| Background:Colorectal Cancer(CRC)is a common malignant tumor of digestive system,which ranks the top three in incidence and mortality in the world.The early treatment is usually surgical resection,but the late treatment is limted and the prognosis is poor,partly due to the lack of effective therapeutic target and complex molecular mechanism of invasion and metastasis in colorectal cancer.Therefore,it is of great clinical significance to study the molecular mechanism of invasion and metastasis of colorectal cancer cells and to design new therapeutic schemes for the prevention or treatment of this disease.Tripartite Motif Containing 21(TRIM21),an E3 ubiquitin ligase,is involved in various important physiological processes for maintaining protein homeostasis,such as cell metabolism,cytokinesis,and redox regulation.Recent studies have shown that TRIM21 plays an important role in tumorigenesis.Although studies have found that TRIM21 plays an important regulatory role in inflammation-associated intestinal carcinogenesis.However,the role of TRIM21 in the progression of colorectal cancer(tumor metastasis stage)remains unclear and needs to be further studied.The abnormal activity of Hippo-Yap signal transduction is closely related to tumorigenesis.Yap is a key coactivator of transcription in Hippo signaling pathway,and its abnormal expression and intracellular activity play an important role in driving tumorigenesis.Therefore,targeting inhibition of the nuclear activity of Yap is an important strategy for the development of anti-tumor drugs.The current anti-Yap drug,verteporfin,is able to inhibit tumor growth by targeting the interaction of YAP/TAZ,however,it causes severe toxicity and side effects in patients for its wide blockage of transcription.Therefore,targeting the upstream modulators of YAP/TAZ would serve as an attractive option for drug development.Methods:In vitro cell culture,in vivo mouse models and tumor organoids were applied to investigate the role of TRIM21 in CRC invasion and metastasis.Molecular assays,such as Western blot,qPCR,co-immunoprecipitation,immunohistochemistry,were performed to study the pathways regulated by TRIM21.A structure-based virtual screening of FDA-approved drugs was performed to identify the potential regulator of TRIM21.The structural and biochemical analysis were used to evaluate the function of vilazodone.Results:We found a direct interaction between TRIM21 and MST2 by co-immunoprecipitation experiments.We identified the PRY-SPRY domain of TRIM21 and the LINKER domain of MST2 as the key domains for the combination by constructing truncated mutations of Trim21 and MST2.Molecular docking and biochemical experiments revealed that MST2 at valine 366(V366)is a key amino acid for maintaining the interaction between TRIM21 and MST2.Mechanistic studies further revealed that TRIM21 directly interacts with and ubiquitinates MST2,which in turn promoted the formation of MST2 homodimer and enhanced the MST2 activity,ultimately suppressing the nuclear localization of YAP and expression of its target genes.Based on this,we established a computer virtual screening model for targeting TRIM21 protein.We are strikingly to find vilazodone,antidepressant,which targets TRIM21 protein and inhibits YAP activity.The organoid model of colorectal cancer patients and the mouse model of lung metastasis further confirmed that vilazodone exhibited effectively anti-metastatic action on CRC progression,which was dependent on TRIM21.Conclusion:In the present study,we revealed the molecular mechanism by which TRIM21 inhibits colorectal cancer cell invasion and metastasis by activating the core kinase MST2 in the Hippo signaling pathway.Moreover,we identified that vilazodone directly bound to TRIM21 to enhance the interaction of TRIM21 with MST2,thereby facilitating the ubiquitination and activation of MST2.This led to the inhibition of YAP’s oncogenic activity and suppression of CRC invasion and metastasis.This study will provide a new theoretical basis for the treatment of advanced colorectal cancer and knowledge accumulation for the future development of targeted anti-tumor drugs with our own intellectual property rights. |
