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Molecular pathways involved in prolactin-dependent JAK2/PAK1 action:

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.

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Molecular pathways involved in prolactin-dependent JAK2/PAK1 action:

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  1. 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

  2. PROLACTIN: the forgotten hormone of breast cancer PROLACTIN PITUTARY Endocrine Blood vessels PROLACTIN STATs and other targets Target genes normal mammary gland development, differentiation and survival

  3. PROLACTIN: the forgotten hormone of breast cancer PROLACTIN PITUTARY Endocrine Autocrine/paracrine Blood vessels PROLACTIN PROLACTIN STATs and other targets STATs and other targets Target genes; hPRL gene normal mammary gland development, differentiation and survival Breast tumorogenesis

  4. Prolactin in breast cancer (1) an autocrine/ paracrine loop for PRL (Clevenger et. al., 1995; Ginsburg et.al., 1995) (2) Prolactin receptoris 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)

  5. PROLACTIN STATs IRS ? PI3K FAK/Src MAPK Grb2 Prolactin signaling pathways CYTOKINE RECEPTOR P JAK2 Target Genes P P P AKT P P SHC BREAST CANCER: Proliferation Survival Metastasis

  6. PROLACTIN STATs IRS PAK1 PI3K FAK/Src MAPK Grb2 Prolactin signaling pathways CYTOKINE RECEPTOR P JAK2 Target Genes P P P AKT P P SHC BREAST CANCER: Proliferation Survival Metastasis

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

  8. 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).

  9. 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

  10. Tyrosine 55 131 142 153 201 270 285 330 334 346 429 441 464 474 SequenceYRSI YNSK YMSF YNSS YTRS YTRF YTAM YLDS YLVG YLAG YWMA YGPK YLNE YLIA Schematic representation of PAK1

  11. JAK2 phosphorylates PAK1 at Tyr(s) 153, 201 and 285 Rider et al., JBC, 2007

  12. Prolactin activates PAK1 +/-PRL SDS-PAGE T47D MCF-7 TMX2-28 cells IP PAK1 kinase assay nitrocellulose [-32P]ATP H4 histone Hammer et al., Mol.Endo, 2013

  13. PRL stimulates kinase activity of PAK1 and PAK1 ability to form protein-protein interaction Hammer and Diakonova, in press

  14. 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

  15. PRL-activated PAK1 stimulates cell migration PRL Boyden chamber assay Wounding assay Hammer&Rider, 2013

  16. Filamin A is actin-binding protein M2 A7 • Dimerizes at C-terminus • Most binding partners bind at C-terminus • Cross-links actin • Required for cell motility PAK1 phosphorylates Filamin A at Ser 2152. Filamin A stimulates PAK1 kinase activity by a positive feedback loop. Vadlamudi et.al., 2002 Cunningham et. al., 1997

  17. JAK2-dependent phosphorylation of PAK1 increase Filamin A Ser2152 phosphorylation Hammer & Rider et al., 2013

  18. prolactin pY-201 pY-153 PRL-R PAK1 pY-285 JAK2 integrins Filamin A pSer2152 cell migration

  19. Maximal invasion of TMX2-28 cells in response to PRL requires tyrosyl phosphorylation of PAK1 ECM PRL Oladimeji & Rider et al., 2013

  20. Collagens and PRL-dependent tyrosyl phosphorylatoin of PAK1 regulates transcription and secretion of MMP1 and 2 Col IV Col I (by MMP1 and 3)

  21. Filamin A Ser2152 pY-201 pY-153 PAK1 pY-285 b integrins collagen IV prolactin ? ? ? PRL-R DDR JAK2 collagen IV cell migration JNK ERK p38 MMP1 MMP3 AP-1 c-fos Jun MMP1 and MMP3 secretion invasion

  22. PAK1 tyrosyl phosphorylation regulates cell spreading and adhesion Hammer et.al., submitted

  23. PAK1 tyrosyl phosphorylation contributes to PAK1 activity

  24. PIX PAK1 GIT1 PAK1 PAK1 P Paxillin FAK FAK Talin Talin P S273 ECM Focal Complex Nascent Adhesion

  25. PIX PAK1 GIT1 PAK1 Adhesion Turnover PAK1 P Paxillin FAK FAK Talin Talin P S273 ECM Focal Complex Nascent Adhesion

  26. PRL PRL-R PRL-R ? PIX PAK1 GIT1 P P PAK1 P P P JAK2 JAK2 Y285 P P P P Adhesion Turnover PAK1 P Paxillin FAK FAK Talin Talin P S273 ECM Focal Complex Nascent Adhesion

  27. PAK1 tyrosyl phosphorylation regulates binding to βPIX/GIT1 complex GIT1

  28. JAK2 phosphorylates tyrosine 285 of PAK1 in response to PRL Hammer et.al., submitted

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

  30. Phosphorylation of tyrosine 285 of PAK1 regulates adhesion turnover GFP WT Y3F Hammer et.al., submitted

  31. Filamin A Ser2152 pY-201 pY-153 PAK1 pY-285 b integrins collagen IV prolactin ? ? ? PRL-R DDR JAK2 collagen IV integrins paxillin GIT1 bPIX PAK1 pY-285 MMP1 and MMP3 secretion invasion adhesion turnover motility

  32. PAK1 tyrosyl phosphorylation on Y285 is highest in breast carcinoma Hammer et.al., submitted

  33. 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

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