Etk/Bmx regulates proteinase-activated-receptor1 (PAR1) in breast cancer invasion: signaling partners, hierarchy and physiological significance.

BACKGROUND: While protease-activated-receptor 1 (PAR(1)) plays a central role in tumor progression, little is known about the cell signaling involved. METHODOLOGY/PRINCIPAL FINDINGS: 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/SIGNIFICANCE: 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.

Protease activated receptor-1, PAR1, promotes placenta trophoblast invasion and beta-catenin stabilization.

Despite extensive efforts toward elucidation of the molecular pathway controlling cytotrophoblast (CTB) invasion to the uterine decidua, it remains poorly defined. There are striking similarities between tumor cell invasion and cytotrophoblast implantation to the deciduas whereby the role of Protease Activated Receptors (PARs) and wnt signaling is well recognized. We examine here consequences of modulation of PAR1 and PAR2 expression and function on CTB invasion and beta-catenin stabilization. Toward this end, we utilized a model system of extravillous trophoblast (EVT) organ culture and various placenta cell lines (e.g., JAR and HTR-8/Svneo). Activation of PAR1 induces EVT invasion while hPar1-SiRNA and PAR1 antagonist SCH79797--effectively inhibited it. In parallel, the Wnt inhibitor Dickkopf-1 (Dkk1) similarly inhibited it. Nuclear localization of beta-catenin is seen only after PAR1 activation, and is markedly reduced following the application of hPar1-SiRNA construct and PAR1 antagonist in CTBs. In contrast, PAR2 elicited a low cytoplasmic beta-catenin level as also proliferation and invasion. In the non-activated CTBs in-comparison, beta-catenin appeared limited to the membrane pools. Concomitantly, a temporal regulated pattern of Wnt-4, 5a, 7b, 10a, 10b expression is seen along PAR1 appearance. Enforced expression of Wnt antagonists, Secreted Frizzled Related Proteins; SFRP2 & 5; into HTR-8/Svneo, resulted with a markedly reduced nuclear beta-catenin levels, similar to the effect obtained by hPar1-SiRNA treatment. Identification of PAR1 downstream target/s may nonetheless contribute to the formation of a future platform system for eliciting a firm placenta-uterus interactions and to the definition of late pregnancy outcomes.