Molecular Mechanism Of SMURF2 And USP13 Regulating STAT1 In Prostate Cancer Cells

Prostate cancer(PCa)is one of the most common malignant tumors among men in western countries with high mortality rate.Currently,the treatment of PCa can be divided into surgical castration and drug castration.However,patients with advanced PCa treated by castration will inevitably develop into metastatic castration resistant prostate cancer(m CRPC)within five years.The mechanisms of drug resistance include androgen receptor(AR)amplification and mutation,ligand free but active AR splicing variants,mutation of ancillary transcription factors,intratumoral androgen synthesis,etc.There is no curable treatment.Epithelial-mesenchymal transition(EMT)is the first step of tumor invasion and migration.Previous studies in our laboratory found that the expression of TNS4(Tensin 4)in normal prostate cells was significantly higher than in prostate cancer cells,and it was confirmed that TNS4 could inhibit the ubiquitination degradation of STAT1,stabilize STAT1 protein,and then inhibit the EMT process.However,the molecular mechanism of its regulation in STAT1 ubiquitination degradation has not been reported.In order to analyze the signaling pathways that influence the ubiquitination degradation of STAT1,in this study,based on bioinformatics website analysis,we identified the E3 ubiquitin ligase SMURF2 of HECT family,which regulated STAT1,by using reverse transcription PCR(RT-PCR),gene overexpression and knockdown assay,immunofluorescence and Co-immunoprecipitation(Co-IP)assay.At the same time,it was reported that the deubiquitinase USP13 affected STAT1 in other literatures.Then,CCK-8 assay,colony formation assay,wound-healing assay and transwell migration assay were used to further explore their roles in the growth,cloning and migration of prostate cancer cells and their molecular mechanisms,the main results are as follows.1.Overexpression of SMURF2 downregulated the level of STAT1 protein,while knockdown of SMURF2 upregulated the level of STAT1 protein;Knockdown of SMURF2 inhibited the growth,cloning and migration of prostate cancer cells;Immunofluorescence and Co-IP assay showed co-localization and interaction between SMURF2 and STAT1;SMURF2 was mainly distributed in the nucleus of normal cells,but was increased in the cytoplasm of cancer cells.Knockdown of SMURF2 in prostate cancer cells increased the accumulation of STAT1 in the cytoplasm.2.Overexpression of USP13 upregulated STAT1 protein level without affecting its transcription level,while knockdown of USP13 downregulated the level of STAT1protein;USP13 inhibited the growth,cloning and migration of prostate cancer cells;Ubiquitination and Co-IP experiments showed that USP13 reduced the ubiquitination of STAT1 and the binding between SMURF2 and STAT1;Immunofluorescence assay showed that USP13 increased the content of STAT1 in cytoplasm.In conclusion,SMURF2 regulated STAT1 ubiquitination and degradation as an E3 ubiquitin ligase,while USP13 stabilized STAT1 by deubiquitination in prostate cancer cells.These results can lay a foundation for further study on the molecular mechanism of TNS4 inhibiting STAT1 ubiquitination and degradation in prostate cancer,and also provided theoretical basis for the development of new ideas for the treatment of prostate cancer.