Galectine-4 (gal-4), encoded by the LGALS4 gene, was recently shown to exhibit a tumor suppressive effect in colorectal carcinoma and pancreatic adenocarcinoma, although how the expression of this gene is regulated remains unknown. No reports describe the significance of gal-4 in the malignant potential of urothelial tumors. Thus, we analyzed LGALS4 methylation and gene expression and their clinical relevance and biological function in urothelial carcinoma (UC).LGALS4 methylation was initially identified as a progression biomarker for UC patients through genomewide DNA methylation profiling of 16 tumor samples. Bisulfite sequencing PCR and immunohistochemistry were performed to validate the promoter methylation and expression of LGALS4. We used quantitative methylation-specific PCR to determine the methylation levels of LGALS4 normalized to ACTB in the tumor samples of 79 UC patients and compared the levels between patients with different clinicopathological characteristics. The association with survival probability was analyzed with the Kaplan-Meier method and Cox regression analysis. The ectopic expression of gal-4 in cancer cell lines was used to address its biological function in UC in vitro. The promoter hypermethylation of LGALS4 (>2.51, log 10 scale) revealed a positive correlation with high levels of both histological grade and tumor T category and with lymph node metastasis (all P≤0.001). In addition, LGALS4 hypermethylation was an independent predictor of inferior survival in UC patients (P<0.05). The ectopic expression studies demonstrated that gal-4 suppressed urothelial cancer cell growth, migration, and invasion. Thus, LGALS4 may function as a tumor suppressor gene in UC progression. Our findings provide evidence that methylation-mediated LGALS4 gene repression may be involved in urothelial tumor progression.

DNA methylation profiles are one of valuable biomarkers to molecularly define cancer subtypes that may serve as better indicators for disease progression, treatment and prognosis. In the present study, we investigated the aberrant promoter methylation of 5 tumor-related genes, CCND1, CHFR, FAM51A1, LGALS4, and RBBP7, which were selected from pooled DNA methylation profiles of urothelial carcinoma (UC) with various clinicopathological parameters. DNA was extracted from 90 patients with confirmed UC and 10 adjacent non-tumor controls. Promoter aberrant methylation of these 5 genes was determined by quantitative methylation-specific PCR (qMSP). Our results showed that methylation levels were higher in UC as compared with controls in promoter of CHFR, FAM51A1, LGALS4, and RBBP7 (6.24 vs. 4.27; 0.66 vs. 0.01; 7.47 vs. 2.14; 0.45 vs. 0.01, respectively). Among UC patients, the levels of promoter methylation of CCND1, CHFR and LGALS4 were significantly associated with tumor stages (p&lt; 0.01), whereas that of FAM51A1 was inversely associated with tumor stages (p=0.012). Tumors with poor differentiation showed higher levels of promoter methylation on CCND1, CHFR and LGALS4 genes than tumors with moderate differentiation (p&lt;0.05). UC patients with highly methylated CCND1, CHFR, LGALS4 or RBBP7 tended to have a poor survival than patients with low levels of methylated gene (p&lt;0.02). This is the first report to show the association of promoter methylation of these 5 genes with UC tissues. We anticipate that the methylation status of these novel marker genes may have great clinical implications for further classifying UC patients. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 102nd Annual Meeting of the American Association for Cancer Research; 2011 Apr 2-6; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2011;71(8 Suppl):Abstract nr LB-183. doi:10.1158/1538-7445.AM2011-LB-183