RESUMEN
Cell adhesion molecules (CADMs) comprise a protein family whose functions include maintenance of cell polarity and tumor suppression. In this report, we show that the CADM2 gene is repressed in human clear renal cell carcinoma by DNA promoter hypermethylation and/or loss of heterozygosity. Moreover, the loss of CADM2 expression is associated with a higher tumor pathology stage (p<0.05). The re-expression of CADM2 in the renal cancer cell line 786-O significantly suppressed tumor cell growth in vitro and in mouse xenografts by a G1 phase cell cycle arrest and the induction of apoptosis. Lentivirus-mediated CADM2 expression also significantly suppressed cancer cell anchorage-independent growth and invasion. Furthermore, the inhibition of endogenous CADM2 expression using siRNAs induced a tumorigenic phenotype in polarized non-tumorigenic MDCK cells. Thus, we conclude that CADM2 functions as a novel tumor suppressor and may serve as a potential therapeutic target for human renal cell carcinoma.
Asunto(s)
Carcinoma de Células Renales/genética , Carcinoma de Células Renales/patología , Moléculas de Adhesión Celular/metabolismo , Metilación de ADN/genética , Neoplasias Renales/genética , Neoplasias Renales/patología , Animales , Moléculas de Adhesión Celular/genética , Línea Celular Tumoral , Proliferación Celular , Regulación hacia Abajo/genética , Regulación Neoplásica de la Expresión Génica/genética , Humanos , Ratones , Ratones Endogámicos BALB CRESUMEN
PURPOSE: Kidney cancer is notoriously chemo-resistant and abundantly expresses the Nox4 NADPH oxidase. To determine if Nox4 superoxide generation contributes to drug resistance, we assayed in vitro drug cytotoxicity following Nox4 shRNA silencing in human renal cancer cells. MATERIALS AND METHODS: Human conventional kidney cell lines, 786-0 and RCC4 expressing Nox4-specific shRNA or a non-targeting, control shRNA were grown in serial dilutions of cisplatin, vincristine, doxorubicin, or etoposide. Cell viability curves were generated and the concentration required to kill 50% of the cells (IC50) calculated for each drug. Apopotosis was estimated by TUNEL assay. Quantitative RT-PCR and Western blots were used to confirm Nox4 silencing and evaluate expression of apoptotic pathway proteins. RESULTS: Silencing significantly lowered the IC50 for cisplatin, vincristine and etoposide, and promoted drug-induced apoptosis by TUNEL assay. Improved sensitivity to cisplatin was reproduced by Nox inhibiton with diphenyliodonium, whereas induction of intracellular superoxide by dithiothreitol superoxide enhanced chemo-resistance. RT-PCR and Western blot revealed decreased expression of anti-apoptotic Bcl-XL and Bcl-2 and increased expression of pro-apoptotic Bax following Nox4 knockdown. CONCLUSION: Nox4 contributes to RCC chemo-resistance through modulation of pro-apoptotic and anti-apoptotic signaling, suggesting that Nox4 inhibition might enhance the efficacy of conventional cytotoxic drugs against RCC.
Asunto(s)
Antineoplásicos/farmacología , Apoptosis/genética , Cisplatino/farmacología , Resistencia a Antineoplásicos/genética , NADPH Oxidasas , Apoptosis/efectos de los fármacos , Línea Celular Tumoral , Supervivencia Celular/efectos de los fármacos , Supervivencia Celular/genética , Regulación Neoplásica de la Expresión Génica/efectos de los fármacos , Regulación Neoplásica de la Expresión Génica/genética , Silenciador del Gen , Humanos , Neoplasias Renales/genética , Neoplasias Renales/metabolismo , NADPH Oxidasa 4 , NADPH Oxidasas/antagonistas & inhibidores , NADPH Oxidasas/genética , NADPH Oxidasas/metabolismo , Especies Reactivas de Oxígeno/metabolismoRESUMEN
PURPOSE: Vesicular stomatitis virus has been investigated as an oncolytic agent for cancer therapy because it preferentially replicates in tumor but not in normal cells due to the lack of a robust interferon antiviral system in transformed cells. However, wild-type vesicular stomatitis virus can induce a strong systemic immunological response and replicate in the central nervous system, potentially limiting its clinical usefulness. We report the construction of the recombinant, replication restricted vesicular stomatitis virus encoding SV5-F, which can induce syncytial formation with enhanced oncolytic properties against TRAMP-C2 tumors in an immunocompetent mouse model of prostate cancer. MATERIALS AND METHODS: We constructed the SV5-F recombinant restricted virus vector by replacing the vesicular stomatitis virus G gene with that of the SV5-F transgene to generate rVSV-DeltaG-SV5-F. Morphological changes and DNA fragmentation induced by rVSV-DeltaG-GFP or rVSV-DeltaG-SV5-F were determined by phase contrast microscopy and gel electrophoresis. In vitro cytotoxicity by recombinant vesicular stomatitis virus was done by MTT assay. In vivo study of rVSV treatment was done in immunocompetent mice by subcutaneous administration of TRAMP-C2 cells. RESULTS: In vitro characterization of the recombinant fusogenic VSV-DeltaG vector on TRAMP-C2 cells showed significantly enhanced apoptotic and cytotoxic effects relative to a similar virus encoding green fluorescent protein, that is rVSV-DeltaG-GFP. Regardless of initial tumor size intratumor rVSV-DeltaG-SV5-F administration in mice bearing subcutaneous TRAMP-C2 tumors resulted in a significantly reduced tumor load over that of the nonfusogenic green fluorescent control virus and of heat inactivated recombinant vesicular stomatitis virus in treated animals (p <0.01). CONCLUSIONS: Results show that G complemented recombinant VSV-DeltaG vectors, especially rVSV-DeltaG-SV5-F, are an effective oncolytic agent against mouse prostate cancer cells in vitro and in an in vivo immunocompetent mouse model system.
Asunto(s)
Viroterapia Oncolítica , Neoplasias de la Próstata/terapia , Vesiculovirus/genética , Proteínas Virales de Fusión/genética , Animales , Línea Celular Tumoral , Humanos , Masculino , Ratones , Células Tumorales CultivadasRESUMEN
Most sporadically occurring renal tumors include a functional loss of the tumor suppressor von Hippel Lindau (VHL). Development of VHL-deficient renal cell carcinoma (RCC) relies upon activation of the hypoxia-inducible factor-2α (HIF2α), a master transcriptional regulator of genes that drive diverse processes, including angiogenesis, proliferation, and anaerobic metabolism. In determining the critical functions for HIF2α expression in RCC cells, the NADPH oxidase NOX4 has been identified, but the pathogenic contributions of NOX4 to RCC have not been evaluated directly. Here, we report that NOX4 silencing in VHL-deficient RCC cells abrogates cell branching, invasion, colony formation, and growth in a murine xenograft model RCC. These alterations were phenocopied by treatment of the superoxide scavenger, TEMPOL, or by overexpression of manganese superoxide dismutase or catalase. Notably, NOX4 silencing or superoxide scavenging was sufficient to block nuclear accumulation of HIF2α in RCC cells. Our results offer direct evidence that NOX4 is critical for renal tumorigenesis and they show how NOX4 suppression and VHL re-expression in VHL-deficient RCC cells are genetically synonymous, supporting development of therapeutic regimens aimed at NOX4 blockade.
Asunto(s)
Factores de Transcripción con Motivo Hélice-Asa-Hélice Básico/metabolismo , Carcinoma de Células Renales/patología , Neoplasias Renales/patología , NADPH Oxidasas/genética , Proteína Supresora de Tumores del Síndrome de Von Hippel-Lindau/metabolismo , Animales , Factores de Transcripción con Motivo Hélice-Asa-Hélice Básico/biosíntesis , Catalasa/biosíntesis , Línea Celular Tumoral , Núcleo Celular/metabolismo , Transformación Celular Neoplásica/genética , Transformación Celular Neoplásica/patología , Óxidos N-Cíclicos/farmacología , Femenino , Regulación Neoplásica de la Expresión Génica , Humanos , Riñón/patología , Neoplasias Renales/genética , Ratones , Ratones SCID , NADPH Oxidasa 4 , Trasplante de Neoplasias , Inhibidores de la Síntesis de la Proteína/farmacología , Interferencia de ARN , Marcadores de Spin , Superóxido Dismutasa/biosíntesis , Superóxidos/metabolismo , Proteína Supresora de Tumores del Síndrome de Von Hippel-Lindau/genéticaRESUMEN
Prostate cancer is the most frequently diagnosed cancer and the second leading cause of cancer deaths in men today. Although virus-based gene therapy is a promising strategy to combat advanced prostate cancer, its current effectiveness is limited partially due to inefficient cellular transduction in vivo. To overcome this obstacle, conditional oncolytic viruses (such as conditional replication adenovirus (CRAD)) are developed to specifically target prostate without (or with minimal) systemic toxicity due to viral self-replication. In this study, we have analyzed and compared three prostate-specific promoters (PSA, probasin, and MMTV LTR) for their specificity and activity both in vitro and in vivo. Both mice model with xenograft prostate tumor model and canine model were used. The best PSP was selected to construct a prostate-specific oncolytic adenovirus (CRAD) by controlling the adenoviral E1 region. The efficacy and specificity of CRAD on prostate cancer cells were examined in cell culture and animal models.
Asunto(s)
Adenoviridae , Viroterapia Oncolítica/métodos , Virus Oncolíticos , Regiones Promotoras Genéticas , Neoplasias de la Próstata/terapia , Proteína de Unión a Andrógenos/genética , Animales , Línea Celular Tumoral , Perros , Humanos , Masculino , Virus del Tumor Mamario del Ratón/genética , Ratones , Ratones Endogámicos BALB C , Ratones Desnudos , Antígeno Prostático Específico/genética , Neoplasias de la Próstata/patología , Ratas , Secuencias Repetidas Terminales/genéticaRESUMEN
PURPOSE: Cell adhesion molecules (CADM) comprise a newly identified protein family whose functions include cell polarity maintenance and tumor suppression. CADM-1, CADM-3, and CADM-4 have been shown to act as tumor suppressor genes in multiple cancers including prostate cancer. However, CADM-2 expression has not been determined in prostate cancer. EXPERIMENTAL DESIGN: The CADM-2 gene was cloned and characterized and its expression in human prostatic cell lines and cancer specimens was analyzed by reverse transcription-PCR and an immunohistochemical tissue array, respectively. The effects of adenovirus-mediated CADM-2 expression on prostate cancer cells were also investigated. CADM-2 promoter methylation was evaluated by bisulfite sequencing and methylation-specific PCR. RESULTS: We report the initial characterization of CADM-2 isoforms: CADM-2a and CADM-2b, each with separate promoters, in human chromosome 3p12.1. Prostate cancer cell lines, LNCaP and DU145, expressed negligible CADM-2a relative to primary prostate tissue and cell lines, RWPE-1 and PPC-1, whereas expression of CADM-2b was maintained. Using immunohistochemistry, tissue array results from clinical specimens showed statistically significant decreased expression in prostate carcinoma compared with normal donor prostate, benign prostatic hyperplasia, prostatic intraepithelial neoplasia, and normal tissue adjacent to tumor (P < 0.001). Adenovirus-mediated CADM-2a expression suppressed DU145 cell proliferation in vitro and colony formation in soft agar. The decrease in CADM-2a mRNA in cancer cell lines correlated with promoter region hypermethylation as determined by bisulfite sequencing and methylation-specific PCR. Accordingly, treatment of cells with the demethylating agent 5-aza-2'-deoxycytidine alone or in combination with the histone deacetylase inhibitor trichostatin A resulted in the reactivation of CADM-2a expression. CONCLUSIONS: CADM-2a protein expression is significantly reduced in prostate cancer. Its expression is regulated in part by promoter methylation and implicates CADM-2 as a previously unrecognized tumor suppressor gene in a proportion of human prostate cancers.