Stem cells and cancer: treatment resistance and novel therapeutic targets Rob Clarke Manchester, UK Q-CROC, Montreal, 6 th November, 2010 Cancer
Dec 16, 2015
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
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
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