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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: the forgotten hormone of breast cancer PROLACTIN PITUTARY Endocrine PROLACTIN STATs and other targets Target genes normal mammary gland development, differentiation and survival PROLACTIN: the forgotten hormone of breast cancer PROLACTIN PITUTARY Endocrine Autocrine/paracrine PROLACTIN STATs and other targets PROLACTIN STATs and other targets Target genes; hPRL gene normal mammary gland development, differentiation and survival Breast tumorogenesis 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 signaling pathways PROLACTIN CYTOKINE RECEPTOR JAK2 P P P STATs P P P IRS PI3K SHC Grb2 FAK/Src ? Target Genes AKT MAPK BREAST CANCER: Proliferation Survival Metastasis Prolactin signaling pathways PROLACTIN CYTOKINE RECEPTOR JAK2 P P P STATs P P P IRS PI3K SHC Grb2 FAK/Src PAK1 Target Genes AKT MAPK BREAST CANCER: Proliferation Survival Metastasis 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 upregulated 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 Nb2 cells ON Dep 10 nM PRL, 10’ +/- 50 mM AG490, 24h IP with aPAK1 SDS-PAGE transfer to NC IB with aPY re-IB with aJAK2 and aPAK1 Rider et al., JBC, 2007 Schematic representation of PAK1 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 JAK2 phosphorylates PAK1 at Tyr(s) 153, 201 and 285 Rider et al., JBC, 2007 Prolactin activates PAK1 T47D +/-PRL IP MCF-7 aPAK1 TMX2-28 cells kinase assay [-32P]ATP H4 histone SDS-PAGE nitrocellulose 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 Metastatic spread to different organs > 90% of mortality from cancer is attributable to metastases, not the primary tumors from which these malignant lesions arise Schroeder er.al., 2012 PRL-activated PAK1 stimulates cell migration PRL Wounding assay Boyden chamber assay Hammer&Rider, 2013 Filamin A is actin-binding protein • Dimerizes at C-terminus • Most binding partners bind at C-terminus • Cross-links actin • Required for cell motility M2 A7 PAK1 phosphorylates Filamin A at Ser 2152. Filamin A stimulates PAK1 kinase activity by a positive feedback loop. Cunningham et. al., 1997 Vadlamudi et.al., 2002 JAK2-dependent phosphorylation of PAK1 increase Filamin A Ser2152 phosphorylation Hammer & Rider et al., 2013 prolactin integrins PRL-R Filamin A JAK2 pY-153 pSer2152 pY-201 PAK1 pY-285 cell migration Maximal invasion of TMX2-28 cells in response to PRL requires tyrosyl phosphorylation of PAK1 ECM PRL Oladimeji & Rider et al., 2013 Collagens and PRL-dependent tyrosyl phosphorylatoin of PAK1 regulates transcription and secretion of MMP1 and 2 Col IV Col I (by MMP1 and 3) integrins prolactin b collagen IV ? Filamin A PRL-R ? Ser2152 ? DDR pY-153 JAK2 pY-201 collagen IV PAK1 pY-285 cell migration ERK p38 JNK AP-1 c-fos Jun MMP1 and MMP3 secretion invasion MMP1 MMP3 PAK1 tyrosyl phosphorylation regulates cell spreading and adhesion Hammer et.al., submitted PAK1 tyrosyl phosphorylation contributes to PAK1 activity PIX PAK1 GIT1 PAK1 P Paxillin FAK Talin S273 P PAK1 FAK Talin ECM Focal Complex Nascent Adhesion PIX PAK1 PAK1 GIT1 PAK1 P Paxillin FAK Talin S273 P Adhesion Turnover FAK Talin ECM Focal Complex Nascent Adhesion PRL-R PRL-R PRL P P P P P P P JAK2 JAK2 P ? PIX PAK1 P PAK1 GIT1 Y285 PAK1 P Paxillin FAK Talin S273 P Adhesion Turnover FAK Talin ECM Focal Complex Nascent Adhesion PAK1 tyrosyl phosphorylation regulates binding to βPIX/GIT1 complex 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 integrins prolactin b collagen IV ? Filamin A PRL-R ? ? Ser2152 DDR pY-153 JAK2 pY-201 collagen IV PAK1 pY-285 integrins GIT1 paxillin bPIX PAK1 pY-285 MMP1 and MMP3 secretion invasion adhesion turnover motility PAK1 tyrosyl phosphorylation on Y285 is highest in breast carcinoma Hammer et.al., submitted Acknowledgements Current lab members: Jenny Jay Peter Oladimeji Alan Hammer Saba Barezi Hamad Yadikar Rose Henry Rebekah Skerl Courtney Rusch Former lab members: Leah Rider, Ph.D. Xiaofeng Zhou, Ph.D. Jing Tao Leslie Webb Sneha Laghate Luis De Las Casas – Dpt. Pathology, UT Daniel J. Lindner, M.D., Ph.D. - Taussig Cancer Institute, Cleveland Clinic Edward Feener, Ph.D. - Harvard University NIH/NIDDK R01 DK88127 NIH/NCI R15 CA135378 NIH/NIDDK R21 DK074689 NIH/NIAID R21, AI05778 deArce Memorial Endowment Fund
