Stem cells and cancer: treatment resistance and novel therapeutic targets

Rob ClarkeManchester, UK

Q-CROC, Montreal, 6th 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 recurrence

Current model Cancer stem cell model

No tumour recurrence

CSC

CSC inhibitors

THERAPY

Tumour models

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, 2004Ricci-Vitiani et al., Nature, 2007O’Brien et al., Nature, 2007

Self-renewal

Proliferation

Stem cell

Sphere colonies grow in vitro from cancerstem-like cells

• 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

Hannah Harrison Ciara O’Brien Gillian Farnie

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+

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

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 Survival 34 mo

60%Response rate

EARLY DISEASE

ADVANCED DISEASE

33% 37%

24 mo31 mo

Breast Cancer

Subtype

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

0

20

40

60

80

100

All ce

lls

% M

amm

osp

her

e s

urv

ival

aft

er 6

Gy

Pre-invasivetreatment naive

cancer

Advanced invasive cancer

Gillian Farnie

Mam

mos

pher

e

-form

ing

cells

Mam

mos

pher

e

-form

ing

cells

All ce

lls

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)

CD

44+

/CD

24-

low

No

. of

MS

/10,

000

cells

Initial Week 3 Week 12 Initial Week 3 Week 12

Li et al, JNCI, 2008

P <0.001 P <0.001

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

• 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+

ER+

ER+

ER +

ER +ER + ER+ breast

cancer

TAMOXIFEN

Cancer stem cell (CSC)

Experimental overview

Day 1Estradiol and tumour cell

implant

Tumour growth

Day 90

14 days

TreatmentDay 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 sorting3. Limiting dilution secondary transplants

HARVEST TUMOUR FOR:

p = 0.0049

BB7

*

placebo tamoxifen

BB9

placebo tamoxifen

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

Mam

mos

pher

e Fo

rmati

on (%

)

0

0.5

Mam

mos

pher

e Fo

rmati

on (%

)

Ciara O’Brien

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

What are the treatment options for targeting cancer stem cells?

Current therapy

RelapseLoss of

tumour bulkCSC 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

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

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

Notch

Self-Renewal& Survival

Stem Cell

Stem cell signalling pathway

• First described nearly 100 yearsago 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 Receptor Signalling Pathway

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

RBPJk

CoRNICD

HesHey

MAML

-Secretase

-secretase inhibitorsDAPT or DBZ

Notch or DLL antibodies

DLL/JAGNOTCH

ADAM10

Cell 1Cell 2

Notch activation (NICD) protects normal breast cells from chemotherapy

Stylianou et al, 2006, Cancer Res

(Notch Intra-Cellular Domain)

MCF10A

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

Meurette et al, 2009, Cancer Res

Melphelan-secretase inhibitor

Notch inactivation using -secretase inhibitor (DAPT) reduces mammosphere formation

PE – pleural effusionIDC – 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

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

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 AblettJagdeep SinghAndré VieiraAngelica Gomez-Santiago

CANCER STEM CELL RESEARCHGillian Farnie Pam Willans

MOLECULAR PATHOLOGYHannah Harrison

MEDICAL ONCOLOGYSacha Howell

LIFE SCIENCESKeith Brennan

SURGERYNigel Bundred

UNIVERSITY OF OXFORDAdrian Harris