E3 Ubiquitin Ligase FBXW5 Promotes Migration And Invasion Of Gastric Cancer Through The Dysregulation Of Hippo Pathway

Background and Purpose: Dysregulation of the Hippo signaling pathway leads to the tumorigenesis and progression of gastric cancer(GC).The core components of the Hippo signaling pathway are mainly regulated by the ubiquitin-proteasome system.For example,the PPx Y sequence of the LATS1/2 kinase domain can directly bind to the WD40 domain then be ubiquitinated and degraded.The F-box protein family is an important subunit of the SCF(Skp1-Cullin1-F-box protein)E3 ligase complex and works on substrates identification.FBXW5,as a new member of the F-box protein family,contains repeated WD40 domains is widely expressed in human malignancies.This study aims to explore the relationship between FBXW5 and Hippo signaling pathway.Methods: Bioinformatics analysis was used to analyze the relationship between FBXW5 m RNA and the expressions of related oncogenes.Immunohistochemistry(IHC)was used to analyze the expression of FBXW5 protein and other oncoproteins in clinical and xenografted paraffin samples.Western blot analysis of the FBXW5 protein expression in fresh clinical GC specimens and xenografts,and the regulatory relationship between FBXW5 protein and Hippo pathway-related proteins in clinical GC samples or GC cell lines.The regulatory relationship of FBXW5 on Hippo pathway-related oncogenes at the m RNA level was investigated by q PCR.Immunofluorescence(IF)was used to explored the intracellular localization of YAP1.Transwell assay and Wounding healing assay were used to evaluate the invasion and migration abilities of GC cells.CCK-8 and clonogenic assay were used to study the effects of FBXW5 on cell chemoresistance.Co-immunoprecipitation(co-IP)was conducted to identify the binding between FBXW5 and Hippo pathway.The promoting effect of FBXW5 on LATS1 ubiquitination was validated by in vivo ubiquitination assay.Xenograft models were used to verify the biological function of FBXW5 in GC cells.Results: FBXW5 protein is highly expressed in GC,elevated FBXW5 expression leads to tumor invasion,lymph node metastasis,higher TNM stage and predicts poor prognosis of GC patients.FBXW5 promotes the invasion,metastasis and EMT of GC cells.And FBXW5 silencing will slow the growth of xenografts.Moreover,FBXW5 induces GC resistance to 5-fluorouracil and cisplatin.Western blot and real-time quantitative PCR revealed that FBXW5 regulates the Hippo signaling pathway.The results of immunofluorescence experiments suggested that FBXW5 promotes the transfer of YAP1 protein to the nucleus.Co-immunoprecipitation and in vivo ubiquitination assays suggested FBXW5 binds to LATS1 protein and promotes the ubiquitination process of LATS1.Conclusions: Elevated expression of FBXW5 protein is related to GC progression resulting in poor prognosis.FBXW5 promotes the invasion,metastasis and chemoresistance of GC cells by promoting ubiquitination and degradation of LATS1 which results in inactivation of the Hippo pathway.