Malignant pleural mesothelioma is an asbestos-related neoplasm with poor prognosis, refractory to current therapies, the incidence of which is expected to increase in the next decades. Female gender was identified as a positive prognostic factor among other clinical and biological prognostic markers for malignant mesothelioma, yet a role of estrogen receptors (ERs) has not been studied. Our goal was to investigate ERs expression in malignant mesothelioma and to assess whether their expression correlates with prognosis. Immunohistochemical analysis revealed intense nuclear ERβ staining in normal pleura that was reduced in tumor tissues. Conversely, neither tumors nor normal pleura stained positive for ERα. Multivariate analysis of 78 malignant mesothelioma patients with pathologic stage, histologic type, therapy, sex, and age at diagnosis indicated that ERβ expression is an independent prognostic factor of better survival. Moreover, studies in vitro confirmed that treatment with 17β-estradiol led to an ERβ-mediated inhibition of malignant mesothelioma cell proliferation as well as p21^CIP1 and p27^KIP1 up-regulation. Consistently cell growth was suppressed by ERβ overexpression, causing a G₂-M-phase cell cycle arrest, paralleled by cyclin B1 and survivin down-regulation. Our data support the notion that ERβ acting as a tumor suppressor is of high potential relevance to prediction of disease progression and to therapeutic response of malignant mesothelioma patients. [Cancer Res 2009;69(11):4598–604]

The 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. We modulated ERβ expression in REN and MSTO-211H MMe cell lines and evaluated cell proliferation and EGF receptor (EGFR) activation. Our 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. This 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.

Estrogen receptor (ER) β has growth inhibitory and chemo drug potentiating effect on ovarian cancer cells. We studied the dependence of ERβ function on the presence of KDM6B and SIRT1 in human ovarian cancer cells in vitro. Activation of ERβ with the subtype-selective agonist KB9520 resulted in significant inhibition of human ovarian cancer cell growth. KB9520-activated ERβ had an additive effect on growth inhibition in combination with cisplatin and paclitaxel, respectively. Loss of KDM6B expression had a negative effect on ERβ function as a ligand-dependent inhibitor of ovarian cancer cell growth. In contrast, loss or inhibition of SIRT1 deacetylase activity restored ligand-activated ERβ functionality. Presented data suggest that selective targeting of ERβ with an agonist potentiate chemotherapy efficacy for the treatment of ovarian cancer and that downregulation or inhibition of SIRT1 may further enhance its therapeutic effect.

PI3K/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. We 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. We 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. This 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.

Human 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. We 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. We 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. Here we demonstrate that SUMO-1 modification improving EGR1 ubiquitination is involved in the modulation of its stability upon EGF mediated induction.

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.