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

BACKGROUND: 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. METHODS: We modulated ERβ expression in REN and MSTO-211H MMe cell lines and evaluated cell proliferation and EGF receptor (EGFR) activation. RESULTS: 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. CONCLUSIONS: 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

Molecular pharmacology of malignant pleural mesothelioma: Challenges and perspectives from preclinical and clinical studies

Malignant pleural mesothelioma (MPM) is one of the deadliest and most heterogeneous tumors, highly refractory to multimodal therapeutic approach, including surgery, chemo- and radiotherapy. Preclinical and clinical studies exploring the efficacy of drugs targeting tyrosine kinases, angiogenesis and histone deacetylases, did not fulfil the expected clinical benefits. Thus, novel molecular targets should be identified from a definite knowledge of the unique biology and most relevant transduction pathways of MPM cells. Cancer stem cells (CSCs) are a subset of malignant precursors responsible for initiation, progression, resistance to cytotoxic drugs, recurrence and metastatic diffusion of tumor cells. CSCs are putative driving factors for MPM development and contribute to its clinical and biological heterogeneity; hence, targeted eradication of CSCs represents an ineludible goal to counteract MPM aggressiveness. In this context, innovative preclinical models could be exploited to identify novel intracellular pathway inhibitors able to target CSC viability. Novel drug targets have been identified among key factors responsible for the oncogenic transformation of mesothelial cells, often directly induced by asbestos. These include mitogenic and anti-apoptotic signaling that may also be activated by autocrine and paracrine cytokine pathways controlling cell plasticity. Both signaling pathways affecting proto-oncogene and transcription factor expression, or genetic and epigenetic alterations, such as mutations in cell cycle genes and silencing of tumor suppressor genes, represent promising disease-specific targets. In this review we describe current knowledge of MPM cell biology, focusing on potential targets to be tested in pharmacological studies, and highlighting results and challenges of clinical translation

Comparison between novel steroid‐like and conventional nonsteroidal antioestrogens in inhibiting oestradiol‐ and IGF‐I‐induced proliferation of human breast cancer‐derived cells

This study has two specific aims: (a) to compare the antioestrogenic activity of two steroidal analogues of 17β‐oestradiol, the 7α‐alkylamide, ICI 164, 384 and the 7α‐alkylsulphinylamide, ICI 182, 780, with that of the triphenylethylene‐derived compound 4OH‐tamoxifen on a pool of human breast cancer cell lines (HBCCL) with a range of hormonal responsiveness and acquired antioestrogen resistance and (b) to investigate the ability of such antioestrogens to modulate the potent breast carcinoma growth‐stimulatory activity of the ‘IGF‐I system’. For the chemosensitivity investigations we used a long‐term colorimetric and the short‐term thymidine incorporation assay; we analysed IGF‐I in conditioned media by a radioimmunoassay, IGF‐I mRNA in the cells by RT‐PCR and molecular species of IGF‐I‐binding proteins, secreted in conditioned media, by Western ligand blot. IGF‐I receptors were assayed on cell monolayers by binding studies and by Scatchard analysis, we calculated KD, Bmax and sites/cell. Our results indicate that ICI 182, 780 and ICI 164, 384 are 1.5‐5.5 fold more potent than 40H‐tamoxifen in inhibiting the basal proliferation of oestrogen‐receptor positive (ER +) breast cancer cell lines. Moreover we demonstrate the capacity of ICI 182, 780 and ICI 164, 384 to reduce, in a time‐dependent fashion, oestrogen‐ and/or IGF‐I‐stimulated growth of ER + cell lines, possibly by negatively interfering with an IGF‐I‐like material secretion and IGF‐I‐receptor number. Our data provide the first evidence that, on ER+ human breast carcinoma cell lines, steroidal antioestrogens inhibit cell growth and modulate the IGF‐I mitogenic system. The mechanism of this latter effect has yet to be identified. 1995 British Pharmacological Societ

Anti-insulin-like growth factor-I activity of a novel polysulphonated distamycin A derivative in human lung cancer cell lines

1. The purpose of this study was to investigate the antiproliferative effect and the modulation of the mitogenic insulin-like growth factor-I (IGF-I) system by FCE 26644 and FCE 27784, two polyanionic sulphonated distamycin A derivative compounds, on two human non-small cell lung cancer (N-SCLC) cell lines. 2. For cell growth studies the colorimetric MTT and the thymidine incorporation assays were performed; the presence of IGF-I and IGF-binding proteins in conditioned media was revealed by radioimmunoassay and Western ligand blot, respectively. Variations at the IGF-I-receptor level were tested by binding studies on cell monolayers. 3. A significant concentration- and time-dependent cytostatic activity of FCE 26644 (IC(50)≈200 μg ml(−1) at 72 h) compared to its analogue FCE 27784 (IC(50)>800 μg ml(−1)) was observed in both cell lines studied. The IGF-I-stimulated proliferation of the IGF-I-responsive A549 cell line was abolished by 24 h of FCE 26644 treatment whereas FCE 27784 was inactive. FCE 26644 increased (4 to 6 fold) the secretion of IGF-I-like material and reduced the IGF-I binding (IC(50)>100 μg ml(−1)) in both A549 and Ca-Lu-1 cell lines. FCE 26644 (100 μg ml(−1)) did not affect the K(D) (≈0.5 nM) but reduced the B(max) and the number of receptor sites (50%). 4. Our findings demonstrate that the ability to down-regulate the cell proliferation of N-SCLC cell lines, shown by FCE 26644, depends at least partially, on interference with the ‘IGF-I mitogenic system'

Sorafenib selectively depletes human glioblastoma tumor-initiating cells from primary cultures

none7noGlioblastomas are grade IV brain tumors characterized by high aggressiveness and invasiveness, giving patients a poor prognosis. We investigated the effects of the multi-kinase inhibitor sorafenib on six cultures isolated from human glioblastomas and maintained in tumor initiating cells-enriching conditions. These cell subpopulations are thought to be responsible for tumor recurrence and radio- and chemo-resistance, representing the perfect target for glioblastoma therapy. Sorafenib reduces proliferation of glioblastoma cultures, and this effect depends, at least in part, on the inhibition of PI3K/Akt and MAPK pathways, both involved in gliomagenesis. Sorafenib significantly induces apoptosis/cell death via downregulation of the survival factor Mcl-1. We provide evidence that sorafenib has a selective action on glioblastoma stem cells, causing enrichment of cultures in differentiated cells, downregulation of the expression of stemness markers required to maintain malignancy (nestin, Olig2 and Sox2) and reducing cell clonogenic ability in vitro and tumorigenic potential in vivo. The selectivity of sorafenib effects on glioblastoma stem cells is confirmed by the lower sensitivity of glioblastoma cultures after differentiation as compared with the undifferentiated counterpart. Since current GBM therapy enriches the tumor in cancer stem cells, the evidence of a selective action of sorafenib on these cells is therapeutically relevant, even if, so far, results from first phase II clinical trials did not demonstrate its efficacy.noneCarra E; Barbieri F; Marubbi D; Pattarozzi A; Favoni RE; Florio T; Daga ACarra, Elisa; Barbieri, Federica; Marubbi, Daniela; Pattarozzi, Alessandra; Favoni, Re; Florio, Tullio; Daga, A

Receptor tyrosine kinase inhibitors and cytotoxic drugs affect pleural mesothelioma cell proliferation: insight into EGFR and ERK1/2 as antitumor targets

Malignant pleural mesothelioma (MPM) is an aggressive chemotherapy-resistant cancer. Up-regulation of epidermal growth factor receptor (EGFR) plays an important role in MPM development and EGFRtyrosine kinase inhibitors (TKIs) may represent novel therapeutic options. We tested the effects of the EGFR TKIs gefitinib and erlotinib and TKIs targeted to other growth factors (VEGFR and PDGFR), in comparison to standard antineoplastic agents, in two human MPM cell lines, IST-Mes2 and ZL55. All drugs showed IC50 values in the micromolar range: TKIs induced cytostatic effects at concentrations up to the IC50, while conventional drug growth-inhibitory activity was mainly cytotoxic. Moreover, the treatment of IST-Mes2 with TKIs (gefitinib and imatinib mesylate) in combination with cisplatin and gemcitabine did not show additivity. Focusing on the molecular mechanisms underlying the antiproliferative and pro-apoptotic effects of EGFR-TKIs, we observed that gefitinib induced the formation and stabilization of inactive EGFR homodimers, even in absence of EGF, as demonstrated by EGFR Bmax and number of sites/cell. The analysis of downstream effectors of EGFR signaling demonstrated that EGF-induced proliferation, reverted by gefitinib, involved ERK1/2 activation, independently from Akt pathway. Gefitinib inhibits MPM cell growth and survival, preventing EGF-dependent activation of ERK1/2 pathway by blocking EGFR-TK phosphorylation and stabilizing inactive EGFR dimers. Along with the molecular definition of TKIs pharmacological efficacy in vitro, these results may contribute to delve deep into the promising but still controversial role for targeted and conventional drugs in the therapy of MPM