CLDN6 Inhibits Migration And Invasion In Colon Cancer Sw1116 Cell Via Tyk2/stat3 Signaling Pathway

CLDN6, a member of tight junction proteins family, is involved in intracellular signal transduction. The PDZ domain located at the C terminal of CLDN6 gene is associate with the process of extracellular signals transducing to inside, regulation of cell proliferation, differentiation and metabolism, cell adhesion and polarity maintaining, and control of cell migration and invasion. We previously found that migration and invasion was suppressed in MCF-7 cells over-expressing CLDN6.Microarray analysis showed that Tyk2/Stats signaling pathway proteins Tyk2, Stat1,Stat3 and Stat4 were increased in breast cancer cells over-expressing CLDN6. We demonstrated that migration and invasion was suppressed in colon cancer cell sw1116 stably over-expressing CLDN6. However, the exact mechanism remains unclear.Purpose: To demonstrate the mechanism that tight junction protein CLDN6 regulates metastasis in colon cancer cell sw1116 via Tyk2/Stat3 signaling pathway, providing new target in colon cancer therapy.Methods:1. Western-blot was used to detect Tyk2/Stat3 signaling pathway proteins expressing and activating levels;2. MTT was used to calculate the maximum safe dose of tyrosine kinase inhibitor AG490 to sw1116 cells over-expressing CLDN6;3. Western-blot was used to observe optimal concentration and action time of AG490 when inhibiting phosphorylation of Stat3 in sw1116 cells over-expressing CLDN6;4. Observe the morphological change of clone cells after treatment with the AG490 in Inverted fluorescence microscope.5. Wound healing assay and transwell assay were used to observe the effect of AG490 to migration and invasion of sw1116 cells over-expressing CLDN6.Results:1. Phosphorylation of Tyk2 and Stat3 in sw1116 cells over-expressing CLDN6 was significantly increased;2. IC50 of AG490 to sw1116 cells was 96μM;3. The optimal concentration and action time of AG490 was 50μM and 48 h respectively when inhibiting phosphorylation of Stat3 in sw1116 cells over-expressing CLDN6;4. Compared with DMSO treatment group,the morphology of clone cell into spindle cell by irregular slabs of stone after deal with AG490;5. AG490 increased migration and invasion of sw1116 cells over-expressing CLDN6.Nick experiments show that 24 h cell migration rate increased,P=0.012<0.05.Transwell migration experiment show that the number of cells migration increased,P=0.001<0.01.Transwell invasion experiments show that the number of cells through the Matrigel increased,P=0.0001<0.01.Conclusion:1. sw1116 cells over-expressing CLDN6 promote Phosphorylation of Tyk2 and Stat3.2. Tyk2 and Stat3 activation was related to the inhibition of CLDN6 to the migration and invasion of sw1116 cells.