Protease Activated Receptor-1 (PAR-1) Impedes Tumor Progression

Activation of cell signaling by thrombin through Protease Activated Receptor-1 (PAR-1) represents one important interface between blood coagulation and cell activation in response to injury and inflammation. In the context of cancer, PAR-1 has been suggested to promote tumor growth through mechanisms coupled to tumor cell proliferation, tumor cell migration, and the development of a supportive tumor stroma. Consistent with this view, both tumor cells and stromal cells express high levels of PAR-1, and elevated PAR-1 expression has been correlated with a poor prognosis across several tumor types. In the current studies, we tested the hypothesis that PAR-1 is a critical driver of tumorigenesis and tumor growth using murine models of genetically-induced prostate and intestinal tumor growth. To define the role of PAR-1 in prostate tumor progression, we interbred mice expressing the TRAMP transgene (transgenic adenocarcinoma of the mouse prostate; SV40 Large T antigen under the control of a probasin promoter) to PAR-1-deficient mice (PAR-1^-/-) in order to generate male TRAMP mice with and without PAR-1 expression for detailed analyses of prostate tumor growth. Surprisingly, prostate tumors harvested from PAR-1^-/- mice were significantly larger than those harvested from PAR-1^+/+ mice. In order to begin to address the PAR-1 expressing cellular compartments responsible for prostate tumor inhibition, we subcutaneously inoculated immunocompetent C57Bl/6-derived PAR-1^-/- and control mice with tumor cells derived from a C57Bl/6 TRAMP mouse. TRAMP-derived tumors grew indistinguishably in PAR-1^-/- and control mice, suggesting that stromal-cell associated PAR-1 is dispensable for prostate tumor growth. We next tested the effect of tumor cell-intrinsic inhibition of PAR-1 in TRAMP tumor cells by viral transduction with a construct containing an shRNA against murine PAR-1 in parallel to a non-specific shRNA construct. Diminishing tumor cell-associated PAR-1 expression resulted in significantly more rapid tumor growth in vivo. In order to better define the role of tumor cell-intrinsic PAR-1 we harvested TRAMP tumor cells from a PAR-1 deficient mouse and grew these cells in vitro. We transduced these PAR-1-deficient prostate tumor cells with viral vectors conferring expression of WT murine PAR-1 (PAR-1⁺), a PAR-1 mutant lacking the thrombin cleavage (R41A mutant) or empty vector (PAR-1^-). PAR-1^- cells grew robustly and similarly to the parental cells in vitro with a doubling time of approximately 48 hours. Cells expressing the R41A mutant PAR-1 also grew robustly and similarly to PAR-1 deficient cells. However, PAR-1⁺ cells failed to show any signs of cell proliferation over the span of a 4 day observation period. Furthermore, PAR-1 expression dramatically altered the ability of TRAMP cells to demonstrate signs of cell spreading as measured by the frequency of pseudopodia per cell. As a means of determining the role of PAR-1 in tumorigenesis and tumor growth in another spontaneously occurring setting, we interbred PAR-1^-/- mice with APC^Min/+ mice genetically predisposed to intestinal adenoma formation due to loss of heterozygosity of the tumor suppressor adenomatous polyposis coli gene. Blinded quantitative histological analyses of the intestinal tracts of PAR-1^-/- and PAR-1^+/+ APC^Min/+ mice revealed that PAR-1 deficiency resulted in a significant 2-fold increase in the number of adenomas observed. Furthermore, the adenomas observed in PAR-1^-/- mice were significantly larger based on morphometric analyses of adenoma surface area in histological sections. In sum, these data demonstrate a surprising and unexpected role for PAR-1 in the inhibition of tumor growth in the context of two distinct tumor types.