Pin1 Regulates Chemoresistance Through Ubiquitination-mediated Activation Of Akt Signaling Pathways In Breast Cancer

Objective:Doxorubicin is one of the classic drugs for the treatment of breast cancer,and its efficacy is poor due to its chemotherapy resistance and high dose toxicity.However,the molecular mechanisms remain poorly understood.The purpose of this study was to investigate the role of Pin1 in regulating chemosensitivity of breast cancer cells and to explore the mechanism of Pin1 regulating the activation of Akt signaling pathway.Methods:In this study,human breast cancer cell line MCF-7 and breast cancer doxorubicin resistant cell line MCF-7 ADR were used as research objects.The expression of Pin1 in breast cancer cell lines and drug-resistant cell lines was detected by Western blot.After Si RNA was transfected into breast cancer cell lines and drug-resistant cell lines,the effects of Pin1 on the growth,proliferation and chemotherapeutic resistance of breast cancer cells were observed by MTT,clone formation and EdU assay.Western blot detection was used to explore the changes of Akt signaling pathway and MDR-1 expression.The interaction between Pin1 and Akt was verified by immunoprecipitation method.The ubiquitin experiment was used to explore the effect of Pin1 on the ubiquitin of Akt.Pin1 knockout lentivirus stable cell line was constructed and BALB/c nude mouse tumor transplantation model was established by subcutaneous injection,to explore the effect of Pin1 on tumor growth in vivo.Results:(1)The expression of Pin1 in drug-resistant breast cancer cell line MCF-7ADR was significantly higher than that in breast cancer cell line MCF-7.Immunohistochemistry showed that the expression of Pin1 in breast cancer tissues was significantly higher than that in paracancerous tissues.Kaplan-Meier analysis showed that the overall survival time of patients with high expression of Pin1 was significantly shorter than that of patients with low expression of Pin1.(2)Knockdown Pin1 can inhibit cell proliferation,increase apoptosis,decrease IC50 and reverse the drug resistance of breast cancer cells to doxorubicin.(3)Western blot assay showed that Pin1 regulated the expression of MDR1 through Akt/mTOR signaling pathway to reverse drug resistance in breast cancer.(4)Pin1 interacted with Akt and regulated the expression of p-Akt without changing the abundance of Akt,and the ubiquitin level of Akt was significantly decreased in Pin1 knockdown cells by ubiquitin assay.These results suggest that Pin1 regulates the phosphorylation and activation of Akt through ubiquitin.(5)In vivo experiments confirmed that knockout of Pin1 could inhibit the growth of tumor.Conclusions: Through the above studies,we found that the expression of Pin1 in breast cancer is up-regulated and associated with drug resistance and poor prognosis.Knockdown of Pin1 can regulate the expression of MDR1 through Akt/mTOR signaling pathway and reverse the drug resistance of breast cancer.Pin1 binds to Akt and regulates Akt phosphorylation and activation via the ubiquitination.Therefore,this study suggests that the inhibition of Pin1 expression may be a therapeutic strategy to reverse drug resistance in breast cancer.