Role And Mechanism Of Cytidine Triphosphate Synthase-1(CTPS1) In Triple Negative Breast Cancer

Triple negative breast cancer(TNBC)is the most heterogeneous subtype of breast cancer with unfavorable outcome and uncertain efficacy.Therefore,exploring the inherent heterogeneity of TNBC and searching potential effective therapeutic targets to further classify and treat TNBC are not only the main path to solve the current clinical problems but also the hot points of clinical and basic research.Objective: To explore the role and potential regulation mechanism of CTPS1 in triple negative breast cancer.Methods: Differentially expressed proteins between carcinoma and adjacent normal tissues were identified by proteomics methodology.We analyzed the relevance and prognostic correlation between the expression of CTPS1 and TNBC based on TCGA database,GEO databases and immuno-histochemistry(IHC)of 210 patients who were treated in our department and with complete follow-up information.CTPS1over-expression and RNA interference vector were further constructed to regulate the expression of CTPS1 in MDA-MB-231 and BT-549 TNBC cell lines.The effects of CTPS1 on proliferation,migration and invasion of those cells were detected by cell counting kit-8(CCK-8)assay,cloning assay,Transwell assay,wound-healing assay and xenograft model.In addition,JASPAR and PROMO databases were used to predict the upstream transcription factors of CTPS1.Dual luciferase reporter assay was used to screen the transcription factor and the binding sites of promoter region.Chromatin immunoprecipitation(Ch IP)assays was utilized to verify the interaction between YBX1 and CTPS1 promoter region.The function of YBX1 in TNBC were verified by RNAi test and rescue experiment.We constructed WCGNA to analyze the CTPS1 related genes and co-expression network in TNBC and used RNA-seq to seek the enrichment of downstream genes and pathways.Protein mass spectrometry combined with Co-Immunoprecipitation(Co-IP)was performed to identify the binding proteins of CTPS1.Combining the results of above experiment,we tried to look for the downstream action factor of CTPS1 and verify the regulatory relationship between them through Western blot and cell function experiment.Results: Based on the clinical samples and proteomics methodology,we found that CTPS1 showed significant difference in cancer and adjacent tissues.Furthermore,CTPS1 expression was signifcantly upregulated in TNBC tissues and cell lines.Higher CTPS1 expression was correlated with a poorer disease-free survival(DFS)and overall survival(OS)in TNBC patients.Silencing of CTPS1 dramatically inhibited the proliferation and invasion ability in MDA-MB-231 and BT-549 cells.Xenograft tumor model also indicated that CTPS1 knockdown remarkably reduced tumor growth in mice.Predicted by bioinformatics methods,YBX1 could bind to the promoter of CTPS1 to promote its transcription.Dual luciferase reporter and Ch IP assays confirmed that YBX1 exerts transcription factor activity by binding to the-1967 bp sequence of the upstream promoter region of CTPS1.Rescue experiments confirmed that the enhanced cell proliferation and invasion ability induced by YBX1 over-expression could be reversed by CTPS1 knockdown.Furthermore,the expression of YBX1 was positively correlated with CTPS1 in TNBC tissues and associated with the prognosis of TNBC patients.P53 pathway was detected in both WCGNA and RNA-seq enrichment analysis.CTPS1 might interact with EEF1A1 though the results of Co-IP.Additional experiments showed that CTPS1 could influence the proliferation and invasion of TNBC by regulating EEF1A1.According to the previous reports,EEF1A1 was involved in the regulation of P53 pathway.As the results,it was showed that the important proteins Bcl-2 and BAX in p53 signaling pathway could be regulated by CTPS1 and EEF1A1.The rescue experiment confirmed the existence of this regulator chain.Conclusion:CTPS1 was highly expressed in TNBC.It could affect the biological function of TNBC and was associated with poor prognosis.CTPS1 could be up-regulated by transcription factor YBX1,and further regulated P53 pathway through binding EEF1A1.Our data demonstrated that YBX1/CTPS1/EEF1A1 axis might play an important role in the progression of TNBC.CTPS1 could be a promising prognosis biomarker and potential therapeutic target for patients with triple negative breast cancer.