The Study Of CHFR Regulating Chemoresistance In Triple Negative Breast Cancer Through Destabilizing ZEB1

Breast cancer is more and more morbidity and mortality as the tumor in women in China and the world in recent years.The three targets of estrogen receptor(ER),progesterone receptor(PR)and human epidermal growth factor receptor(HER2)are all negatively expressed in triple negative breast cancer(TNBC).TNBC accounts for 15-20% of the total breast cancer.It has the characteristics of high drug resistance and metastasis and lacks therapeutic targets.It is prone to metastasis and relapse,making it difficult to cure through clinical treatment.Currently,TNBC is divided into four subtypes: a luminal androgen receptor subtype,an immunomodulatory subtype,a basal-like and immune-suppressive subtype and a mesenchymal-like subtype.Based on this molecular typing,triple-negative breast cancer patients have received precise treatment options such as immunotherapy and targeted therapy in addition to conventional chemotherapy.However,treatment options for some patients,especially those with mesenchymal-related subtypes of TNBC,are still very limited,and still face the dilemma of drug resistance and relapse.There is an urgent need to investigate the new drug resistance mechanism of the mesenchymal subtype of TNBC and develop a new treatment plan.Therefore,research on this issue is being conducted.Zinc finger E-box binding homeobox 1(ZEB1),as an important activator of Epithelial mesenchymal transition(EMT),not only modulates the migration and invasion of tumor cells,but also confers tumor cells stem cell-like property and plasticity.Our previous work found that mesenchymal-like breast cancer cells with high expression of ZEB1 were strongly resistant to irradiation treatment.Here,through cell cycle synchronization experiments,we found that mesenchymal breast cancer cells in different cell cycle phases showed various resistance to conventional chemotherapeutic drugs such as doxorubicin(DOX).Compared to cells in G1 phase,Cells in G2/M phase were strongly resistant to the drug.In order to uncover the mechanism by which ZEB1 is highly expressed in G2/M phase,we used affinity purification-mass spectrometry to identify the interacting proteins of ZEB1,and found that the cell cyclerelated ubiquitin ligase(Checkpoint with forkhead and ring finger domains,CHFR)can interact with ZEB1 proteins.To determine whether CHFR inhibits the expression of ZEB1,we transfected CHFR into HEK293 T and TNBC mesenchymal cells(SUM159 cells)and observed a significant reduction in the half-life of both exogenous and endogenous ZEB1 expression.In contrast,stable expression of CHFR sh RNA in TNBC mesenchymal cells(LM2 breast cancer cells)effectively inhibits CHFR expression and leads to a significant upregulation of ZEB1 expression.These results suggest that CHFR may act as the ubiquitin E3 ligase of ZEB1.Further ubiquitination experiments showed that CHFR is indeed the ubiquitin ligase of ZEB1.In conclusion,CHFR interacts with ZEB1 to promote its ubiquitination and then inhibit the expression of ZEB1.Although ZEB1,which induces drug resistance in multiple tumors,has been reported,there is no research on whether CHFR is involved in chemotherapy resistance in the mesenchymal subtype of TNBC.Here,by stably expressing CHFR in mesenchymal TNBC cell line LM2,we found that CHFR can significantly improve the therapeutic effect of clinical chemotherapy drugs such as doxorubicin on TNBC mesenchymal type by inhibiting the expression of ZEB1.DOX can inhibit the synthesis of RNA and DNA,affect the cell cycle of tumor cells and the expression of cycle-related proteins,and can induce apoptosis.Based on the low expression of CHFR in TNBC,this study further searched for small molecule compounds that could induce the expression of CHFR.It is reported that Trichostatin A(TSA)treatment activates CHFR transcription in colon cancer cell lines.To determine whether TSA plays the same role in TNBC,we treated five TNBC mesenchymal cell lines and observed that while TSA did not regulate the mRNA level of CHFR,it significantly upregulated CHFR protein level and downregulated ZEB1 expression.However,in CHFR knockdown cells,TSA could not downregulate ZEB1,these results suggest that TSA may inhibit the expression of ZEB1 by upregulating CHFR.In addition,we found that TSA treatment can significantly increase the sensitivity of TNBC mesenchymal cells to chemotherapeutic drugs,which in turn promotes cell death.Numerous studies have demonstrated that the up-regulation of fatty acid synthase(FASN)expression plays a pivotal role in the epithelial-mesenchymal transition(EMT).Furthermore,overexpression of FASN in human breast cancer has been shown to be associated with drug resistance.Here,it was found that ZEB1 can be positively correlated with FASN,and the significant down-regulation of FASN can reduce drug resistance.At this time,the apoptosis factor Caspase-3 was significantly up-regulated in TNBC mesenchymal cells LM2.Analysis of the database revealed that patients with high CHFR expression in breast cancer and the mesenchymal subtype of TNBC exhibited significantly higher recurrencefree survival rates compared to patients with low CHFR expression.Further analysis of the correlation between the survival of 177 patients with mesenchymal breast cancer who had the expression of CHFR protein and received conventional chemotherapy,and found that patients with mesenchymal subtype breast cancer with low CHFR expression were resistant to chemotherapy.These results suggest that downregulation of CHFR may lead to the overexpression of ZEB1 in human malignant breast tumors,which in turn leads to chemoresistance and ultimately promotes breast cancer metastasis and recurrence.In conclusion,this study identifies the chemoresistance mechanism of TNBC mesenchymal type,and finds that ZEB1 and CHFR can be used as potential drug therapy targets,which provides a new solution for the treatment options for TNBC mesenchymal type.