PartⅠ:Molecular Mechanism Of USP49 Regulating Lipid Metabolism Reprogramming To Promote Malignant Proliferation Of Triple Negative Breast Cancer PART Ⅱ:Evaluation Of A Beneficial Effect Of Adjuvant Chemotherapy In Patients With StageⅠ Triple-negative Br

Breast cancer is the most common malignancy among women in the world,which seriously threatens the health of women.In terms of current breast cancer treatment,Due to the low expression of ER,PR and HER-2 in patients with triple-negative breast cancer,endocrine therapy and targeted therapy are ineffective,and only chemotherapy can be used.However,the overall prognosis is very poor,which is the difficulty of current treatment.Tumor metabolic reprogramming belongs to the hall marks of tumors.Warburg first discovered that cancer cells prefer energy supply through aerobic glycolysis in 1956,which is called the "Warburg effect".In recent years,metabolic reprogramming(glucose metabolism,lipid metabolism,amino acid metabolism)in malignant tumors have gradually become the focus of researchers.Studies have found that there is a special lipid metabolism pattern in triple-negative breast cancer,which may lead to its biological characteristics of high proliferation and high aggressiveness.ACAT1(Acetyl-CoA Acetyltransferase 1)transfers long-chain fatty acids to cholesterol and converts them to cholesterol esters to form lipid droplets.There are many evidences that phenotypic lipid droplets are associated with malignant progression and poor prognosis in a variety of tumors.Meanwhile,there is a special metabolic pattern of fatty acid metabolism in triple negative breast cancer,which includes up-regulation of FAO and down-regulation of fatty acid synthesis(FAS).Previous studies have reported that high levels of FAO play a key role in the malignant proliferation of TNBC.Therefore,in this study,from the point of lipid metabolism,ACAT1 was first found to be highly expressed in basal-like breast cancer cell lines through fluorescence staining of lipid droplet probe,which can cause the formation of lipid droplet aggregation.Then we search the gene which can enables ACAT1 gene expression changes through the ubiquitin enzyme plasmid library.Because the basal-like subtypes highly coincidence with triple negative breast cancer in gene expression profiles,We used TCGA database to analyze the results of differential genes combined with cell RNA-seq in patients with triple-negative breast cancer and non-triple-negative breast cancer.Finally,USP49 was selected as a candidate gene.Western Blot analysis showed that USP49 was highly expressed in triple negative breast cancer cell lines.Protein Co-Immunoprecipitation(Co-IP)combined with mass spectrometry showed that USP49 interacted with fatty acid β oxidation(FAO)ratelimiting enzyme CPT1A in lipid metabolism.In triple negative breast cancer,there is a special metabolic pattern of fatty acid metabolism with up-regulation of FAO and downregulation of fatty acid synthesis(FAS).Previous studies have reported that high levels of FAO play a key role in the malignant proliferation of TNBC.Through Co-IP and in vitro ubiquitination experiments,we found that USP49 could stabilize the protein expressions of AC AT1 and CPT1A through deubiquitylation,resulting in a relatively special lipid metabolism pattern in triple-negative breast cancer.Through lipid metabolization-related phenotype experiments,we found that USP49 could up-regulate the level of FAO metabolism,acetyl CoA production,cholesterol synthesis and lipid droplet aggregation in cells.Through Xcelligence RTCA MP system,EdrU assay,clone formation assay and Transwell assay,USP49 was significantly associated with proliferation,invasion,migration,and clone formation of breast cancer cells.In conclusion,the high expression of USP49 in triple-negative breast cancer can stabilize the expression of ACAT1 and CPT1A through deubiquitylation enzyme.By affecting the lipid metabolism phenotype of breast cancer cells,the proliferation,invasion and migration ability of breast cancer cells can be enhanced,and the tumor growth can be promoted.Triple-negative breast cancer is a subtype of breast cancer with negative estrogen progesterone receptor(ER PR)and HER2 receptor,accounting for about 15%of the population.Due to the lack of therapeutic targets,hormone receptor antagonism therapy and HER2-targeting therapy cannot be carried out,and the current treatment regimen is mainly adjuvant chemotherapy.In this study,our aim is to evaluate the effect of adjuvant chemotherapy on improving the prognosis of patients with stage I triple-negative breast cancer(TNBC).Firstly,the early stage TNBC patients diagnosed in the SEER 18 database from 2010 to 2015 were included.All patients were divided into T1a,T1b,Tlc and IB groups.Kaplan-Meier plots and log-rank tests were used to compare the differences in breast cancer-specific survival(BCSS)and overall survival(OS)between subgroups of variables.A Cox proportional hazard model was used to determine the prognostic factors affecting BCSS and OS.A total of 9256 patients were enrolled.Among these patients,380 died from breast cancer,and 703 died from other reasons.Patients who received chemotherapy had significantly better BCSS and OS than those who did not receive chemotherapy for stage T1cN0M0(BCSS,hazard ratio(HR)=0.68,95%confidence interval(CI)0.51-0.90;OS,HR=0.54,95%CI 0.44-0.67)and stage IB(BCSS,HR=0.39,95%CI 0.16-0.95;OS,HR=0.41,95%CI 0.19-0.87)disease.Patients who received chemotherapy did not have significantly better BCSS or OS than those who did not receive chemotherapy for stage T1aN0M0 or T1bN0M0 disease.The patients who received chemotherapy in the poorly differentiated and undifferentiated groups had better BCSS(HR=0.68,95%CI 0.52-0.88)and OS(HR=0.54,95%CI 0.44-0.66)than the patients who did not receive chemotherapy.The main conclusion of this study is that in the current treatment for early triplenegative breast cancer,the treatment strategies for patients with T1bN0M0 may be overly aggressive,and some patients may even be overtreated.The adjuvant chemotherapy strategies similar to those used for more advanced triple-negative breast cancer do not significantly improve prognosis,meanwhile patients are subjected to side effects of chemotherapy that may not be necessary.According to current clinical guidelines,patients with stage T1bN0M0 TNBC are probably overtreated.The prognosis of these patients with stage T1aN0M0 or T1bN0M0 disease is good enough that adjuvant chemotherapy cannot improve it further.In this study,data analysis was conducted in a large database for the prognosis of patients with early triple-negative breast cancer treated by chemotherapy.In view of the possible problems existing in the current guidelines,some suggestions were put forward for reference,which provided favorable evidence for the clinical treatment of early patients in the future.