Taurodeoxycholic Acid-YAP1 Upregulate OTX1 In Promoting Gallbladder Cancer Malignancy Through IFITM3-Dependent AKT Activation

Background:Orthodenticle homeobox(OTX1)is reported to be involved in numerous cancers,but the expression level and molecular function of OTX1 in gallbladder cancer(GBC)remain unknow.Objective:(1)To explore the expression of OTX1 in GBC patients and the correlation with the prognosis.(2)To explore the mechanism of OTX1 on the proliferation of GBC cells in vitro and in vivo.(3)To explore the mechanism of the correlation between TDCA and OTX1.(4)To explore the mechanism of OTX1 in regulating the expression of IFITM3.(5)To explore the effect of IFITM3 on the proliferation of GBC cells and the internal mechanism.Methods:(1)q PCR,immunohistochemical staining and western blot were performed to detect the level of OTX1.(2)CCK-8 assay,Brd U staining,and colony formation assay were used to detect the proliferation of GBC cells with OTX1 knocking down or overexpression.(3)Transcriptome sequencing was used to analyze the genes influenced by OTX1 in GBC cells with OTX1 overexpression.(4)The binding and functional verification of transcription factors were determined by luciferase reporter gene assay and chromatin immunoprecipitation assay.(5)The transplanted tumor model in nude mice was performed to determine the role of TDCA,YAP1,OTX1 and IFITM3 in the proliferation of GBC cells in vivo.Results:(1)Highly expressed OTX1 in GBC tissues.(2)Elevated OTX1 correlated with the poor prognosis of GBC patients.(3)OTX1 promoted GBC cell proliferation in vitro and in vivo.(4)Taurodeoxycholic acid(TDCA)induced YAP1 activation in elevating OTX1 expression in GBC.(5)TDCA induced YAP1-OTX1 signaling in promoting GBC progression.(6)High expression levels of OTX1 influenced the genes related to interferon responses.(7)OTX1 regulated AKT signaling pathway via directly transactivating IFITM3.(8)IFITM3 knockdown attenuates the tumor promoting role of OTX1 on the proliferation of GBC cells in vitro and in vivo.Conclusion: Here,we found the elevated level of OTX1 associate with poor prognosis in human gallbladder cancer.In vitro and in vivo studies of human gallbladder cancer cell lines demonstrated that overexpression of OTX1 promoted cell proliferation,whereas the downregulation inhibited it.Additionally,we found the tight correlation between the serum level of taurodeoxycholic acid(TDCA)and OTX1 level.TDCA-induced activation of YAP1 contributed the expression of OTX1.Mechanistically,we identified that OTX1 activated AKT signaling pathway by transactivating the expression of IFITM3 and thus promoted the proliferation of GBC cells.Taken together,our results showed that TDCA-YAP1-dependent expression of OTX1 regulated IFITM3 and affected GBC proliferation via AKT signaling pathway.Our experiments also suggested that OTX1 is a novel therapeutic target for GBC.