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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
• 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
If cancers arise in self-renewing cells then:
characteristics of stem cells
• self renewal • differentiation into multiple cell
types
• types of stem cells embryonic adult • cancer
DIF
FER
EN
TIAT
ION
Early Progenitor
Late Progenitor
Luminal cells
Myoepithelial cells
Alveolar cells
Stem cell
Quiescent pool of stem cell
Cancer stem cell
Cancer stem cell
Stem cells and breast carcinogenesis
Suspension culture (Serum-free, EGF, …)
Mammospheres
Dissociated to single cells Dissociated to single cells
Reduction mammoplasties
N generation of spheres
A. Self - renewal in vitro
(Suspension)
N generation of spheres
A. Self - renewal in vitro
(Suspension)
Dontu & Wicha, 2003
Mixed Epithelial Myoepithelial
B. Differentiation in vitro in 2 - D culture
(Collagen)
B. Differentiation in vitro in 2 - D culture
(Collagen)
Mixed Epithelial Myoepithelial
B. Differentiation in vitro in 2 - D culture
(Collagen)
Epithelial Myoepithelial
Dontu & Wicha, 2003
Acinar structure
Ductal - acinar structure
C. Differentiation in vitro in 3 - D culture
( Matrigel )
Acinar structure
Ductal - acinar structure
C. Differentiation in vitro in 3 - D culture
( Matrigel )
Weaver & Bissell, 1999 Dontu & Wicha, 2003
D. Differentiation and self - renewal in vivo (NOD/SCID mice)
Regeneration of the mammary gland in the cleared fat - pad
D. Differentiation and self - renewal in vivo (NOD/SCID mice)
Regeneration of the mammary gland in the cleared fat - pad
Kuperwasser & Weinberg, 2004 Liu & Wicha, 2006
“Mammosphere” Culture Systems
ALDH
BAAA
DEAB
ALDEFLUOR + cells in Normal breast epithelium
With DEAB Without DEAB
6% of ALDEFLUOR positive cells in Normal breast epithelium
6% Ginestier et al Cell Stem Cell 2007
With DEAB Without DEAB
ALDEFLUOR+ cells have stem cell properties
Culture in suspension
Only ALDEFLUOR + cells
generate mammospheres
in suspension
Without DEAB
ALDEFLUOR+ cells have stem cell properties
Luminal cells CK18+
Myoepithelial cells SMA+
ALDEFLUOR- ALDEFLUOR+
ALDEFLUOR-
ALDEFLUOR+
CK18 SMA
Ginestier, 2007
Models of Tumor Heterogeneity
Stochastic model Cancer stem cell model
CSC
CSC
CSC
CSC
Cancer cells are heterogeneous, but most cells can proliferate extensively and form new tumors.
Cancer cells are heterogeneous, and only rare cancer stem cells have the ability to proliferate extensively and form new tumors.
Reya et al 2001
20,000
CD44- CD24+ Cells
200
CD44+ CD24-
Cells
Tumor Formation by Human Breast Cancer “Stem Cells”
Al Hajj et al PNAS 2003
Stem Cell Markers • CD44 • CD24 • Aldehyde Dehydrogenase ( Christophe Ginestier) • Oct-4 • Bmi-1 • Nuclear B-Catenin
With DEAB Without DEAB
R1 R2 R1 R2
ALDEFLUOR- 50,000 cells 5000 cells 500 cells
ALDEFLUOR+ 50,000 cells 5000 cells 500 cells
ALDEFLUOR + 500 cells
ALDEFLUOR – 50,000 cells
ALDEFLUOR- ALDEFLUOR+
ALDEFLUOR+ population and tumorigenicity
5% to 10% of ALDEFLUOR + population in Breast tumors
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
Invasive carcinoma
Breast Cancer Development
Hyperplasia In situ carcinoma Normal duct
Luminal Myoepithelial
0
100
200
300
400
500
600
1 2 3 4
Rel
ativ
e in
vasi
on ra
te
BrCa-MZ-01 MDA-MB-453 SUM159
MDAMB453 (100k)
0
50
100
150
200
250
300
350
400
450
500
1 2 3 4 5
Weeks after injection
Norm
aliz
ed P
hoto
n Fl
ux
Positive
Negative
Unsorted
SUM159 (100k)
0
5
10
15
20
25
30
1 2 3 4 5
Weeks after injection
Nor
mal
ized
Pho
ton
Flux
PositiveNegativeUnsorted
HCC1954 (100k)
050
100150200250300350400450500
1 2 3 4 5 6 7 8
Weeks after injection
No
rmal
ized
Ph
oto
n F
lux
Positive
Negative
Unsorted
Nor
mal
ized
Pho
ton
Flux
N
orm
aliz
ed P
hoto
n Fl
ux
Nor
mal
ized
Pho
ton
Flux
Weeks after injection
Weeks after injection
Weeks after injection
MDA-MB-453
SUM159
HCC1954
100,000 ALDELFUOR-positive
100,00 ALDEFLUOR-negative
100,000 Unseparated
100,000 ALDELFUOR-positive
100,00 ALDEFLUOR-negative
100,000 Unseparated
100,000 ALDELFUOR-positive
100,00 ALDEFLUOR-negative
100,000 Unseparated
C
G
H
D
I
J
B
E
F
K
L
M
U (-) (+)
A HCC1954 MDA-MB-453 SUM159
IL8 - - - + + + - - - + + + ALDEFLUOR + + + + + + - - - - - -
Aldefluor + cells are invasive and metastatic
CSC with PARTIAL malignant potential
CSC
TDC
TDC
TDC
TDC
TDC
TDC
No Metastases CSC=Cancer Stem Cell
TDC=Terminally Differentiated Cell
Cancer Stem Cells: Implications For Metastasis
1o Tumor CSC with FULL
malignant potential
Secondary Oncogenic “Hits” and/or Changes in Microenvironment
Subsequently to other sites
Metastases in months to few years
Dormancy followed by Metastases after many years:
TDC only
NO CSC
Dysregulation
Stem
cell
regulation
Carcinogenesis
Cancer
Stem Cell
Generation
Microenvironment TUMOR
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)
0
4
8
12
16
SUM159alone
Control 100 ug/mlanti-CXCL5
50 ug/mlanti-CXCL6
20 ug/mlanti-CXCL7
100 ug/mlanti-IL6
10 ug/mlanti-IL8
ALDE
FLOU
R-po
sitiv
e ce
lls (%
ALD
EFLO
UR
-pos
itive
SU
M15
9 ce
lls (%
)
SUM159 alone
N.T. 100 ug/ml anti-CXCL5
50 ug/ml anti-CXCL6
20 ug/ml anti-CXCL7
100 ug/ml anti-IL6
10 ug/ml anti-IL8
Co-culture
0
2
4
6
8
10
12
14
Co-culture
Control 2 ug/mlCXCL1
1 ug/mlCXCL5
10 ng/mlCXCL6
10 ng/mlCXCL7
100 ng/mlIL6
100 ng/mlIL8
ALDE
FLOU
R-po
sitive
cells
(%)
ALD
EFLO
UR
-pos
itive
SU
M15
9 ce
lls (%
)
Co- culture
N.T. 2 ug/ml CXCL1
1 ug/ml CXCL5
10 ng/ml CXCL6
10 ng/ml CXCL7
100 ng/ml IL8
100 ng/ml IL6
SUM159
A
B
Effects of Cytokines on breast cancer stem/progenitor cell population
Cancer Stem Cell
Bulk Tumor Cells Mesenchymal
Cells
CXCL7
CXCR1
Stem cell self renewal
IL-6
IL-6R gp130
CXCL5 CXCL1
CXCL6 IL-8 IL-6
ALDH-
ALDH+
IL-6R gp130
ALDH-
ALDH-
CXCR2
Model of cytokine networks between MSC and breast cancer cells
S. Liu Canc. Res. 2011
Regulation of CSC’s by cytokine loops in the tumor microenviornment
Korkaya & Wicha JCI, 121(10):3804-09, 2011
Neilson H K et al. Cancer Epidemiol Biomarkers Prev 2009;18:11-27
Diet ,exercise and breast cancer risk
IL-8 IL-8
IL-8 IL-8
CXCR1
AKT
GSK3β β-catenin
β-catenin TCF
Wnt pathway targets, Self-renewal
Fas
Fas-Ligand
FOXO3a
FAK
FADD
IL-8 Fas-Ligand
Fas
Fas-Ligand
Apoptosis
FADD
IL-8 IL-8
IL-8
Fas-Ligand
Chemotherapy
Bystander effect
PI3-K
PTEN
CXCR1
AKT
Fas
Fas-Ligand
Apoptosis FOXO3a
FAK
FADD
Repertaxin
FOXO3a Fas-Ligand
IL-8 Fas-Ligand
Fas
Fas-Ligand
Apoptosis
FADD
IL-8 IL-8
IL-8
Fas-Ligand
Chemotherapy
Bystander effect
Cancer stem cell Bulk tumor cells
Cancer stem cell Bulk tumor cells
A
B
Self- Renewal
Death
Stem Cell Equilibria
MET EMT
miR-93
TGF-b
Il6
Epcam (ESA) -
Quiescent Cycling
Vimentin +
p53
p21
CD44+/CD24-
Vimentin -
Epcam (ESA) +
E Cadherin -
p53
p21
Aldefluor +
E Cadherin +
Differentiation
44+/24-
Aldefluor+
Highly Tumorigenic
Recapitulation of Tumors Via CSC Metastasis
Micrometastasis
CD44+/CD24-
EMT MET
Invasive Edge of Tumor
Necrotic Zone
Aldefluor +
Breast Tumor Macrometastasis
Vasculature
Time
Distal Healthy Organ Distal Colonized Organ
Estimated Lifetime Risk
Toxicity
Sporadic
Haplotype-SNP I1307K
Familial Syndrome (BRCA)
<10%
10%-40%
>40%
I N C
R E A S E
I N C
R E A S E
Genetic Background
NUTRITION BOTANICAL BASED
PHARMACEUTICAL BASED
Strategic Approach to Preventive Therapeutics
Kakalara
SC Self renewal
Notch Hedgehog BRCA-1
PTEN
Progenitor cells
ER- stem cells
ER+
Myoepithelial cells
Ductal epithelial cells
Alveolar epithelial cells
Bmi-1
Differentiation
Her2 SC SC SC
Self Renewal and Differientiation Pathways In Breast Stem Cells
A Conceptual Link Between Hereditary & Sporatic Breast Cancers
Breast Tumor
IL-6R gp130
IL-8 IL-8
IL-8 IL-8
CXCR1
AKT GSK3β
β-catenin
β-catenin TCF
FAK PI3-K
PTEN
Cancer stem cell
CXCR2
CXCL7
IL6
Frizzled
Wnt Wnt
LRP
DSH
Src RTKs
HH HH
HH
GLI
PTCH
SMO
FU
SUFU
GLI
NOTCH DSL
DSL
γ-secretase NICD
HES
Bmi-1
HER2
Trastuzumab
Repertaxin
Perifosine
GSI
IL-8 IL-6
Cyclopamine
IL6 IL6
gp130 IL-6R
JAK3
STAT3
STAT3
Anti-DLL4
Mesenchymal Cells
HER2
Monoclonal Abs
Targeting Self-Renewal Pathways in CSC’S
Broccoli contains sulforaphane, which seemed to kill off cancer stem cells in tests
Broccoli could stop breast cancer spreading by targeting stem cells
Primary Mammospheres
2nd Passage 3rd Passage
Mammosphere Size 0.5 µM SF
0 µM SF
Figure 2.
A B
C
MCF7
SUM159
1 µM SF
5 µM SF
0.0
20.0
40.0
60.0
80.0
100.0
120.0
140.0
0 0.5 1 5
Sph
ere
form
atio
n
nor
mal
ized
to
con
trol
(%
)
Concentration of SF (µM)
MCF7 SUM159
*
**
0.0
20.0
40.0
60.0
80.0
100.0
120.0
0 0.5 1 5
Sph
ere
form
atio
n
nor
mal
ized
to
con
trol
(%
)
Concentration of SF (µM)
MCF7 SUM1…
** ** **
**
** **
0.0
20.0
40.0
60.0
80.0
100.0
120.0
0 0.5 1 5
Sph
ere
form
atio
n
nor
mal
ized
to
con
trol
(%
)
Concentration of SF (µM)
MCF7 SUM159
**
** **
**
**
**
**
*
Li CCR 2010
Control 1 µM SF 5 µM SF
Figure 3.
0 0.5
1 1.5
2 2.5
3 3.5
4
0 1 5
% A
LDH
-pos
itiv
e ce
lls
Concentration of SF (µm
A
B
P=0.008
3.01% 1.47% 0.49%
P=0.003
0.00
0.50
1.00
1.50
2.00
2.50
3.00
% A
LDH
-pos
itiv
e ce
lls
Control Treatment
A B
C D P=0.003
0
100
200
300
400
500
13 18 23 28 33
Tum
or v
olum
e (m
m3 )
Days after inoculation
1st Generation
Control 50 mg/kg SF
5
10
15
20
25
13 18 23 28 33
Body
wei
ght (
g)
Days after inoculation
Control 50 mg/kg SF
1st Generation
P=0.018
2.39% 1.08%
A
B
0
20
40
60
80
100
120
0 20 40
% T
um
or-f
ree
mic
e
Days after inoculation
Control
2nd Generation
2nd Generation
0.1
1
10
100
1000
5 15 25 35Days after inoculation
Tum
or v
olum
e
(mm
3 )
Control group 1Control group 2SF group 1SF group 2
P=0.007 P=0.0006
C
Control SF group
0.00 2.00 4.00 6.00 8.00
10.00 12.00 14.00 16.00 18.00
% d
GFP
-pos
itiv
e ce
lls
MCF7
β-catenin cyclin D1
β-actin
SF (µM) 4 d 0 1 5 10
SF (5 µm) 0 1 2 4 d
SUM159 SF (µM) 4 d
0 1 5 10 SF (5 µm)
0 1 2 4 d β-catenin
cyclin D1
β-actin
A
B C TOP-dGFP TOP-dGFP+SF
TOP-dGFP+BIO TOP-dGFP+BIO+SF
P<0.0001 P<0.0001
3.04% 2.13%
16.56% 6.92%
P=0.002
Stages of Cancer Progression and Its Suppression by Curcumin
Normal Cells
Tumor cells
Tumor growth
Tumor metastasis
Curcumin
transformation proliferation invasion
Constitutive activation of transcription factors
-AP -1, NFκB
-Tumor Suppressor Genes
Modulation of Signaling
- Wnt/β catenin
-Notch
Overexpression of
-Oncogenes
-Her 2
-Growth factors eg. EGF, PDGF
-Survival factors eg. Survivin, bcl 2, bcl-xl
-Cyclin D1
Overexpression of
-MMPs
-Cox 2
-adhesion molecules
-chemokines
-TNF
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
10 µM curcumin
Kakarala et al, Breast Cancer Res Treat; 2010.
DMSO control
Effect of Curcumin and Piperine on ALDH+ cells (%)
0123456789
10
NT C5C10 P10
C10+P10
Curcumin and Piperine Concentration (µM)
% A
LDH
1A1+
Cel
ls
Kakarala et al, Breast Cancer Res Treat; 2010.
Breast Cancer Prevention Strategy Curcumin/Piperine Targeting Stem Cells
Phase I Clinical Trial Bioavailability/Toxicity (Spring 2010)
Phase II Clinical Trial BRCA1 Patients
Curcumin/Piperine (2011)
Preclinical Research: Stem cell self renewal, differentiation, signaling (ongoing)
Kakalara & Brenner
Risk reduced tissueHyperplasia
Polyphenols
Example: curcumin genistein
Early detection Detection of expanded stem cells or secreted markers
Example: EZH2
Primary preventionApoptosis or differentiation of initiated stem cellsExample: Vitamin D Residual non-tumorigenic cells
Inhibitors of Hedgehog, Notch, Wnt signalingExample: Gamma secretaseinhibitor
Treatment
Residual non-tumorigenic cells
Residual non-tumorigenic cells at distant sites
Conventional therapy
RecurrenceResidual metastases
Recurrence
Cure
Secreted cancer stem cell markers
Risk reduction
Targeting cancer stem cells
Stem Cells in Breast Cancer Risk reduction,Early detection,Prevention and Therapy
Acknowledgements
• Hasan Korkaya • Madhuri Kakarala • Kathy Day • Suthinee Ithimakin • Suling Liu • Shawn Clouthier • Mark Diebel • Amanda Paulson • Wissam Abdallah • Min Hee Hur • Kyle Jackson • Jessica Foley • Mari Kawamur
• Duxin Sun • Dan Hayes • Dafydd Thomas • Tom Bersano • Dean Brenner
• Greg Hannon • Emmanuelle Jauffret • Daniel Birnbaum