title: Etk/Bmx regulates proteinase-activated-receptor1 (PAR1) in breast cancer invasion: signaling partners, hierarchy and physiological significance.
abstract: BACKGROUND
  While protease-activated-receptor 1 (PAR(1)) plays a central role in tumor progression, little is known about the cell signaling involved.
  
  
  RESULTS
  We show here the impact of PAR(1) cellular activities using both an orthotopic mouse mammary xenograft and a colorectal-liver metastasis model in vivo, with biochemical analyses in vitro. Large and highly vascularized tumors were generated by cells over-expressing wt hPar1, Y397Z hPar1, with persistent signaling, or Y381A hPar1 mutant constructs. In contrast, cells over-expressing the truncated form of hPar1, which lacks the cytoplasmic tail, developed small or no tumors, similar to cells expressing empty vector or control untreated cells. Antibody array membranes revealed essential hPar1 partners including Etk/Bmx and Shc. PAR(1) activation induces Etk/Bmx and Shc binding to the receptor C-tail to form a complex. Y/A mutations in the PAR(1) C-tail did not prevent Shc-PAR(1) association, but enhanced the number of liver metastases compared with the already increased metastases obtained with wt hPar1. We found that Etk/Bmx first binds via the PH domain to a region of seven residues, located between C378-S384 in PAR(1) C-tail, enabling subsequent Shc association. Importantly, expression of the hPar1-7A mutant form (substituted A, residues 378-384), which is incapable of binding Etk/Bmx, resulted in inhibition of invasion through Matrigel-coated membranes. Similarly, knocking down Etk/Bmx inhibited PAR(1)-induced MDA-MB-435 cell migration. In addition, intact spheroid morphogenesis of MCF10A cells is markedly disrupted by the ectopic expression of wt hPar1. In contrast, the forced expression of the hPar1-7A mutant results in normal ball-shaped spheroids. Thus, by preventing binding of Etk/Bmx to PAR(1) -C-tail, hPar1 oncogenic properties are abrogated.
  
  
  CONCLUSIONS
  This is the first demonstration that a cytoplasmic portion of the PAR(1) C-tail functions as a scaffold site. We identify here essential signaling partners, determine the hierarchy of binding and provide a platform for therapeutic vehicles via definition of the critical PAR(1)-associating region in the breast cancer signaling niche.
authors.affiliation: Department of Oncology, Hadassah-University Hospital, Jerusalem, Israel.
authors.forename: Irit
authors.initials: I
authors.lastname: Cohen
authors.forename: Myriam
authors.initials: M
authors.lastname: Maoz
authors.forename: Hagit
authors.initials: H
authors.lastname: Turm
authors.forename: Sorina
authors.initials: S
authors.lastname: Grisaru-Granovsky
authors.forename: Bella
authors.initials: B
authors.lastname: Maly
authors.forename: Beatrice
authors.initials: B
authors.lastname: Uziely
authors.forename: Einat
authors.initials: E
authors.lastname: Weiss
authors.forename: Rinat
authors.initials: R
authors.lastname: Abramovitch
authors.forename: Eithan
authors.initials: E
authors.lastname: Gross
authors.forename: Oded
authors.initials: O
authors.lastname: Barzilay
authors.forename: Yun
authors.initials: Y
authors.lastname: Qiu
authors.forename: Rachel
authors.initials: R
authors.lastname: Bar-Shavit
mesh_terms: D000595:Amino Acid Sequence; D000818:Animals; D001483:Base Sequence; D001706:Biopsy; D001943:Breast Neoplasms / Q000378:metabolism / Q000473:pathology*; D045744:Cell Line, Tumor; D017931:DNA Primers; D005260:Female; D006801:Humans; D007150:Immunohistochemistry; D008113:Liver Neoplasms / Q000556:secondary; D051379:Mice; D008819:Mice, Nude; D008969:Molecular Sequence Data; D009362:Neoplasm Metastasis; D011505:Protein-Tyrosine Kinases / Q000502:physiology*; D044463:Receptor, PAR-1 / Q000737:chemistry / Q000378:metabolism*; D020133:Reverse Transcriptase Polymerase Chain Reaction; D015398:Signal Transduction
chemical_list: D017931:DNA Primers; D044463:Receptor, PAR-1; C492367:BMX protein, human; D011505:Protein-Tyrosine Kinases