Downregulation of P-cadherin expression in hepatocellular carcinoma induces tumorigenicity

Abstract

P-cadherin is a major contributor to cell-cell adhesion in epithelial tissues, playing pivotal roles in important morphogenetic and differentiation processes and in maintaining tissue integrity and homeostasis. Alterations of P-cadherin expression have been observed during the progression of several carcinomas where it appears to act as tumor suppressive or oncogenic in a context-dependent manner. Here, we found a significant downregulation of P-cadherin in hepatocellular carcinoma (HCC) cell lines and tissues compared to primary human hepatocytes and non-malignant liver tissues. Combined immunohistochemical analysis of a tissue microarray containing matched pairs of HCC tissue and corresponding non-tumorous liver tissue of 69 patients confirmed reduced P-cadherin expression in more than half of the cases. In 35 human HCC tissues, the P-cadherin immunosignal was completely lost which correlated with tumor staging and proliferation. Also in vitro, P-cadherin suppression in HCC cells via siRNA induced proliferation compared to cells transfected with control-siRNA. In summary, downregulation of P-cadherin expression appears to induce tumorigenicity in HCC. Therefore, P-cadherin expression may serve as a prognostic marker and therapeutic target of this highly aggressive tumor.

Keywords: P-cadherin; hepatocellular carcinoma; proliferation; tumor staging.

Figure 1

P-cadherin expression in HCC cells and tissues. A. Quantitative realtime RT-PCR of P-cadherin expression in primary human hepatocytes (PHH) of three different donors and four HCC cell lines (HepG2, PLC, Huh7 and Hep3B) (*: P < 0.05 compared to PHH); B. P-cadherin expression in human HCC tissues and corresponding non-tumorous (NT) liver tissue (*: P < 0.05 compared to NT); C. Representative images of strong cytoplasmatic P-cadherin staining in non-tumorous tissue and missing P-cadherin expression in corresponding HCC tissue (upper panel). Percentage of tissues with and without detectable P-cadherin immunosignal (lower panel); D. Percentage of Ki67-positice cells in HCC tissues with and without positive P-cadherin immunosignal (*: P < 0.05).

Figure 2

Inhibition of P-cadherin expression in HCC cells with siRNA in vitro. A. Quantitative realtime RT-PCR analysis of P-cadherin expression in HCC cells transiently transfected with siRNA directed against P-cadherin (Pcad-siRNA) or control cells transfected with scrambled RNA (ctrl siRNA) (*: P < 0.05); B. Analysis of proliferation of HCC cells with and without P-cadherin via siRNA over time (day 1, day 2 and 3) using a WST proliferation assay (*: P < 0.05); C. Analysis of E-cadherin, N-cadherin and vimentin expression in HCC cells transfected with siRNA directed against P-cadherin (Pcad-siRNA) compared to control cells transfected with scrambled siRNA (ctrl siRNA). qPCR analysis without cDNA (ctr) was performed as negative control.