Breast Cancer and Activated Signaling Pathways: Avenues for New Therapies

Michael Reedijk MD PhD FRCSC Surgical Oncology

Princess Margaret Cancer Center University Health Network

• No conflicts of interest

Basal-like, closely-associated with

TNBC (ER-/PR-/ErbB2-negative)

Sorlie et al., 2001

Basal-like breast cancer

Current management of TNBC

• Most TNBC are high-grade invasive ductal carcinomas (IDC) of no special type (NST)

• Chemotherapy is the main systemic treatment Anthracyclines

o Meta-analysis of anthracycline-containing regimens vs CMF showed 23%↑ DFS (Di Leo et al. 2010)

o But FEC not superior to CMF (MA5) Taxanes

o FEC-D (docetaxel) vs FEC in LN +ve BLBC showed 35%↑ DFS (PACS 01)

o T (taxotere)AC vs CAF in TNBC showed 50%↑DFS (BCIRG 001) Platinum

o BRCA gene dysfunction (germline and non-germline mutations; promoter methylation)

o Up-regulation of cell cycle and cell division genes o Conflicting Phase I and II results (effective in BRCA1 mutants)

Androgen receptor

Immune response

Cell cycle + DNA repair

Angiogenesis

Growth Factors + Receptors

Intracellular signaling

Cytoskeleton Motility Metastases

Tumor Initiating cells

Targets in TNBC

Hedgehog Wnt Notch

RAS, RAF, PI3K, PTEN AKT, mTOR

PARP CCND1

EGFR, FGFR1+2, IGFR1, KIT, MET PDGFRA

New strategies in the management of TNBC

• DNA repair mechanisms Poly (ADP ribose) polymerase (PARP) inhibitors

o Phase II in metastatic TNBC: gemcatabine/carboplatinum +/- iniparib showed ↑ response rate, ↑ DFS and OS by months (O’Shaughnessy et al., 2011)

o Failed in Phase III metastatic TNBC (BSI-201) (perhaps ↑DFS and OS in second and third-line setting)

• Angiogenesis Bevacizumab in TNBC may improve response rate (GerarQuinto

vs NSABP-B40) but no good evidence on improving OS (not funded in any province in Canada)

New strategies in the management of TNBC

• Kinase signaling cascades: EGFR (activating mutations, ↑EGFR gene copy number,

overexpression) o Cisplatinum +/- cetuximab in metastatic TNBC showed ↑DFS (no

difference in RR or OS) (BALI-1) o A phase II trial of carboplatin/docetaxel/erlotinib in metastatic TNBC

showed pCR of 40% (Sharma et al., 2010) PI3K activation is common in TNBC o PI3K-CA mutation/amplification (10%) o pAkt overexpression o PTEN loss

Ligand

PI3kinase

PI(4,5)P

PI(3,4,5)P

Receptor PTK

PTEN

P D K

AKT

Proliferation Survival Migration

mTOR

Hedgehog Wnt Notch

New strategies in the management of TNBC

Hh signaling

Reedijk and McGlade. 2013 .

Cellular Signaling. The basic

science of oncology (ed:

Tannock, Hill, Bristow and

Harrington).

Hh signaling and TNBC

Hh in cancer • Ptc1 loss or Smo gain: BCC and medulloblastoma (Pasca di

Magliano and Hebrok, 2003) • Gorlin syndrome Breast cancer • Hh and Gli expression are associated with TNBC and poor

outcome (O’Toole et al., 2011; Li et al., 2012) • Ptc1 loss or Smo gain are seen in >50% of DCIS and IDC

(Moraes et al., 2007) • SHh promoter hypomethylation and SHh up-regulation are

frequently observed in TNBC (Wolf et al., 2007) • Mouse models: Hh signaling promotes an interaction

between malignant epithelial cells and stromal cells → EMT and metastases (Lewis et al. 1999)

Targeting Hh signaling

Hh inhibitors -Robotikinin; -Hh blocking mAb, 5E1 Preclinical Smo inhibitors

-Classic: cyclopamine -Vismodegib Approved for LA or metastatic BCC -GDC-0449 Advanced BCC Prohibitive SE in NCT01071564 -LDE225 BKM120 metatastatic BC

Gli inhibitors -GANT58, GANT61

Phase 1

Wnt signaling

Reedijk and McGlade. 2013. Cellular Signaling.

The basic science of oncology (ed: Tannock,

Hill, Bristow and Harrington).

Wnt signaling and TNBC

Wnt in cancer • Familial adenomatous polyposis coli (FAP): mutant APC

Breast cancer • The WNT1 locus is a common MMTV proviral integration site in

MMTV-induced BC (named INT)

• WNT1 and ∆Nβ-catenin transgenic mice develop BLBC (Ayyanan et al., 2006)

• Wnt pathway genes (FZD7, LRP6, TCF) up-regulation is associated with TNBC (Yang et al., 2011)

• Loss of membranous β-catenin is a feature of TICs (CD44/CD24lo) and associated with TNBC and poor outcome (Khramstova et al., 2010; Lopez-Knowles et al., 2010; Geyer et al., 2011)

Targeting Wnt signaling

Fz blocking mAb -OMP-18R5

Phase 1 in solid malignancies

Wnt antagonists -IWP-4 Wnt blocking mAb Soluble receptor decoys -F8CRdhFc

β-catenin Degradation -XAV-939

β-catenin/Tcf Inhibitors -PRI-724

Phase 1 in solid malignancies

Dsh antagonists

Axin

GSK3 -Genistein

Notch signaling

Reedijk and McGlade. 2013. Cellular Signaling.

The basic science of oncology (ed: Tannock,

Hill, Bristow and Harrington).

Notch signaling and breast cancer

Notch in cancer • >50% of T-ALL have activating mutations of Notch-1 (Weng et al., 2004) Breast cancer • The Notch-4 locus is a common proviral integration site in MMTV-

induced murine BC (Gallahan and Callahan, 1987)

• N1,3,4IC transgenic mice develop BC (Jhappan et al., 1992 Dievart et al., 1999; Hu et al., 2006)

• High-level co-expression of JAG1 and NOTCH1 are associated with TNBC and poor survival (Reedijk et al., 2005, 2007)

• Tumor-promoting Notch target genes (CCND1, slug, survivin, uPA) are expressed in TNBC (Leong et al., 2007; Lee et al., 2008; Cohen et al. 2010; Shimizu et al., 2011)

• Notch is required for the maintenance of breast cancer TICs (Farnie et al., 2007; Sansone et al., 2007a,b; Korkaya et al., 2009)

uPA is a direct Notch target

BA

Set 1

Set 2

IgG

Anti-N1

Input

Anti-N3

uPA set 1

uPA set 2

GAPDH

C

F

E

D

A

B

C

D

probe C

cold wt C

cold mut C

lysate

cold wt B

cold mut B

-

-

-

-

lysate - + + + + + + +

complex

probe B

-

-

scr

CBF-1

siR

NA

scr

CBF-1

probe B

lysate

cold wt B

- + +

- - - +

E

+

-

-

-

+

-

-

+

+

-

+

-

+

BA

Set 1

Set 2

IgG

Anti-N1

Input

Anti-N3

uPA set 1

uPA set 2

GAPDH

C

F

E

D

A

B

C

D

probe C

cold wt C

cold mut C

lysate

cold wt B

cold mut B

-

-

-

-

lysate - + + + + + + +

complex

probe B

-

-

scr

CBF-1

siR

NA

scr

CBF-1

probe B

lysate

cold wt B

- + +

- - - +

E

+

-

-

-

+

-

-

+

+

-

+

-

+

-YGTGRGAA- RBPjĸ binding site “B”

Shimizu et al., 2011.

uPA

Actin

M2Ø

MB231

Scr

uP

A

NO

TC

H1

/3

JA

G1

Scr

uP

A

NO

TC

H1

/3

JA

G1

Scr

Scr

Scr

Scr

uP

A

NO

TC

H1

/3

JA

G1

Scr

uP

A

NO

TC

H1

/3

JA

G1

Scr

Scr

Scr

- - - -

- - - -

A

1 2 3 4 5 6 7 8 9 10 11 12 13 14 15

50-

40-

B

M2Ø-derived TGFβ promotes JAG1- Notch mediated uPA expression in tumor cells

Smad4

Actin

TGFbR1

MB231

M2ø

siS

cr

siT

GF

bR

1

siS

ma

d4

siS

cr

siT

GF

bR

1

siS

ma

d4

αT

GF

b

uPA

Shen et al., unpublished

TGFBR1

Actin

siS

cr

siN

OT

CH

1/3

siJ

AG

1

A B C

51-

22-

29-

latent TGFβ

mature TGFβ

M2Ø

MB231

siS

cr

siS

cr

siu

PA

siN

OT

CH

1/3

siJ

AG

1

62- 51-

22-

29-

latent TGFβ

mature TGFβ

MB231 + + - + + -

M2Ø - + + - + +

24hr 48hr

Notch potentiates TGFβ signaling between tumor cells and M2Ø

Shen et al., unpublished

TGFβR1

Notch-dependant interactions in the TME

Inflammatory Tumor

Microenvironment

uPA Shimizu, 2011

TGFβ

(1)

Rac1

(2)

uPA

monocyte

TAM

Latent-TGFβ

(3) ECM

Trb3 Izrailit, 2013

Targeting Notch signaling

Dll4 blocking mAb -demcizumab -Enoticumab Phase 1 in advanced malignancies

mAb to ADAM

ɣ-secretase Inhibitor -MK-0752 Advanced breast cancer : plus docetaxel ↓tumor size, TICs (Schott et al., 2013) -RO4929097

α-helical peptide to MAMAL1

uPA

uPA inhibitor -Mesupron Phase II in pancreatic ca - ↑RR, DFS, OS (Heinemann et al., 2010) Metastatic HER2-neg BC

• TNBC is a heterogeneous group of breast cancers with limited treatment options

• Predictive biomarkers. “Who to treat”

• Heavily pre-treated patients with advanced disease for which there is no remaining standard curative therapy and no therapy with a demonstrated survival benefit

• Side effects – developmental pathways are ubiquitous so toxicity is an important consideration (e.g. GI toxicity with GSIs)

Important considerations

Acknowledgements

• Qiang Shen

• Mamiko Shimizu

• Brenda Cohen

• CIHR

• CBCF

• Komen for the cure

• Campbell family