Breast Cancer Stem cells : Stemming the tide of resistance - Emmanuelle CHARAFE-JAUFFRET
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Breast Cancer Stem cells :
Stemming the tide of resistance
Emmanuelle CHARAFE-JAUFFRET
Epithelial Stem Cell and Cancer team
Centre de Recherche en Cancérologie de Marseille
Tumor cell heterogeneity: a complex ecosystem
Conventional Therapies
Genetic diversity Functional diversity CSC resistance
Clone 1 Tumor relapse
Clone 2 Metastatic progression
Clone 3
Adapted from Kreso & Dick et al., Cell Stem Cell, 2014
Tumor cell heterogeneity: a complex CSC system
Identify sources
of heterogeneity
within cancer
Understanding CSC’s How essential is the
biology to cure niche in regulating
cancer CSC metastatic
spreading?
Conventional
therapies
Anti-CSC
therapies
Targeting CSC strategy
CSC Screening Cancer stem cells Characterization
CSC Profiling CSC Cell lines
CSC Tumorspheres Markers and targets
Existing
PDXs
therapies
Primary tumors
Descriptive
genomic
Drug library
CSC drug (FDA/Experimental)
screening Drugs
Targeted drugs/Chemo
Ab
Xenograft assay
shRNA
Clinical trials Preclinical step
Deciphering CSC biology to discover anti-CSC therapies
Repertaxin
TGFb epigenetic CXCR1/IL8 axis
Charafe-Jauffret et al., Cancer Research, 2009
network
Ginestier et al., JCI, 2010
El Helou et al., Stem cells, 2014
Fas-Ligand IL-8 IL-8
Fas IL-8 IL-8
TGFbR
FADD CXCR1 FRIDA Clinical Trial ,
TGFb
TGFb Phase II
FAK
TGFb PI3-K
SCD1
TGFb
SMADs PTEN
SMADs
PORCN
AKT Mir-600 molecular
FOXO3a
β-catenin
GSK3β switcher of bCSC
β-catenin
El Helou et al., Cell rep, 2017
TCF
Acetyl-CoA
HMG-CoA GGTI
citrate
Mevalonate
Farnesylation Geranylation
Cholesterol
Mevalonate Metabolism
Ginestier, (…), Charafe-jauffret, Stem cells,
2012
Cancer Stem Cell
ALDEFLUOR+ population and tumorigenicity
ALDEFLUOR- ALDEFLUOR+
ALDEFLUOR assay
R1 R2
ALDEFLUOR- ALDEFLUOR+ ALDEFLUOR – ALDEFLUOR +
50,000 cells 50,000 cells 50,000 cells 500 cells
5000 cells 5000 cells
500 cells 500 cells
Ginestier et al. Cell stem cell, 2007
ALDEFLUOR+ population and tumorigenicity
ALDEFLUOR - ALDEFLUOR +
ALDEFLUOR+ population regenerates
heterogeneity of the initial tumor
Ginestier et al. Cell stem cell, 2007
Targeting CSC strategy
CSC Screening Cancer stem cells Characterization
CSC Profiling CSC Cell lines
CSC Tumorspheres Markers and targets
Existing
PDXs
therapies
Primary tumors
Descriptive
genomic
Drug library
CSC drug (FDA/Experimental)
screening Drugs
Targeted drugs/Chemo
Ab
Xenograft assay
shRNA
Clinical trials Preclinical step
Patient Tumors‐derived xenografts(PDXs)
Primary Tumor
«Clearing»
Dissociation
Tumoral growth monitoring
+ Matrigel
-oestradiol
Time from birth
(weeks)
3 5 7 12
Human fibroblasts
Humanization
Patient Tumors‐derived xenografts (PDXs)
50 PDXs obtained
Proportion of tumor outgrowth
Normal-like
Basal
p=0.006e-2
ERBB2-like
Luminal B 1. Conservation of histology
and phenotype
Luminal A
Days after PT implantation
Molecular subtypes
Charafe-Jauffret et al, Cancer Research 2013CRCM 181 PT
HES ER PR ERBB2
CRCM 181X
HES ER PR ERBB2CRCM 224 PT
HES ER PR ERBB2
CRCM 224X
HES ER PR ERBB2Patient Tumors‐derived xenografts (PDXs)
200 Tm injected 50 PDXs obtained
Proportion of tumor outgrowth
Normal-like
Basal
p=0.006e-2
ERBB2-like 1. Conservation of histology
Luminal B and phenotype
Luminal A
Days after PT implantation
Molecular subtypes 2. Conservation of molecular
phenotype
3. Maintain genomic profile
4. Maintain a hierarchical
organization
Charafe-Jauffret et al, Cancer Research 201325%
engrafment rate
High grade Expression of CSC marker
PT non-engrafter
Metastasis-free survival
Variable Hazard ratio (95% CI) p-value
ER 0.78 [0.27-2.31] 0,66
PR 0.67 [0.23-1.92] 0,45
SBR Grade 2.94 [1.02-8.44] 4,60E-02 PT engrafter
KI67 1.32 [0.46-3.82] 0,61
ALDH1 3.61 [1.12-11.65] 3,20E-02
p=0.00352
Charafe-Jauffret et al., Cancer Research 2013Breast CSC transcriptional programs
8 PDXs
Tumor dissociation
Gene expression
profiling
dNTP metabolism HR machinery
Charafe-jauffret et al., Cancer Res, 2013in vivo in vitro
Cell tracker
ALDH1/Ki67/DNA
Cell tracking
R1 R2
8 6 4 2 0 Days
Cell tracking
8 6 4 2 0 Days
non-CSC
Cell proliferation
CSC
Tracking time (days)bCSC transcriptional programs as a response to replicative stress?
Macharet & Halazonetis, 2015
dNTPs pool DSB
Genomic
instability
HR
Fork‐restart pathway
DNA replication Stalled fork
initiation
Completed
Fork restart replication
HR
Replication fork « escort »ɣH2AX
ALDHlow
ɣH2AX positive cell (%) count
G1
S
DNA
G2
ɣH2AX
ALDHbr
count
ɣH2AX positive cell (%)
G1
S
DNA
Breast CSC present a limited replication stress
G2in vivo
ALDH1/γH2AX/DNABreast CSC present a limited replication stress
IDU
ALDHlow
20 min IdU
Replication cluster
ALDHbr
*Breast CSC present a limited replication stress
DNA combing
*
O
ALDHlow
Fork velocity w)
ALDHbr
SUM159RAD51 expression in bCSC
RAD51 positive cell (%)
RAD51 positive cell (%)
G1 S G2/M G1 S G2/M
DNA/RAD51
DNA/RAD51
ALDHlow ALDHbr
Restart of halted fork?
Protection of nascent forks by the formation of a stable Rad51 filament?RAD51 inhibition induces replication stress in bCSC
RAD51 inhibitor
(Huang & Mazin, 2014) DNA/RAD51 ALDH1/γH2AX/DNA
CTRL BO2 CTRL BO2
***RAD51 inhibition induces replication stress in bCSC
***bCSC Targeting
Tumor regrowth
Tumor debulking
B02 Conventional therapy
Cisplatin
Tumor
eradicationBO2 sensitize bCSC to cisplatin
*** < 0,001
Proportion of ALDHbr cells
(normalized withcontrol)
CTRL CisplatinBO2 sensitize bCSC to cisplatin
*** < 0,001
Proportion of ALDHbr cells
(normalized withcontrol)
CTRL Cisplatin BO2 Cisplatin
+
BO2Xenograft assay
D1 D33 D35 D37 D39 D41 D43 D45 D47
BO2
Cisplatin
300,0
225,0
Volume (mm3)
BO2
150,0
Cisplatine
Combi
CTRL
75,0
0,0
33 36 40 44 48
Days post-graftTake home message
CSC: limited Replicative Stress compare to mature cells
Are bCSC less
tolerant to RS ?
RS is limited by HR activation
HR inibition increase RS in bCSC
In vivo validation
Inhibition of HR sensitize bCSC to chemotherapies ? in PDX is ongoingTargeting CSC strategy
CSC Screening Cancer stem cells Characterization
CSC Profiling CSC Cell lines
CSC Tumorspheres Markers and targets
Existing
PDXs
therapies
Primary tumors
Descriptive
genomic
Drug library
CSC drug (FDA/Experimental)
screening Drugs
Targeted drugs/Chemo
Ab
Xenograft assay
shRNA
Clinical trials Preclinical stepPersonalized medicine: genome‐based approaches
Rubio-Perez et al., Cancer Cell, 201544‐Drugs Panel
To improve Personalized medicine
Dual Ex vivo
drug screening
Bulk drug
screening
PDX
Short Term Culture
(STC) assay IC50
CSC drug screening
Chemogram/ Mininiaturized Aldefluor
assay
Stemogram
Bulk drug screening
CSC drug screeningPDX genomic background
MYC
TP53
PDGFRA
ERBB2
PIK3CA Amplification
PTEN Deletion
FGFR1/ZNF703 mutation 18.7
JAK2
ROR2
CCNE1
HTERT
ch1q21
MYH9
STK11
PIK3R1
RB1
AKT1
CCND1
CDKN1B
NOTCH2
MYH2
DMD
ACVR1B
4.9
ANK3
POLD2
IRF4
ICAM1
PSMA6PI3K/AKT pathway
PDXs ID
Doxorubicin Salinomycin
5-FU
chemotherapies Cisplatin
Docetaxel Temsirolimus (MTOR) PRIMA1 (p53mut)
Gemcitabine
Eribulin
Crizotinib
LY2157299
Simvastatin
Lovastatin
Mevalonate inhibitors Pravastatin
GGTI-298
FTI-277
Tamoxifen
ATRA
vemurafenib
RAF inhibitors Sorafenib
Dabrafenib
Trastuzumab-DM1
Gefitinib
EGFR-family inhibitors Lapatinib
Cetuximab
Trastuzumab
BKM120 sensitivity resistance
MK-2206
PI3K/AKT/mTOR inhibitors Everolimus
Temsirolimus
SAHA ERBB2
S78454
Epidrugs Decitabine
Vidaza
Salinomycin
Gefitinib (EGFR)
Squalamine
Crenolanib
RTK (class III) Inhibitors Imatinib
Sunitinib
Masitinib
RO-3306 cx-4945 (CK2a)
BI2536 MK-2206 (AKT)
cx-4945 Doxorubicin (DNA)
smi4a
GSK1120212
MK-0752
TMI1
vismodegib
Olaparib
PRIMA-1
Nutlin-3
SRC Inhibitors Dasatinib
Trodusquemine
sensitivity resistance
R SPDXs ID PDXs ID
Doxorubicin
5-FU
chemotherapies Cisplatin
Docetaxel
Gemcitabine
Eribulin
Crizotinib
LY2157299
Simvastatin
Lovastatin
Mevalonate inhibitors Pravastatin
GGTI-298
FTI-277
Tamoxifen
ATRA
vemurafenib
RAF inhibitors Sorafenib
Dabrafenib
Trastuzumab-DM1
Gefitinib
EGFR-family inhibitors Lapatinib
Cetuximab
Trastuzumab
BKM120
MK-2206
PI3K/AKT/mTOR inhibitors Everolimus
Temsirolimus
SAHA
S78454
Epidrugs Decitabine
No correlation between CSC score and genomic alterations
Vidaza
Salinomycin
Squalamine
Crenolanib
RTK (class III) Inhibitors Imatinib
Sunitinib
Masitinib
RO-3306
BI2536
cx-4945
smi4a
GSK1120212
MK-0752
TMI1
vismodegib
Olaparib
PRIMA-1
Nutlin-3
SRC Inhibitors Dasatinib
Trodusquemine
R S CSC
= CSCCSC and non-CSC present identical genomic background
CNAs Mutations
PANDORA6 (H2KD-)
PANDORA21
PANDORA18
PANDORA25
PANDORA11
T404 ALDH+
T404 ALDH-
PANDORA1
PANDORA7
PANDORA6
PANDORA3
T404
T330
T389
T433
T436
T272
T348
T168
T494
T434
T483
T392
T226
T442
PANDORA1
T404
T404 ALDH+
T404 T404 ALDH-
T330
T389
PANDORA21
T433
T404 ALDH- PANDORA18
PANDORA7
T436
PANDORA6
PANDORA6 (H2KD-)
PANDORA3
T404 ALDH+ T272
T348
T168
T494
PANDORA25
T434
Merge PANDORA11
T483
T392
T226
T442pa Pa PA pa Pa PA
T1 nd T4 T4 T4 nd T2 T3 T3 ND T3 T4 T1 nd T4 T4 T49 nd X2 T3 T34 ND T38 T43
68 ora 36 04 94 o2 26 92 48 OR 89 34 68 ora 36 04 4 o2 26 92 8 OR 9 4
1 1 A6 1 1 A6
Doxorubicin
5‐FU
Cisplatin
Docetaxel
Gemcitabine
Eribulin
Crizotinib
LY2157299
Simvastatin
Lovastatin
Pravastatin
GGTI‐298
FTI‐277
Tamoxifen
ATRA
vemurafenib
Sorafenib
Dabrafenib
Trastuzumab‐DM1
Gefitinib
Lapatinib
Cetuximab
Trastuzumab
BKM120
MK‐2206
Everolimus
Temsirolimus
SAHA
S78454
Decitabine
Vidaza
Salinomycin
Squalamine
Crenolanib
Imatinib
Sunitinib
Masitinib
RO‐3306
BI2536
cx‐4945
smi4a
GSK1120212
MK‐0752
TMI1
vismodegib
Olaparib
PRIMA‐1
R S Nutlin‐3
Dasatinib
CSC
= CSC
TrodusqueminePDXs ID PDXs ID
Doxorubicin
5-FU
chemotherapies Cisplatin
Docetaxel
Gemcitabine
Eribulin
Crizotinib
LY2157299
Simvastatin
Lovastatin
Mevalonate inhibitors Pravastatin
GGTI-298
FTI-277
Tamoxifen
ATRA
vemurafenib
RAF inhibitors Sorafenib
Dabrafenib
Trastuzumab-DM1
Gefitinib
EGFR-family inhibitors Lapatinib
Cetuximab
Trastuzumab
BKM120
MK-2206
PI3K/AKT/mTOR inhibitors Everolimus
Temsirolimus
SAHA
S78454
Epidrugs Decitabine
Vidaza
Salinomycin
Squalamine
Crenolanib
RTK (class III) Inhibitors Imatinib
Sunitinib
Masitinib
RO-3306
BI2536
cx-4945
smi4a
GSK1120212
MK-0752
TMI1
vismodegib
Olaparib
PRIMA-1
Nutlin-3
SRC Inhibitors Dasatinib
Trodusquemine
R S CSC
= CSCPA pa PA
pan Pa Pa
T16 T43 T40 T49 T22 T39 T34 ND T38 T43 T16 nd T43 T40 T49 X22 T3 T34 ND T38 T43
dor ndo nd
8 6 4 4 6 2 8 OR 9 4 8 ora 6 4 4 6 92 8 OR 9 4
a1 21 o21
A6 1 A6
Doxorubicin
5‐FU
Cisplatin
Docetaxel
Gemcitabine
Eribulin
Crizotinib
LY2157299
T404 (ALDH+)
T434 (ALDH+)
T404 (ALDH-)
T434 (ALDH-)
Simvastatin
Lovastatin
Pravastatin
GGTI‐298
FTI‐277
Tamoxifen
ATRA
vemurafenib
Sorafenib
Dabrafenib
Trastuzumab‐DM1
- P-src (Y416)
Gefitinib
Lapatinib - GAPDH
Cetuximab
Trastuzumab
BKM120
MK‐2206
Everolimus
Temsirolimus
SAHA
S78454
Decitabine
Vidaza
Salinomycin
Squalamine
Crenolanib
Imatinib
Sunitinib
Masitinib
RO‐3306
BI2536
cx‐4945
smi4a
GSK1120212
MK‐0752
TMI1
vismodegib
Olaparib
R S PRIMA‐1
Nutlin‐3
CSC
= CSC
Dasatinib
Trodusquemine Src inhibitorIn vivo validation
Reimplantation
Dasatinib
Est. number of CSC/10,000 cells
Tumor volume (mm3)
Control
Docetaxel
Days after implantationClinical validation
PERMED1045
PERMED1045
Post‐dasatinib 1
(skin mets)
Dasatinib, 1 month
pre TRT Biopsy
9.4%
ALDH
CD44
79.9%
CD24
implantation
in serial dilutionClinical validation
PERMED1045
PERMED1045
Post‐dasatinib 1
(skin mets)
Dasatinib, 1 month
pre TRT Biopsy Post TRT Biopsy 1
1.8%
9.4%
ALDH ALDH
50,1%
CD44
CD44
79.9%
CD24 CD24
implantation implantation
in serial dilution in serial dilutionClinical validation
PERMED1045
PERMED1045
Post‐dasatinib 1
(skin mets)
Dasatinib, 1 month
1000,
pre TRT (Biopsy 1)
Tumor volume (mm3)
750,
pre TRT Biopsy Post TRT Biopsy 1 500,
post TRT (Biopsy 2)
1.8% 250,
9.4% 0,
ALDH 0 50 100 150 200 250
ALDH
50,1%
Days after reimplantation
CD44
CD44
79.9%
CD24 CD24
implantation implantation
in serial dilution in serial dilutionCSCs assays for personalized therapy: « predictive Medicine »
PDX
CSC personalized
clinical trials
Chemogram
Bulk drug screening
CSC drug screening
From « personalized medicine » to « predictive medicine »Conclusion
Tam et al. Cancer Cell, 2013
• CSCs drug sensitivity follow their own rule
• CSC drug sensitivity profile is INDEPENDANT of CSC genomic background
• CSCs drug sensitivity profile is DEPENDANT of CSC intrinsic signaling pathwaysMolecular Oncology
Daniel Birnbaum
Epithelial stem cell and cancer Lab Francois BERTUCCI
Max CHAFFANET
Pascal FINETTI
Emmanuelle CHARAFE-JAUFFRET Arnaud GUILLE
Christophe GINESTIER Jose ADELAIDE
Geraldine GUASCH-GRANGEON Nadine CARBUCCIA
Véronique CHEVRIER University of Michigan,
Severine GARNIER
Julien WICINSKI Ann Arbor, MI, USA
Marc LOPEZ
Violaine FORISSIER Olivier CABAUD
Violette AZZONI Max WICHA
Emilie MAMESSIER
Abir ARFAOUI Suling LIU
Mary-loup PICOD
Medical Oncology Hasan KORKAYA
Lhoucine MITOYAN Anthony GONCALVES
Patrice VIENS
Former members Oncology Development
Marion SALVADOR J-Fred SAUNIERE
Rita EL HELOU Cancer Research Center of
Simon LAUNAY CiBi platform Lyon,
Ghislain BIDAUT Lyon, France
Claire RIOUALEN
Quentin DA COSTA
Alain PUISIEUX
Anne-Pierre MOREL
TrGET platform Arnaud VIGNERON
Yves COLLETTE
Remy CASTELLANO
Yves TOIRON
Flow Cytometry platform PARi platform,
CEA, Saclay, France
Manon RICHAUD
Françoise MALLET
Annick HAREL-BELLAN
Guillaume PINNA
ICEP
Experimental Pathology platform iRTSV, Grenoble, France
Anne FARINA
Emilie COUQUIAUD Laurent GuyonAcknowledgements CiBi platform Ghislain BIDAUT Claire RIOUALEN Quentin DA COSTA TrGET platform Yves COLLETTE Remy CASTELLANO Yves TOIRON Flow Cytometry platform Marie-Laure THIBULT Françoise MALLET ICEP Experimental Pathology platform Anne FARINA Emilie COUQUIAUD
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