Inhibition of Ca(2+)-activated Cl(-) channel ANO1/TMEM16A expression suppresses tumor growth and invasiveness in human prostate carcinoma

Abstract

The etiology of prostatic adenocarcinoma remains unclear. Prostate cancer cells of varying metastatic potential and apoptotic resistance show altered expression of plasma membrane ion channels and unbalanced Ca2+ homeostasis. Ca(2+)-activated Cl(-) channels (CaCCs) are robustly expressed in epithelial cells and function to regulate epithelial secretion and cell volume for maintenance of ion and tissue homeostasis in proliferation, differentiation and apoptosis. ANO1/TMEM16A was recently identified as a CaCC, and it is of interest to determine whether ANO1 plays a role in development and metastasis of prostate carcinoma. Here we show that ANO1 mRNA and protein are highly expressed in human metastatic prostate cancer LNCaP and PC-3 cells by quantitative analysis of real-time PCR and Western blot. These findings were confirmed by whole-cell patch clamp recording of LNCaP and PC-3 cells with increased current density of ANO1 channels. Immunohistochemistry staining further revealed overexpression of ANO1 in human prostate cancer tissues, which correlated with the clinical TNM stage and Gleason score. Experiments with small hairpin RNAs (shRNAs) targeting human ANO1 resulted in a significant reduction of proliferation, metastasis and invasion of PC-3 cells using WST-8, colony formation, wound-healing and transwell assays. Moreover, intratumoral injection of ANO1 shRNA completely inhibited established tumor growth and survival in orthotopic nude mice implanted with PC-3 cells. Our findings provide compelling evidence that upregulation of CaCC ANO1 is involved in the proliferation, progression and pathogenesis of metastatic prostate cancer. Membrane ANO1 protein may therefore serve as a biomarker, and inhibition of overexpressed ANO1 has potential for use in prostate cancer therapy.