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

Oncogene volume 31, pages 1376–1388 (2012)Cite this article

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.

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Acknowledgements

We thank Yves Chrétien, UMRS_938, for statistical analysis, Anne-Marie Faussat, IFR65, for flow cytometry experiments and the Nikon Imaging Center, Institut Curie-CNRS Paris, for time-lapse microscopy. Confocal imaging was performed at the Cellular and Molecular Imaging facility of the IFR71-IMTCE (Faculty of Pharmacy, Paris Descartes University, Paris, France). This work was supported by grants from the Institut National du Cancer, Association pour la Recherche sur le Cancer and Fonds CSP (to CH). LF dedicates this work to RB Doctor and JG Fitz.

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Author notes

  1. A Clapéron, N Guedj and M Mergey: These authors contributed equally to this work.

Authors and Affiliations

  1. INSERM, UMRS_938, Paris, France

    A Clapéron, M Mergey, C Desbois-Mouthon, M Boissan, C Housset & L Fouassier

  2. UPMC Univ Paris 06, Paris, France

    A Clapéron, M Mergey, C Desbois-Mouthon, M Boissan, C Housset & L Fouassier

  3. INSERM, UMRS_U773 and AP-HP, Service de Pathologie, Hôpital Beaujon, Clichy, France

    N Guedj & V Paradis

  4. CNRS UMR 144, Institut Curie, Paris, France

    D Vignjevic

  5. AP-HP, Service de Biochimie et Hormonologie, Hôpital Tenon, Paris, France

    M Boissan

  6. INSERM U705, CNRS UMR 8206, Faculté de Pharmacie, Université Paris Descartes, Paris, France

    B Saubaméa

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  1. A Clapéron
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Correspondence to L Fouassier.

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Clapéron, A., Guedj, N., Mergey, M. et al. Loss of EBP50 stimulates EGFR activity to induce EMT phenotypic features in biliary cancer cells. Oncogene 31, 1376–1388 (2012). https://doi.org/10.1038/onc.2011.334

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