RESUMEN
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, â¼2500 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.
Asunto(s)
Efrina-A2/genética , Ácido Hialurónico/fisiología , Neoplasias/patología , Neovascularización Patológica/fisiopatología , Proteínas Tirosina Quinasas Receptoras/genética , Activación Transcripcional , Animales , Progresión de la Enfermedad , Efrina-A2/fisiología , Células Endoteliales de la Vena Umbilical Humana , Humanos , Ácido Hialurónico/química , Ratones , Ratones Endogámicos C57BL , Peso Molecular , Proteínas Tirosina Quinasas Receptoras/fisiologíaRESUMEN
RATIONALE: An increased cancer aggressiveness and mortality have been recently reported among patients with obstructive sleep apnea (OSA). Intermittent hypoxia (IH), a hallmark of OSA, enhances melanoma growth and metastasis in mice. OBJECTIVES: To assess whether OSA-related adverse cancer outcomes occur via IH-induced changes in host immune responses, namely tumor-associated macrophages (TAMs). MEASUREMENTS AND MAIN RESULTS: Lung epithelial TC1 cell tumors were 84% greater in mice subjected to IH for 28 days compared with room air (RA). In addition, TAMs in IH-exposed tumors exhibited reductions in M1 polarity with a shift toward M2 protumoral phenotype. Although TAMs from tumors harvested from RA-exposed mice increased TC1 migration and extravasation, TAMs from IH-exposed mice markedly enhanced such effects and also promoted proliferative rates and invasiveness of TC1 cells. Proliferative rates of melanoma (B16F10) and TC1 cells exposed to IH either in single culture or in coculture with macrophages (RAW 264.7) increased only when RAW 264.7 macrophages were concurrently present. CONCLUSIONS: Our findings support the notion that IH-induced alterations in TAMs participate in the adverse cancer outcomes reported in OSA.
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Hipoxia/inmunología , Neoplasias Pulmonares/patología , Macrófagos/patología , Melanoma Experimental/patología , Apnea Obstructiva del Sueño/fisiopatología , Animales , Línea Celular Tumoral , Proliferación Celular , Modelos Animales de Enfermedad , Citometría de Flujo , Hipoxia/etiología , Neoplasias Pulmonares/inmunología , Masculino , Melanoma Experimental/inmunología , Ratones , Ratones Endogámicos C57BL , Invasividad Neoplásica , Fenotipo , Apnea Obstructiva del Sueño/inmunología , Linfocitos T Citotóxicos/patologíaRESUMEN
BACKGROUND: Recent epidemiologic studies suggesting that there were differences in cancer recurrence contingent on anesthetic regimens have raised the possibility that µ-opioid agonists can influence cancer progression. Based on our previous studies indicating the µ-opioid receptor (MOR) is up-regulated in several types of non-small cell lung cancer, this study examined the functional significance of MOR overexpression to elucidate a possible mechanism for the epidemiologic findings. METHODS: Stable vector control and MOR1 overexpressing human bronchioloalveolar carcinoma cells were evaluated using immunoblot analysis, proliferation and transendothelial extravasation assays with or without Akt inhibitor, mTOR inhibitor (temsirolimus), or the peripheral MOR antagonist, methylnaltrexone. In human lung cancer xenograft models, primary tumor growth rates and lung metastasis were analyzed using consecutive tumor volume measurements and nestin immunoreactivity in lungs of the nude mouse model. RESULTS: The authors provide evidence that MOR is an important regulator of lung cancer progression. MOR overexpression increased Akt and mTOR activation, proliferation, and extravasation in human bronchioloalveolar carcinoma cells. In vivo, overexpression of MOR in human bronchoalveolar carcinoma cells increased primary tumor growth rates in nude mice by approximately 2.5-fold and lung metastasis by approximately 20-fold compared with vector control cells (n = 4 per condition). CONCLUSIONS: The overexpression data suggest a possible direct effect of MOR on Akt and mTOR activation and lung cancer progression. Such an effect provides a plausible explanation for the epidemiologic findings. The authors' observations further suggest that exploration of MOR in non-small cell lung carcinoma merits further study both as a diagnostic and therapeutic option.
Asunto(s)
Carcinoma de Pulmón de Células no Pequeñas/genética , Carcinoma de Pulmón de Células no Pequeñas/secundario , Neoplasias Pulmonares/genética , Proteínas Proto-Oncogénicas c-akt/metabolismo , Receptores Opioides mu/biosíntesis , Serina-Treonina Quinasas TOR/metabolismo , Animales , Carcinoma de Pulmón de Células no Pequeñas/metabolismo , Línea Celular Tumoral , Progresión de la Enfermedad , Humanos , Neoplasias Pulmonares/metabolismo , Neoplasias Pulmonares/patología , Masculino , Ratones , Ratones Desnudos , Proteínas Proto-Oncogénicas c-akt/fisiología , Receptores Opioides mu/genética , Receptores Opioides mu/fisiología , Serina-Treonina Quinasas TOR/fisiología , Ensayos Antitumor por Modelo de Xenoinjerto/métodosRESUMEN
Non-small cell lung cancer (NSCLC) has a poor prognosis and improved therapies are needed. Expression of EphA2 is increased in NSCLC metastases. In this study, we investigated EphA2 mutations in NSCLC and examined molecular pathways involved in NSCLC. Tumor and cell line DNA was sequenced. One EphA2 mutation was modeled by expression in BEAS2B cells, and functional and biochemical studies were conducted. A G391R mutation was detected in H2170 and 2/28 squamous cell carcinoma patient samples. EphA2 G391R caused constitutive activation of EphA2 with increased phosphorylation of Src, cortactin, and p130(Cas). Wild-type (WT) and G391R cells had 20 and 40% increased invasiveness; this was attenuated with knockdown of Src, cortactin, or p130(Cas). WT and G391R cells demonstrated a 70% increase in focal adhesion area. Mammalian target of rapamycin (mTOR) phosphorylation was increased in G391R cells with increased survival (55%) compared with WT (30%) and had increased sensitivity to rapamycin. A recurrent EphA2 mutation is present in lung squamous cell carcinoma and increases tumor invasion and survival through activation of focal adhesions and actin cytoskeletal regulatory proteins as well as mTOR. Further study of EphA2 as a therapeutic target is warranted.
Asunto(s)
Carcinoma de Células Escamosas/metabolismo , Adhesiones Focales/metabolismo , Regulación Neoplásica de la Expresión Génica , Péptidos y Proteínas de Señalización Intracelular/metabolismo , Neoplasias Pulmonares/metabolismo , Mutación , Proteínas Serina-Treonina Quinasas/metabolismo , Receptor EphA2/biosíntesis , Línea Celular Tumoral , Supervivencia Celular , Análisis Mutacional de ADN , Humanos , Inmunohistoquímica/métodos , Invasividad Neoplásica , Análisis de Secuencia por Matrices de Oligonucleótidos , Serina-Treonina Quinasas TOR , TransfecciónRESUMEN
Hyaluronan (HA) has diverse functions in normal lung homeostasis and pulmonary disease. HA constitutes the major glycosaminoglycan in lung tissue, with HA degradation products, produced by hyaluronidase enzymes and reactive oxygen species, being implicated in several lung diseases, including acute lung injury, asthma, chronic obstructive pulmonary disease, and pulmonary hypertension. The differential activities of HA and its degradation products are due, in part, to regulation of multiple HA-binding proteins, including cluster of differentiation 44 (CD44), Toll-like receptor 4 (TLR4), HA-binding protein 2 (HABP2), and receptor for HA-mediated motility (RHAMM). Recent research indicates that exogenous administration of high-molecular-weight HA can serve as a novel therapeutic intervention for lung diseases, including lipopolysaccharide (LPS)-induced acute lung injury, sepsis/ventilator-induced lung injury, and airway hyperreactivity. This review focuses on the regulatory role of HA and HA-binding proteins in lung pathology and discusses the capacity of HA to augment and inhibit various lung diseases.
Asunto(s)
Receptores de Hialuranos/metabolismo , Ácido Hialurónico/metabolismo , Enfermedades Pulmonares/metabolismo , Pulmón/metabolismo , Lesión Pulmonar Aguda/inducido químicamente , Lesión Pulmonar Aguda/prevención & control , Animales , Hiperreactividad Bronquial/tratamiento farmacológico , Hiperreactividad Bronquial/prevención & control , Bronquitis/prevención & control , Enfermedad Crónica , Proteínas de la Matriz Extracelular/metabolismo , Glucuronosiltransferasa/metabolismo , Humanos , Hialuronano Sintasas , Ácido Hialurónico/química , Ácido Hialurónico/uso terapéutico , Lipopolisacáridos , Enfermedades Pulmonares/inducido químicamente , Enfermedades Pulmonares/tratamiento farmacológico , Enfermedades Pulmonares/prevención & control , Peso Molecular , Isoformas de Proteínas/metabolismo , Enfisema Pulmonar/prevención & control , Especies Reactivas de Oxígeno/metabolismo , Receptor Toll-Like 4/metabolismo , Lesión Pulmonar Inducida por Ventilación Mecánica/prevención & controlRESUMEN
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 1x10(6) 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. CONCLUSIONS: 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.
Asunto(s)
Lesión Pulmonar Aguda/metabolismo , Permeabilidad de la Membrana Celular/fisiología , Endotelio Vascular/metabolismo , Serina Endopeptidasas/metabolismo , Lesión Pulmonar Aguda/etiología , Lesión Pulmonar Aguda/fisiopatología , Animales , Permeabilidad de la Membrana Celular/efectos de los fármacos , Modelos Animales de Enfermedad , Endotelio Vascular/citología , Endotelio Vascular/efectos de los fármacos , Humanos , Ácido Hialurónico/farmacología , Lipopolisacáridos/efectos adversos , Masculino , Ratones , Ratones Endogámicos C57BL , Receptores Proteinasa-Activados/metabolismo , Transducción de Señal/fisiología , Ventiladores Mecánicos/efectos adversos , Quinasas Asociadas a rho/metabolismo , Proteína de Unión al GTP rhoA/metabolismoRESUMEN
BACKGROUND: The possibility that µ 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 µ 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 â¼5- 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-Ala(2), N-MePhe(4), 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 â¼65% 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.
Asunto(s)
Carcinoma Pulmonar de Lewis/metabolismo , Carcinoma de Pulmón de Células no Pequeñas/metabolismo , Progresión de la Enfermedad , Neoplasias Pulmonares/metabolismo , Receptores Opioides mu/fisiología , Animales , Carcinoma Pulmonar de Lewis/patología , Carcinoma de Pulmón de Células no Pequeñas/patología , Línea Celular , Línea Celular Tumoral , Femenino , Humanos , Neoplasias Pulmonares/patología , Ratones , Ratones Endogámicos C57BL , Ratones Noqueados , Invasividad Neoplásica/patología , Ensayos Antitumor por Modelo de XenoinjertoRESUMEN
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.
Asunto(s)
Vasos Sanguíneos/metabolismo , Permeabilidad Capilar , Ácido Hialurónico/química , Ácido Hialurónico/metabolismo , Pulmón/irrigación sanguínea , Lesión Pulmonar Aguda , Animales , Caveolina 1/metabolismo , Línea Celular , Células Endoteliales/citología , Células Endoteliales/metabolismo , Endotelio Vascular/citología , Endotelio Vascular/metabolismo , Humanos , Receptores de Hialuranos/genética , Receptores de Hialuranos/metabolismo , Inflamación/metabolismo , Ratones , Ratones Noqueados , Datos de Secuencia Molecular , Peso Molecular , Proteoma/análisis , ARN Interferente Pequeño/metabolismoAsunto(s)
Progresión de la Enfermedad , Neoplasias/metabolismo , Neoplasias/patología , Receptores Opioides mu/fisiología , Animales , Humanos , Antagonistas de Narcóticos/farmacología , Antagonistas de Narcóticos/uso terapéutico , Invasividad Neoplásica/patología , Invasividad Neoplásica/prevención & control , Neoplasias/tratamiento farmacológico , Receptores Opioides mu/antagonistas & inhibidoresRESUMEN
Pancreatic ductal adenocarcinoma (PDAC) is a devastating cancer in large part due to late diagnosis and a lack of effective screening tests. In spite of recent progress in imaging, surgery and new therapeutic options for pancreatic cancer, the overall five-year survival still remains unacceptably low. Numerous studies have shown that focal adhesion kinase (FAK) is activated in many cancers including PDAC and promotes cancer progression and metastasis. Paxillin, an intracellular adaptor protein that plays a key role in cytoskeletal organization, connects integrins to FAK and plays a key role in assembly and disassembly of focal adhesions. Here, we have reviewed evidence in support of FAK as a potential therapeutic target and summarized related combinatorial therapies.
Asunto(s)
Carcinoma Ductal Pancreático/metabolismo , Quinasa 1 de Adhesión Focal/metabolismo , Neoplasias Pancreáticas/metabolismo , Paxillin/metabolismo , Biomarcadores de Tumor/antagonistas & inhibidores , Biomarcadores de Tumor/metabolismo , Carcinoma Ductal Pancreático/tratamiento farmacológico , Carcinoma Ductal Pancreático/patología , Quinasa 1 de Adhesión Focal/antagonistas & inhibidores , Humanos , Modelos Biológicos , Terapia Molecular Dirigida/métodos , Neoplasias Pancreáticas/tratamiento farmacológico , Neoplasias Pancreáticas/patología , Paxillin/antagonistas & inhibidores , Inhibidores de Proteínas Quinasas/uso terapéutico , Transducción de Señal/efectos de los fármacosRESUMEN
Malignant pleural mesothelioma (MPM) is an aggressive cancer that is commonly associated with prior asbestos exposure. Receptor tyrosine kinases (RTKs) such as MET and its downstream target PI3K are overexpressed and activated in a majority of MPMs. Here, we studied the combinatorial therapeutic efficacy of the MET/ALK inhibitor crizotinib, with either a pan-class I PI3K inhibitor, BKM120, or with a PI3K/mTOR dual inhibitor, GDC-0980, in mesothelioma. Cell viability results showed that MPM cells were highly sensitive to crizotinib, BKM120 and GDC-0980 when used individually and their combination was more effective in suppressing growth. Treatment of MPM cells with these inhibitors also significantly decreased cell migration, and the combination of them was synergistic. Treatment with BKM120 alone or in combination with crizotinib induced G2-M arrest and apoptosis. Both crizotinib and BKM120 strongly inhibited the activity of MET and PI3K as evidenced by the decreased phosphorylation of MET, AKT and ribosomal S6 kinase. Using a PDX mouse model, we showed that a combination of crizotinib with BKM120 was highly synergetic in inhibiting MPM tumor growth. In conclusion our findings suggest that dual inhibition of PI3K and MET pathway is an effective strategy in treating MPM as compared to a single agent.
Asunto(s)
Antineoplásicos/farmacología , Neoplasias Pulmonares/tratamiento farmacológico , Neoplasias Pulmonares/metabolismo , Mesotelioma/tratamiento farmacológico , Mesotelioma/metabolismo , Inhibidores de las Quinasa Fosfoinosítidos-3 , Neoplasias Pleurales/tratamiento farmacológico , Neoplasias Pleurales/metabolismo , Proteínas Proto-Oncogénicas c-met/antagonistas & inhibidores , Aminopiridinas/administración & dosificación , Aminopiridinas/farmacología , Animales , Antineoplásicos/administración & dosificación , Apoptosis/efectos de los fármacos , Compuestos Bicíclicos Heterocíclicos con Puentes/farmacología , Puntos de Control del Ciclo Celular/efectos de los fármacos , Línea Celular Tumoral , Movimiento Celular/efectos de los fármacos , Proliferación Celular/efectos de los fármacos , Crizotinib , Sinergismo Farmacológico , Femenino , Humanos , Neoplasias Pulmonares/patología , Mesotelioma/patología , Mesotelioma Maligno , Ratones , Ratones Desnudos , Microtúbulos/efectos de los fármacos , Morfolinas/administración & dosificación , Morfolinas/farmacología , Neoplasias Pleurales/patología , Inhibidores de Proteínas Quinasas/farmacología , Pirazoles/administración & dosificación , Pirazoles/farmacología , Piridinas/administración & dosificación , Piridinas/farmacología , Pirimidinas/farmacología , Transducción de Señal/efectos de los fármacos , Ensayos Antitumor por Modelo de XenoinjertoRESUMEN
Malignant mesothelioma (MM), is an intractable disease with limited therapeutic options and grim survival rates. Altered metabolic and mitochondrial functions are hallmarks of MM and most other cancers. Mitochondria exist as a dynamic network, playing a central role in cellular metabolism. MM cell lines display a spectrum of altered mitochondrial morphologies and function compared to control mesothelial cells. Fractal dimension and lacunarity measurements are a sensitive and objective method to quantify mitochondrial morphology and most importantly are a promising predictor of response to mitochondrial inhibition. Control cells have high fractal dimension and low lacunarity and are relatively insensitive to mitochondrial inhibition. MM cells exhibit a spectrum of sensitivities to mitochondrial inhibitors. Low mitochondrial fractal dimension and high lacunarity correlates with increased sensitivity to the mitochondrial inhibitor metformin. Lacunarity also correlates with sensitivity to Mdivi-1, a mitochondrial fission inhibitor. MM and control cells have similar sensitivities to cisplatin, a chemotherapeutic agent used in the treatment of MM. Neither oxidative phosphorylation nor glycolytic activity, correlated with sensitivity to either metformin or mdivi-1. Our results suggest that mitochondrial inhibition may be an effective and selective therapeutic strategy in mesothelioma, and identifies mitochondrial morphology as a possible predictor of response to targeted mitochondrial inhibition.
Asunto(s)
Fractales , Neoplasias Pulmonares/patología , Mesotelioma/patología , Mitocondrias/metabolismo , Línea Celular Tumoral , Cisplatino/farmacología , Glucólisis , Humanos , Neoplasias Pulmonares/metabolismo , Mesotelioma/metabolismo , Mesotelioma Maligno , Metformina/farmacología , Mitocondrias/efectos de los fármacos , Proteínas Mitocondriales/metabolismoRESUMEN
Defects in vascular integrity are an initiating factor in several disease processes. We have previously reported that high molecular weight hyaluronan (HMW-HA), a major glycosaminoglycan in the body, promotes rapid signal transduction in human pulmonary microvascular endothelial cells (HPMVEC) leading to barrier enhancement. In contrast, low molecular weight hyaluronan (LMW-HA), produced in disease states by hyaluronidases and reactive oxygen species (ROS), induces HPMVEC barrier disruption. However, the mechanism(s) of sustained barrier regulation by HA are poorly defined. Our results indicate that long-term (6-24 hours) exposure of HMW-HA induced release of a novel type of extracellular vesicle from HLMVEC called enlargeosomes (characterized by AHNAK expression) while LMW-HA long-term exposure promoted release of exosomes (characterized by CD9, CD63, and CD81 expression). These effects were blocked by inhibiting caveolin-enriched microdomain (CEM) formation. Further, inhibiting enlargeosome release by annexin II siRNA attenuated the sustained barrier enhancing effects of HMW-HA. Finally, exposure of isolated enlargeosomes to HPMVEC monolayers generated barrier enhancement while exosomes led to barrier disruption. Taken together, these results suggest that differential release of extracellular vesicles from CEM modulate the sustained HPMVEC barrier regulation by HMW-HA and LMW-HA. HMW-HA-induced specialized enlargeosomes can be a potential therapeutic strategy for diseases involving impaired vascular integrity.
RESUMEN
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.
RESUMEN
Fractals are mathematical constructs that show self-similarity over a range of scales and non-integer (fractal) dimensions. Owing to these properties, fractal geometry can be used to efficiently estimate the geometrical complexity, and the irregularity of shapes and patterns observed in lung tumour growth (over space or time), whereas the use of traditional Euclidean geometry in such calculations is more challenging. The application of fractal analysis in biomedical imaging and time series has shown considerable promise for measuring processes as varied as heart and respiratory rates, neuronal cell characterization, and vascular development. Despite the advantages of fractal mathematics and numerous studies demonstrating its applicability to lung cancer research, many researchers and clinicians remain unaware of its potential. Therefore, this Review aims to introduce the fundamental basis of fractals and to illustrate how analysis of fractal dimension (FD) and associated measurements, such as lacunarity (texture) can be performed. We describe the fractal nature of the lung and explain why this organ is particularly suited to fractal analysis. Studies that have used fractal analyses to quantify changes in nuclear and chromatin FD in primary and metastatic tumour cells, and clinical imaging studies that correlated changes in the FD of tumours on CT and/or PET images with tumour growth and treatment responses are reviewed. Moreover, the potential use of these techniques in the diagnosis and therapeutic management of lung cancer are discussed.
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Fractales , Procesamiento de Imagen Asistido por Computador , Neoplasias Pulmonares/patología , Humanos , Modelos BiológicosRESUMEN
INTRODUCTION: Lung cancer mortality rates remain at unacceptably high levels. Although mitochondrial dysfunction is a characteristic of most tumor types, mitochondrial dynamics are often overlooked. Altered rates of mitochondrial fission and fusion are observed in lung cancer and can influence metabolic function, proliferation and cell survival. AREAS COVERED: In this review, the authors outline the mechanisms of mitochondrial fission and fusion. They also identify key regulatory proteins and highlight the roles of fission and fusion in metabolism and other cellular functions (e.g., proliferation, apoptosis) with an emphasis on lung cancer and the interaction with known cancer biomarkers. They also examine the current therapeutic strategies reported as altering mitochondrial dynamics and review emerging mitochondria-targeted therapies. EXPERT OPINION: Mitochondrial dynamics are an attractive target for therapeutic intervention in lung cancer. Mitochondrial dysfunction, despite its molecular heterogeneity, is a common abnormality of lung cancer. Targeting mitochondrial dynamics can alter mitochondrial metabolism, and many current therapies already non-specifically affect mitochondrial dynamics. A better understanding of mitochondrial dynamics and their interaction with currently identified cancer 'drivers' such as Kirsten-Rat Sarcoma Viral Oncogene homolog will lead to the development of novel therapeutics.
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Antineoplásicos/uso terapéutico , Neoplasias Pulmonares/tratamiento farmacológico , Mitocondrias/fisiología , Dinámicas Mitocondriales/fisiología , Animales , Antineoplásicos/farmacología , Humanos , Neoplasias Pulmonares/metabolismo , Mitocondrias/efectos de los fármacos , Dinámicas Mitocondriales/efectos de los fármacosRESUMEN
Recent epidemiologic studies implying differences in cancer recurrence based on anesthetic regimens raise the possibility that the mu opioid receptor (MOR) can influence cancer progression. Based on our previous observations that overexpression of MOR in human non-small cell lung cancer (NSCLC) cells increased tumor growth and metastasis, this study examined whether MOR regulates growth factor receptor signaling and epithelial mesenchymal transition (EMT) in human NSCLC cells. We utilized specific siRNA, shRNA, chemical inhibitors and overexpression vectors in human H358 NSCLC cells that were either untreated or treated with various concentrations of DAMGO, morphine, fentanyl, EGF or IGF. Cell function assays, immunoblot and immunoprecipitation assays were then performed. Our results indicate MOR regulates opioid and growth factor-induced EGF receptor signaling (Src, Gab-1, PI3K, Akt and STAT3 activation) which is crucial for consequent human NSCLC cell proliferation and migration. In addition, human NSCLC cells treated with opioids, growth factors or MOR overexpression exhibited an increase in snail, slug and vimentin and decrease ZO-1 and claudin-1 protein levels, results consistent with an EMT phenotype. Further, these effects were reversed with silencing (shRNA) or chemical inhibition of MOR, Src, Gab-1, PI3K, Akt and STAT3 (p<0.05). Our data suggest a possible direct effect of MOR on opioid and growth factor-signaling and consequent proliferation, migration and EMT transition during lung cancer progression. Such an effect provides a plausible explanation for the epidemiologic findings.
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Analgésicos Opioides/farmacología , Carcinoma de Pulmón de Células no Pequeñas/patología , Movimiento Celular/efectos de los fármacos , Factor de Crecimiento Epidérmico/farmacología , Transición Epitelial-Mesenquimal/efectos de los fármacos , Neoplasias Pulmonares/patología , Receptores Opioides mu/metabolismo , Anestésicos/efectos adversos , Línea Celular Tumoral , Proliferación Celular/efectos de los fármacos , Progresión de la Enfermedad , Receptores ErbB/metabolismo , Silenciador del Gen , Humanos , Receptores Opioides mu/deficiencia , Receptores Opioides mu/genética , Transducción de Señal/efectos de los fármacosRESUMEN
Malignant pleural mesothelioma (MPM) is an aggressive disease with a poor prognosis. Studies have shown that both MET and its key downstream intracellular signaling partners, PI3K and mTOR, are overexpressed in MPM. Here we determined the combinatorial therapeutic efficacy of a new generation small molecule inhibitor of MET, ARQ 197, and dual PI3K/mTOR inhibitors NVP-BEZ235 and GDC-0980 in mesothelioma cell and mouse xenograft models. Cell viability results show that mesothelioma cell lines were sensitive to ARQ 197, NVP-BEZ235 and GDC-0980 inhibitors. The combined use of ARQ 197 with either NVP-BEZ235 or GDC-0980, was synergistic (CI<1). Significant delay in wound healing was observed with ARQ 197 (p<0.001) with no added advantage of combining it with either NVP-BEZ235 or GDC-0980. ARQ 197 alone mainly induced apoptosis (20±2.36%) that was preceded by suppression of MAPK activity, while all the three suppressed cell cycle progression. Both GDC-0980 and NVP-BEZ235 strongly inhibited activities of PI3K and mTOR as evidenced from the phosphorylation status of AKT and S6 kinase. The above observation was further substantiated by the finding that a majority of the MPM archival samples tested revealed highly active AKT. While the single use of ARQ 197 and GDC-0980 inhibited significantly the growth of MPM xenografts (p<0.05, p<0.001 respectively) in mice, the combination of the above two drugs was highly synergistic (p<0.001). Our results suggest that the combined use of ARQ 197/NVP-BEZ235 and ARQ 197/GDC-0980 is far more effective than the use of the drugs singly in suppressing MPM tumor growth and motility and therefore merit further translational studies.
Asunto(s)
Antineoplásicos/farmacología , Neoplasias Pulmonares/genética , Mesotelioma/genética , Inhibidores de las Quinasa Fosfoinosítidos-3 , Proteínas Proto-Oncogénicas c-met/antagonistas & inhibidores , Serina-Treonina Quinasas TOR/antagonistas & inhibidores , Animales , Apoptosis/efectos de los fármacos , Compuestos Bicíclicos Heterocíclicos con Puentes/farmacología , Ciclo Celular/efectos de los fármacos , Línea Celular Tumoral , Movimiento Celular/efectos de los fármacos , Proliferación Celular/efectos de los fármacos , Sinergismo Farmacológico , Femenino , Humanos , Imidazoles/farmacología , Neoplasias Pulmonares/tratamiento farmacológico , Mesotelioma/tratamiento farmacológico , Mesotelioma Maligno , Ratones , Ratones Desnudos , Fosfohidrolasa PTEN/metabolismo , Fosfatidilinositol 3-Quinasas/genética , Fosfatidilinositol 3-Quinasas/metabolismo , Fosforilación/efectos de los fármacos , Proteínas Proto-Oncogénicas c-met/genética , Proteínas Proto-Oncogénicas c-met/metabolismo , Pirimidinas/farmacología , Pirrolidinonas/farmacología , Quinolinas/farmacología , Serina-Treonina Quinasas TOR/genética , Serina-Treonina Quinasas TOR/metabolismo , Cicatrización de Heridas/efectos de los fármacos , Cicatrización de Heridas/genética , Ensayos Antitumor por Modelo de XenoinjertoRESUMEN
Cytoskeletal and focal adhesion abnormalities are observed in several types of cancer, including lung cancer. We have previously reported that paxillin (PXN) was mutated, amplified, and overexpressed in a significant number of lung cancer patient samples, that PXN protein was upregulated in more advanced stages of lung cancer compared with lower stages, and that the PXN gene was also amplified in some pre-neoplastic lung lesions. Among the mutations investigated, we previously found that PXN variant A127T in lung cancer cells enhanced cell proliferation and focal adhesion formation and colocalized with the anti-apoptotic protein B Cell Lymphoma 2 (BCL-2), which is known to localize to the mitochondria, among other sites. To further explore the effects of activating mutations of PXN on mitochondrial function, we cloned and expressed wild-type PXN and variants containing the most commonly occurring PXN mutations (P46S, P52L, G105D, A127T, P233L, T255I, D399N, E423K, P487L, and K506R) in a GFP-tagged vector using HEK-293 human embryonic kidney cells. Utilizing live-cell imaging to systematically study the effects of wild-type PXN vs. mutants, we created a model that recapitulates the salient features of the measured dynamics and conclude that compared with wild-type, some mutant clones confer enhanced focal adhesion and lamellipodia formation (A127T, P233L, and P487L) and some confer increased association with BCL-2, Dynamin-related Protein-1 (DRP-1), and Mitofusion-2 (MFN-2) proteins (P233L and D399N). Further, PXN mutants, through their interactions with BCL-2 and DRP-1, could regulate cisplatin drug resistance in human lung cancer cells. The data reported herein suggest that mutant PXN variants play a prominent role in mitochondrial dynamics with direct implications on lung cancer progression and hence, deserve further exploration as therapeutic targets.
Asunto(s)
Adhesiones Focales/genética , Neoplasias Pulmonares/genética , Dinámicas Mitocondriales/genética , Paxillin/genética , Adhesiones Focales/metabolismo , Células HEK293 , Humanos , Neoplasias Pulmonares/metabolismo , Mutación , Paxillin/metabolismoRESUMEN
Vascular integrity or the maintenance of blood vessel continuity is a fundamental process regulated, in part, by the endothelial glycocalyx and cell-cell junctions. Defects in endothelial barrier function are an initiating factor in several disease processes including atherosclerosis, ischemia/reperfusion, tumor angiogenesis, cancer metastasis, diabetes, sepsis and acute lung injury. The glycosaminoglycan, hyaluronan (HA), maintains vascular integrity through endothelial glycocalyx modulation, caveolin-enriched microdomain regulation and interaction with endothelial HA binding proteins. Certain disease states increase hyaluronidase activity and reactive oxygen species (ROS) generation which break down high molecular weight HA to low molecular weight fragments causing damage to the endothelial glycocalyx. Further, these HA fragments can activate specific HA binding proteins upregulated in vascular disease to promote actin cytoskeletal reorganization and inhibition of endothelial cell-cell contacts. This review focuses on the crucial role of HA in vascular integrity and how HA degradation promotes vascular barrier disruption.