Abstract
Abstract Introduction: Non-small cell lung cancer (NSCLC) has a very poor prognosis and improved therapies are needed. Expression of the mu opioid receptor (MOR) is increased in metastatic sites of patients with NSCLC. Previous work by our laboratory and others suggested a role of opioids in several aspects of tumor growth (Wang et al., Anticancer Res, 2009; Mol Cancer Ther, 2008; Gupta et al., Cancer Res, 2002). In this study, we investigated whether inhibition of MOR by the peripheral MOR antagonist, methylnaltrexone (MNTX), attenuates EGF- and IGF-mediated human bronchioloalveolar carcinoma (BAC) molecular pathways involved in NSCLC oncogenic potential. Methods: Human H358 BAC cells were pretreated with various siRNAs targeting MOR, Akt, cortactin, Gab1, PLC 1 or STAT3 or pretreated with MNTX (1.0–250 nM), 10-(4 -(N-diethylamino)butyl)-2-chlorophenoxazine (Akt inhibitor, 5 µM), U-73122 (PLC inhibitor, 1 µM) or 6-Nitrobenzo[b]thiophene-1,1-dioxide (STAT3 inhibitor, 1 µM) prior to the addition of EGF or IGF (10 ng/ml). Functional (cell proliferation and migration) and biochemical studies (immunoprecipitation, immunoblotting) were then conducted. Results: 14 NSCLC cell lines (7 adenocarcinomas, 3 SCC, 2 bronchoioloalveolar carcinoma, 1 large cell carcinoma, 1 adenosquamous carcinoma) were examined for MOR expression using immunoblotting techniques. MOR expression was increased in most NSCLC cell lines with BAC having the highest levels of MOR expression (7–9 fold higher that control primary lung epithelial or BEAS-2B cells). EGF and IGF treatment of BAC (10 ng/ml, 5–30 minutes) stimulated MOR association with the EGF and IGF receptor, respectively. MNTX inhibited EGF- and IGF-mediated BAC, but not control BEAS-2B, proliferation and migration in a dose-dependent manner (IC50 = ∼10 and 100 nM). On a mechanistic level, we observed that MNTX inhibited both EGF- and IGF-induced recruitment of the adaper protein, Gab1 (GRB2-associated binding protein 1), to the plasma membrane and complex formation with the EGF/IGF receptor in BAC. Silencing (siRNA) MOR, Gab1 or MNTX treatment inhibited EGF/IGF-mediated Akt (serine/threonine kinase), cortactin (actin regulatory protein), PLC 1 (phospholipase) and STAT3 (Signal Transducer and Activator of Transcription protein) phosphorylation/activation, events which are involved in BAC oncogenic signaling. In addition, inhibiting (siRNA and/or chemical inhibitors) Akt, cortactin, PLC 1 and/or STAT3 attenuated both EGF- and IGF-induced BAC growth and migration (50–90%). Conclusion: We have shown that MNTX inhibits EGF/IGF-mediated oncogenic signaling through inhibition of MOR-mediated Gab1 recruitment to the plasma membrane and consequent attenuation of Akt, cortactin, PLC 1 and STAT3 activation leading to reduced BAC proliferation and migration. Further study of this molecule as a potential therapeutic agent is warranted. Citation Information: Mol Cancer Ther 2009;8(12 Suppl):C79.
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