Etk/Bmx Regulates Proteinase-Activated-Receptor1 (PAR1) in Breast Cancer Invasion: Signaling Partners, Hierarchy and Physiological Significance

Abstract

BackgroundWhile protease-activated-receptor 1 (PAR1) plays a central role in tumor progression, little is known about the cell signaling involved.Methodology/Principal FindingsWe show here the impact of PAR1 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. PAR1 activation induces Etk/Bmx and Shc binding to the receptor C-tail to form a complex. Y/A mutations in the PAR1 C-tail did not prevent Shc-PAR1 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 PAR1 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 PAR1-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 PAR1 -C-tail, hPar1 oncogenic properties are abrogated.Conclusions/SignificanceThis is the first demonstration that a cytoplasmic portion of the PAR1 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 PAR1 -associating region in the breast cancer signaling niche.

Deletion of the Endothelial Bmx Tyrosine Kinase Decreases Tumor Angiogenesis and Growth

“…Although the abrogation of tumor cell dissemination may be secondary to the decreased tumor growth or vascularization, previous data have implicated Bmx signals in cell migration (32)(33)(34). Bmx promotes integrin signaling via binding to focal adhesion kinase (FAK; ref.…”

A functional siRNA screen identifies genes modulating angiotensin II-mediated EGFR transactivation

“…Tec family kinases have been directly implicated in GPCR signalling, where G q and/ or bc subunits of the heterotrimeric G protein complex can bind to and activate the kinase (Bence et al, 1997;LanghansRajasekaran et al, 1995;Tsukada et al, 1994). BMX, through its PH domain, binds to the thrombin-activated GPCR, PAR1, and this is a requirement for association with Shc and oncogenic activity (Cohen et al, 2010). Together, this evidence suggests that Tec family kinases can modulate GPCR function by acting as signalling scaffolds that allow recruitment of other binding partners and effector proteins, or as second messengers that modulate downstream signalling activities.…”

Endothelial Bmx tyrosine kinase activity is essential for myocardial hypertrophy and remodeling

“…However, Bmx deletion does not result in any obvious developmental phenotype in mice (5), suggesting that it has a redundant function during embryogenesis. In contrast, Bmx has been shown to be important in a variety of pathological states, including tumor growth (6)(7)(8), and its overexpression promotes ischemia-induced arteriogenesis and inflammatory angiogenesis (9).…”