Nurbek Mambetsariev

The novel role of the mu opioid receptor in lung cancer progression: a laboratory investigation.

BACKGROUND:The possibility that &mgr; opioid agonists can influence cancer recurrence is a subject of recent interest. Epidemiologic studies suggested that there were differences in cancer recurrence in breast and prostate cancer contingent on anesthetic regimens. In this study, we identify a possible mechanism for these epidemiologic findings on the basis of &mgr; opioid receptor (MOR) regulation of Lewis lung carcinoma (LLC) tumorigenicity in cell and animal models. METHODS:We used human lung tissue and human non–small cell lung cancer (NSCLC) cell lines and evaluated MOR expression using immunoblot and immunohistochemical analysis. LLC cells were treated with the peripheral opioid antagonist methylnaltrexone (MNTX) or MOR shRNA and evaluated for proliferation, invasion, and soft agar colony formation in vitro and primary tumor growth and lung metastasis in C57BL/6 and MOR knockout mice using VisEn fluorescence mediated tomography imaging and immunohistochemical analysis. RESULTS:We provide several lines of evidence that the MOR may be a potential target for lung cancer, a disease with high mortality and few treatment options. We first observed that there is ∼5- to 10-fold increase in MOR expression in lung samples from patients with NSCLC and in several human NSCLC cell lines. The MOR agonists morphine and [D-Ala2, N-MePhe4, Gly-ol]-enkephalin (DAMGO) increased in vitro LLC cell growth. Treatment with MNTX or silencing MOR expression inhibited LLC invasion and anchorage-independent growth by 50%–80%. Injection of MOR silenced LLC lead to a ∼65% reduction in mouse lung metastasis. In addition, MOR knockout mice do not develop significant tumors when injected with LLC in comparison with wild-type controls. Finally, continuous infusion of the peripheral opioid antagonist MNTX attenuates primary LLC tumor growth and reduces lung metastasis. CONCLUSIONS:Taken together, our data suggest a possible direct effect of opiates on lung cancer progression, and provide a plausible explanation for the epidemiologic findings. Our observations further suggest a possible therapeutic role for opioid antagonists.

High-molecular-weight hyaluronan is a novel inhibitor of pulmonary vascular leakiness

Endothelial cell (EC) barrier dysfunction results in increased vascular permeability, a perturbation observed in inflammatory states, tumor angiogenesis, atherosclerosis, and both sepsis and acute lung injury. Therefore, agents that enhance EC barrier integrity have important therapeutic implications. We observed that binding of high-molecular-weight hyaluronan (HMW-HA) to its cognate receptor CD44 within caveolin-enriched microdomains (CEM) enhances human pulmonary EC barrier function. Immunocytochemical analysis indicated that HMW-HA promotes redistribution of a significant population of CEM to areas of cell-cell contact. Quantitative proteomic analysis of CEM isolated from human EC demonstrated HMW-HA-mediated recruitment of cytoskeletal regulatory proteins (annexin A2, protein S100-A10, and filamin A/B). Inhibition of CEM formation [caveolin-1 small interfering RNA (siRNA) and cholesterol depletion] or silencing (siRNA) of CD44, annexin A2, protein S100-A10, or filamin A/B expression abolished HMW-HA-induced actin cytoskeletal reorganization and EC barrier enhancement. To confirm our in vitro results in an in vivo model of inflammatory lung injury with vascular hyperpermeability, we observed that the protective effects of HMW-HA on LPS-induced pulmonary vascular leakiness were blocked in caveolin-1 knockout mice. Furthermore, targeted inhibition of CD44 expression in the mouse pulmonary vasculature significantly reduced HMW-HA-mediated protection from LPS-induced hyperpermeability. These data suggest that HMW-HA, via CD44-mediated CEM signaling events, represents a potentially useful therapeutic agent for syndromes of increased vascular permeability.

Hyaluronic Acid Binding Protein 2 Is a Novel Regulator of Vascular Integrity

Objective—The disruption of the endothelial cell barrier is a critical feature of inflammation and an important contributing factor to acute lung injury (ALI), an inflammatory condition that is a major cause of morbidity and mortality in critically ill patients. We evaluated the role of the extracellular serine protease, hyaluronic acid binding protein 2 (HABP2), in vascular barrier regulation. Methods and Results—By using immunoblot and immunohistochemical analysis, we observed that lipopolysaccharide (LPS) induces HABP2 expression in murine lung endothelium in vivo and in human pulmonary microvascular endothelial cells (ECs) in vitro. High-molecular-weight hyaluronan (HMW-HA, approximately 1×106 Da) decreased HABP2 protein expression in human pulmonary microvascular ECs and decreased purified HABP2 enzymatic activity, whereas low-molecular-weight HA (LMW-HA, approximately 2500 Da) increased these activities. The effects of LMW-HA, but not HMW-HA, on HABP2 activity were inhibited with a peptide of the polyanion-binding domain of HABP2. Silencing (small interfering RNA) HABP2 expression augmented HMW-HA–induced EC barrier enhancement and inhibited LPS and LMW-HA–mediated EC barrier disruption, results that were reversed with overexpression of HABP2. Silencing protease-activated receptor 1 and 3, RhoA, or Rho kinase expression attenuated LPS-, LMW-HA–, and HABP2-mediated EC barrier disruption. By using murine models of acute lung injury, we observed that LPS- and ventilator-induced pulmonary vascular hyperpermeability was significantly reduced with vascular silencing (small interfering RNA) of HABP2. Conclusion—HABP2 negatively regulates vascular integrity via activation of protease-activated receptor/RhoA/Rho kinase signaling and represents a potentially useful therapeutic target for syndromes of increased vascular permeability.

Dynamin 2 and c-Abl Are Novel Regulators of Hyperoxia-mediated NADPH Oxidase Activation and Reactive Oxygen Species Production in Caveolin-enriched Microdomains of the Endothelium

Reactive oxygen species (ROS) generation, particularly by the endothelial NADPH oxidase family of proteins, plays a major role in the pathophysiology associated with lung inflammation, ischemia/reperfusion injury, sepsis, hyperoxia, and ventilator-associated lung injury. We examined potential regulators of ROS production and discovered that hyperoxia treatment of human pulmonary artery endothelial cells induced recruitment of the vesicular regulator, dynamin 2, the non-receptor tyrosine kinase, c-Abl, and the NADPH oxidase subunit, p47phox, to caveolin-enriched microdomains (CEMs). Silencing caveolin-1 (which blocks CEM formation) and/or c-Abl expression with small interference RNA inhibited hyperoxia-mediated tyrosine phosphorylation and association of dynamin 2 with p47phox and ROS production. In addition, treatment of human pulmonary artery endothelial cells with dynamin 2 small interfering RNA or the dynamin GTPase inhibitor, Dynasore, attenuated hyperoxia-mediated ROS production and p47phox recruitment to CEMs. Using purified recombinant proteins, we observed that c-Abl tyrosine-phosphorylated dynamin 2, and this phosphorylation increased p47phox/dynamin 2 association (change in the dissociation constant (Kd) from 85.8 to 6.9 nm). Furthermore, exposure of mice to hyperoxia increased ROS production, c-Abl activation, dynamin 2 association with p47phox, and pulmonary leak, events that were attenuated in the caveolin-1 knock-out mouse confirming a role for CEMs in ROS generation. These results suggest that hyperoxia induces c-Abl-mediated dynamin 2 phosphorylation required for recruitment of p47phox to CEMs and subsequent ROS production in lung endothelium.

Methylnaltrexone Potentiates the Anti-Angiogenic Effects of mTOR Inhibitors

BackgroundRecent cancer therapies include drugs that target both tumor growth and angiogenesis including mammalian target of rapamycin (mTOR) inhibitors. Since mTOR inhibitor therapy is associated with significant side effects, we examined potential agents that can reduce the therapeutic dose.MethodsMethylnaltrexone (MNTX), a peripheral mu opioid receptor (MOR) antagonist, in combination with the mTOR inhibitors temsirolimus and/or rapamycin, was evaluated for inhibition of VEGF-induced human pulmonary microvascular endothelial cell (EC) proliferation and migration as well as in vivo angiogenesis (mouse Matrigel plug assay).ResultsMNTX inhibited VEGF-induced EC proliferation and migration with an IC50 of ~100 nM. Adding 10 nM MNTX to EC shifted the IC50 of temsirolimus inhibition of VEGF-induced proliferation and migration from ~10 nM to ~1 nM and from ~50 to ~10 nM respectively. We observed similar effects with rapamycin. On a mechanistic level, we observed that MNTX increased EC plasma membrane-associated tyrosine phosphate activity. Inhibition of tyrosine phosphatase activity (3,4-dephostatin) blocked the synergy between MNTX and temsirolimus and increased VEGF-induced tyrosine phosphorylation of Src with enhanced PI3 kinase and mTOR Complex 2-dependent phosphorylation of Akt and subsequent activation of mTOR Complex 1 (rapamycin and temsirolimus target), while silencing Src, Akt or mTOR complex 2 components blocked VEGF-induced angiogenic events.ConclusionsOur data indicate that MNTX exerts a synergistic effect with rapamycin and temsirolimus on inhibition of VEGF-induced human EC proliferation and migration and in vivo angiogenesis. Therefore, addition of MNTX could potentially lower the dose of mTOR inhibitors which could improve therapeutic index.

Transactivation of the Receptor-tyrosine Kinase Ephrin Receptor A2 Is Required for the Low Molecular Weight Hyaluronan-mediated Angiogenesis That Is implicated in Tumor Progression

Background: Hyaluronan (HA)-mediated angiogenesis has been implicated in tumor progression. Results: LMW-HA-mediated transactivation of EphA2 is required for PATJ and Dbs membrane recruitment and subsequent RhoA activation required for angiogenesis. Conclusion: EphA2 plays a crucial role in HA-mediated angiogenesis. Significance: Targeting downstream effectors of LMW-HA could be a useful therapeutic intervention for angiogenesis-associated diseases including various malignancies. Angiogenesis or the formation of new blood vessels is important in the growth and metastatic potential of various cancers. Therefore, understanding the mechanism(s) by which angiogenesis occurs can have important therapeutic implications in numerous malignancies. We and others have demonstrated that low molecular weight hyaluronan (LMW-HA, ∼2500 Da) promotes endothelial cell (EC) barrier disruption and angiogenesis. However, the mechanism(s) by which this occurs is poorly defined. Our data indicate that treatment of human EC with LMW-HA induced CD44v10 association with the receptor-tyrosine kinase, EphA2, transactivation (tyrosine phosphorylation) of EphA2, and recruitment of the PDZ domain scaffolding protein, PATJ, to the cell periphery. Silencing (siRNA) CD44, EphA2, PATJ, or Dbs (RhoGEF) expression blocked LMW-HA-mediated angiogenesis (EC proliferation, migration, and tubule formation). In addition, silencing EphA2, PATJ, Src, or Dbs expression blocked LMW-HA-mediated RhoA activation. To translate our in vitro findings, we utilized a novel anginex/liposomal targeting of murine angiogenic endothelium with either CD44 or EphA2 siRNA and observed inhibition of LMW-HA-induced angiogenesis in implanted Matrigel plugs. Taken together, these results indicate LMW-HA-mediated transactivation of EphA2 is required for PATJ and Dbs membrane recruitment and subsequent RhoA activation required for angiogenesis. These results suggest that targeting downstream effectors of LMW-HA could be a useful therapeutic intervention for angiogenesis-associated diseases including tumor progression.

HABP2 is a Novel Regulator of Hyaluronan-Mediated Human Lung Cancer Progression.

Background Lung cancer is a devastating disease with limited treatment options. Many lung cancers have changes in their microenvironment including upregulation of the extracellular matrix glycosaminoglycan, hyaluronan (HA), which we have previously demonstrated can regulate the activity of the extracellular serine protease, hyaluronan binding protein 2 (HABP2). This study examined the functional role of HABP2 on HA-mediated human lung cancer dynamics. Methods Immunohistochemical analysis was performed on lung cancer patient samples using anti-HABP2 antibody. Stable control, shRNA, and HABP2 overexpressing human lung adenocarcinoma cells were evaluated using immunoblot analysis, migration, extravasation, and urokinase plasminogen activator (uPA) activation assays with or without high-molecular weight HA or low-molecular weight HA (LMW-HA). In human lung cancer xenograft models, primary tumor growth rates and lung metastasis were analyzed using consecutive tumor volume measurements and nestin immunoreactivity in nude mouse lungs. Results We provide evidence that HABP2 is an important regulator of lung cancer progression. HABP2 expression was increased in several subtypes of patient non-small cell lung cancer samples. Further, HABP2 overexpression increased LMW-HA-induced uPA activation, migration, and extravasation in human lung adenocarcinoma cells. In vivo, overexpression of HABP2 in human lung adenocarcinoma cells increased primary tumor growth rates in nude mice by ~2-fold and lung metastasis by ~10-fold compared to vector control cells (n = 5/condition). Conclusion Our data suggest a possible direct effect of HABP2 on uPA activation and lung cancer progression. Our observations suggest that exploration of HABP2 in non-small cell lung carcinoma merits further study both as a diagnostic and therapeutic option.

Abstract C78: The mu opioid receptor regulates Lewis lung carcinoma tumor growth and metastasis

Introduction: Expression of the mu opioid receptor (MOR) is increased in patients with non‐small cell lung cancer (NSCLC), a disease with poor prognosis and limited therapies. Previous work by our laboratory and others suggested a role of opioids in several aspects of tumor growth (Anticancer Res 2009;29:3195–3205, Mol Cancer Ther 2008;7:1669–1679, Cancer Res 2002;62:4491–4498). In this study, we investigated MOR as a potential therapeutic target for lung carcinoma. Methods: Lewis lung carcinoma (LLC) cells were treated with MOR siRNA or the peripheral MOR antagonist, methylnaltrexone (MNTX, 250 nM), prior to the addition of EGF (10 ng/ml), IGF (10 ng/ml), DAMGO (1 nM), morphine (1 nM) or serum (1, 5 or 10%). In vitro functional (cell proliferation and invasion) and biochemical studies (immunoblotting) were then conducted. For in vivo assays, C57BL/6J mice were injected intravenously with dual color (GFP‐RFP labeled) LLC cells, with or without MOR siRNA treatment. In another experiment, MOR knockout as well as wildtype control mice were injected with LLC cells (subcutaneously into the flank) and followed for 12 weeks. In a parallel experiment, LLC tumor bearing C57BL/6J mice received continuous infusion of MNTX (10 mg/kg/day) for 2 weeks. Tumor growth and lung metastasis were evaluated using tumor volume measurements and/or in vivo fluorescent microscopy (Olympus OV‐100) with or without intravenous injection of ProSense and/or MMP Sense probes. Results: Our data indicate that LLC cells have ∼5 fold increased expression of MOR (compared to primary lung epithelial or BEAS‐2B cells). Inhibition of MOR with siRNA or MNTX treatment reduced in vitro LLC proliferation (90%) and invasion (50–75%). In vivo lung metastasis was reduced by ∼75% in wildtype C57BL/6J mice with LLC lacking MOR (siRNA) versus control siRNA‐treated LLC (3 weeks post‐i.v. injection, quantitated with OV‐100). In addition, primary LLC tumor volume was substantially reduced (>90%) in MOR knockout mice compared to wildtype mice (flank injection). Finally, we observed that continuous infusion of MNTX (10 mg/kg/day) in mice with pre‐existing LLC primary tumors attenuates further tumor growth. Conclusion: We have shown that MOR is a potential therapeutic target for inhibition of LLC proliferation, invasion and metastasis. In addition, MNTX can attenuate LLC primary tumor growth. Further study of MNTX as a potential therapeutic agent is warranted. Citation Information: Mol Cancer Ther 2009;8(12 Suppl):C78.

Abstract C79: Methylnaltrexone inhibits EGF‐ and IGF‐induced human bronchioloalveolar carcinoma proliferation and migration

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.