ERb Function in Mesothelioma Figure 3. ERb over-expression influences EGFR mediated signaling and internalization. A) The graph show the growth curves of mock- and ERb-transfected REN cell treated for 24 and 48 hours with 5 ng/ml of EGF in 2% FBS culture medium. At each time point, the cells were assayed for proliferation. Each value represents mean 6 SD (n = 3). Adjacent to the graph is reported a representative Western blot analysis that documents ERb expression. Tubulin staining indicates equal loading of the proteins. B) Mock- and ERb- transfected REN cells made quiescent for 2 hours were treated with 5 ng/ml of EGF for 5 minutes and detergent extracted. Levels of phosphorylated EGFR, ERK 1/2 MAP kinases and Akt were analyzed by immunoblotting. Membranes were also blotted with antibodies to EGFR, Erk1/2 and Akt to evaluate protein expression. Tubulin was blotted to show equal amount of loading. Western blot analysis with anti ERb antibodies documents its expression in transfected cells. Representative of three separate experiments. C) Evaluation of EGFR internalization was performed by Flow cytometry analysis on wild type and ERb expressing REN cells treated 60 or 120 minutes with 10 ng/ml of human recombinant EGF. Histograms represent percentage of positive cells following incubation with anti-EGFR antibody indicated for each condition 6 SD. Data are representative of three separate experiments. D) Representative immunoprecipitation experiment of membrane associated EGFR performed on mock and ERb over-expressing REN cells, treated 60 or 120 minutes with 10 ng/ml of human recombinant EGF. Membrane was blotted with anti-pY and anti-EGFR antibodies. doi:10.1371/journal.pone.0014110.g003 a greater reliance of the cells upon EGFR-coupled signaling pathways to support proliferation. These data were reinforced by concordant data obtained with Gefitinib treatment of wild type and ERb positive MSTO-211H cells (Fig. 5A). Because EGFR signals through PI3-kinase/Akt and MAP/ERK effectors pathways, phosphorylation of Akt and ERK 1/2 were analyzed in ERb silenced REN and in MSTO-211H cells treated with EGF in the absence or in the presence of Gefitinib. The basal level of phosphorylated EGFR was increased in both cell types upon EGF treatment and this resulted in increased Akt and ERK1/2 phosphorylation. Gefitinib addition abrogated both basal and EGF induced EGFR and ERK1/2 phosphorylation, but only the EGF induced amount of phosphorylated Akt (Fig. 5B). Discussion ERa and ERb act as ligand-regulated transcription factors that positively and negatively regulate gene expression, directly or indirectly, through the activation of protein kinase signaling. Models of action have been described that involve synergism, as well as competition between the two receptors, which is further refined by other transcription factor interactions [32]. ERa and ERb display differential expression profiles in estrogen-responsive tissues [33] and shifts in their expression profile have also been identified in malignant as compared to normal tissue and also through the stages of cancer progression [34]. We have found that ERb is the predominant isoform expressed in the pleural mesothelial cells and patients with ERb over expressing tumors have a better survival [5]. Evidence points to ERb having a significant anti-neoplastic role in MMe but the mechanisms underpinning this role remain to be elucidated. In the present study we transfected the ERb-expressing REN MMe cell line with ERb-specific shRNA to suppress expression. This resulted in the promotion of anchorage-indepen- dent cell growth and transition to a less epithelioid phenotype. The mechanisms responsible for the increased cell growth and the phenotypic shift that correlates with the loss of ERb expression are important in understanding the role of ERb as a tumor suppressor. Conversely, exogenous expression of ERb by the ERb-negative, MSTO-211H MMe cell line resulted in suppression of anchorage- independent cell growth and transition to a more epithelioid phenotype. We previously demonstrated that in vitro ERb over- expression caused a G2/M cell cycle phase arrest of MMe cells, both in a ligand-dependent and -independent manner. The fact that exogenous expression of ERb leads to inhibition of proliferation correlates with in vivo data showing that ERb expression was lost in the more aggressive sarcomatoid forms of the malignancy. In this present study we found that MMe cells silenced or constitutively devoid of ERb expression also display a more aggressive phenotype, with the enhanced formation of foci when cultured in vitro and the development of more abundant colonies when cultured in soft agar. The modulation of cell cycle regulating proteins through ERb is compatible with rapidly induced signaling and ablation of ERb impacts upon the activation but not the expression of multiple signaling intermediates in the MMe cells including Akt and Erk1/2. Cross-talk between ERs and growth factor receptor-mediated pathways at the plasma membrane has been described [35–37] and functional interactions between ERb and the epidermal growth factor receptor (EGFR) is documented [20]. Over expression of EGFR has been detected in up to 68% of MMe tumors, however, the EGFR expression level is itself not a good prognostic indicator. In the present study we investigated the interaction between EGFR and ERb in MMe cells. In cells which express high levels of ERs, ERb but not ERa constitutively co-localizes with EGFR in caveolin 1 enriched regions. This clustering interferes with EGFR phosphorylation in response to its ligand, and also results in delayed internalization of the receptor and activation of coupled signaling cascades following stimulation. As a consequence, ERb over-expressing cells are insensitive to treatment with the EGFR inhibitor, Gefitinib, while cells silenced in ERb expression display basal EGFR phosphorylation and are more sensitive to Gefitinib. Our data give a possible explanation for the inefficacy of EGFR inhibitors in phase II clinical trials for ERb positive epithelioid MMe patients and opens the possibility of a more successful employment of these drugs in more aggressive, ERb negative, tumors either as a single agent or in combination with Akt inhibitors. Materials and Methods Reagents and antibodies The monoclonal antibody specific for a-tubulin and the polyclonal antibodies for ERa,ERb, EGFR, caveolin-1, ERK1/2 MAP kinase and cyclin D1 were from Santa Cruz Biotechnology (Santa Cruz, CA). The monoclonal antibody specific for Akt and the phosphor- ylation site-specific polyclonal antibodies for ERK1 (pThr202 and pTyr204), ERK2 (pThr185 and pTyr187) MAP kinases, and Akt (pSer473 and pThr308) were from Cell Signaling Technology (Beverly, MA). The polyclonal antibodies for EGFR and ERb, used in immunofluorescence analysis were obtained from Calbiochem (Darmstadt, Germany) and Zymed-Invitrogen (Carlsbad, CA), respectively. Protein A-Sepharose and ECL were from Amersham Pharmacia Biotech (Uppsala, Sweden). Nitrocellulose membranes and protein assay kits were from Bio-Rad (Hercules, CA). The polyclonal phosphorylation site-specific antibody for EGFR (pTyr1086), anti-mouse and anti-rabbit IgG horseradish peroxidase conjugated antibodies, human recombinant EGF and all other chemical reagents unless otherwise specified were from Sigma- Aldrich (St Louis, MO). All reagents were of analytical grade. Culture media, sera, antibiotics, and LipofectAMINE were from Invitrogen (Carlsbad, CA). Gefitinib is an EGFR inhibitor also used clinically as a chemotherapeutic agent and it is marketed by AstraZeneca. PLoS ONE | www.plosone.org 6 November 2010 | Volume 5 | Issue 11 | e14110