Targeting mammary stem cells for breast cancer prevention · TDC TDC . TDC TDC . TDC . No Metastases . CSC=Cancer Stem Cell . TDC=Terminally Differentiated Cell . Cancer Stem Cells:

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