Molecular pathways involved in prolactin-dependent JAK2/PAK1 action: implication in breast cancer cell motility and invasion Maria Diakonova, University.

Molecular pathways involved in prolactin-dependent JAK2/PAK1 action: implication in breast cancer cell motility

and invasion

Maria Diakonova,University of Toledo

Dep. of Biological Sciences

PROLACTIN

PITUTARY

Blood vessels

STATs and other targets

PROLACTIN

Target genes

normalmammary gland development, differentiation and survival

Endocrine

PROLACTIN: the forgotten hormone of breast cancer

PROLACTINPROLACTIN

PITUTARY

Blood vessels

STATs and other targets

PROLACTIN

Target genes; hPRL gene

STATs and other targets

normalmammary gland development, differentiation and survival

Breast tumorogenesis

Autocrine/paracrineEndocrine

PROLACTIN: the forgotten hormone of breast cancer

Prolactin in breast cancer (1) an autocrine/ paracrine loop for PRL (Clevenger et. al., 1995; Ginsburg

et.al., 1995)(2) Prolactin receptor is detected in 80% of human breast cancers (Bonneterre

et.al., 1987) and is overexpressed in breast cancer samples (Touraine et.al., 1998, Kelly et.al., 1991)

(3) PRL has a mitogenic action in breast cells (Clevenger et.al., 2003). PRL alters the expression of cyclins D1 and B1 (Brockman et.al., 2002, 2005). PRL activates MAPK pathways in T47D cells (Das et.al., 1996). The interactions between PRL interacts with Her-2 (Yamauchi et.al., 2000) and BRCA1 (Favy

et.al., 1999)

(4) PRL acts as a potent survival factor (Perks et.al., 2004)

(5) PRL is involved in tumor vascularization (Struman et.al., 1999; Goldhar et.al., 2005)

(6) gain-of-function PRL receptor (Bernichtein et.al., 2003; Bogorad et.sl., 2008)

(7) PRL increases cell migration/invasion in breast cancer cells (Maus et.al., 1999, Miller et.al., 2007, Gutzman et.al., 2007)

PROLACTIN

Prolactin signaling pathways

P PP

P

P

P

BREAST CANCER:ProliferationSurvival Metastasis

?

Target GenesJAK2

STATs

Grb2 MAPK

SHC

IRSPI3K AKT

CYTOKINERECEPTOR

FAK/Src

PROLACTIN

Prolactin signaling pathways

P PP

P

P

P

BREAST CANCER:ProliferationSurvival Metastasis

PAK1

Target GenesJAK2

STATs

Grb2 MAPK

SHC

IRSPI3K AKT

CYTOKINERECEPTOR

FAK/Src

PAKs are serine-threonine protein kinases activated by a variety of GTPase-dependent and -independent mechanisms

PAK1 participates in breast cancer

• The PAK1 gene is amplified and/or PAK1 protein is up-regulated in breast cancer (Bekri et. al., 1997)

• Overexpression of PAK1 was observed in 34 of 60 breast tumor specimens (Balasenthil et. al., 2004)

• Higher levels of PAK1 have been found in higher grade tumors (Holm et. al,. 2006)

• Highly proliferating human breast cancer cell lines and tumor tissues contain hyperactive PAK1 (Mira et. al. 2000)

• In a transgenic mouse model, PAK1 hyperactivation (PAK1 T423E mutant) leads to the formation of mammary gland tumors (Wang et.al., 2006).

Prolactin-activated endogenous JAK2 phosphorylates endogenous PAK1 on tyrosines in vivo

SDS-PAGE

transfer to NC

Nb2 cells

IP with PAK1

IB with PY

re-IB with JAK2 and PAK1

ON Dep

10 nM PRL, 10’

+/- 50 M AG490, 24h

Rider et al., JBC, 2007

Tyrosine 55 131 142 153 201 270 285 330 334 346 429 441 464 474

Sequence YRSI YNSK YMSF YNSS YTRS YTRF YTAM YLDS YLVG YLAG YWMA YGPK YLNE YLIA

Schematic representation of PAK1

JAK2 phosphorylates PAK1 at Tyr(s) 153, 201 and 285

Rider et al., JBC, 2007

Prolactin activates PAK1

T47DMCF-7TMX2-28cells

+/-PRL IP PAK1 [-32P]ATP

SDS-PAGEnitrocellulose

kinase assay

H4 histone

Hammer et al., Mol.Endo, 2013

PRL stimulates kinase activity of PAK1 and PAK1 ability to form protein-protein interaction

Hammer and Diakonova, in press

Schroeder er.al., 2012

Metastatic spread to different organs

> 90% of mortality from cancer is attributable to metastases, not the primary tumors from which these malignant lesions arise

PRL-activated PAK1 stimulates cell migration

Wounding assay Boyden chamber assay

Hammer&Rider, 2013

PRL

Filamin A is actin-binding protein

• Dimerizes at C-terminus• Most binding partners bind at

C-terminus• Cross-links actin• Required for cell motility

Cunningham et. al., 1997

M2 A7

PAK1 phosphorylates Filamin A at Ser 2152. Filamin A stimulates PAK1 kinase activity by a positive feedback loop.

Vadlamudi et.al., 2002

JAK2-dependent phosphorylation of PAK1 increase Filamin A Ser2152 phosphorylation

Hammer & Rider et al., 2013

integrins

Filamin ApSer2152

JAK2

prolactin

PRL-R

PAK1

pY-153

pY-285

pY-201

cell migration

Maximal invasion of TMX2-28 cells in response to PRL requires tyrosyl phosphorylation of PAK1

ECM

Oladimeji & Rider et al., 2013

PRL

Collagens and PRL-dependent tyrosyl phosphorylatoin of PAK1 regulates transcription and secretion of MMP1 and 2

Col IV

Col I (by MMP1 and 3)

invasion

p38

JAK2

ERK JNK

c-fos

Jun

MMP1MMP3AP-1

collagen IVintegrins

?

?prolactin

DDR

?

PAK1

pY-153

pY-285

pY-201

Filamin ASer2152PRL-R

collagen IV

MMP1 and MMP3 secretion

cell migration

PAK1 tyrosyl phosphorylation regulates cell spreading and adhesion

Hammer et.al., submitted

PAK1 tyrosyl phosphorylation contributes to PAK1 activity

ECM

GIT1

Paxillin FAKTalin

PAK1

P

PS273TalinFAK

Focal Complex Nascent Adhesion

PAK1PAK1

PIX

ECM

GIT1

Paxillin FAKTalin

PAK1

P

PS273TalinFAK

AdhesionTurnover

Focal Complex Nascent Adhesion

PAK1PAK1

PIX

PRL

JAK

2

PR

L-R

P PP P

P P

P P

PR

L-R

JAK

2

ECM

GIT1

Paxillin FAKTalin

PAK1

P

PS273TalinFAK

AdhesionTurnover

Focal Complex Nascent Adhesion

PAK1PAK1

PY285

PIX

?

PAK1 tyrosyl phosphorylation regulates binding to βPIX/GIT1 complex

GIT

1

JAK2 phosphorylates tyrosine 285 of PAK1 in response to PRL

Hammer et.al., submitted

PAK1 phosphorylated on Tyr 285 localizes to small paxillin-containing adhesion complexes

Phosphorylation of tyrosine 285 of PAK1 regulates adhesion turnover

GFP

WT

Y3F

Hammer et.al., submitted

invasion

JAK2

collagen IVintegrins

?

?prolactin

DDR

?

PAK1

pY-153

pY-285

pY-201

Filamin ASer2152PRL-R

collagen IV

MMP1 and MMP3 secretion

PAK1

pY-285

PIX

GIT1

motility

adhesion turnover

paxillin

integrins

PAK1 tyrosyl phosphorylation on Y285 is highest in breast carcinoma

Hammer et.al., submitted

AcknowledgementsCurrent lab members:Jenny JayPeter OladimejiAlan HammerSaba BareziHamad Yadikar  Rose HenryRebekah SkerlCourtney RuschFormer lab members:Leah Rider, Ph.D.Xiaofeng Zhou, Ph.D. Jing TaoLeslie WebbSneha Laghate

Luis De Las Casas – Dpt. Pathology, UTDaniel J. Lindner, M.D., Ph.D. - Taussig Cancer

Institute, Cleveland Clinic Edward Feener, Ph.D. - Harvard University

NIH/NIDDK R01 DK88127

NIH/NCI R15 CA135378NIH/NIDDK R21 DK074689NIH/NIAID R21, AI05778deArce Memorial Endowment Fund