RESUMEN
PURPOSE: Prostate cancer is a major cause of cancer death among men and the development of new biomarkers is important to augment current detection approaches. EXPERIMENTAL DESIGN: We identified hypermethylation of the ssDNA-binding protein 2 (SSBP2) promoter as a potential DNA marker for human prostate cancer based on previous bioinformatics results and pharmacologic unmasking microarray. We then did quantitative methylation-specific PCR in primary prostate cancer tissues to confirm hypermethylation of the SSBP2 promoter, and analyzed its correlation with clinicopathologic data. We further examined SSBP2 expression in primary prostate cancer and studied its role in cell growth. RESULTS: Quantitative methylation-specific PCR results showed that the SSBP2 promoter was hypermethylated in 54 of 88 (61.4%) primary prostate cancers versus 0 of 23 (0%) in benign prostatic hyperplasia using a cutoff value of 120. Furthermore, we found that expression of SSBP2 was down-regulated in primary prostate cancers and cancer cell lines. Hypermethylation of the SSBP2 promoter and its expression were closely associated with higher stages of prostate cancer. Reactivation of SSBP2 expression by the demethylating agent 5-aza-2'-deoxycytidine in prostate cancer cell lines confirmed epigenetic inactivation as one major mechanism of SSBP2 regulation. Moreover, forced expression of SSBP2 inhibited prostate cancer cell proliferation in the colony formation assay and caused cell cycle arrest. CONCLUSION: SSBP2 inhibits prostate cancer cell proliferation and seems to represent a novel prostate cancer-specific DNA marker, especially in high stages of human prostate cancer.
Asunto(s)
Adenocarcinoma/genética , Proliferación Celular , Metilación de ADN , Proteínas de Unión al ADN/genética , Proteínas de Unión al ADN/fisiología , Neoplasias de la Próstata/genética , Adenocarcinoma/patología , Anciano , Azacitidina/análogos & derivados , Azacitidina/farmacología , Secuencia de Bases , Biomarcadores de Tumor/genética , Biomarcadores de Tumor/fisiología , Ciclo Celular/efectos de los fármacos , Ciclo Celular/genética , Análisis Mutacional de ADN , Decitabina , Regulación Neoplásica de la Expresión Génica/efectos de los fármacos , Humanos , Masculino , Persona de Mediana Edad , Datos de Secuencia Molecular , Estadificación de Neoplasias , Hiperplasia Prostática/genética , Neoplasias de la Próstata/patología , Células Tumorales CultivadasRESUMEN
BACKGROUND: DNA methylation has emerged as a promising biomarker for prostate cancer detection. In this report, we screened 36 candidate genes generated by a bioinformatic analysis of the human genome, and found that the melanoma cell adhesion molecule (MCAM) was an excellent candidate for cancer-specific methylation in prostate cancer. METHODS: Direct sequencing of bisulfite-treated genomic DNA, conventional methylation-specific PCR (MSP), real-time quantitative methylation-specific PCR, immunohistochemistry, colony formation assay, and statistical analysis. RESULTS: We found that the melanoma cell adhesion molecule (MCAM) gene promoter was specifically methylated in prostate cancer cell lines and primary prostate cancer (PCa) but not in non-neoplastic prostate (BPH) tissues by direct sequencing of bisulfite-treated genomic DNA and conventional methylation-specific PCR (MSP). Further analysis with quantitative MSP showed greater hypermethylation of the MCAM promoter (80%, 70/88) in primary prostate cancer compared to 12.5% (3/24) in BPH. Prostatic intraepithelial neoplasias (PIN), potential precursors of prostate carcinoma, showed an intermediate methylation rate of 23% (7/30). We further observed that MCAM promoter methylation was directly correlated with tumor stage (pT3+pT4) (P = 0.001) and Gleason score (P = 0.018) in primary prostate carcinoma. CONCLUSIONS: Our results suggest that MCAM promoter hypermethylation deserves further attention as a potential diagnostic prostatic DNA marker in human prostate cancer.
