FBXO22 Inhibits Hippo Signaling Pathway To Promote The Growth Of Pancreatic Cancer Through Ubiquitination And Degradation Of LATS2

Background and Objective:Pancreatic cancer is a common malignant tumor of digestive system.Its incidence rate and mortality are increasing year by year in the world,and it is also one of the main causes of cancer related deaths in the world.Although the diagnosis and treatment of pancreatic cancer have improved in recent years,the prognosis of patients is still poor,with a total 5-year survival rate <10%.The main reasons for poor prognosis of pancreatic cancer are asymptomatic progression,molecular heterogeneity and early dissemination of the tumor.Therefore,a better understanding of the mechanism in the occurrence and development of pancreatic cancer will provide a new direction for the treatment of pancreatic cancer and play a positive role in improving the prognosis of patients.Hippo signaling pathway is an evolutionarily highly conserved inhibitory cell signaling pathway.The key to activation of this pathway is to promote the phosphorylation of YAP/TAZ through kinase cascade reaction,thus limiting cell proliferation and tissue growth.The key regulators of Hippo pathway are LATS1 and LATS2 kinases.LATS1/2 can directly phosphorylate YAP and TAZ at several different sites,inhibit their entry into the cell nucleus,interact with 14-3-3 protein outside the nucleus and degrade them through ubiquitin proteasome pathway.The non-phosphorylated YAP /TAZ can enter into cell nucleus and interact with DNA-binding proteins to trigger the transcription of genes such as cell proliferation,apoptosis and migration..Ubiquitin proteasome system plays an important role in the occurrence and development of various human tumors.FBXO22 protein is an E3 ubiquitin ligase,which has been reported to be involved in many aspects of tumor progression.Multiple gene database data show that FBXO22 is highly expressed in pancreatic cancer.However,the clinical significance and biological function of FBXO22 protein in pancreatic cancer are still unclear.This topic has certain clinical significance for early diagnosis and precise treatment of pancreatic cancer by in-depth studying the expression of FBXO22 in pancreatic cancer,analyzing its clinical significance,and exploring the role and mechanism of FBXO22 in the occurrence and development of pancreatic cancer.Methods:1.The expression of FBXO22 in pancreatic cancer was analyzed by GEPIA database.At the same time,the expression of FBXO22 in pancreatic cancer and corresponding adjacent tissues was detected by immunohistochemistry,real-time fluorescent quantitative PCR and Western blot.The relationship between the different expression levels of FBXO22 and the clinicopathological characteristics and survival time of postoperative patients with pancreatic cancer was statistically analyzed.2.Pancreatic cancer cell lines with low and high expression of FBXO22 were established by interference and transfection techniques.CCK-8 and Colony formation assay were used to detect the cell proliferation and growth ability of each group in vitro.The effect of FBXO22 expression on the growth of pancreatic tumor was studied in vivo by constructing a nude mouse model of transplanted tumor.3.The m RNA expression of FBXO22,LATS2 and Hippo pathway related genes CYR61,CTGF and C-Myc were detected by q RT PCR in cell lines with different levels of FBXO22 expression,pancreatic cancer and corresponding paracancerous tissues;WB was used to detect the expression of FBXO22,LATS2,YAP1 and p-YAP1 proteins;The localization of YAP1 in pancreatic cancer cells was detected by immunofluorescence.4.The direct interaction between FBXO22 and LATS2 in pancreatic cancer cells was detected by Co-IP assay.After inhibiting protein synthesis and degradation of pancreatic cancer cells,we use WB to detected the effect of different FBXO22 expression on LATS2 protein expression.The effect of overexpression or under-expression of FBXO22 on the ubiquitination level of LATS2 was detected by ubiquitination test.5.Knock down FBXO22 and down regulate the expression of LATS2 in pancreatic cancer cells with high expression of FBXO22.Observe the changes of tumor cell proliferation through CCK8 and Colony formation assay;Observation of tumor growth in nude mice.Results:1.High expression of FBXO22 in pancreatic cancer.GEPIA database search showed that the expression of FBXO22 m RNA in pancreatic cancer tissues was significantly higher than that in paracancerous tissues.The results of q RT PCR and WB showed that the levels of FBXO22 m RNA and protein in pancreatic cancer tissues were significantly higher than those in adjacent tissues.IHC data showed that FBXO22 positive staining mainly appeared in pancreatic cancer tissue,while the adjacent tissues were weak staining or negative staining.2.The prognosis of patients with high expression of FBXO22 was worse.Statistical analysis of IHC results shows that: The high expression of FBXO22 is closely related to tumor size and lymph node metastasis.But,there was no significant correlation with gender,age,TNM grade,vascular and nerve infiltration.The patients with high expression of FBXO22 had poorer overall survival and shorter disease-free survival.3.FBXO22 promotes pancreatic cancer cell proliferation and tumor growth.CCK-8,Colony formation and nude mouse tumorigenesis experiments showed that knocking down the expression of FBXO22 inhibited the proliferation and tumor growth of pancreatic cancer cells;Overexpression of FBXO22 promotes the proliferation of pancreatic cancer cells and tumor growth.4.FBXO22 inhibits Hippo signal pathway through LATS2.q RT-PCR showed that FBXO22 overexpression promoted the m RNA expression of CYR61,CTGF,C-myc in pancreatic cancer cells,but had no significant effect on the level of LATS2 m RNA.WB showed that overexpression of FBXO22 significantly reduced the expression of p-YAP1 and LATS2 proteins;However,total YAP1 protein was increased.LATS2 protein was significantly low expressed in pancreatic cancer tissues,and was negatively correlated with the level of FBXO22.Immunofluorescence showed that after overexpression of FBXO22,the expression of YAP1 in the cytoplasm decreased,while the expression of YAP1 in the nucleus increased.5.FBXO22 can directly binds to LATS2,promotes the ubiquitination and degradation of LATS2.Co-IP assay showed that FBXO22 protein in pancreatic cancer cells can directly bind to LATS2 protein.CHX inhibited protein synthesis,and FBXO22 silencing significantly inhibited the degradation of LATS2 protein in pancreatic cancer cells.After MG132 blocked the proteasome pathway,there was no significant change in LATS2 protein in pancreatic cancer cells with high expression of FBXO22,the effect of FBXO22 on LATS2 was eliminated in pancreatic cancer cells.Ubiquitination experiment showed that knocking down the expression of FBXO22 reduced the ubiquitination of LATS2 in pancreatic cancer cells,while up regulating the expression of FBXO22 increased the ubiquitination of LATS2.6.Knocking down LATS2 can partially reverse the carcinogenesis mediated by FBXO22: CCK-8,Colony formation and nude mouse tumorigenesis experiments showed that FBXO22 knockdown significantly inhibited the proliferation and growth of pancreatic cancer cells;With the knockdown of LATS2,the role of FBXO22 in promoting the growth of pancreatic tumors was partially reversed.Conclusions:1.FBXO22 is highly expressed in pancreatic cancer,and is closely related to the clinical characteristics and prognosis of pancreatic cancer patients;2.FBXO22 can promote pancreatic cancer cell proliferation and tumor growth in vivo and vitro;3.FBXO22 inhibits Hippo pathway activity and affects downstream target genes through LATS2;4.FBXO22 can directly bind to LATS2,and after the ubiquitin modification of LATS2,it can be degraded through the ubiquitin-proteasome system;5.FBXO22 inhibits Hippo signaling pathway through ubiquitination of LATS2,thus promoting the proliferation of pancreatic cancer cells and tumor growth.