Methylation of the γ-Catenin Gene Is Associated With Poor Prognosis of Renal Cell Carcinoma

Purpose: γ-Catenin is a cell adhesion protein, and its functional loss is associated with tumor invasion and metastasis. We hypothesize that (1) promoter CpG methylation regulates the expression and function of the γ -catenin gene in renal cell carcinoma (RCC) and (2) methylation of the γ -catenin gene is associated with poor prognosis of RCC. To test these hypotheses, we analyzed the CpG methylation status of the γ -catenin gene and its correlation with clinical outcome in RCC. Experimental Design: Genomic DNA and total RNA were extracted from three renal cancer cell lines (A498, Caki-1, and Caki-2) and 54 RCC tissue samples with their corresponding normal kidney tissue samples. Expression of γ -catenin gene was analyzed by reverse transcription-PCR and immunostaining. Promoter methylation was analyzed by two different methylation-specific PCR (MSP-A and MSP-B), and the results were verified by DNA sequencing. Results: The demethylating agent (5-aza-2′-deoxycytidine) increased levels of mRNA transcript of the γ -catenin gene in three renal cancer cell lines. γ-Catenin mRNA and protein expression were significantly reduced in RCC samples compared with normal kidney samples, respectively ( P < 0.05). MSP-A and MSP-B bands were detected in 45 of 54 (83.3%) and 49 of 54 (90.7%) RCC samples, respectively. In normal kidney, weak products of MSP-A and MSP-B were detected in 5 of 54 (9.3%) and 6 of 54 (11.1%) samples, respectively. Likewise, both MSP-A and MSP-B ratios were significantly higher in RCC samples compared with normal kidney samples, respectively ( P < 0.01). Multivariate analysis revealed that the MSP-B ratio was a powerful and independent predictor superior to nuclear grade and Robson stage with respect to survival and disease progression ( P = 0.029 and 0.0071, respectively). No mutations in the NH 2 -terminal region of γ-catenin were found in this study. Conclusion: Expression of γ-catenin is regulated by promoter CpG methylation, and the balance between methylated and unmethylated RCC cell populations could determine its functional role. Because the conventional nuclear grade and/or staging system have some limitations to predict precise clinical outcome, this is the first report demonstrating that promoter CpG methylation of γ -catenin can be an independent and superior predictor for survival and disease progression.