Estrogen Receptor β Exerts Tumor Repressive Functions in Human Malignant Pleural Mesothelioma via EGFR Inactivation and Affects Response to Gefitinib

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 Estrogen Receptor b Exerts Tumor Repressive Functions in Human Malignant Pleural Mesothelioma via EGFR Inactivation and Affects Response to Gefitinib Giulia Pinton 0 Warren Thomas 0 Paolo Bellini 0 Arcangela Gabriella Manente 0 Roberto E. Favoni 0 Brian J. Harvey 0 Luciano Mutti 0 Laura Moro 0 Joseph Alan Bauer, Bauer Research Foundation, United States of America 0 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 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. - 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. . These authors contributed equally to this work. 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 912 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) [711]. 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 been shown that a pool of ERa resides in or associates with the plasma membrane and utilizes the membrane EGFR to rapidly signal through various kinase cascades that influence both transcriptional and non-transcriptional actions of estrogen in breast cancer cells [14,17]. Moreover, the activation of ERK1/2 through EGFRs and IGFR changes the phosphorylation state of ERa to modulate receptor localization and transcriptional activity [18,19]. More recently, it has become clear that ERb function can also be modulated by phosphorylation in its N-terminal region, so coupling ERb activity to growth factor signaling [20]. A large number of studies have focused on the expression of growth factor receptors in MMe. EGFR is over-expressed in MMe and this correlates significantly with increased tumor cell proliferation and with the promotion of angiogenesis [21,22]. Despite these evidences two phase II studies with Erlotinib and Gefitinib, two anti-phospho tyrosine kinase EGFR specific molecules, did not show efficacy suggesting that further characteristic apart from EGFR expression could be involved in determining sensitivity to these agents [23,24]. The aim of this study is to achieve a better knowledge on the molecular mechanism by which ERb exerts its tumor repressor effects on MMe progression, in view of potential novel patienttailored therapies. ERb expression in ERs negative MMe cells reduces their growth rate To confirm the tumor repressor role of ERb in the modulation of MMe cell growth, we expressed ERb in the constitutively ERsnegative MSTO-211H MMe cell line, by using a pCXN2 based plasmid expressing ERb. ERb expression conferred a more epithelioid phenotype on the MSTO-211H cells compared to mock transfected cells, characterized by a more cortical actin distribution (Fig. 1B). 48 hours after transfection, total protein extracts were prepared from mock- and ERb -transfected (...truncated)