Cx32 exerts anti-apoptotic and pro-tumor effects via the epidermal growth factor receptor pathway in hepatocellular carcinoma

Yuke Xiang,Liang Tao,Qin Wang,Yile Fu,Hui Ge,Hui Ge,Yunquan Guo,Xiyan Wang,Xiyan Wang

doi:10.1186/s13046-019-1142-y

Abstract

BackgroundAbnormal expression or distribution of connexin 32 (Cx32) is associated with hepatocarcinogenesis, but the role of Cx32 and the underlying mechanisms are still unclear.MethodsThe expression level of Cx32 in 96 hepatocellular carcinoma (HCC) specimens was determined using western blotting and immunohistochemistry. The correlation between Cx32 expression and clinicopathological parameters was analyzed. The cell apoptosis rate was examined using flow cytometry and western blotting. The role of Cx32 in the Src kinase and epidermal growth factor receptor (EGFR) signaling pathways was measured by quantitative real-time PCR, western blotting and coimmunoprecipitation (CO-IP). The effect of Cx32 overexpression on the streptonigrin (SN)-induced tumor growth suppression and apoptosis was assessed in nude mice.ResultsOur study showed that overexpressed Cx32 accumulated in the cytoplasm and that Cx32-containing gap junctions (GJs) were nearly absent in HCC specimens. Upregulated Cx32 expression was highly correlated with advanced tumor-node-metastasis (TNM) stage and poor tumor differentiation and was an independent predictive marker for poor prognosis in HCC. Overexpression of Cx32 significantly inhibited SN-induced apoptosis by activating the EGFR signaling pathway in vitro and in vivo. Moreover, the expression levels of Cx32 and EGFR were positively correlated in HCC specimens. The CO-IP experiments demonstrated that Cx32 could bind to Src kinase, and the western blotting results revealed that Cx32 increased the levels of EGFR and p-EGFR by upregulating Src expression.ConclusionThe present study demonstrated that overexpressed and internalized Cx32 was associated with advanced TNM stage and poor tumor differentiation and predicted poor prognosis in HCC. Cx32 facilitated HCC progression by blocking chemotherapy-induced apoptosis in vitro and in vivo via interacting with Src and thus promoting the phosphorylation of EGFR, subsequently activating the EGFR signaling pathway. Cx32 may be a potential biomarker and a new therapeutic target for HCC.

Highlights

Abnormal expression or distribution of connexin 32 (Cx32) is associated with hepatocarcinogenesis, but the role of Cx32 and the underlying mechanisms are still unclear
Cx32 is overexpressed and internalized in hepatocellular carcinoma (HCC) tissues Initially, we evaluated the expression of three major hepatocellular Cx isoforms— Cx32, Cx43 and Cx26—in specimens of human HCC tissues (n = 96), peritumoral tissues (n = 57) and remote normal liver tissues (n = 43) by western blotting
Cx32 was prominently localized in cytomembranes in normal liver tissues (40/42, 95.2%) and peritumoral tissues (85/96, 88.5%), and dense distributions were observed in the junctions of adjacent cells, which were identified as Cx32-constituted gap junction (GJ) plaques

Summary

Introduction

Abnormal expression or distribution of connexin 32 (Cx32) is associated with hepatocarcinogenesis, but the role of Cx32 and the underlying mechanisms are still unclear. During carcinogenesis, GJs generally reduced in number or lost due to decreased expression levels and/ or internalization of Cxs [3]. Restoration of GJ by increasing cytomembrane Cx expression suppresses tumor development [4] and sensitizes cells to radiotherapy/chemotherapy [5]. Cx-containing GJs are believed to suppress tumor progression [6, 7]. Emerging evidence has demonstrated that Cxs per se might play a proper nonchannel role in the development of carcinoma [8] by altering the expression of different genes [9, 10]. The nonjunctional role of Cxs in carcinogenesis remains unclear

Methods

Results

Discussion

Conclusion