Estrogen Receptor b Exerts Tumor Repressive Functions in Human Malignant Pleural Mesothelioma via EGFR Inactivation and Affects Response to Gefitinib Giulia Pinton 1 , Warren Thomas2, Paolo Bellini1, Arcangela Gabriella Manente1, Roberto E. Favoni3, Brian J. Harvey2, Luciano Mutti4., Laura Moro1*. 1 Department of Chemical, Food, Pharmaceutical and Pharmacological Sciences, Drug and Food Biotechnology Center, University of Piemonte Orientale A. Avogadro, Novara, Italy, 2 Department of Molecular Medicine, Royal College of Surgeons in Ireland and Education and Research Centre, Beaumont Hospital, Dublin, Ireland, 3 Laboratory of Experimental Pharmacology, National Cancer Institute, Genoa, Italy, 4 Department of Medicine, Local Health Unit 11, Vercelli, Italy Abstract Background: The role of estrogen and estrogen receptors in oncogenesis has been investigated in various malignancies. Recently our group identified estrogen receptor beta (ERb) expression as an independent prognostic factor in the progression of human Malignant Pleural Mesothelioma (MMe), but the underlying mechanism by which ERb expression in tumors determines clinical outcome remains largely unknown. This study is aimed at investigating the molecular mechanisms of ERb action in MMe cells and disclosing the potential translational implications of these results. Methods: We modulated ERb expression in REN and MSTO-211H MMe cell lines and evaluated cell proliferation and EGF receptor (EGFR) activation. Results: Our data indicate that ERb knockdown in ER positive cells confers a more invasive phenotype, increases anchorage independent proliferation and elevates the constitutive activation of EGFR-coupled signal transduction pathways. Conversely, re-expression of ERb in ER negative cells confers a more epithelioid phenotype, decreases their capacity for anchorage independent growth and down-modulates proliferative signal transduction pathways. We identify a physical interaction between ERb, EGFR and caveolin 1 that results in an altered internalization and in a selective reduced activation of EGFR-coupled signaling, when ERb is over-expressed. We also demonstrate that differential expression of ERb influences MMe tumor cell responsiveness to the therapeutic agent: Gefitinib. Conclusions: This study describes a role for ERb in the modulation of cell proliferation and EGFR activation and provides a rationale to facilitate the targeting of a subgroup of MMe patients who would benefit most from therapy with Gefitinib alone or in combination with Akt inhibitors. Citation: Pinton G, Thomas W, Bellini P, Manente AG, Favoni RE, et al. (2010) Estrogen Receptor b Exerts Tumor Repressive Functions in Human Malignant Pleural Mesothelioma via EGFR Inactivation and Affects Response to Gefitinib. PLoS ONE 5(11): e14110. doi:10.1371/journal.pone.0014110 Editor: Joseph Alan Bauer, Bauer Research Foundation, United States of America Received July 6, 2010; Accepted October 22, 2010; Published November 29, 2010 Copyright: 2010 Pinton et al. This is an open-access article distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited. Funding: This work was supported by MARF (Mesothelioma Applied Research Foundation), Fondazione Buzzi Unicem and Higher Education Authority of Ireland (PRTLI4) through the National Biophotonics and Imaging Platform. G. Pinton was supported by a PhD fellowship from Progetto Lagrange (Cassa di Risparmio di Torino). The funders had no role in study design, data collection and analysis, decision to publish, or preparation of the manuscript. Competing Interests: The authors have declared that no competing interests exist. * E-mail: moro@pharm.unipmn.it . These authors contributed equally to this work. Introduction Malignant pleural mesothelioma (MMe) is a highly aggressive tumor, most often associated with asbestos exposure, although a role for SV40 and genetic susceptibility have also been proposed [1]. The delayed clinical diagnosis of this tumor is due to the slow progression of the malignancy [2]. The clinical prognosis is generally poor, with a reported median survival from presentation of 9–12 months. Several clinical prognostic factors have been tentatively correlated to patient survival; these include histological type (epithelioid, sarcomatoid or biphasic) and tumor grade [3,4]. We recently published data demonstrating that estrogen receptor beta (ERb) is linked with better prognosis in MMe patients and is likely to act as tumor repressor [5]. Estrogens exert their biological effects through two distinct receptors: ERa and ERb. The ERs are transcribed from two different genes and display specific tissue expression patterns as well as distinct ligand specificities even though both bind the most biologically active estrogen, 17b-estradiol [6]. This is confirmed by the fact that mice lacking ERb (bER KO) display a very different phenotype to those devoid of ERa (aERKO) [7–11]. In addition to ligand binding ERb activity and sub-cellular distribution is also regulated through its post-translational modification [12,13]. Evidences accumulated over the past decade describe a cross-talk between ERs and EGFRs [14]. Work in this area has established a requirement of ERs for some EGFR actions [15,16]. Recent findings suggest the important role of EGFR (or similar receptors) for estrogen signaling from the membrane in breast cancer. It has PLoS ONE | www.plosone.org 1 November 2010 | Volume 5 | Issue 11 | e14110