The Expression of CLDN6 in Hepatocellular Carcinoma Tissue and the Effects of CLDN6 on Biological Phenotypes of Hepatocellular Carcinoma Cells.

CLDN6, a member of claudin (CLDN) family, was found to be a breast cancer suppressor gene in our early experiments. However, CLDN6 was highly expressed in human hepatocellular carcinoma (hHCC) (TCGA database), and the role of CLDN6 in hHCC is still unclear. To investigate the expression of CLDN6, immunohistochemical staining was performed in hHCC tissues. As a result, hHCC tissues highly expressed CLDN6, and the expression was related to the degree of tumor's differentiation. To research the role of CLDN6 in hHCC cells, CLDN6 was silenced in HepG2 and Hep3B cells which highly expressed CLDN6 through liposome transfection. Results showed that after silencing of CLDN6, the proliferation, migration and invasion abilities of hHCC cells were inhibited. Meanwhile, the expression of E-cadherin was upregulated, and the expression of N-cadherin and Vimentin was downregulated. All the results above indicated that CLDN6 promoted the development of hHCC, and could be a potential target for the treatment of it.

CLDN6 enhances chemoresistance to ADM via AF-6/ERKs pathway in TNBC cell line MDAMB231.

Claudin-6 (CLDN6), a critical tight junction protein acting as a tumor suppressor in breast cancer, is also considered to be a stem cell marker. Triple-negative breast cancer (TNBC) is a subtype of claudin-low and stem cell-like breast cancer which is chemoresistant to multiple anti-cancer drugs. The aim of our study was to determine whether CLDN6 plays a role in chemoresistance of TNBC. We found that overexpression of CLDN6 in TNBC cell line MDAMB231 significantly inhibited cell growth, migration, and invasion. The expression of CLDN6 increased the IC50 of adriamycin (ADM) and promoted the clonogenic survival. CLDN6 inhibited ADM-induced apoptosis and senescence in MDAMB231 cells. However, P-gp, a resistance-related protein highly associated with chemoresistance, was downregulated by CLDN6 overexpression in MDAMB231 cells. Epithelial mesenchymal transition (EMT) marker E-cadherin was increased, and vimentin was decreased by CLDN6. In addition, stem cell markers OCT4, SOX2, and Nanog were dramatically increased. CLDN6 colocalized and interacted with AF-6. Overexpression of CLDN6 increased the expression of afadin (AF-6) and hampered the activation of ERK signaling. PMA, a specific ERK activator, reversed the expression of EMT and stem cell markers, and decreased chemoresistance of MDAMB231 cells to ADM with a decreased IC50 and an increased apoptosis resulting from CLDN6. Together, we conclude that CLDN6 enhances the chemoresistance to ADM via activating the AF-6/ERK signaling pathway and up-regulating cancer stem cell characters in MDAMB231 cells.

Bioinformatic analysis reveals potential properties of human Claudin-6 regulation and functions.

Claudin-6 (CLDN6) is an integral component of the tight junction proteins in polarized epithelial and endothelial cells and plays a crucial role in maintaining cell integrity. Deregulation of CLDN6 expression and distribution in tumor tissues have been widely documented and correlated with cancer progression and metastasis. However, a complete mechanistic understanding of CLDN6 regulation and function remains to be studied. Herein, we show new potential properties of CLDN6 regulation and functions from bioinformatics analysis. Using numerous algorithms to characterize the CLDN6 gene promoter elements and the CLDN6 protein structure, physio-chemical and localization properties, and its evolutionary relationships. CLDN6 is regulated by a diverse set of transcription factors (SP1, SPR, AML-1a, CdxA, CRE-BP and CREB) and associated with the levels of methylation of CpG islands in promoters. The structural properties of CLDN6 indicate that it promotes cancer cell behavior via the ASK1-p38/JNK MAPK secretory signaling pathway. In conclusion, this information from bioinformatics analysis will help future attempts to better understand CLDN6 regulation and functions.

DNA methylation of claudin-6 promotes breast cancer cell migration and invasion by recruiting MeCP2 and deacetylating H3Ac and H4Ac.

BACKGROUND: Claudin-6 (CLDN6), a member of claudin transmembrane protein family, has recently been reported to be undetectable or at low levels in human breast cancer cell lines and tissues and plays a role in suppression of migration and invasion in breast cancer cells. In addition, it is reported that CLDN6 expression is regulated by DNA methylation in various human cancers and cell lines. However, it is unclear how DNA methylation regulates CLDN6 expression. Here we show the mechanism by which DNA methylation regulates CLDN6 expression in human breast cancer cell line MCF-7. METHODS: RT-PCR, Western blot and immunofluorescent staining were utilized to investigate CLDN6 expression in breast cancer tissues and MCF-7 cells. Methylation-Specific PCR (MSP) was applied to determine DNA methylation status in CLDN6 gene promoter region. Wound-healing assay and invasion assay were utilized to test mobility of MCF-7 cells treated with 5-aza-dC (DNA methyltransferase inhibitor). MeCP2 binding, H3Ac and H4Ac in CLDN6 promoter region were analyzed by ChIP assay. Nuclease accessibility assay was performed for analysis of the chromatin conformation of CLDN6 gene. To study the role of CLDN6 in malignant progression, we used RNAi to knockdown CLDN6 expression in MCF-7 cells treated with 5-aza-dC, and examined the mobility of MCF-7 cells by wound-healing assay and invasion assay. RESULTS: 5-aza-dC and TSA (histone deacetylase inhibitor) application induced CLDN6 expression in MCF-7 cells respectively and synergistically. 5-aza-dC treatment induced CLDN6 demethylation, inhibited MeCP2 binding to CLDN6 promoter and increased H3Ac and H4Ac in the promoter. In addition, TSA increased H4Ac, not H3Ac in the promoter. The chromatin structure of CLDN6 gene became looser than the control group after treating with 5-aza-dC in MCF-7 cells. 5-aza-dC up-regulated CLDN6 expression and suppressed migration and invasion in MCF-7 cells, whereas CLDN6 silence restored tumor malignance in MCF-7 cells. CONCLUSIONS: DNA methylation down-regulates CLDN6 expression through MeCP2 binding to the CLDN6 promoter, deacetylating H3 and H4, and altering chromatin structure, consequently promoting migratory and invasive phenotype in MCF-7 cells.