CLDN6 Inhibits The Invasion Of Cervical Carcinoma Cell Via Down-regulating AF-6/ERK Signaling Pathway

Cervical carcinoma is one of the most common gynecologic malignancies which late metastasis is the main reason for poor prognosis. The main way of metastasis in cervical carcinoma is local invasion and infiltration into the surrounding tissues, organs and lymph nodes. Therefore, research of the mechanism involved in tumor invasion is necessary for improving the prognosis of cervical carcinoma.Research shows that tight junction proteins including claudins(CLDNs) may play an important role in the occurance and metastasis of epithelium originated tumor through affecting tight junction structure, function and cell signaling pathways. CLDN6, a member of 27 CLDNs family, consists of four transmembrane domains. Its carboxy terminus in cytoplasm can directly interact with ZOs, AF-6 which contains PDZ domain and involve with cellular responses to external signals and signal transduction. AF-6, a Ras/Raf/MEK/ERK signaling pathway regulation protein, can inhibit ERK pathway activation though binding with Ras. In our previous work, we have cloned and identified CLDN6 gene which functioned as a potential breast cancer suppressor in rat breast cancer resistant copenhegan mammary epithelial cells. We found that CLDN6 inhibited the capabilities of metastasis and invasion of breast cancer cells. In order to study the mechanism of CLDN6 inhibition on metastasis and invasion, we use the gene microarray to analysis and screen the target gene and related signaling pathways of CLDN6. It is showed that overexpression of CLDN6 inhibited ERK signaling pathway. However, whether CLDN6 can affect ERK signaling pathway by AF-6 in cervical carcinoma cells has not been reported yet. In previous work, it revealed that CLDN6 was low expressed in cervical carcinoma cell lines, suggesting that low CLDN6 expression may involve with the occurrence and development of cervical carcinoma. So far, the role of CLDN6 in human cervical carcinoma invasion and the involved mechanism is unclear. We aim to study the effect of CLDN6 on human cervical carcinoma cell invasion with molecular biology technology, aiming at providing a new target in cervical carcinoma treatment and control of cervical carcinoma early metastasis.Method:1. Establishment and identification of human cervical carcinoma cell clones with stable CLDN6 expression.Constructed p-EGFP-C1/CLDN6 eukaryotic expression vector, transfected the plasmid into human cervical carcinoma cells HELA and C33 A utilizing liposome method, appropriate concentration of G418 was used to screen single cell clones, identified the expression of CLDN6 by RT-PCR and Western-blot, and detected CLDN6 expression localization by immunofluorescence.2. The influence of CLDN6 expression on metastatic phenotype of human cervical carcinoma cell(1) Transepithelial electrical resistance(TEER) assay was applied to detect the TEER of monolayer cervical carcinoma cells.(2) Wound healing assay was conducted to detect the migration ability of cervical carcinoma cells in vitro, and the invasive ability was tested by Transwell chamber assay.(3) Western-blot and immunofluorescence methods were applied to detect expression and localization of epithelial markers E-Cadherin and mesenchymal marker N-Cadherin, Vimentin; Xenografts model in nude mice was conducted to detect the tumor growth ability in vivo, expression of Ki67 and E-Cadherin in xenograft tumor was examined by immunohistochemistry.3. The mechanisms of CLDN6 effect on cervical carcinoma cell invasion(1) The effect of CLDN6 on Ras-Raf-MEK-ERK signaling pathway in cervical carcinoma cell: detected the activation status of ERK, the upstream gene Ras and MEK1/2 by Western-blot. Used immunofluorescence assay to detect the localization and expression of ERK protein.(2) The effect of ERK signaling pathway activator PMA on cell invasion ability:(1) treated the cervical carcinoma clone cells with PMA: tested the optimum concentration and time of PMA treatment on ERK signaling pathway activation by Western-blot assay.(2) the effect of PMA treatment on cell invasion ability: detected cell invasion ability by Transwell chamber assay. Detected the epithelial markers and mesenchymal marker expression and localization by immunofluorescence assay.(3) CLDN6 inhibition on ERK signaling pathway via AF-6 and its effect on cell invasion:(1)used immunofluorescence method to detect the AF-6 localization; used the immunoprecipitation assay to detect the interaction of CLDN6 and AF-6 protein.(2)AF-6 expression was knockdowned by RNAi method; Western-blot assay and immunofluorescence were conducted to verify AF-6 knockdown effect; Western-blot were applied to detect phosphorylation level of ERK1/2 and MEK1/2; Transwell chamber assay were applied to detect invasion ability of cervical carcinoma cells; immunofluorescence method was used to detect expression and localization of epithelial marker and mesenchymal markers.4. CLDN6, p-ERK and E-Cadherin expression in cervical carcinoma tissuesImmunohistochemistry was used to detect CLDN6, p-ERK and E-Cadherin expression in 56 cases of cervical carcinoma and adjacent cervical tissues, and analyzed the correlation of p-ERK and E-Cadherin expression with CLDN6.Result1. Establishment of human cervical carcinoma cell clones with stable CLDN6 expressionTransfected p-EGFP-C1/CLDN6 vector into human cervical carcinoma cells HELA and C33 A, obtained the single cell clone after 14 days of G418 screening. The expression of CLDN6 in HELA and C33 A cervical carcinoma cell clones were significantly higher than vector group and located in the cell membrane.2. The influence of CLDN6 expression on human cervical carcinoma cell metastatic phenotypeCLDN6 significantly increased the TEER of cervical carcinoma cells; inhibited the invasion and metastasis ability of cervical carcinoma cells in vitro; up-regulated membrane expression of E-Cadherin and down-regulated the cytoplasmic expression of N-Cadherin and Vimentin; inhibited xenograft tumor growth in vivo, upregulated E-Cadherin expression and downregulated Ki67 expression in xenograft tumor tissues.3. The mechanisms of CLDN6 effect on cervical carcinoma cell invasion(1) The effect of CLDN6 on Ras-Raf-MEK-ERK signaling pathway in cervical carcinoma cell: phosphorylation levels of ERK1/2 and upstream element MEK1/2 was significantly decreased after CLDN6 transfection and the Ras activity was significantly reduced. ERK expression localization translocated from nucleus to cytoplasm.(2) The effect of ERK signaling pathway activation on the invasion ability of cervical carcinoma cells: phosphorylation levels of ERK1/2 and MEK1/2 was significantly increased after PMA treatment. ERK expression localization translocated from cytoplasm to nucleus. Activation of ERK signaling pathway downregulated membrane expression of E-Cadherin; upregulated the cytoplasmic expression of mesenchymal markers N-Cadherin, Vimentin; enhanced the invasive ability of cervical carcinoma cells.(3) The effect of CLDN6 inhibition on ERK pathway by AF-6 on cell invasion:(1) after CLDN6 transfection, expression of AF-6 position transferred from the cytoplasm to the cell membrane. co-immunoprecipitation assay revealed that the combination between CLDN6 and AF-6 can be detected.(2) after AF-6 knockdown in cervical carcinoma cell clones, the membrane expression of AF-6 was significantly downregulated; phosphorylation levels of ERK1/2 and MEK1/2 was increased; ERK expression localization translocated from cytoplasm to nucleus; membrane expression of E-Cadherin was downregulated; the cytoplasmic expression of mesenchymal markers N-Cadherin, Vimentin was upregulated; cell invasion ability was significantly enhanced.4. CLDN6, p-ERK and E-Cadherin expression in cervical carcinoma cervical tissuesCLDN6 expression was downregulated in cervical carcinoma tissues and negatively correlated with lymph node metastasis.p-ERK1/2 expression was significantly up-regualted in cervical carcinoma tissues, correlated with the differentiation degree and lymph node metastasis. p-ERK1/2 expression negatively correlated with CLDN6 expression.E-Cadherin expression in cervical carcinoma tissues was significantly downregulated and correlated with the differentiation degree and lymph node metastasis. E-Cadherin expression positively correlated with CLDN6 expression.Conclusion:1. CLDN6 re-expression in cervical carcinoma cells can inhibit cell invasion ability and reverse the process of epithelial-mesenchymal transdifferentiation;2. CLDN6 inhibited the activation of ERK signaling pathway by binding with AF-6 may be an important mechanism for the role of CLDN6 in cervical carcinoma cell invasion.3. The down-regulation of CLDN6, E-Cadherin and upregultion of p-ERK may play a positive role in development of cervical carcinoma.