RESUMEN
Macrophage inhibitory factor 1 (MIC1) is frequently altered in various cancers. The aim of this study was to investigate the clinical significance of MIC1 for esophageal squamous cell carcinoma (ESCC). Serum MIC1 of 286 ESCC and 250 healthy subjects was detected, the diagnostic performance was assessed and compared with SCC, CEA, CA199 and CA724, and the value as a prognostic indicator was also evaluated. The expression of MIC1 in ESCC cell lines, tissues were detected, and the inhibition of MIC1 antibody on ESCC was carried out in vitro and in vivo. The results showed that the serum MIC1 of ESCC was significantly higher than normal groups (P < 0.001), and was positively associated with tumor invasion (P = 0.030) as well as lymph node metastasis (P = 0.007). The sensitivity of MIC1 was significantly better than SCC, CEA, CA199 and CA724, especially for stage I ESCC. Patients with higher serum MIC1 also had a poorer prognosis in relapse-free (P = 0.050) and tumor-specific survival (P = 0.005). In vitro studies showed that the expression of MIC1 was upregulated in 37.5% (3/8) ESCC cell lines and 45% (18/40) tissues, and the transcription of MIC1 in tumor tissues was significantly higher than paired adjacent normal tissues (P = 0.001). The antibody of MIC1 inhibited the tumor growth (P < 0.001), and showing preference for tumor tissues in xenograft model. The decreased formation of neovascularization lumen may be involved in the mechanism. We conclude that MIC1 plays an important role in the progression of ESCC and can serve as a potential biomarker and therapeutic target for ESCC.
Asunto(s)
Anticuerpos/administración & dosificación , Biomarcadores de Tumor/sangre , Biomarcadores de Tumor/inmunología , Carcinoma de Células Escamosas/metabolismo , Carcinoma de Células Escamosas/terapia , Neoplasias Esofágicas/metabolismo , Factor 15 de Diferenciación de Crecimiento/inmunología , Animales , Biomarcadores de Tumor/metabolismo , Carcinoma de Células Escamosas/inmunología , Carcinoma de Células Escamosas/patología , Línea Celular , Línea Celular Tumoral , Progresión de la Enfermedad , Neoplasias Esofágicas/sangre , Neoplasias Esofágicas/tratamiento farmacológico , Neoplasias Esofágicas/inmunología , Neoplasias Esofágicas/patología , Neoplasias Esofágicas/terapia , Carcinoma de Células Escamosas de Esófago , Femenino , Factor 15 de Diferenciación de Crecimiento/sangre , Factor 15 de Diferenciación de Crecimiento/metabolismo , Células Endoteliales de la Vena Umbilical Humana , Humanos , Metástasis Linfática , Masculino , Ratones , Ratones Endogámicos BALB C , Ratones Desnudos , Persona de Mediana Edad , Recurrencia Local de Neoplasia/sangre , Recurrencia Local de Neoplasia/metabolismo , Recurrencia Local de Neoplasia/patología , Neovascularización Patológica/sangre , Neovascularización Patológica/tratamiento farmacológico , Neovascularización Patológica/metabolismo , Pronóstico , Distribución Aleatoria , Transcripción Genética/efectos de los fármacos , Regulación hacia Arriba/efectos de los fármacos , Ensayos Antitumor por Modelo de XenoinjertoRESUMEN
OBJECTIVE: To investigate the mechanism of loss of human esophageal cancer-related gene 4 (ECRG4) expression in esophageal squamous cell carcinoma (ESCC.) METHODS: PCR-SSCP and DNA sequencing analysis were used to detect the mutation of ECRG4 exons in esophageal cancer and matched adjacent normal tissues of 80 patients. DNA bisulfite-modifying ssPCR sequencing assay was used to examine the methylation status of ECRG4 promoter in human esophageal squamous cell carcinoma EC9706 cells. The re-expression of ECRG4 mRNA was examined by RT-PCR in EC9706 cells, after treatment with either demethylation drug 5-aza-2'-deoxycytidine or arsenic trioxide. RESULTS: No mutation in the four ECRG4 exons was found in all the ESCC and matched normal adjacent tissues. RT-PCR showed that 11 of 16 CpG islands of ECRG4 promoter were hypermethylated, while ECRG4 mRNA expression level was undetectable in the EC9706 cells. The ECRG4 mRNA was re-expressed after treatment with either demethylation drug 5-aza-2'-deoxycytidine or arsenic trioxide. CONCLUSION: The epigenetic mechanism of methylation is a reason of loss of ECRG4 gene expression in the ESCC cell line EC9706.
Asunto(s)
Carcinoma de Células Escamosas/metabolismo , Islas de CpG/genética , Metilación de ADN , Neoplasias Esofágicas/metabolismo , Proteínas de Neoplasias/metabolismo , Antimetabolitos Antineoplásicos/farmacología , Antineoplásicos/farmacología , Trióxido de Arsénico , Arsenicales/farmacología , Azacitidina/análogos & derivados , Azacitidina/farmacología , Carcinoma de Células Escamosas/genética , Carcinoma de Células Escamosas/patología , Línea Celular Tumoral , Decitabina , Epigénesis Genética , Neoplasias Esofágicas/genética , Neoplasias Esofágicas/patología , Exones , Regulación Neoplásica de la Expresión Génica/efectos de los fármacos , Humanos , Mutación , Proteínas de Neoplasias/genética , Óxidos/farmacología , Regiones Promotoras Genéticas , ARN Mensajero/metabolismo , Proteínas Supresoras de TumorRESUMEN
The ECRG4 gene was initially identified and cloned in our laboratory from human normal esophageal epithelium (GenBank accession no. AF325503). We revealed the expression of ECRG4 protein was downregulated in 68.5% (89/130) ESCC samples using tissue microarray. The low ECRG4 protein expression was significantly associated with regional lymph node metastasis, primary tumor size, and tumor stage in ESCC (p < 0.05). ECRG4 mRNA expression was downregulated in ESCC due to the hypermethylation in the gene promoter. The treatment with 5-aza-2'-deoxycytidine, which is a DNA methyltransferase inhibitor restored ECRG4 mRNA expression in ESCC cells. The result indicated that promoter hypermethylation may be 1 main mechanism leading to the silencing of ECRG4. The high expression of ECRG4 in patients with ESCC was associated with longer survival compared with those with low ECRG4 expression by Kaplan-Meier survival analysis (p < 0.05). ECRG4 protein was an independent prognostic factor for ESCC by multivariable Cox proportional hazards regression analysis (p < 0.05). The restoration of ECRG4 expression in ESCC cells inhibited cell proliferation, colony formation, anchorage-independent growth, cell cycle progression and tumor growth in vivo (p < 0.05). The transfection of ECRG4 gene in ESCC cells inhibited the expression of NF-kappaB and nuclear translocation, in addition to the expression of COX-2, a NF-kappaB target gene, was attenuated. Taken together, ECRG4 is a novel candidate tumor suppressor gene in ESCC, and ECRG4 protein is a candidate prognostic marker for ESCC.
Asunto(s)
Biomarcadores de Tumor/genética , Carcinoma de Células Escamosas/genética , Neoplasias Esofágicas/genética , Genes Supresores de Tumor , Proteínas de Neoplasias/genética , Anciano , Biomarcadores de Tumor/aislamiento & purificación , Western Blotting , Carcinoma de Células Escamosas/metabolismo , Carcinoma de Células Escamosas/patología , Línea Celular Tumoral , Proliferación Celular , Ciclooxigenasa 2/metabolismo , Metilación de ADN , Neoplasias Esofágicas/metabolismo , Neoplasias Esofágicas/patología , Femenino , Citometría de Flujo , Silenciador del Gen , Humanos , Inmunohistoquímica , Masculino , Persona de Mediana Edad , Análisis Multivariante , FN-kappa B/metabolismo , Proteínas de Neoplasias/aislamiento & purificación , Pronóstico , Regiones Promotoras Genéticas , Reacción en Cadena de la Polimerasa de Transcriptasa Inversa , Factores de Riesgo , Transfección , Proteínas Supresoras de TumorRESUMEN
Human riboflavin transporter 2 (RFT2, also termed as SLC52A3) was recently identified as a susceptibility gene to esophageal squamous cell carcinoma (ESCC), however, its expression and biologic function has remained unclear in ESCC. In this study, we demonstrated that RFT2 was frequently overexpressed in tumor samples compared with normal adjacent tissue in ESCC patients. Knockdown of RFT2 in ESCC cells resulted in decreases of intracellular flavin status, mitochondrial membrane potential and cellular ATP levels, and inhibitions of cell proliferation, colony formation and anchorage-independent growth. Knockdown of RFT2 increased p21 and p27 protein levels, decreased their downstream targets cyclin E1 and Cdk2 protein levels and caused pRb hypophosphorylation, leading to cell cycle arrest at G1-G1/S. Knockdown of RFT2 also reduced anti-apoptotic proteins Bcl-2, Bcl-xl and survivin levels, caused activation of caspase-3 and apoptosis. In contrast, ectopic overexpression of RFT2 in ESCC cells promoted cell proliferation under restricted conditions (soft agar), conferred resistance to cisplatin, and enhanced tumorigenicity in nude mice. These results suggest that RFT2 contributes to ESCC tumorigenesis and may serve as a potential therapeutic target.