Molecular Mechanism Of USP11-Mediated Proliferation Of Breast Cancer Cells By Stabilizing Cytoplasmic P21

USP11 is a deubiquitinase.USP11 regulates DNA damage repair,proliferation and metastasis in multiple cancer types by specifically interacting with and deubiquitinating target proteins.The tumor-suppressive activities of USP11 were found in non-small cell lung cancer,brain tumors and squamous cell carcinoma.However,USP11 is mainly localized in the cytoplasm and promotes breast cancer cell malignant proliferation.The mechanisms that regulate the localization of USP11 and the relationship between the localization of USP11 and its biological functions remain obscure.The function of p21 is related to its cellular localization.Nuclear p21 functions as a tumor suppressor protein,but cytoplasmic p21 functions as an oncoprotein.Cytoplasmic p21 is positively associated with breast cancer malignancy and a potential molecular target for breast cancer.p21 is a labile protein,but high levels of p21expression in the cytoplasm of breast cancer.p21 protein levels are mainly regulated by two posttranslational modifications,namely phosphorylation and ubiquit ination.Phosphorylation events mainly impact the subcellular localization of p21.For instance,ERK2-and AKT-mediated phosphorylation leads to cytoplasmic localization of p21.Moreover,ubiquitination is primarily involved in the control of p21 protein levels.In the cytoplasm,the E3 ubiquitin ligase compl ex CRL2^LRR1 has been revealed to promote p21 degradation via ubiquitination.However,it remains unknown the molecular mechanism leading to direct regulation of cytoplasmic p21 stability.Given USP11 and p21 similar subcellular localization and biological function in breast cancer cell,we hypothesized that there might be a significant positive correlation between p21 and USP11 in breast cancer,and post-translational modifications of USP11 play a significant role in its subcellular localization,which resulted in breast cancer cell malignant proliferation via regulating p21 levels.The following conclusions can be obtained from this study:（1）A strong association between USP11 and p21 in breast cancer.Using database and breast cancer tissue microarrays,we identified that significantly higher p21 and USP11 levels were observed in breast cancer tissues than in adjacent normal tissues,and increased p21 or USP11 expression was associated with higher clinical stages.Moreover,p21 and USP11 expression showed the same trend in breast cancer tissues and cell.Further research using immunofluorescence and co-immunoprecipitation experiments,we found that USP11 interacts and colocalizes with p21 in the cytoplasm of breast cancer cells.（2）USP11 stabilizes cytoplasmic p21 through deubiquitination.Breast cancer cells were transfected with wild-type USP11(USP11^WT),nuclear localization signal deletion mutants of USP11（USP11-ΔNLS）and a catalytically inactive USP11(USP11^C275S/C283S),and we found that USP11 led to an increase in cytoplasmic p21levels,which is dependent on its enzymatic activity.However,USP11 overexpression,USP11 knockdown and USP11 inhibition with mitoxantrone markedly decreased cytoplasmic p21.USP11 overexpression,knockdown and inhibition had no effects on the m RNA levels of p21,which indicated that the regulation of USP11 on p21 is not on the transcriptional level.Proteasome inhibition,ubiquitination and half-life assays indicated that USP11 prolonged the half-life of cytoplasmic p21 by cleaving its polyubiquitin chains.（3）ERK1/2-mediated phosphorylation leads to cytoplasmic localization of USP11.A strong correlation between p21 and USP11 in th e cytoplasm of breast cancer tissues and cells.Hyperactivation of the ERK1/2 and AKT pathways in breast cancer often contributes to cytoplasmic localization of p21.To elucidate the potential regulatory mechanism underlying of USP11 localization in breast cancer cytoplasm,we examined the effects of p21,ERK1/2 and AKT on USP11 localization.The findings suggested that p21 did not affect the subcellular localization of USP11 in breast cancer cells.ERK1/2 promoted USP11 retention in breast cancer cytoplasm by phosphorylating the Ser905 site of USP11,thereby stabilizing cytoplasmic p21.AK T did not affect the subcellular localization of USP11,but regulated the expression level of USP11 through the ubiquitin-proteasome pathway.（4）USP11 contributes to breast cancer cell proliferation by stabilizing cytoplasmic p21.The results of clone formation and tumorigenicity experiments in nude mice showed that USP11 depletion severely suppressed MCF-7 cells proliferation,and that p21-ΔNLS reintroduction could reversed the effect induced by USP11depletion.The results of tumorigenic experiments in Usp11 KO mice showed that the microenvironment of Usp11 knockout was not favorable for breast cancer cells growth.In addition,the USP11 inhibitor mitoxantrone can effectively inhibit the growth of breast cancer cells in mice.In conclusion,our study elucidated that USP11 promotes breast cancer cell proliferation by stabilizing cytoplasmic p21,uncovered ERK1/2 promotes USP11retention in breast cancer cytoplasm by phosphorylating the Ser905 site of USP11,expanded the knowledge of the anticancer mechanism of mitoxantrone and indicated that cytoplasmic USP11-p21 axis may be a potential therapeutic target for the treatment of breast cancer.