The Role OfTRIM21-regulated Hippo-YAP Signaling In The Invasion And Metastasis Of Colorectal Cancer

Background:Colorectal cancer(CRC)is a common malignant tumor,ranking among the top three malignant tumors worldwide in terms of incidence and mortality.In recent years,with the popularization of colonoscopy,the proportion of early colon cancer diagnoses in China has been increasing yearly.However,the early symptoms of colon cancer are more insidious,and most patients are already in the middle and late stages when they are diagnosed,so the treatment effect is not ideal and the recurrence rate after surgery is high.As an important point in the development of colorectal cancer,invasion and metastasis of colorectal cancer cells are closely related to the poor prognosis of patients in the middle and late stages of the disease.They are also a major cause of cancer-related deaths.Similar to other tumors,colon cancer metastasis is a multistage process involving the coordinated action of multiple factors and genes.Therefore,the screening and identification of key molecules that regulate the infiltration and metastasis of colon cancer cells,and the detailed elucidation of their regulatory mechanisms are of paramount clinical importance for targeted intervention in mid-to late-stage colon cancer.TRIM21(Triartite Motif 21)is an important member of the Triartite Motif(TRIM)family with E3 ubiquitin ligase activity.TRIM21 was first identified as an autoantigen,and studies have confirmed that TRIM21 is involved in the regulation of various physiopathological processes such as cell proliferation,cell migration,apoptosis,autophagy,and differentiation.Recent studies have revealed that TRIM21 plays an important role in tumorigenesis.However,the specific function depends on the specific tumor cells and microenvironment.However,the role of TRIM21 in colorectal cancer invasion and metastasis and its regulatory mechanisms are still unclear.Hippo signaling is a kinase chain consisting of a number of protein kinases and transcription factors;it is highly conserved from lower to higher animals and plays an important role in maintaining tissue growth and organ size.YAP,known as a key transcriptional coactivator in the Hippo signaling pathway,is involved in intracellular signal transduction and transcriptional regulation of its downstream target genes in a phosphorylated form,which plays an important role in maintaining the normal size of organs as well as the homeostasis of the body’s internal environment.Recent studies have shown that the abnormally high expression of YAP and its significant aberrant nuclear localization are closely related to the development of various tumors.However,the underlying regulatory mechanisms remain to be further investigated.METHODS:By collecting RNA-seq data of colon cancer from the TCGA and GEO databases,we analyzed the expression of TRIM21 in colorectal cancer tissues and revealed its clinical significance.With in vitro migration and invasion assays,a tail vein injection lung metastasis model in nude mice,in situ implantation of colon cancer tissue to induce liver metastasis in mice,an AOM/DSS-induced intestinal tumor model in TRIM21 knockout mice,and a colon tumor organoid model,we investigated the role of TRIM21 in colon cancer cell migration and invasion in vitro and infiltration and metastasis in vivo.On this basis,we further investigated the molecular mechanism by which TRIM21 regulates Hippo/YAP signaling by using RNA-seq,immunoprecipitation,Western blot,qPCR and other experimental methods.RESULTS:Clinically,the expression of TRIM21 showed a significant negative correlation with the progression of colon cancer.Patient prognostic analysis suggested that low TRIM21 expression was strongly associated with poor prognosis in colon cancer patients.Gene Set Enrichment Analysis(GSEA)further revealed that TRIM21 expression was strongly associated with recurrence and metastasis of colon cancer.RNA-seq enrichment analysis reveals the involvement of TRIM21 in the regulation of epithelial-mesenchymal transition(EMT)and Hippo signaling pathways.In vitro and in vivo functional experiments confirmed that TRIM21 regulates Yap-dependent EMT in colon cancer cells.Mechanistic studies revealed that TRIM21 catalyzes K63-linked polyubiquitination of MST2 through direct interaction with MST2,and promotes MST2 homodimer formation and autophosphorylation,which in turn enhances MST2 kinase activity,and reduces Yap nuclear accumulation and its oncogenic activity to inhibit colon cancer development.CONCLUSIONS:In this study,we revealed the important role of TRIM21 in the infiltration and metastasis of colon cancer cells,which relies on its regulation of the Hippo-YAP signaling pathway.Mechanistic studies revealed that MST2,the core kinase of Hippo signaling,is a novel interacting protein of TRIM21.TRIM21 is able to catalyze its K63-linked ubiquitination through interaction with MST2,which in turn promotes the formation of the MST2 homodimer and enhances its kinase activity,which is most likely an important molecular pathological basis for TRIM21 to regulate the epithelial-mesenchymal transition in colon cancer cells.This study further advances the understanding of the molecular mechanism of fine invasion and metastasis in colorectal cancer,and provides new ideas and strategies for the treatment of intermediate and advanced colon cancer.