experimental overview In vivo assay of stem cell activity in primary breast cancer xenografts after 14 days tamoxifen or vehicle control treatment

Day 1 Estradiol and tumour cell implant

Tumour growth 14 days Treatment

Day 90 Day 104 1.

2.

3.

HARVEST TUMOUR FOR: Mammosphere assay CD44/CD24/ESA cell sorting Limiting dilution secondary transplants Ciara O’Brien

1

Tamoxifen treatment of ER+ primary breast cancer xenografts enriches for CSC activity

BB7 p = 0.0049

*

0.5

BB9 p = 0.015

*

0 0 placebo tamoxifen placebo tamoxifen 14 days treatment of tumour xenograft in vivo Ciara O’Brien

Cancer stem cells are enriched for by radio-, chemo- and endocrine therapies

Current therapy

CSC re-grows Loss of tumour Relapse tumour bulk What are the treatment options for targeting cancer stem cells?

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

1) Targeting cancer stem cell resistance

Targeting CSC resistance alongside current therapy

Loss of CSC and differentiated cancer cells Tumour shrinkage with current therapy Cure 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

2) Targeting cancer stem cell self-renewal

Targeting CSC self-renewal alongside current therapy

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

Stem cell signalling pathway

Notch Self-Renewal & Survival

• 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

Stem Cell

Notch Receptor Signalling Pathway Notch or DLL antibodies ADAM10 NICD NOTCH DLL/JAG



-Secretase Cell 1 Cell 2



-secretase inhibitors DAPT or DBZ RBPJk

• •

5 ligands: Jagged1/2 and Delta-like (DLL) 1/3/4 4 receptors: Notch1-4 Hes Hey

Notch activation (NICD) protects normal breast cells from chemotherapy MCF10A

(Notch Intra-Cellular Domain) Stylianou et al, 2006, Cancer Res

Notch inhibition using the  -secretase inhibitor DAPT sensitises breast cancer cells to chemotherapy

Melphelan



-secretase inhibitor Meurette et al, 2009, Cancer Res

Notch inactivation using  -secretase inhibitor (DAPT) reduces mammosphere formation PE – pleural effusion IDC – invasive ductal carcinoma

Harrison et al, Cancer Res, 2010

Notch 4 activation is highest in the breast CSC-enriched population (P1) P1 = CD44+/CD24lo = breast CSC-enriched

Harrison et al, 2010, Cancer Res

Notch4 but not Notch1 inhibition prevents tumour initiation in nude mice

Notch 1 shRNA Notch 4 shRNA

X

Harrison et al, Cancer Res, 2010

Model of Notch signalling in breast cancer

Harrison et al, Cancer Res, 2010

Are cancer stem cells ready for the clinic?

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

Neoadjuvant trial

Clinical biomarker endpoints for novel cancer stem cell (CSC) therapies CSC Expression CSC Function Tissue Biopsies Blood Samples

Microarray RT-PCR Gene profiling Identify CSC population CSC markers IHC FACS In vitro: Colony formation 2D 3D In vivo: Tumour formation Serial transplantation Advanced cancer trial

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

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