Aberrant expression of proteinase‐activated receptor 4 promotes colon cancer cell proliferation through a persistent signaling that involves Src and ErbB‐2 kinase

Abstract

Thrombin is now recognized as an important factor in many cancers. Here, we examined the expression and role of the recently discovered thrombin receptor PAR4, in human colon cancer cells. PAR4 mRNA was found in 10 out of 14 (71%) human colon cancer cell lines tested but not in epithelial cells isolated from normal human colon. This finding is in line with immunostaining results of PAR4 in human colon tumors and its absence in normal human colonic mucosa. Investigation of the functional significance of the aberrant expression of PAR4 in colon cancer cells revealed (i) a prompt increase in intracellular calcium concentration on challenge with PAR4-specific agonist AP4 (100 lM) and (ii) marked mitogenic response (2.5-fold increase in cell number) in a dose-dependent manner on treatment with AP4 (0.1-300 lM). Analysis of the signaling pathways downstream of PAR4 activation in HT29 cells showed (i) a sustained phosphorylation of extracellular signal-related kinase 1/2 (ERK1/2) and (ii) the involvement of epidermal growth factor receptor B-2 (ErbB-2) but not of epidermal growth factor receptor in PAR4-induced mitogen-activated protein kinase activation. Tyrphostin AG1478, the ErbB inhibitor, reversed the action of AP4 on ERK1/2 and ErbB-2 phosphorylation and HT29 cell growth. Finally, the Src inhibitor PP2 abrogated ErbB-2 and ERK phosphorylation and HT29 cell proliferation, suggesting the essential role of Src activity in PAR4-induced phosphorylation of ErbB-2. These data highlight the role of PAR4 as a new important player in the control of colon tumors and underline the critical role of ErbB-2 transactivation. ' 2008 Wiley-Liss, Inc.Key words: colon cancer; thrombin; protease; receptor /PAR4; signal transduction; proliferation; ErbB-2; EGFR The progression from normal colonic mucosa to malignant tumor is a multistep process whereby genetic alterations in prooncogenes and/or tumor suppressor genes can lead to aberrant growth. 1,2 Proteases have long been associated with colon cancer progression because of their ability to degrade extracellular matrices, which facilitates invasion and metastasis. 3 However, recent studies have shown that these enzymes target diverse substrates and favor all steps of tumor evolution. 4 Recently, the traditional view of the role of proteases in tumor growth and progression has significantly changed. Besides their contribution to cancer progression by degrading extracellular matrix proteins, it is now clear that a subclass of proteases serve as signal molecules controlling cell functions through specific membrane receptors, the proteaseactivated receptors (PARs). 5,6 This new class of receptors belongs to the superfamily of the serpentine G protein-coupled receptors (GPCRs). The PARs are activated by low subnanomolar concentrations of serine proteases that cleave a single peptide bond in the N-terminal extracellular domain of the receptor. The resulting new N-terminal domain serves as a receptor agonist. 5,6 This intramolecular ligand binds to the core of the receptor and initiates sig...

Protease-activated receptors in cancer: A systematic review

“…The expression of PAR1 has been correlated with the malignant phenotype. For PAR1, a role in the progression of epithelial tumors, including breast (75,82,94,97,98), colon (76,99), kidney (100), pulmonary tumor (101), melanoma (102) and hepatocellular carcinoma (80,103) has been shown.…”

Kallikrein-Related Peptidase 14 Acts on Proteinase-Activated Receptor 2 to Induce Signaling Pathway in Colon Cancer Cells

“…Indeed, we previously demonstrated that in colonic tumors, trypsin acting through up-regulated PAR-2 and thrombin acting through aberrantly expressed PAR-1 and PAR-4 are very robust growth factors that control mitogen-activating protein kinase (MAPK) activation and subsequent cell proliferation and migration of human colon cancer cells. 8,[11][12][13] Furthermore, PARs participate in cell invasion and metastasis of many cancers. 7,14,15 However, despite an intense research to discover novel PAR activators, 4,5,16 -18 the endogenous enzymes responsible for activating PARs in colon cancer remain unknown.…”

Down-regulation of Protease-activated Receptor 4 in Lung Adenocarcinoma is Associated with a More Aggressive Phenotype

“…Positive expression of PAR4 in some tumors have been found in previous studies and is thought to be involved in hepatocellular carcinoma cell migration and colon cancer progression (Kaufmann et al, 2007;Gratio et al, 2009). It was also reported that PAR4 activation contributed to the hematogenous metastasis of melanoma using the knockout mice (Camerer et al, 2004).…”