Stem cells and cancer:
treatment resistance and
novel therapeutic targets
Rob Clarke
Manchester, 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 models
Current model
Cancer stem cell model
CSC
THERAPY
CSC inhibitors
Tumour recurrence
No tumour recurrence
Proliferation
Self-renewal
Cancer stem-like cells
Stem cell
(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
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 cells
ii. Mammosphere colonies in vitro
iii. Tumour formation in vivo
Breast tumour resistance:
Opportunities to improve therapy
Breast Cancer
Subtype
Hormone
HER2 +ve
Receptor Positive
(eligible for Herceptin)
(ER &/or PR+ve)
10-15%
70-80%
Triple Negative
(ER/PR/HER2-ve)
10-15%
EARLY DISEASE
5 year DFS*
87%
75%
64%
Response rate
33%
60%
37%
Overall Survival
34 mo
31 mo
24 mo
TREATMENT
Endocrine
+/chemotherapy
Herceptin +
chemotherapy
Chemotherapy
ADVANCED DISEASE
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
% Mammosphere
survival after 6Gy
preferentially survive 6Gy irradiation
Pre-invasive
treatment naive
cancer
Advanced
invasive
cancer
100
80
60
40
20
0
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 chemoresistant in human tumours in vivo
No. of MS/10,000 cells
CD44+/CD24-low
• Human breast cancer biopsies assayed after neoadjuvant chemotherapy
(docetaxel or doxorubicin and cyclophosphamide)
P <0.001
Initial Week 3
Week 12
P <0.001
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)
• CSCs in ER+ BC may
respond indirectly to or
function independently
of estrogen.
ER+ ER +
Cancer stem ER +ER ER+
cell (CSC)
ER+ER +
TAMOXIFEN
ER+ breast
cancer
• ER- breast CSCs
represent a novel
mechanism of
resistance to endocrine
therapy.
Experimental overview
In vivo assay of stem cell activity in primary breast cancer
xenografts after 14 days tamoxifen or vehicle control treatment
14 days
Tumour growth
Day 1
Estradiol and
tumour cell
implant
Treatment
Day 104
Day 90
HARVEST TUMOUR FOR:
1.
2.
3.
Mammosphere assay
CD44/CD24/ESA cell sorting
Limiting dilution secondary transplants
Ciara O’Brien
Tamoxifen treatment of ER+ primary breast
cancer xenografts enriches for CSC activity
BB9
p = 0.0049
*
1
0
placebo
tamoxifen
Mammosphere Formation (%)
Mammosphere Formation (%)
BB7
p = 0.015
*
0.5
0
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
tumour
Loss of
tumour bulk
Relapse
What are the treatment options for targeting cancer stem cells?
Cancer Stem Cell
Differentiated Tumour Cell
Endothelium
Fibroblast
Potential resistance mechanisms:
• Radio and chemo: Efficient DNA damage repair
• Chemo: Drug efflux pumps
• Endocrine: Lack of ER in stem cells
Collagen Fibres
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
Tumour shrinkage with
current therapy
Differentiation
of CSC
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
• First described nearly 100 years
ago as a wing mutant in Drosophila
Notch
Self-Renewal
& Survival
Stem Cell
• 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
Notch or DLL
antibodies
ADAM10
NOTCH
DLL/JAG
NICD
CoR
MAML
-Secretase
RBPJk
Cell 1
Cell 2
-secretase inhibitors
DAPT or DBZ
• 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
Clinical biomarker endpoints for novel
cancer stem cell (CSC) therapies
Neoadjuvant trial
CSC Function
CSC Expression
Microarray
RT-PCR
In vitro:
Tissue Biopsies
Gene profiling
Colony
formation
2D
3D
Identify CSC
population
CSC markers
Blood Samples
IHC
Advanced cancer trial
FACS
Tumour
formation
In vivo:
Serial
transplantation
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