The Role Of Hippo/YAP For 5-FU Resistance In Colonrectal Cancer

Background:At present,the colorectal cancer is leading cause of cancer-related death in the world.5-FU is a main chemotherapy drug for treatment of advanced colorectal cancer.However,resistance to 5-FU occurs after several rounds of treatment.Thus,identifying the molecular mechanism underlying drug resistance can help design novel strategies for colorectal cancer treatment.Purposes:The mechanisms for 5-FU resistance in colorectal cancer is presently not known.Therefore,it is necessary to study the mechanism of tumor resistance on the basis of drug resistance model.This project mainly focuses on the relationship between the Hippo-YAP1 signaling and 5-FU drug resistance in colorectal cancer.We will determine whether we can make drug resistant cells sensitive to the 5-FU,hoping to develop a novel therapeutic strategy.Methodology:We compared the genes expression differences between LoVo＿Resistant cells and LoVo Sensitive cells by Next-generation sequencing.We determined the Hippo-YAP1 signaling by examining the target gene expression,YAP1 protein accumulation in the nucleus as well as phosphorylation of upstream signaling molecules.We measured IC50 values of LoVo＿R and LoVo＿S cells to determine the drug resistance and determined the effects of YAP1 knockdown on the IC50 level and target gene expression in the resistant cells.We observed the the tumor formation by injecting LoVo＿shNC and LoVo＿shYAP1 cells into NSG mice.Results:We found that 5-FU drug resistant LoVo cells have spindle cell shape and the cell size was relatively big in comparison with the sensitive cells.While cell proliferation rate of drug resistant cells was slow,LoVo＿R cells tolerate more 5-FU in culture.We discovered that The Hippo-YAP1 pathway is highly activated in in LoVo＿R(5-FU)resistant cells as indicated by elevated expression of Hippo-YAP1 target genes ANKRD1 and CTGF,nuclear accumulation of YAP1 protein and we treated LoVo-R cells with AZD6244 and BEZ235,observed an increase in the ratio of phosphor-LATS1/2 vs.total LATS1/2.We found that down-regulation of YAP1 by shRNAs rendered 5-FU resistant LoVo＿R＿cells more sensitive both in cultured cells and in mice.YAP1 and target gene expression level in colonrectal cancer of human correlates with progression.YAP1 target genes expression levels in colonrectal cancer of human were positive correlation with the poor prognosis of5-FU treatment.Conclusion:In human colonrectal cancer,the Hippo-YAP1 signaling pathway was found to be responsible for 5-FU resistane.We detected a high level of nuclear YAP1 protein and phosphorylation of cytoplasm YAP1 protein as well as elevated CTGF,ANKRD1 of YAP1 target gene expression in 5-FU resistant cells in comparison with the sensitive cells.We demonstrated that down-regulation of YAP1 by shRNAS sensitize resistrant cells to 5-FU treatment.This is the first report on the role of Hippo-YAP1 signaling for colonrectal cancer drug resistance.Our study suggests that down-regulation of Hippo-YAP1 signaling may be effective in promoting 5-FU-based chemotherapy in colorectal cancer.YAP1 and target gene expression level in colonrectal cancer of human correlates with progression.YAP1 target genes expression levels in colonrectal cancer of human were positive correlation with the poor prognosis of 5-FU treatment.