Mechanism Of MEK2 Targeting MTSS1 To Regulate Invasion And Migration Of Colon Cancer

Background Colon cancer is the third most malignant tumor in the world and endangers human health.Although surgery combined with adjuvant therapy improves the survival rate of cancer patients,more than 50% of metastatic patients have a high recurrence rate within 5 years after surgery,so finding effective inhibitory treatment strategies becomes the key to prolong the survival of patients.The mitogen-activated protein kinase kinase（MEK）pathway is one of the most characteristic kinases in cancer cell biology,which plays an important role in the carcinogenesis and maintenance of various cancers.However,the relationship between MEK and colon cancer metastasis is not clear.MTSS1（cancer metastasis suppressor protein-1）has been shown to inhibit the process of metastasis in a variety of tumors.The purpose of this paper is to discuss the regulatory relationship between MEK kinase and MTSS1.ObjectiveTo clarify the effect of MEK2 on migration and invasion of colon cancer,and to clarify the mechanism of MEK2 regulating migration and invasion of colon cancer cells by targeting MTSS1 protein,so as to find new targets and strategies for clinical treatment of metastatic colon cancer.Methods1.Colon cancer cell lines HCT-116 with stable low expression of MEK1（sh MEK1）and MEK2（sh MEK2）were constructed using lentiviral plasmids,and the protein expression levels of MEK1 and MEK2 in the stable knockout cell lines were detected by Western Blotting（WB）assay.2.Cell scratch assay and invasion assay were used to detect the effects of sh MEK1,sh MEK2 and MEK1/2 inhibitor binimetinib on the invasion and migration of colon cancer cells.WB assay was used to detect the effect of sh MEK1,sh MEK2 and MEK1/2 inhibitor binimetinib on MTSS1 expression in colon cancer cells.3.GEPIA 2.0 database analyzed the correlation between MEK 1,MEK2 and MTSS1.4.The migration and invasion of colon cancer RKO and HCT-116 cells treated by sh MEK2 and Binimetinib with down-regulated MTSS1 si RNA were determined by cell scratches and invasion assay.5.HCT-116 cells stably transfected with sh NC and sh MEK2 were used to construct the in situ model of human xenografted nude mouse colon cancer.Liver tissue was stained with HE（eosin-hematoxylin）to observe the liver metastasis of colon cancer.The subcutaneous tumor model of nude mice was established by HCT-116 cells,and binimetinib was injected intratumoral to observe the tumor growth.MTSS1 protein expression level in binimetinib-treated tumor tissues was detected by WB,and MTSS1 expression level in tumor tissues was detected by immunohistochemistry.6.The relationship between MEK and MTSS1 was detected by immunoprecipitation;The modification mode and site of MEK2 to MTSS1 were analyzed by mass spectrometry.Results1.WB test results showed that compared with the control group,the expression levels of MEK1 and MEK2 in sh MEK1 and sh MEK2 colon cancer cells were reduced.2.Compared with the control group,inhibition of MEK1 and MEK2 expression significantly down-regulated the migration and invasion ability of colon cancer cells;At the same time,the migration and invasion of colon cancer cells were significantly inhibited by binimetinib,which was treated with different concentrations of MEK1/2 inhibitor.Statistical analysis of the results showed that the difference was statistically significant（P< 0.05）.3.Western Blotting results showed that the expression level of MTSS1 protein in colon cancer cells increased after treatment with different concentrations of Binimetinib on RKO,HCT-116,Lo Vo and SW480.4.GEPIA 2.0 database analysis showed that MEK2 was negatively correlated with MTSS1,with statistically significant results（P < 0.05）.5.Western Blotting results showed that sh MEK2 could increase MTSS1 protein expression,while sh MEK1 could not change MTSS1 protein expression.6.MTSS1 si RNA could significantly enhance the migration and invasion ability of colon cancer hc T-116 cells treated by sh MEK2 cells and Binimetinib.The results were statistically analyzed,and the difference was statistically significant（P < 0.05）.7.In the nude mouse model of orthotopic xenotransplantation for colon cancer,liver metastasis occurred in the sh NC group and no liver metastasis occurred in the sh MEK2 group.The results were statistically analyzed,and the difference was statistically significant（P < 0.05）.8.Western Blotting and immunohistochemistry showed that MTSS1 protein expression increased in the nude mouse HCT-116 cell subcutaneous transplanted tumor model given me K1/2 inhibitor Binimetinib compared with the control group.9.Immunoprecipitation results showed that MEK2 protein could significantly bind to MTSS1.10.Ms analysis suggested that MEK2 may phosphorylate MTSS1 threonine at sites 595 and 603 to regulate its intracellular expression.Conclusions1.Inhibition of MEK2 can significantly down-regulate the migration and invasion ability of colon cancer cells.2.Inhibition of MEK2 inhibits invasion and migration of colon cancer cells by up-regulating MTSS1 expression in cells3.MEK2 regulates MTSS1 expression by phosphorylation.