Abstract 4104: Protease-activated receptor 2, a KLK14 target in colon cancer cells

Abstract

Abstract Serine proteinases signal through proteinase-activated receptors (PARs). Our studies have implicated PAR1 and PAR4 (thrombin receptors) and PAR2 (trypsin receptor) in human colon cancer growth. However, endogenous activators of PARs in colon tumors are still unknown. Recently, members of the kallikrein-related peptidase (KLK) family have been shown to activate PARs in many cell systems. Thus, our aim was to determine if KLK14, a KLK family member, is expressed in colonic tumors and to see if this KLK can activate PARs in human colon cancer cells. The presence of KLK14 in colon cancer cell lines was assessed by RT-PCR, ELISA and immunofluorescence, and its expression in human colonic tissue was assessed by immunohistochemistry. The effects of recombinant KLK14 in parallel with PAR1/2 agonists (SFLLRN-NH2, an activating peptide for both PAR1 and PAR2; TFLLR-NH2, a selective activating peptide for PAR1 and the selective PAR2 agonists, 2-furoyl-LIGRLO-NH2; SLIGRL-NH2) were assessed for calcium flux, receptor internalization and ERK1/2 phosphorylation in a human colon cancer-derived HT29 cell line that expresses PAR1 and PAR2. We found that: a) KLK14 mRNA (RT-PCR) was present in 16 out of 16 human colon cancer cell lines, b) KLK14 protein is present (ELISA) in HT29 conditioned media and other colon cancer cell lines; c) there was strong staining of KLK14 in the cytoplasmic compartment of HT29 cells and of human colonic tumors (immunofluorescence and immunohistochemistry; paraffin sections) d) KLK14 (0.1 µM) caused prompt increases in intracellular calcium ([Ca2+]i) in HT29 cells (Microspectrofluorimetry with Fura 2/fluo-3). The KLK14-induced Ca2+-flux was abrogated after an initial challenge of the cells with SFLLRN-NH2 (100 µM), which desensitizes PAR1 and PAR2 simultaneously. Desensitization with 2-furoyl-LIGRLO-NH2 (10 µM) a potent PAR2 agonist, attenuated most of the Ca2+ flux induced by KLK14, whereas responses to TFLLR-NH2, the PAR1 agonist, were minimally affected. Consistently, a first challenge with thrombin did not affect KLK14-induced Ca2+ flux. These results strongly suggest that KLK14 activates preferentially PAR2 in HT29 cells. e) KLK14 initiates a dramatic loss of cell surface PAR2 (microscopic analysis) due to its internalization and f) KLK14 induces a rapid (within 5-10 min) and significant ERK1/2 phosphorylation in HT29 cells. This KLK14-PAR signaling pathway may represent a novel therapeutic target for colon tumorigenesis. Note: This abstract was not presented at the AACR 101st Annual Meeting 2010 because the presenter was unable to attend. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 101st Annual Meeting of the American Association for Cancer Research; 2010 Apr 17-21; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2010;70(8 Suppl):Abstract nr 4104.