Targeting mammary stem cells for breast cancer prevention
Max Wicha M.D. Cancer Stem Cell Hypothesis
Cancers Arise from Cells with Dysregulated Self-Renewal
Cancers Are “Driven” By Cells With Stem Cell Properties Stem cells as targets for cancer prevention HYPOTHESIES If cancers arise in self-renewing cells then: • Stem/Progenitor cell number may be a cancer risk factor • Targeting tissue stem/progenitor cell self- renewal pathways may reduce cancer risk • Tissue stem cell and self-renewal pathway components may be useful biomarkers for cancer prevention studies
characteristics of stem cells
• self renewal • differentiation into multiple cell types
• types of stem cells embryonic adult • cancer
Alveolar cells Quiescent pool Luminal cells ofcell stem
carcinogenesis and breast cells Stem
DIFFERENTIATION Myoepithelial cells Late Progenitor Early Progenitor Stem cell
Cancercell stem
Cancercell stem
Reduction mammoplasties
“Mammosphere” Suspension culture (Serum-free, EGF, …) Culture Systems
Mammospheres
Dissociated to single cells
Acinar Ductal- acinar Regeneration of the mammary N generation of spheres Mixed Myoepithelial Epithelial structure structure gland in the cleared fat- pad
B. Differentiation in C. Differentiation in A. Self- renewal D. Differentiation and in vitro vitro in 2- D culture vitro in 3- D culture self- renewal in vivo (Suspension) (Collagen) (Matrigel ) (NOD/SCID mice) Dontu & Wicha, 2003 Dontu & Wicha, 2003 Weaver & Bissell, 1999 Kuperwasser & Weinberg, 2004 Dontu & Wicha, 2003 Liu & Wicha, 2006 ALDEFLUOR + cells in Normal breast epithelium
BAAA
ALDH
DEAB
With DEAB Without DEAB
6% of ALDEFLUOR positive cells in Normal breast epithelium
6% Ginestier et al Cell Stem Cell 2007 ALDEFLUOR+ cells have stem cell properties
Culture in suspension
With DEAB Without DEAB
Only ALDEFLUOR + cells
generate mammospheres
in suspension ALDEFLUOR+ cells have stem cell properties
Without DEAB
CK18 SMA ALDEFLUOR+ Luminal cells CK18+ Myoepithelial cells SMA+
ALDEFLUOR-
ALDEFLUOR- ALDEFLUOR+
Ginestier, 2007 Models of Tumor Heterogeneity
Stochastic model Cancer stem cell model
CSC
CSC
CSC
CSC
Cancer cells are heterogeneous, Cancer cells are heterogeneous, but most cells can proliferate and only rare cancer stem cells extensively and form new tumors. have the ability to proliferate extensively and form new tumors.
Reya et al 2001 Tumor Formation by Human Breast Cancer “Stem Cells”
20,000 200
CD44- CD44+ CD24+ CD24- Cells Cells
Al Hajj et al PNAS 2003 Stem Cell Markers
• CD44 • CD24 • Aldehyde Dehydrogenase ( Christophe Ginestier) • Oct-4 • Bmi-1 • Nuclear B-Catenin
ALDEFLUOR+ population and tumorigenicity
With DEAB Without DEAB ALDEFLUOR- ALDEFLUOR+
R1 R2 R1 R2
5% to 10% of ALDEFLUOR + population in Breast tumors
ALDEFLUOR- ALDEFLUOR+ 50,000 cells 50,000 cells ALDEFLUOR – ALDEFLUOR + 5000 cells 5000 cells 50,000 cells 500 cells 500 cells 500 cells ALDEFLUOR+ population and tumorigenicity
ALDEFLUOR - ALDEFLUOR +
ALDEFLUOR+ population regenerates heterogeneity of the initial tumor Overlap between CD44+CD24- and ALDEFLUOR+ Populations
Ginestier et al Cell Stem Cell 2007 Breast Cancer Development Luminal Myoepithelial
Normal duct Hyperplasia In situ carcinoma Invasive carcinoma Aldefluor + cells are invasive and metastatic U (-) (+) HCC1954HCC1954 (100k)
500 100,000 ALDELFUOR-positive 450 100,00 ALDEFLUOR-negative 400 350 100,000 Unseparated 300 Positive E 250 Negative 200 Unsorted 150 100 50 Normalized Flux Photon Normalized Photon Flux Photon Normalized 0 1 2 3 4 5 6 7 8 F WeeksWeeks after after injection injection B
600 MDAMDAMB453-MB-453 (100k) K
500 500 450 100,000 ALDELFUOR-positive 400 400 100,00 ALDEFLUOR-negative 350 300 300 100,000 Unseparated 250 Positive G 200 200 Negative 150 Unsorted 100 100 Relative invasion rate Normalized Flux Photon 50
Normalized Photon Flux Photon Normalized 0 0 L 1 2 3 4 1 2 3 4 5 ALDEFLUOR + + + + + + ------Weeks after injection BrCa-MZ-01 MDA-MB-453 SUM159 Weeks after injection C H
IL8 - - - + + + - - - + + + SUM159SUM159 (100k) HCC1954 MDA-MB-453 SUM159 30 A 100,000 ALDELFUOR-positive 25 100,00 ALDEFLUOR-negative 20 100,000 Unseparated Positive 15 Negative Unsorted I M 10
5 Normalized Flux Photon
Normalized Photon Flux Photon Normalized 0 1 2 3 4 5 Weeks after after injection injection D J Cancer Stem Cells: Implications For Metastasis
TDC only No Metastases CSC=Cancer Stem Cell TDC=Terminally Differentiated Cell NO CSC
1o Tumor CSC with FULL malignant potential Metastases in months to few years TDC TDC Subsequently to other sites TDC CSC TDC TDC TDC
CSC with PARTIAL malignant potential Dormancy followed by Metastases after many years:
Secondary Oncogenic “Hits” and/or Changes in Microenvironment Carcinogenesis
Stem cell regulation
Dysregulation
Cancer TUMOR Microenvironment Stem Cell Generation Tumor Microenviornment and the “CSC Niche”
• The Microenvironment (“Stem cell niche”) contains fibroblasts, endothelial cells, inflammatory cells and mesenchymal stem cells recruited from bone marrow. • The Microenvironment plays an important role in normal mammary development as well as tumor growth and metastasis.
> Bone marrow derived mesenchymal stem cells are recruited to murine breast carcinomas (Karnoub 2007). • Cytokines regulate breast cancer stem cells. > IL6 increases breast cancer stem cells (Sanson 2008). > IL8/CXCR1 axis regulates breast cancer stem cells (Charafe-Jaffret Cancer Research 2009)
Effects of Cytokines on breast cancer stem/progenitor cell population
A 14 12
10
- positive 8
6
4 SUM159 cells (%) ALDEFLOUR-positive cells (%)
ALDEFLOUR 2
0 Co- Control 2 ug/ml 1 ug/ml 10 ng/ml 10 ng/ml 100 ng/ml 100 ng/ml Co- 100 ng/ml culture N.T. CXCL12 ug/ml CXCL51 ug/ml CXCL610 ng/ml CXCL710 ng/ml IL6 100IL8 ng/ml culture CXCL1 CXCL5 CXCL6 CXCL7 IL6 IL8 SUM159
B 16
12 - positive 8
4 SUM159 cells (%) ALDEFLOUR ALDEFLOUR-positive cells (% cells ALDEFLOUR-positive 0 10 ug/ml SUM159SUM159 ControlN.T. 100100 ug/ml ug/ml 5050 ug/ml ug/ml 2020 ug/ml ug/ml 100100 ug/ml ug/ml 10 ug/ml anti-IL8 alonealone anti-CXCL5anti-CXCL5 anti-CXCL6anti-CXCL6 anti-CXCL7anti-CXCL7 anti-IL6anti-IL6 anti-IL8 Co-culture Model of cytokine networks between MSC and breast cancer cells
CXCL7 Bulk Tumor Mesenchymal Cells Cells ALDH- CXCR2 ALDH+ ALDH- IL-6R ALDH- gp130 IL-6
CXCL1 CXCL5 CXCL6 IL-8 IL-6
CXCR1 IL-6R gp130 Stem cell self Cancer renewal Stem Cell
S. Liu Canc. Res. 2011 Regulation of CSC’s by cytokine loops in the tumor microenviornment
Korkaya & Wicha JCI, 121(10):3804-09, 2011 Diet ,exercise and breast cancer risk
Neilson H K et al. Cancer Epidemiol Biomarkers Prev 2009;18:11-27 Fas-Ligand IL-8 IL-8 Fas-Ligand IL-8 IL-8 Chemotherapy Fas CXCR1 Fas
FADD PI3-K FADD FAK PTEN
AKT Apoptosis Self- FOXO3a IL-8 IL-8 β-catenin IL-8 Renewal GSK3β
β-catenin Wnt pathway IL-8 TCF targets, Fas-Ligand Self-renewal Fas-Ligand
A Cancer stem cell Bystander effect Bulk tumor cells
Repertaxin Fas-Ligand Fas-Ligand Chemotherapy Fas Fas CXCR1
FADD FADD
FAK
FOXO3a Apoptosis Apoptosis IL-8 IL-8 AKT Death IL-8
FOXO3a IL-8 Fas-Ligand Fas-Ligand Fas-Ligand
Cancer stem cell Bystander effect Bulk tumor cells B Stem Cell Equilibria
miR-93
EMT MET Differentiation
TGF-b Il6 Quiescent Cycling
Vimentin + Vimentin - Epcam (ESA) - Epcam (ESA) + p53 p53 p21 p21
CD44+/CD24- Aldefluor +
E Cadherin - E Cadherin +
44+/24- Highly Tumorigenic
Aldefluor+ Recapitulation of Tumors Via CSC Metastasis
Breast Tumor Micrometastasis Macrometastasis
Vasculature
Aldefluor + Invasive Edge of Tumor
Time
CD44+/CD24- Distal Healthy Organ
Necrotic Distal Colonized Organ Zone
EMT MET Strategic Approach to Preventive Therapeutics
Genetic Estimated Toxicity Background Lifetime Risk I N C R E A S E
NUTRITION I N C R E A S E BOTANICAL Sporadic <10% BASED
Haplotype-SNP 10%-40% I1307K
PHARMACEUTICAL Familial Syndrome >40% BASED (BRCA)
Kakalara Self Renewal and Differientiation Pathways In Breast Stem Cells
ER- stem cells
SC SC SC Her2 Self renewal SC Notch Bmi-1 BRCA-1 Hedgehog PTEN
ER+ Progenitor cells
Differentiation Myoepithelial cells Alveolar epithelial cells
Ductal epithelial cells
A Conceptual Link Between Hereditary & Sporatic Breast Cancers Wnt Wnt IL-8 IL-8 IL-8 IL-8 LRP CXCR1 Repertaxin HH PI3-K HH PTCH Frizzled Perifosine HH FAK
DSH GSK3β AKT FU
SMO PTEN Cyclopamine β-catenin RTKs Src HER2 SUFU β-catenin
GLI TCF Trastuzumab
GLI Bmi-1
STAT3 NOTCH HES DSL HER2
DSL
NICD STAT3 Anti-DLL4 γ-secretase JAK3 gp130 IL-6R GSI CXCR2 IL6 IL6 IL6 Cancer stem cell Monoclonal Abs Breast Tumor IL-8 CXCL7 IL-6 IL-6R gp130 Targeting Self-Renewal Pathways in CSC’S
Mesenchymal Cells Broccoli could stop breast cancer spreading by targeting stem cells
Broccoli contains sulforaphane, which seemed to kill off cancer stem cells in tests
Figure 2. A Primary Mammospheres B Mammosphere Size
120.0 MCF7 0 µM 0.5 µM 1 µM 5 µM 100.0 SUM1… SF SF SF SF 80.0 MCF7 ** 60.0 ** ** **
40.0 ****
Sphere formation Sphere formation 20.0 SUM159
normalized to control (%) 0.0 0 0.5 1 5 Concentration of SF (µM)
C 2nd Passage 3rd Passage
120.0 140.0 MCF7 MCF7 100.0 SUM159 120.0 SUM159 100.0 80.0 ** 80.0 * 60.0 ** 60.0 * 40.0 ** ** 40.0
Sphere formation Sphere formation 20.0 ** Sphere formation 20.0 ** ** ** normalized to control (%) 0.0 normalized to control (%) 0.0 0 0.5 1 5 0 0.5 1 5 Li CCR 2010 Concentration of SF (µM) Concentration of SF (µM) Figure 3. A P=0.003
4 P=0.008 3.5 3 2.5 2
positive cells - positive 1.5 1 0.5 % ALDH % 0 0 1 5 B Concentration of SF (µm Control 1 µM SF 5 µM SF
3.01% 1.47% 0.49% B
A 25 ) 500 1st Generation 1st Generation 3
400 Control 20 50 mg/kg SF 300 P=0.018 15 200 10 100 Control Body weight (g) weight Body 50 mg/kg SF Tumor volume (mm volume Tumor 0 5 13 18 23 28 33 13 18 23 28 33 Days after inoculation Days after inoculation
3.00 C P=0.003 D 2.50 2.00 Control Treatment 1.50 cells positive - 1.00 0.50
% ALDH 0.00
2.39% 1.08% A 2nd Generation 1000
100 )
3 P=0.007 10 Control group 1 P=0.0006 (mm Control group 2 1 SF group 1 Tumor volume SF group 2 0.1 5 15 25 35 Days after inoculation C B 2nd Generation
120 100 80 60 40 Control 20 0 free mice Tumor - free % 0 20 40 Control SF group Days after inoculation SF (µM) 4 d SF (5 µm) A MCF7 0 1 5 10 0 1 2 4 d β-catenin cyclin D1 β-actin SF (µM) 4 d SF (5 µm) SUM159 0 1 5 10 0 1 2 4 d β-catenin cyclin D1 β-actin B C TOP-dGFP TOP-dGFP+SF P<0.0001 P<0.0001 18.00 16.00 14.00 12.00 10.00 8.00 6.00 3.04% 2.13%
positive cells positive 4.00 P=0.002 - 2.00 TOP-dGFP+BIO TOP-dGFP+BIO+SF 0.00 % dGFP
16.56% 6.92% Stages of Cancer Progression and Its Suppression by Curcumin
Constitutive activation of Overexpression of Overexpression of transcription factors -Oncogenes -MMPs -AP -1, NFκB -Her 2 -Cox 2 -Tumor Suppressor Genes -Growth factors eg. EGF, PDGF -adhesion molecules Modulation of Signaling -Survival factors eg. Survivin, bcl -chemokines - Wnt/β catenin 2, bcl-xl -TNF -Notch -Cyclin D1
transformation proliferation invasion Normal Tumor Tumor Tumor Cells cells growth metastasis
Curcumin Aggarwal et al Anticancer Research 23, 2003:363-398
Using human stem cells as a screening system for chemopreventive efficacy, in vivo biomarker of efficacy and assay for mechanism
DMSO control 10 µM curcumin
Kakarala et al, Breast Cancer Res Treat; 2010. Effect of Curcumin and Piperine on ALDH+ cells (%)
10 9 8 7 6 5 4 3 2
% ALDH1A1+ Cells % 1 0
NT C5 C10 P10
C10+P10 Curcumin and Piperine Concentration (µM)
Kakarala et al, Breast Cancer Res Treat; 2010. Breast Cancer Prevention Strategy Curcumin/Piperine Targeting Stem Cells
Preclinical Research: Stem cell self renewal, differentiation, signaling (ongoing)
Phase I Clinical Trial Bioavailability/Toxicity (Spring 2010)
Phase II Clinical Trial BRCA1 Patients Curcumin/Piperine (2011) Kakalara & Brenner Stem Cells in Breast Cancer Risk reduction,Early detection,Prevention and Therapy
Risk reduction
Polyphenols
Example: curcumin genistein Hyperplasia Risk reduced tissue
Early detection Detection of expanded stem cells or secreted markers Secreted cancer stem cell markers Example: EZH2
Primary prevention Apoptosis or differentiation of initiated stem cells Residual non-tumorigenic cells Example: Vitamin D
Treatment Recurrence Conventional therapy
Residual metastases Recurrence
Targeting cancer stem cells
Inhibitors of Hedgehog, Notch, Residual non-tumorigenic cells Wnt signaling Example: Gamma secretase Cure inhibitor Residual non-tumorigenic cells at distant sites Acknowledgements
• Hasan Korkaya • Duxin • Madhuri Kakarala Sun • Dan Hayes • Kathy Day • Suthinee Ithimakin • Dafydd Thomas • Suling Liu • Tom Bersano • Shawn Clouthier • Dean Brenner • Mark Diebel • Amanda Paulson • Wissam Abdallah • Min Hee Hur • Greg Hannon • Kyle Jackson • Emmanuelle Jauffret • Jessica Foley • Mari Kawamur • Daniel Birnbaum