RESUMEN
PURPOSE: Intrahepatic cholangiocarcinoma (ICC), a highly malignant hepatobiliary cancer, has a poor prognosis and is refractory to conventional therapies. The aim of this study is to discover a novel molecular target for the treatment of ICC. EXPERIMENTAL DESIGN: To discover novel cancer-associated membrane antigens expressed in ICC cells, we generated monoclonal antibodies (mAb) by immunizing mice with intact ICC cell lines and screened for those that bind to the plasma membrane of ICC cells but not to normal cells. The mAb A10-A3 was selected and its target antigen was identified as the L1 cell adhesion molecule. Expression of L1 in ICC was evaluated by immunohistochemical analysis of tumor samples from 42 ICC patients. The functional significance of L1 expression in the tumor progression of ICC was investigated by L1 suppression, L1 overexpression, and antibody treatment. RESULTS: L1 was not expressed in normal hepatocytes and intrahepatic bile duct epithelium but highly expressed in 40.5% of ICC patients, remarkably at the invasive front of the tumors. Suppression of L1 with short hairpin RNA significantly decreased proliferation, migration, and invasion of ICC cells in vitro. Consistently, L1 overexpression in ICC cells enhanced proliferation, migration, invasion, and apoptosis resistance. In addition, L1 short hairpin RNA or anti-L1 mAb significantly reduced the tumor growth in nude mice bearing ICC xenograft. CONCLUSIONS: We identified that L1 is expressed in ICC. L1 plays an important role in the tumor progression of ICC by enhancing cell proliferation, migration, invasion, and survival. L1 may represent a novel therapeutic target for ICC.
Asunto(s)
Neoplasias de los Conductos Biliares/inmunología , Conductos Biliares Intrahepáticos/inmunología , Colangiocarcinoma/inmunología , Neoplasias Hepáticas/inmunología , Molécula L1 de Adhesión de Célula Nerviosa/biosíntesis , Animales , Anticuerpos Monoclonales/biosíntesis , Anticuerpos Monoclonales/inmunología , Reacciones Antígeno-Anticuerpo , Apoptosis/inmunología , Neoplasias de los Conductos Biliares/patología , Conductos Biliares Intrahepáticos/patología , Western Blotting , Movimiento Celular , Proliferación Celular , Colangiocarcinoma/patología , Progresión de la Enfermedad , Humanos , Inmunohistoquímica , Neoplasias Hepáticas/patología , Ratones , Ratones Desnudos , Trasplante de Neoplasias , Neoplasias Experimentales/inmunología , Molécula L1 de Adhesión de Célula Nerviosa/inmunología , Células Tumorales CultivadasRESUMEN
Caspase 8 and survivin are known as key molecules of apoptosis in hepatocellular carcinoma (HCC). The purpose of the present study was to investigate the relationship between promoter methylation and expression and apoptotic function of caspase 8 and survivin in HCC. Promoter methylation of the caspase 8 and survivin gene was analyzed in 73 primary HCC using methylation-specific polymerase chain reaction. The relationship between immunohistochemical expression of gene products and proliferative/apoptotic indices, and clinicopathological parameters was also investigated. Twenty-five (34%) and 24 (33%) patients had promoter methylation of caspase 8 and survivin gene. Immunohistochemical staining of caspase 8 and survivin was observed in 35 (48%) and 32 (44%). The methylation of caspase 8 and survivin demonstrated a negative correlation with immunohistochemical expression of gene products (P= 0.049 and P= 0.001). Methylation of caspase 8 and positive expression of its gene product was significantly correlated with high apoptotic indices (P= 0.032 and P= 0.026). Nuclear survivin expression was significantly correlated with high proliferative index (P= 0.001). On survival analysis, positive nuclear survivin expression was associated with a poor prognosis in HCC (P= 0.043). In conclusion, epigenetic alteration by promoter methylation of caspase 8 and survivin may constitute an important regulatory mechanism for expression of those genes in HCC.
Asunto(s)
Carcinoma Hepatocelular/genética , Caspasa 8/genética , Metilación de ADN/genética , Neoplasias Hepáticas/genética , Proteínas Asociadas a Microtúbulos/genética , Adulto , Anciano , Análisis de Varianza , Apoptosis/genética , Biomarcadores de Tumor/genética , Biomarcadores de Tumor/metabolismo , Carcinoma Hepatocelular/metabolismo , Carcinoma Hepatocelular/patología , Caspasa 8/metabolismo , Recuento de Células , Proliferación Celular , Distribución de Chi-Cuadrado , Epigénesis Genética/genética , Femenino , Regulación Neoplásica de la Expresión Génica , Humanos , Inmunohistoquímica , Proteínas Inhibidoras de la Apoptosis , Neoplasias Hepáticas/metabolismo , Neoplasias Hepáticas/patología , Masculino , Proteínas Asociadas a Microtúbulos/metabolismo , Persona de Mediana Edad , Pronóstico , Regiones Promotoras Genéticas/genética , SurvivinRESUMEN
PURPOSE: Cholangiocarcinomas (CC) are associated with poor survival, but diagnostic markers and therapeutic targets have not yet been elucidated. We previously found aberrant expression of L1 cell adhesion molecule in intrahepatic CC and a role for L1 in the progression of intrahepatic CC. Here, we analyzed L1 expression in extrahepatic CC (ECC) and evaluated its prognostic significance. EXPERIMENTAL DESIGN: We examined L1 expression in tumors from 75 ECC patients by immunohistochemistry. We analyzed the correlations between L1 expression and clinicopathologic factors as well as patient survival. RESULTS: L1 was not expressed in normal extrahepatic bile duct epithelium but was aberrantly expressed in 42.7% of ECC tumors. High expression of L1 was detected at the invasive front of tumors and was significantly associated with perineural invasion (P < 0.01). Univariate analysis indicated that various prognostic factors such as histologic grade 3, advanced pathologic T stage and clinical stage, perineural invasion, nodal metastasis, and high expression of L1 were risk factors predicting patient survival. Multivariate analyses done by Cox's proportional hazards model showed that high expression of L1 (hazard ratio, 2.171; 95% confidence interval, 1.162-4.055; P = 0.015) and nodal metastasis (hazard ratio, 2.088; 95% confidence interval, 1.159-3.764; P = 0.014) were independent risk factors for patient death. CONCLUSIONS: L1 was highly expressed in 42.7% of ECC and its expression was significantly associated with perineural invasion. High expression of L1 and nodal metastasis were independent poor prognostic factors predicting overall survival in patients with ECC.
Asunto(s)
Neoplasias de los Conductos Biliares/patología , Colangiocarcinoma/patología , Regulación Neoplásica de la Expresión Génica , Molécula L1 de Adhesión de Célula Nerviosa/metabolismo , Anciano , Anciano de 80 o más Años , Neoplasias de los Conductos Biliares/diagnóstico , Neoplasias de los Conductos Biliares/metabolismo , Colangiocarcinoma/diagnóstico , Colangiocarcinoma/metabolismo , Progresión de la Enfermedad , Femenino , Humanos , Masculino , Persona de Mediana Edad , Metástasis de la Neoplasia , Pronóstico , Estudios Retrospectivos , Resultado del TratamientoRESUMEN
Loss or reduced E-cadherin expression and aberrant expression of N-cadherin have been associated with invasiveness of human carcinoma cells and poor prognosis. The role of E- and N-cadherin, however, in hepatocellular carcinoma (HCC) has not yet been elucidated. The aim of the present study was to investigate the expression pattern of E- and N-cadherin in surgically resected HCC specimens according to their relationship with clinicopathological features. The expression patterns of E- and N-cadherin were evaluated on immunohistochemistry in 68 specimens of HCC and adjacent non-tumor tissue. The most different expression pattern between HCC and non-tumor tissue was the decreased staining intensity of E-cadherin (n = 37, 54%) and the dot-like discontinuous staining of N-cadherin (n = 35, 55%). Decreased intensity of E-cadherin and discontinuous staining of N-cadherin in HCC was correlated with advanced stage. The risk factors for expression patterns related to recurrence were loss of E-cadherin expression (odds ratio (OR) = 3.6; 95% confidence interval (CI): 1.1-12.4) and discontinuous staining of N-cadherin (OR = 1.6; 95% CI: 0.8-3.2). In conclusion, discontinuous staining of N-cadherin and loss of E-cadherin expression in HCC predicts a high risk of recurrence after surgical treatment.
Asunto(s)
Antígenos CD/metabolismo , Cadherinas/metabolismo , Carcinoma Hepatocelular/patología , Neoplasias Hepáticas/patología , Adulto , Anciano , Biomarcadores de Tumor/metabolismo , Carcinoma Hepatocelular/metabolismo , Carcinoma Hepatocelular/mortalidad , Carcinoma Hepatocelular/cirugía , Femenino , Técnica del Anticuerpo Fluorescente Indirecta , Humanos , Técnicas para Inmunoenzimas , Neoplasias Hepáticas/metabolismo , Neoplasias Hepáticas/mortalidad , Neoplasias Hepáticas/cirugía , Masculino , Persona de Mediana Edad , Recurrencia Local de Neoplasia , Estadificación de Neoplasias , Tasa de SupervivenciaRESUMEN
BACKGROUND/AIMS: Epigenetic silencing of DNA repair genes, O6-methylguanine-DNA methyltransferase (MGMT), hMLH1 and hMSH3, by promoter hypermethylation have been observed in various cancers. However, the relationship between hypermethylation of DNA mismatch repair genes and microsatellite instability (MSI) has not been studied in hepatocellular carcinoma (HCC) associated with cirrhosis. METHODS: We investigated the methylation pattern of CpG islands of 3 genes using methylation-specific PCR (MSP) and MSI in 40 patients with paired hepatocellular carcinoma and associated cirrhosis. RESULTS: hMSH3 and MGMT were the most methylated genes in both cirrhosis (70% and 68%, respectively) and HCC (75% and 73%, respectively). The methylation of hMLH1 was rarely found in both cirrhosis (8%) and HCC (5%). Gene promoters methylated in cirrhosis were also methylated in HCC with the exception of 9 cases found to be methylated either in cirrhosis or HCC. Of 40 cases of HCC associated with cirrhosis, three had MSI-positive phenotype in which two were MSI-low and one was MSI-high. One MSI-positive phenotype was present both in cirrhosis and in HCC, while two were only in HCC. There was no significant correlation between aberrant DNA methylation of mismatch repair genes and MSI status in HCC associated with cirrhosis. Immunohistochemical expressions of hMLH1, MGMT, and hMSH3 proteins were present in 16 (40%), 6 (15%), and 11 (28%) of 40 cases of HCC respectively. There was no significant correlaton between the aberrant DNA methylation of mismatch repair genes and clinical characteristics such as histological differentiation, postoperative recurrence and mortality. CONCLUSIONS: The methylation of MGMT and hMSH3 among DNA repair genes are frequent, but those of hMLH1 and MSI is very rare in both cirrhosis and HCC. There is no significant correlation between the methylation of DNA repair genes and clinical characteristics of HCC.