The Mechanism Of CENPF In Chemotherapy Resistance Of Triple Negative Breast Cancer

Background:Breast cancer has become the most commonly diagnosed malignancy in women and the second leading cause of cancer-related deaths in women worldwide after lung cancer.According to the latest edition of the World Cancer Statistical Report,the incidence of breast cancer in China has increased year by year,and the number of incidences has increased from 300,000 in 2015 to 420,000 in 2020,and the incidence of the population is gradually younger.The incidence of triple negative breast cancer(TNBC)accounts for about 10-15%of all newly diagnosed breast cancers.It is a special type of breast cancer with the highest degree of malignancy and the worst prognosis.Its main feature is lack of Expression of estrogen(Estrogen receptor,ER),progesterone(Progesterone Receptor,PR)and human epidermal growth factor receptor 2(Human epidermal growth factor receptors-2,HER-2).At present,due to the lack of appropriate treatment strategies for TNBC patients,chemotherapy is still the main treatment modality.However,TNBC patients often develop primary or acquired chemoresistance,which leads to poor chemotherapy effect and seriously affects the survival and prognosis of TNBC patients.Therefore,it is important to explore the molecular mechanism of chemotherapy resistance,find the key molecules of chemotherapy resistance and develop the corresponding drugs targeted,which are always an important direction in triple-negative breast cancer.Centromere Protein F(CENPF),a member of the centromeric protein family,whose expression and localization are strictly cell cycle-dependent,is involved in a variety of cellular processes,including cell division,gene expression,cell morphogenesis,and vesicle trafficking,ciliogenesis,etc.Recent studies have found that CENPF is highly expressed in a variety of malignant tumors,such as prostate cancer,breast cancer,ovarian cancer and lung adenocarcinoma,etc.It mainly promotes cancer cell proliferation,metastasis and chemotherapy resistance by activating signaling pathways related to tumor malignant progression.At present,the mechanism of CENPF in chemotherapy resistance of triple negative breast cancer is rarely reported.In this study,we systematically analyzed the molecular regulation mechanism of CENPF in chemotherapy resistance of triple negative breast cancer and provided a theoretical basis for exploring new mechanisms and therapeutic targets of chemotherapy resistance in triple negative breast cancer.Methods:We Used bioinformatics,immunohistochemical staining and real-time quantitative PCR analysis to explore the expression of CENPF in triple negative breast cancer tissues and cells.Then,we analyze the relationship between CENPF expression with clinicopathological parameters and survival prognosis of patients.The effects of CENPF on the proliferation,apoptosis,cycle,and drug sensitivity of triple negative breast cancer cells were detected in vitro.We used GSEA enrichment analysis,real-time quantitative PCR,Western blot,immunofluorescence co-localization,and co-immunoprecipitation assays to detect CENPF interacting proteins and their mechanisms in chemoresistance.Result:1.Expression and prognosis of CENPF in triple negative breast cancerThe results of GEO.TCGA.and HPA database analysis showed that compared with normal breast tissue,the expression of CENPF was significantly increased in breast cancer tissue,and the increase was more significant in triple negative breast cancer tissue.Survival analysis by Kaplan Meier Plotte website found that high CENPF expression was negatively correlated with breast cancer overall survival and recurrence-free survival.and was correlated with poor prognosis in triple negative breast cancer patients with a history of neoadjuvant therapy.The survival prognosis data of triple negative breast cancer residual tissue(RD)patient after neoadjuvant chemotherapy were obtained from the GEO database.The results of survival analysis showed that high expression of CENPF is correlated with poor prognosis in the residual tissue(RD)of triple negative breast cancer patients after neoadjuvant chemotherapy.In addition,the expression of CENPF in the tissues of 32 triple negative breast cancer patients with neoadjuvant chemotherapy was detected by immunohistochemical staining.The results showed that compared with chemotherapy-sensitive tissues,CENPF was up-regulated in chemotherapy-resistant tissues of triple negative breast cancer.Analysis of clinicopathological parameters suggested that high expression of CENPF was associated with chemotherapy resistance in triple negative breast cancer.We used the CCLE database to analyze the expression of CENPF in various breast cancer cell lines,and the results showed that the expression of CENPF was up-regulated in triple negative breast cancer cells compared with non-triple negative breast cancer cells.The results of real-time quantitative PCR showed that CENPF was up-regulated in chemotherapy-resistant cells MDA-MB-231/ADR compared with chemotherapy-sensitive MDA-MB-231 cells.2.Targeted intervention with CENPF significantly increases the chemosensitivity of triple negative breast cancer cells to doxorubicinThe results of real-time quantitative PCR and Western blot showed that compared with the control group,si-CENPF transfection could significantly down-regulate the expression of CENPF.After the cells were transfected,we carried out cell function experiments such as drug sensitivity,CCK-8 proliferation,apoptosis,and cell cycle.Analysis of the experimental results found that targeted intervention on CENPF expression enhanced the chemosensitivity of TNBC cells to doxorubicin,enhanced doxorubicin-induced apoptosis,and inhibited doxorubicininduced G2/M arrest.However,CENPF had no significant effect on the proliferation of triplenegative breast cancer cells.3.CENPF-Chkl axis regulates doxorubicin sensitivity in Triple negative breast cancer cellsWe found a positive correlation between CENPF and Chkl expression by analyzing the TCGA dataset in the GEPIA database.Subsequently,the results of real-time quantitative PCR and Western blot showed that compared with the control group,targeted inhibition of CENPF could significantly down-regulate the mRNA and protein expression of Chk1.Phosphorylation of Chkl is a key factor in the response to DNA damage caused by doxorubicin.We further tested by Western blot and found that targeting CENPF can negative regulation of Chkl transcription,reduced the total protein level of Chkl,which in turn reduced the Chkl phosphorylation levels under the action of doxorubicin.4.CENPF can transcription regulates Chkl expression via Rb-E2F axisThrough the GSEA enrichment analysis of the GSE86374 dataset in the GEO database,it was found that the high expression of CENPF was mainly enriched in the cell cycle,E2F targets,G2/M phase checkpoints,etc.Real-time quantitative PCR and Western blot showed that knockdown of CENPF could significantly inhibit the phosphorylation of Rb.The results of immunofluorescence colocalization and coimmunoprecipitation experiments showed that CENPF interacts with Rb protein,regulates the transcriptional activity of E2F1 by affecting the phosphorylation of Rb,and inhibits the expression of Chk1.Targeted inhibition of CENPF can regulate the expression of Chk1 through the Rb-E2F axis to promote the chemosensitivity of tumor cells to doxorubicin.Conclusion and significance:The expression of CENPF is significantly increased in triple negative breast cancer,and the high expression of CENPF is closely related to chemotherapy resistance and poor prognosis of triple negative breast cancer.Targeted inhibition of CENPF can significantly increase the sensitivity of triple negative breast cancer cells to doxorubicin,enhance doxorubicin-induced apoptosis,and inhibit doxorubicin-induced G2/M phase arrest.CENPF binds and promotes phosphorylation of Rb protein and promotes Chkl expression through the Rb/E2F1 axis to enhance chemoresistance to doxorubicin in triple negative breast cancer.This study is the first to investigate the role and mechanism of CENPF in chemotherapy resistance of triple negative breast cancer,and propose a new mechanism of CENPF regulating the expression of Chk1,which is expected to provide a new target for the treatment of triple negative breast cancer patients with chemotherapy-resistant.