Downregulation of reversion-inducing-cysteine-rich protein with Kazal motifs (RECK) is associated with enhanced expression of matrix metalloproteinases and cholangiocarcinoma metastases

Abstract

Reversion-inducing-cysteine-rich protein with Kazal motifs (RECK) has been implicated in the attenuation of tumor metastasis by negatively regulating metalloproteinase (MMP) levels. RECK gene expression is downregulated in many solid tumors, with this downregulation being associated with poor prognosis. This study evaluated the role of RECK in cholangiocarcinoma (CCA). The expression of RECK, MMP-2, and MMP-9 in paraffin sections of hamster and human CCA specimens was analyzed by immunohistochemistry. Functional analysis of RECK was performed in RECK small interfering (si) RNA knockdown CCA cell lines. The effect of aspirin on RECK status and function was evaluated using Western blotting, gelatin zymography, invasion and proliferation assays, and PhosphoELISArray analysis of Ras downstream mediators. Hamster tissues showed high RECK expression in hyperplastic biliary duct epithelia, low RECK expression in precancerous lesions, and no RECK expression in CCA. In human specimens, RECK was highly expressed in normal biliary cells, whereas intrahepatic CCA showed low levels of expression. Downregulation of RECK was correlated with tumor metastasis (P < 0.01) and shorter patient survival (P < 0.02). RECK expression levels were inversely correlated with MMP-2 and MMP-9 expression (P < 0.05). SiRNA RECK-depleted M139 CCA cells exhibited increased MMP-2/-9 gelatinase activities and invasiveness. Aspirin (500 μM) demonstrated myriad effects in human CCA cell lines, including growth suppression, reduced phosphorylation of Akt/Erk/c-Jun, elevation of RECK expression, inhibition of MMP-2/MMP-9 activity, and enhanced invasiveness. RECK functions as a metastasis suppressor in CCA; upregulation of RECK expression could provide a potential therapy to improve the prognosis of this type of cancer.