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 charac- teristic 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 patient- tailored therapies. Results 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 ERs- negative 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 cells. Equal amounts of protein from these cell extracts were Western blotted and probed with ERb, phospho-EGFR, EGFR, phospho- Akt, Akt, phospho-ERK1/2, ERK1/2 and cyclin D1-specific antibodies; tubulin was added to confirm equal loading (Fig. 1A). ERb protein expression was not detectable by immunoblot in mock transfected cells, whereas it was in transfected cells. Western blot analysis confirmed the efficacy of ERb expression to down modulate EGFR, Akt and ERK1/2 phosphorylation without a change in the total abundance of these proteins expression, while it resulted in cyclin D1 protein reduction (Fig. 1A). Consistent with our published data [5], ERb expression exerted at any considered time a significant (p,0.05) suppressive effect on MSTO-211H cell proliferation (Fig. 1C), without inducing apoptosis (data not shown); we believe that in these cells PI3K/Akt could act in concert with MAP kinase signaling to modulate cyclin D1 expression and cell cycle progression. Here we found that ERb expression significantly reduced the number and the size of colonies that MSTO-211H cells formed when cultured for 7 days in soft agar. When colonies of more than 15 cells were considered about 50% of reduction was seen in ERb expressing cells (Fig. 1D). ERb silencing promotes MMe cell proliferation We tested whether the suppression of ERb expression could influence the rate of MMe cell proliferation. We previously established that REN cells express moderate levels of ERb [5]. REN cells were transfected with an ERb-specific shRNA (shRNA- ERb) to suppress expression of the receptor. 48 hours after transfection, total protein extracts were prepared from mock- or ERb Function in Mesothelioma shRNA-ERb-transfected cells. Equal amounts of protein from these cell extracts were Western blotted and probed with ERb, phospho-EGFR, EGFR, phospho-Akt, Akt, phospho-ERK1/2, ERK1/2 and cyclin D1-specific antibodies; tubulin was added to confirm equal loading (Fig. 2A). Western blot analysis confirmed the efficacy of the ERb-specific shRNA in suppressing expression of the protein. Silencing of ERb expression in REN cells resulted in increased EGFR, Akt and ERK1/2 phosphorylation without a change in the total abundance of the proteins. However, the abundance of cyclin D1 protein was elevated in cells suppressed in ERb expression. Phase contrast microscopy imaging of cells grown on a solid substrate (Fig. 2B) revealed that silencing of ERb resulted in the loss of contact inhibition by the REN cells, which allowed them to form dense foci rather than a confluent monolayer. The cells were fixed and stained with phalloidin- TRITC to discriminate changes in the actin cytoskeleton of the REN cells. Suppression of ERb resulted in significant remodeling of the actin structure within the REN cells, with a transition from a largely cortical actin polymerization pattern to a highly defined stress fibers organization. We next performed cell proliferation experiments on cells grown on a solid surface and also tested the effect of ERb suppression on the capacity of the REN cells for anchorage-independent growth in semi-solid media. ERb sup- pression significantly (p,0.05) increased the proliferation rate of REN cells compared to wild-type controls at any considered time (Fig. 2C). Moreover, ERb suppression resulted also in a 3 to 4-fold increase in the number of colonies formed by the REN cells after 7 days of culture in soft agar (Fig. 2D). ERb over-expression influences EGFR mediated signaling and internalization Therefore, we sought to investigate EGFR signaling in mock- and ERb-transfected REN cells treated with EGF. Here we show that the proliferation of REN cells is promoted by EGF treatment; while transfection of REN cells with the ERb expression plasmid significantly (p,0.05) inhibited the proliferation rate of these cells both under basal conditions and following EGF exposure (Fig. 3A). In response to EGF treatment of mock cells, EGFR became phosphorylated and the ERK1/2 MAPK and Akt signaling pathways were activated as demonstrated by the phosphorylation state of these kinases (Fig. 3B). In ERb over-expressing cells there was a reduced basal level of EGFR phosphorylation and a diminished response to EGF treatment. This translated into a reduced activation of signal transduction cascades with a slight reduction in EGF induced ERK1/2 phosphorylation, but a complete ablation of the EGF induced Akt phosphorylation (Fig. 3B). To assess whether EGFR internalization was affected by ERb over-expression, we evaluated the process of EGFR internalization at 60 and 120 minutes of EGF treatment, in mock and ERb over-expressing REN cells. As shown in Fig. 3C, EGFR is almost completely internalized in mock-REN cells at both 60 and 120 minutes. In ERb-REN cells, EGFR is internalized at both time points, although the process appears to be slower with respect to mock cells, in particular at 60 minutes, suggesting a different kinetic of internalization/recycling. These data were confirmed by immunoblot analysis with anti-phospho tyrosine and anti-EGFR antibodies of EGFR immunoprecipitated from plasma membrane (Fig. 4D) of EGF treated mock and ERb over-expressing REN cells. ERb associates with EGFR and caveolin 1 Recently, it has been shown that EGFR may also follow the two distinct endocytotic routes: one clathrin-dependent and one clathrin-independent mediated by caveolin [25]. Published PLoS ONE | www.plosone.org 2 November 2010 | Volume 5 | Issue 11 | e14110