Leukemia Inhibitory Factor Receptor Suppresses The Metastasis Of Clear Cell Renal Cell Carcinoma Through Negative Regulation Of The Yes-Associated Protein

Background and objectiveKidney cancer is the seventh most common malignant tumor around the world.,which cause at least 140,000 deaths per year with a serious threat.At present,due to the insensitivity of traditional chemotherapy for advanced kidney cancer,one way of the drug therapies,targeted drugs,for kidney cancer are more and more discovered as research becoming deeper,but the improvement of patients’ survival is still insufficient.In the past 20 years,the treatment of advanced renal cancer has mainly focused on targeted therapy.As a receptor for leukemia inhibitory factor,LIFR participates in the signal transduction process of interleukin-6(IL-6)cytokine family,and plays its role in other cancer research such as breast cancer,lung cancer and melanoma,but it has been reported.Its role in different cancers shows an inconsistent effect,but may even be the opposite.The role of LIFR in clear cell renal cell carcinoma(ccRCC)remains unknown.Based on such situation,this research will focus on the regulatory effect of LIFR on the progression of renal cancer and its regulatory mechanism,and further speculate on its related targeted drugs.Method1.The relationship between LIFR expression and prognosis in ccRCCTCGA and other databases were used to analyze the expression and prognosis correlation of LIFR in ccRCC clinical samples,and immunohistochemical method was also been taken to analyze 25 pathological sections of ccRCC for further verification.2.Related factors affecting LIFR expressionThe upstream methylation level of LIFR and the expression correlation between copy number variation and LIFR were analyzed by MEXPRESS and cBioportal database,and further verified by Western blot experiment.3.Effect of LIFR on ccRCC cell functionAfter knock down the expression of LIFR,MTT test and Transwell test were used to verify the effect of LIFR on cell activity and transfer function,while the expression changes of EMT-related proteins was measured through Western blotting.4.Research on the downstream pathway of LIFRThe TCGA database and GSEA enrichment analysis were used to explore the possible downstream pathway of LIFR preliminarily,and a silent overexpression and double knock model was further established to verify the downstream pathway of LIFR.5.Study on the correlation between the remedial sensitization of targeted drugs and LIFR expression levelThe correlation between LIFR expression and therapeutic sensitivity of different targeted drugs was an7alyzed by MTT assay and CCLE database.Result1.LIFR expression level is negatively correlated with ccRCC progressComparing with ccRCC patients with higher LIFR expression,patients with low LIFR expression showed shorter OS time.Low LIFR expression is correlated with invasive tumor phenotype,such as,TNM stage,pathological grade and clinical stage.2.LIFR expression level is correlated with CNV and the methylation level of its upstreamThe methylation levels of cg03864479,cg06182018 and cg15386377 in ccRCC samples were significantly higher than those in normal tissue samples.The expression level of LIFR was inhibited by the methylation level of its upstream promoter and copy number variation of the gene.3.LIFR affects ccRCC cell migration and EMT transformationLIFR knockdown enhanced the invasion and migration of 786-O and Caki-2 cells,slightly increased the proliferative activity of tumor cells,and also promoted the EMT transformation of tumor cells.4.The Hippo pathway is the downstream of LIFRHippo pathway is one of the main downstream pathways of LIFR.Low expression of LIFR inhibits Hippo pathway and increases the level of Yes-related protein(YAP)in cells.Inhibition of YAP expression reverses the promotion of LIFR inhibition on cell migration,invasion and expression of Cancer Stem Cell markers.5.Low LIFR expression level enhances the sensitivity of some targeted drug therapiesCancer cells with LIFR being knocked down are more sensitive to YAP inhibitors,while cells with low LIFR expression are more sensitive to other two MET-targeted drugs(PHA-665752,PF2341066).ConclusionLIFR acts as a tumor suppressor gene in renal clear cell carcinoma,whose expression level is inhibited by the methylation level of its upstream promoter and the deletion of gene copy number.More over,the tumor progression is regulated by Hippo pathway through inhibiting the metastasis and invasion ability,stem cell nature and EMT transformation of tumor cells.For the clinical application of LIFR,it can be used as a potential biomarker for the prognosis of renal clear cell carcinoma.As for patients with low LIFR expression,the Hippo and MET pathway targeting drugs may be consider preferentially.