Loss of EBP50 stimulates EGFR activity to induce EMT phenotypic features in biliary cancer cells

Abstract

Scaffold proteins form multiprotein complexes that are central to the regulation of intracellular signaling. The scaffold protein ezrin-radixin-moesin-binding phosphoprotein 50 (EBP50) is highly expressed at the plasma membrane of normal biliary epithelial cells and binds epidermal growth factor receptor (EGFR), a tyrosine kinase receptor with oncogenic properties. This study investigated EBP50-EGFR interplay in biliary cancer. We report that in a collection of 106 cholangiocarcinomas, EBP50 was delocalized to the cytoplasm of tumor cells in 66% of the cases. Ectopic expression of EBP50 was correlated with the presence of satellite nodules and with the expression of EGFR, which was at the plasma membrane, implying a loss of interaction with EBP50 in these cases. In vitro, loss of interaction between EBP50 and EGFR was mimicked by EBP50 depletion using a small interfering RNA approach in human biliary carcinoma cells co-expressing the two proteins at their plasma membrane, and in which interaction between EBP50 and EGFR was validated. EBP50 depletion caused an increase in EGFR expression at their surface, and a sustained activation of the receptor and of its downstream effectors (extracellular signal-regulated kinase 1/2, signal transducer and activator of transcription 3) in both basal and EGF-stimulated conditions. Cells lacking EBP50 showed epithelial-to-mesenchymal transition-associated features, including reduction in E-cadherin and cytokeratin-19 expression, induction of S100A4 and of the E-cadherin transcriptional repressor, Slug, and loss of cell polarity. Accordingly, depletion of EBP50 induced the disruption of adherens junctional complexes, the development of lamellipodia structures and the subsequent acquisition of motility properties. All these phenotypic changes were prevented upon inhibition of EGFR tyrosine kinase by gefitinib. These findings indicate that loss of EBP50 at the plasma membrane in tumor cells may contribute to biliary carcinogenesis through EGFR activation.

Epithelial-mesenchymal transition in cholangiocarcinoma: From clinical evidence to regulatory networks

“…In EGFstimulated CCA cells, EMT-TFs (SLUG and ZEB1) and mesenchymal markers (N-cadherin and α-SMA) were induced, favoring cell invasiveness through cytoskeleton remodeling [29,89]. We obtained similar results after down-regulating the PDZ scaffold protein EBP50 which led to the implementation of an EMT program through EGFR activation with the subsequent 13 acquisition of invasive and migratory properties by CCA cells [86]. Besides EBP50, SOX4 transcription factor has been described as a potent inducer of EMT in CCA cells possibly through modulation of EGFR expression [90].…”

“…Epidermal Growth Factor Receptor (EGFR) expression and signaling are strongly associated with CCA development and progression [70,[86][87][88]. We recently showed that ectopic cytoplasmic localization of E-cadherin is correlated with EGFR overexpression in human iCCA and pCCA [29].…”

Reduced EBP50 expression levels are correlated with unfavorable clinicopathological features of extrahepatic bile duct carcinoma and promote the proliferation and migration of QBC939 cells

“…EBP50 is considered to indirectly or directly affect cancer behaviors through the modification of signal transduction (12,14,15,(25)(26)(27), and inhibits EGF-induced breast cancer cell proliferation by blocking the phosphorylation of EGFR (25). This inhibitory role of EBP50 in the EGF/EGFR signaling pathway has also been demonstrated in biliary cancer cells, where the loss of EBP50 promotes EGFR activity and induces the appearance of EMT phenotypic features (15). Regarding the underlying molecular mechanism by which EBP50 may inhibit the proliferation and migration of CC cells, it is possible that EBP50 interacts with signaling molecules that are important in migration and proliferation, and may regulate the functions of its binding partners.…”

“…However, Peng et al (14) demonstrated that EBP50 executed a tumor-suppressor function via the β-catenin/E-cadherin signaling pathway in human hepatocellular cancer. Similarly, in biliary cancer cells, it has been observed that the downregulation of EBP50 stimulates epidermal growth factor receptor (EGFR) activity and is able to induce features of an epithelial-mesenchymal transition (EMT) phenotype (15), suggesting a tumor-suppressing role for EBP50. Therefore, the Reduced EBP50 expression levels are correlated with unfavorable clinicopathological features of extrahepatic bile duct carcinoma and promote the proliferation and migration of QBC939 cells 6 and HUI ZHANG role of EBP50 in liver and biliary-associated cancer has yet to be elucidated.…”

E-cadherin, guardian of liver physiology

“…Ecadherin and EGFR have well documented interactions in the context of cancer [16][17][18]. In CCC cells, E-cadherin colocalizes with EGFR [16], while E-cadherin displays inhibitory activities towards EGFR [18].…”