The Effect And Mechanism Of Knockdown Of PC4 Increases Chemosensitivity Of Oxaliplatin In Triple Negative Breast Cancer

Background and objective: In recent years,the incidence of breast cancer has been increasing.Compared to other breast cancer subtypes,triple negative breast cancer(TNBC)is prone to metastasis and recurrence and has the worst prognosis.Besides,TNBC is not sensitive to endocrine therapy and Her2-targeted therapy due to the lack of the corresponding receptors and is the most difficult type.Therefore,chemotherapy is still the most widely used for advanced TNBC in clinical practice.Oxaliplatin(Oxa)is a third-generation platinum-based anticancer drug,which is popularly applied in the clinical therapeutic schedule of most tumors including TNBC,but the side effects and drug resistance plague the clinical efficiency.Therefore,the discovery of new therapeutic targets to enhance the sensitivity of TNBC to chemotherapy and alleviate chemotherapy-related side effects has become an urgent clinical need.Positive Cofactor 4(PC4),a multifunctional nuclear protein,was initially identified as a general cofactor mediating transcriptional activation.Apart from its roles in transcription,PC4 also plays multiple functions in various cellular processes,such as DNA replication and repair,maintaining genomic stability and in heterochromatin gene silencing。In the past decades,researches have revealed that PC4 plays an important role in tumorigenesis,tumour development and therapy.PC4 has been found to be upregulated in a variety of tumors,and seems to be involved in proliferation,invasion and metastasis.Besides,some studies showed that PC4 might affect the sensitivity of tumour cells to cancer treatments including radiotherapy and chemotherapy.Recently,researchers have identified a small molecule targeting to inhibit PC4 in lung cancer cells,and achieved promising results.It is suggested that PC4 may be a potential target to improve the sensitivity of tumor chemotherapy.However,TNBC is a special malignant tumor,and whether PC4 can be used as a potential target for improving TNBC chemotherapy remains to be further explored.This study aims to investigate the expression of PC4 in TNBC cells and explore its role and preliminary mechanism in the chemosensitivity in TNBC to Oxa,so as to provide a new idea for improving chemotherapy in TNBC patients.Methods:1.The expression characteristics of PC4 in TNBC cells.We selected two TNBC cell lines(MDA-MB-231,MDA-MB-453)and two non-TNBC cell lines(MDA-MB-361,SK-BR-3),q-PCR was used to detect the m RNA level of PC4,Western blot and Immunofluorescent staining were used to detect the protein level of PC4.2.The effect research on down-regulating PC4 increases the chemosensitivity of Oxa in TNBC cells.Cell lines with stable and low expression of PC4 were constructed by lentivirus transfection in MDA-MB-231 and MDA-MB-453 cells.Then,control group and knockdown of PC4 groups(sh PC4-1 group and sh PC4-2 group)were treated with oxaliplatin respectively.CCK-8 assay and colony formation were used to evaluate whether down-regulation of PC4 could enhance the growth-inhibitory effect of TNBC cells with Oxa treatment.Hoechst staining,flow cytometry and Western blot were used to evaluate whether down-regulation of PC4 could enhance the pro-apoptotic effect of TNBC cells with Oxa treatment.Finally,the in vivo effect of down-regulating PC4 on enhancing the sensitivity of TNBC cells to oxaliplatin was further evaluated in tumor-bearing nude mice.3.The preliminary mechanism research on down-regulating PC4 increases the chemosensitivity of Oxa in TNBC cells.Firstly,after PC4 was down-regulated,mTOR related protein expression in TNBC cells and transplanted tumors was detected to evaluate whether down-regulation of PC4 affected mTOR signal pathway.To further investigate whether mTOR is an important mechanism,the PC4 knockdown group was treated with mTOR agonist,MHY1485,and compared the effect.Results:1.The q-PCR showed that the m RNA level of PC4 was significantly upregulated in TNBC cells compared with non-TNBC cells.Moreover,the Western blot and Immunofluorescent staining also confirmed that the expression of PC4 was significantly elevated in TNBC cells than that in non-TNBC cells.2.In vitro experiments,the CCK-8 assay and Colony formation showed that PC4 knockdown enhanced the growth-inhibitory effect of TNBC cells with Oxa treatment.Besides,apoptosis detection by Flow cytometry,the Hoechst staining and Western blot showed that PC4 knockdown promoted the Oxa-induced apoptosis in two TNBC cells.In vivo experiments,PC4 knockdown group exhibited slower tumor growth,smaller and lighter tumor xenografts.In addition,the Immunofluorescent staining for TUNEL and Cleaved caspase 3 showed that more apoptotic cells were also detected in PC4 knockdown group compared with control group after Oxa treatment.3.Our data showed that PC4 knockdown significantly downregulated the phosphorylation of mTOR in two TNBC cells and tumor xenografts.Moreover,MHY1485 rescued the downregulated mTOR signaling and reversed the growth-inhibitory effects and pro-apoptotic effects in PC4 knockdown cells after Oxa treatment.Conclusion:1.PC4 is significantly upregulated in TNBC cells,suggesting its important role in TNBC progression.2.Knockdown of PC4 enhances the chemosensitivity of TNBC cells in vivo and vitro,implying that PC4 might play a critical role in chemotherapeutic resistance of TNBC cells.3.Inhibition of PC4 increases chemosensitivity of Oxa in TNBC by suppressing mTOR pathway.