Aberrant DNA methylation is a potential mechanism underlying the development of colorectal cancer (CRC). Thus, identification of prognostic DNA methylation markers and understanding the related molecular functions may offer a new perspective on CRC pathogenesis. To that end, we explored DNA methylation profile changes in CRC subtypes based on the microsatellite instability (MSI) status through genome-wide DNA methylation profiling analysis. Of 34 altered genes, three hypermethylated (epidermal growth factor, EGF; carbohydrate sulfotransferase 10, CHST10; ependymin related 1, EPDR1) and two hypomethylated (bone marrow stromal antigen 2, BST2; Rac family small GTPase 3, RAC3) candidates were further validated in CRC patients. Based on quantitative methylation-specific polymerase chain reaction (Q-MSP), EGF, CHST10 and EPDR1 showed higher hypermethylated levels in CRC tissues than those in adjacent normal tissues, whereas BST2 showed hypomethylation in CRC tissues relative to adjacent normal tissues. Additionally, among 75 CRC patients, hypermethylation of CHST10 and EPDR1 was significantly correlated with the MSI status and a better prognosis. Moreover, EPDR1 hypermethylation was significantly correlated with node negativity and a lower tumor stage as well as with mutations in B-Raf proto-oncogene serine/threonine kinase (BRAF) and human transforming growth factor beta receptor 2 (TGFβR2). Conversely, a negative correlation between the mRNA expression and methylation levels of EPDR1 in CRC tissues and cell lines was observed, revealing that DNA methylation has a crucial function in modulating EPDR1 expression in CRC cells. EPDR1 knockdown by a transient small interfering RNA significantly suppressed invasion by CRC cells, suggesting that decreased EPDR1 levels may attenuate CRC cell invasion. These results suggest that DNA methylation-mediated EPDR1 epigenetic silencing may play an important role in preventing CRC progression.

We previously reported that the sustained exposure of human urothelial cells (HUCs) to low-dose sodium arsenite induces changes in the gene expression profile and neoplastic transformation. In this study, we used the HumanMethylation27 BeadChip to analyze genome-wide methylation profiles and 5-aza-2'-deoxycytidine to examine the involvement of promoter methylation in gene expression. Because the expression of lipocalin-2 (LCN2) was highly enhanced by promoter hypomethylation in inorganic arsenic (iAs)-HUCs cells as well as bladder cancer tissues, we further showed that mutations at the binding sequences for NF-κB and C/EBP-α significantly reduced LCN2 promoter activity. By chromatin immunoprecipitation assay, we demonstrated the significantly increased binding of RelA (p65) and NF-κB1 (p50) to the hypomethylated promoter of LCN2 in the iAs-HUCs. Furthermore, we also demonstrated that LCN2 overexpression was crucial for the neoplastic characteristics of the iAs-HUCs, such as enhanced anchorage-independent growth, resistance to serum deprivation and activation of NF-κB signaling. In addition, our results indicated that enhanced NF-κB activity in iAs-HUCs was via LCN2-mediated increase in intracellular iron and reactive oxygen species levels. Taken together, our results show that sustained low-dose arsenic exposure results in epigenetic changes and enhanced oncogenic potential via LCN2 overexpression.

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.