OTUD1 Enhances The Chemosensitivity Of Triple Negative Breast Cancer Cells To Doxorubicin By Modulating P16

BackgroundBreast cancer(BC)is the most common cancer among women worldwide and one of the leading causes of death among women worldwide.Triple negative breast cancer(TNBC)is the most metastatic and recurrent breast cancer subtype with the worst prognosis because of its high heterogeneity.Few triple-negative breast cancer patients benefit from endocrine therapy or Her2-targeted therapy because of the lack of corresponding therapeutic receptors.Therefore,chemotherapy remains the mainstay of treatment for triple-negative breast cancer,such as the anthracycline class of classical chemotherapy agents represented by doxorubicin(DOX).However,triple-negative breast cancer patients receiving chemotherapy show frequent resistance to chemotherapy,resulting in poor response to chemotherapy and poor prognosis.At present,the mechanism of chemoresistance in triplenegative breast cancer has not been fully elucidated,and chemoresistance and a series of complications after chemoresistance have become a great challenge for comprehensive treatment.Therefore,it is of great clinical significance to find potential therapeutic targets that can enhance the chemosensitivity of triple-negative breast cancer.A growing body of evidence suggests that deubiquitinating enzymes(DUBs)regulate cell proliferation,differentiation,apoptosis and tumor progression by Deubiquitinating proteins.OTU Domain-Containing Protein 1(OTUD1)is a member of the OTU family of Deubiquitinating enzymes.Recent studies have found that the deubiquitinating enzymes OTUD1 acts as a tumor suppressor in a variety of cancers.The expression level of OTUD1 is down-regulated in many kinds of tumors,which is involved in the process of proliferation and metastasis,oxidative stress and chemotherapy resistance by inhibiting the signal pathway related to malignant progression.Although many studies have suggested that OTUD1 is involved in the regulation of carcinogenesis and development of malignant tumors,the role and mechanism of OTUD1 in chemoresistance of triple-negative breast cancer have not been clarified.PurposeIn this study,we aimed to analyze the expression level of OTUD1 in triple-negative breast cancer and explore its role and molecular regulatory mechanism in chemotherapy resistance of triple-negative breast cancer,it provides a new strategy for finding new targets of targeted therapy and improving the prognosis of patients with triple-negative breast cancer.Methods1.The expression of OTUD1 in breast cancer and its prognosis were analyzed by bioinformatics.2.Two TNBC cell lines,MDA-MB-231 and BT549,were transfected with lentivirus to construct TNBC cell lines with stable overexpression and knockdown of OTUD1.3.The CCK-8 cell proliferation assay and colony formation assay were used to detect whether OTUD1 was involved in the regulation of TNBC cell proliferation.4.The CCK-8 cell viability assay,flow cytometry,Ki67 staining and TUNEL staining were used to analyze whether OTUD1 was involved in the regulation of the sensitivity of TNBC cells to DOX.5.The potential interaction proteins of OTUD1 were analyzed by immunoprecipitation and mass spectrometry,and the interaction between OTUD1 and 16 was further clarified by protein co-immunoprecipitation,the expression levels of OTUD1 and P16 in triple negative breast cancer tissues were analyzed by immunohistochemistry and Western blot.6.The transient transfection assay,Western blot assay,proliferation and viability of CCK-8 cells and Flow cytometry assay were used to determine whether P16 could reverse the effect of OTUD1 on DOX sensitivity of TNBC cells.7.The tumor-bearing nude mouse model was established to further evaluate the role of OTUD1 in regulating the sensitivity of TNBC to doxorubicin in vivo.Results1.OTUD1 expression level and prognostic characteristics in breast cancer: TCGA database,GSE61304,GSE76250,GSE22820 and immunohistochemical assay showed that compared with normal breast tissues,OTUD1 expression was low in breast cancer tissues and significantly lower in triple-negative breast cancer tissues.Survival analysis showed that low expression of OTUD1 was negatively correlated with disease-free survival(DFS),progression-free survival(PFS),overall survival(OS)and relapse-free survival(RFS),high expression of OTUD1 was significantly positively associated with recurrence-free survival in breast cancer patients with a history of neoadjuvant chemotherapy.2.OTUD1 inhibited TNBC cell proliferation and enhances the chemosensitivity of TNBC cells to DOX: The results of RT-q PCR and Western blot showed that OTUD1 was successfully knocked down and overexpressed in TNBC cells.The results of in vitro functional experiments showed that overexpression of OTUD1 significantly inhibited the proliferation of TNBC cells and enhanced the cytotoxicity of DOX on TNBC cells,the apoptosis induced by DOX was enhanced and the level of Cleaved-caspase 3 and Cleavedcaspase 9 was up-regulated.In contrast,knockdown of OTUD1 significantly promoted the proliferation and clonogenic ability of triple-negative breast cancer cells and inhibited the cytotoxic effect of DOX on TNBC cells,down-regulated the level of Cleaved-caspase 3 and Cleaved-caspase 9.3.OTUD1 enhanced the sensitivity of TNBC cells to DOX in vivo: The tumor weight of nude mice with over-expression of OTUD1 was lower than that of the control group after administration of both normal saline and DOX,the staining intensity of Ki67 was lower and the percentage of TUNEL positive cells was higher.Moreover,in the group of OTUD1 overexpression combined with DOX,the tumor weight was the lowest in the four groups,the Ki67 staining intensity was the weakest,and the TUNEL-positive cell rate was the highest.4.The OTUD1-P16 axis may serve as a key signaling axis regulating the sensitivity of TNBC cells to DOX: Immunoprecipitation combined with mass spectrometry analysis suggested that P16 might be a potential interaction protein of OTUD1,and coimmunoprecipitation further confirmed the interaction between OTUD1 and P16.In addition,the expression of OTUD1 was positively correlated with the expression of P16 protein in TNBC tissues.Western blot assay showed that overexpression of OTUD1 upregulated P16 expression,while knockdown of OTUD1 inhibited P16 expression.The results of functional recovery experiments showed that knockdown of P16 reversed the sensitivity of OTUD1-overexpressing MDA-MB-231 cells to DOX,whereas overexpression of P16 increased the sensitivity of OTUD1-knockdown MDA-MB-231 cells to DOX.ConclusionsThe expression level of OTUD1 in triple-negative breast cancer was significantly decreased and the low expression was closely related to the poor prognosis of the patients.In vitro,OTUD1 overexpression significantly inhibited the proliferation of TNBC cells and increase the sensitivity of TNBC cells to doxorubicin,while OTUD1 knockdown resulted in the opposite effect.The results of animal experiments showed that overexpression of OTUD1 could enhance the chemosensitivity of TNBC cells to DOX in vivo.Mechanistically,OTUD1 interacts with P16 to inhibit TNBC cell proliferation and enhance the chemosensitivity of TNBC cells to DOX by upregulating the expression of P16.This study aimed to investigate the role and mechanism of OTUD1 in chemoresistance of triplenegative breast cancer,and proposed for the first time that OTUD1 participates in chemoresistance of triple-negative breast cancer by regulating the expression of P16,it may provide a new idea for neoadjuvant chemotherapy and targeted therapy of breast cancer.