BackgroundThe role of estrogen and estrogen receptors in oncogenesis has been investigated in various malignancies. Recently our group identified estrogen receptor beta (ERβ) expression as an independent prognostic factor in the progression of human Malignant Pleural Mesothelioma (MMe), but the underlying mechanism by which ERβ expression in tumors determines clinical outcome remains largely unknown. This study is aimed at investigating the molecular mechanisms of ERβ action in MMe cells and disclosing the potential translational implications of these results.MethodsWe modulated ERβ expression in REN and MSTO-211H MMe cell lines and evaluated cell proliferation and EGF receptor (EGFR) activation.ResultsOur data indicate that ERβ 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 ERβ 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 ERβ, EGFR and caveolin 1 that results in an altered internalization and in a selective reduced activation of EGFR-coupled signaling, when ERβ is over-expressed. We also demonstrate that differential expression of ERβ influences MMe tumor cell responsiveness to the therapeutic agent: Gefitinib.ConclusionsThis study describes a role for ERβ 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.

BackgroundPI3K/AKT signalling pathway is aberrantly active and plays a critical role for cell cycle progression of human malignant pleural mesothelioma (MMe) cells.AKT is one of the important cellular targets of perifosine, a novel bio-available alkylphospholipid that has displayed significant anti-proliferative activity in vitro and in vivo in several human tumour model systems and is currently being tested in clinical trials.MethodsWe tested Perifosine activity on human mesothelial cells and different mesothelioma cell lines, in order to provide evidence of its efficacy as single agent and combined therapy.ResultsWe demonstrate here that perifosine, currently being evaluated as an anti-cancer agent in phase 1 and 2 clinical trials, caused a dose-dependent reduction of AKT activation, at concentrations causing MMe cell growth arrest. In this study we firstly describe that MMe cells express aside from AKT1 also AKT3 and that either the myristoylated, constitutively active, forms of the two proteins, abrogated perifosine-mediated cell growth inhibition. Moreover, we describe here a novel mechanism of perifosine that interferes, upstream of AKT, affecting EGFR and MET phosphorylation. Finally, we demonstrate a significant increase in cell toxicity when MMe cells were treated with perifosine in combination with cisplatin.ConclusionsThis study provides a novel mechanism of action of perifosine, directly inhibiting EGFR/MET-AKT1/3 axis, providing a rationale for a novel translational approach to the treatment of MMe.

Estrogen receptor (ER)-β has been shown to possess a tumor suppressive effect, and is a potential target for cancer therapy. Using gene-expression meta-analysis of human malignant pleural mesothelioma, we identified an ESR2 (ERβ coding gene) signature. High ESR2 expression was strongly associated with low succinate dehydrogenase B (SDHB) (which encodes a mitochondrial respiratory chain complex II subunit) expression. We demonstrate that SDHB loss induced ESR2 expression, and that activated ERβ, by over-expression or by selective agonist stimulation, negatively affected oxidative phosphorylation compromising mitochondrial complex II and IV activity. This resulted in reduced mitochondrial ATP production, increased glycolysis dependence and impaired cell proliferation. The observed in vitro effects were phenocopied in vivo using a selective ERβ agonist in a mesothelioma mouse model. On the whole, our data highlight an unforeseen interaction between ERβ-mediated tumor suppression and energy metabolism that may be exploited to improve on the therapy for clinical management of malignant mesothelioma.

Malignant Pleural Mesothelioma (MMe) is a rare but increasingly prevalent, highly aggressive cancer with poor prognosis. The aetiology of MMe is essentially a function of previous exposure to asbestos fibres, which are considered to be an early-stage carcinogen. Asbestos is toxic to human mesothelial cells (HMCs), that activate the nuclear enzyme poly(ADP-ribose) polymerase-1 (PARP1) to repair DNA. The targeting of PARP1 is showing considerable potential for delivering selective tumour cell kill while sparing normal cells, and offers a scientifically rational clinical application. We investigated PARP1 expression in normal mesothelial and MMe tissues samples. Immunohistochemical analysis revealed low PARP1 staining in peritumoural mesothelium. As opposite, a progressive increase in epithelioid and in the most aggressive sarcomatoid MMe tissues was evident. In MMe cell lines, we correlated increased PARP1 expression to sensitivity to its inhibitor CO-338 and demonstrated that CO-338 significantly reduced cell viability as single agent and was synergistic with cis-platin. Interestingly, we described a new correlation between PARP1 and the AKT/mTOR axis regulated by SIRT1. SIRT1 has a role in the modulation of AKT activation and PARP1 has been described to be a gatekeeper for SIRT1 activity by limiting NAD+ availability. Here, we firstly demonstrate an inverse correlation between AKT acetylation and phosphorylation modulated by SIRT1 in MMe cells treated with CO-338. In conclusion, this study demonstrates that PARP1 overexpression defines increased responsiveness to its inhibition, then these results imply that a substantial fraction of patients could be candidates for therapy with PARP inhibitors.

BackgroundHuman early growth response-1 (EGR1) is a member of the zing-finger family of transcription factors induced by a range of molecular and environmental stimuli including epidermal growth factor (EGF). In a recently published paper we demonstrated that integrin/EGFR cross-talk was required for Egr1 expression through activation of the Erk1/2 and PI3K/Akt/Forkhead pathways. EGR1 activity and stability can be influenced by many different post-translational modifications such as acetylation, phosphorylation, ubiquitination and the recently discovered sumoylation. The aim of this work was to assess the influence of sumoylation on EGF induced Egr1 expression and/or stability.MethodsWe modulated the expression of proteins involved in the sumoylation process in ECV304 cells by transient transfection and evaluated Egr1 expression in response to EGF treatment at mRNA and protein levels.ResultsWe demonstrated that in ECV304 cells Egr1 was transiently induced upon EGF treatment and a fraction of the endogenous protein was sumoylated. Moreover, SUMO-1/Ubc9 over-expression stabilized EGF induced ERK1/2 phosphorylation and increased Egr1 gene transcription. Conversely, in SUMO-1/Ubc9 transfected cells, EGR1 protein levels were strongly reduced. Data obtained from protein expression and ubiquitination analysis, in the presence of the proteasome inhibitor MG132, suggested that upon EGF stimuli EGR1 sumoylation enhanced its turnover, increasing ubiquitination and proteasome mediated degradation.ConclusionsHere we demonstrate that SUMO-1 modification improving EGR1 ubiquitination is involved in the modulation of its stability upon EGF mediated induction.