Aberrant and function of protease activated receptor 4 (PAR4) in human colon cancer cells

AACR Annual Meeting-- Apr 14-18, 2007; Los Angeles, CA 4379 The thrombin receptor, Proteinase-activated receptor-4 (PAR4, belongs to a new G protein-coupled receptor subfamily activated by serine proteinases. The presence and role of PAR4 in human gastrointestinal system functions have not been fully investigated. The aim of this study is to investigate the expression and the potential role of the thrombin receptor PAR4 in human colon cancer. The presence of PAR4 in colon cancer cell lines was assessed by immunofluorescence detection and RT-PCR, and its presence in resected human colonic tumors and normal colon was assessed by immunohistochemistry. By using the serine protease thrombin and the specific receptor-activating peptide (AYPGKF-NH) AP4, we tested the functionality of PAR-4 on calcium mobilization and cell proliferation in human colon cancer cells HT29. The following results were obtained: 1) immunofluorescence studies using polyclonal anti-PAR4 antibodies revealed expression of PAR-4 in HT29 cells . Immunohistochemical studies on paraffin embedded section of human colonic cancer showed a strong staining with the PAR4 antibody in some dysplastic areas and cancers. In contrast no staining was observed in normal human colonic epithelium; 2) RT-PCR analysis showed expression of PAR-4 mRNA in most human colon cancer cell lines (10 out of 14). In contrast, no mRNA was detected in normal epithelial cells from human colon; 3) microspectrofluorimetry using Fura 2-loaded cells demonstrated a prompt increase in intracellular calcium ([Ca2+]i) transients in HT29 in response to thrombin (10 nM) or AP4 (10-200 µM) challenges; 4) HT-29 cells exhibited dramatic mitogenic responses when grown in serum-deprived media supplemented with thrombin or AP4. This stimulatory effect on cell proliferation was observed also in several other human colon cancer cell lines; 5) we also show here that the PAR4-dependent increase in HT29 cell proliferation isdependent on src activity and transactivation of epidermal growth factorreceptor (EGFR). Indeed, PAR4-mediated cell proliferation wasblocked by the src inhibitor PP2, the metalloproteinase inhibitor batimastat and the specificEGFR tyrosine kinase inhibitors AG1478 and PD 168393. In conclusion thrombin exerts proliferative responses in colon cancer cells through aberrant expression of PAR4.