Preliminary Study On The Effect And Mechanism Of ATXN3 On Angiogenesis In Breast Cancer

ObjectiveAt present,the global incidence and mortality of breast cancer are increasing year by year,which is a serious threat to women’s health.The growth and metastasis of breast cancer depend on angiogenesis,and the ability to induce angiogenesis is one of the basic characteristics of tumor cells.In addition,more and more studies have shown that deubiquitin enzyme(Deubiquitinatingenzymes,DUBs)participates in different stages of breast cancer through deubiquitination activity on important substrates,and most DUBs plays a promoting role in tumorigenesis and development.However,the role and mechanism of DUBs in breast cancer angiogenesis is not clear.Therefore,we made a preliminary study on the role and mechanism of DUBs in breast cancer angiogenesis,hoping to provide a new direction for the role of DUBs in breast cancer angiogenesis and help clinical anti-angiogenesis therapy and improve prognosis of breast cancer.ATXN3(also known as Aaxin-3,ATX3,AT3)is a member of the Machado-Joseph protein domain protease(Machado-Josephdisease,MJD)family.Many studies have confirmed that ATXN3 has many substrates,including Chk1,p53,BECN1,CHIP,histone H2 B,etc.,indicating that ATXN3 is a widespread deubiquitination enzyme that regulates the deubiquitination and stability of various proteins.The purpose of this study is to explore the role and mechanism of ATXN3 in breast cancer angiogenesis,and to propose and verify the ubiquitin-stabilized mitochondrial-related apoptosis inducing factor 2(AIFM2)to promote breast cancer angiogenesis.Methods1.The analysis of the protein genomics panorama based on targeted therapy found that angiogenesis-related proteins in specific types of BC patients in TCGA were associated with RNA,and the breast cancer angiogenesis-related DUBs: ATXN3 were determined by combining the breast cancer metastasis-related DUBs molecules screened in the previous TCGA database.2.Construction of stable knockdown and overexpression of ATXN3 breast cancer MCF-7 and MDA-MB-231 cell models,using in vitro angiogenesis assays(including angiogenesis correlation factor detection in cell supernatant;endothelial cell ring formation experiment;endothelial cell chemotaxis experiments;mouse aortic ring experiment),and mouse in vivo model of breast cancer in situ,combined with bevacizumab,verified that ATXN3 can induce breast cancer angiogenesis.3.The differentially expressed proteins between MCF-7 cells overexpressed ATXN3 and control cells were quantitatively analyzed by the i TRAQ technique,and then the deubiquitination substrate mitochondrial associated apoptosis inducing factor 2(AIFM2)of ATXN3 was verified by Western blot;Immunoprecipitation(IP)assay and ubiquitin assay.The recovery experiment was used to verify that ATXN3 participates in breast cancer angiogenesis through substrate AIFM2.4.The serum of patients with clinical breast cancer was collected,and the expression of ATXN3,VEGF-A in serum was detected by ELISA.The tissues of patients with clinical breast cancer were collected,and the expressions of ATXN3 and CD31 were detected by immunohistochemistry assay.Results1.The analysis of the protein genomics panorama based on BC occurrence and targeted therapy in the early stage found that the angiogenesis-related proteins of specific types of BC in TCGA were poorly correlated with RNA,suggesting that post-transcriptional regulation such as protein stability regulation plays a role in it,and the expression of a variety of deubiquitinases in such patients is different from that of other types of patients,suggesting that these enzymes play a role in the angiogenesis process.In conjunction with previous studies,a series of breast cancer metastasis-associated DUBs,including ATXN3,were initially screened in the TCGA database.2.After overexpression of ATXN3 in breast cancer MCF-7 and MDA-MB-231 cells,the results of in vitro angiogenesis experiments showed that ATXN3 could promote the secretion of VEGF-A into the cell supernatant by breast cancer cells.The use of breast cancer cell supernatant overexpressing ATXN3 can induce endothelial cell ring formation and migration,and can also promote the neovascularization of the aortic ring in mice.Knocking down ATXN3,the above results are reversed.At the same time,overexpression of ATXN3 was able to promote breast cancer tumorigenesis and lung metastasis in mice,and this advantage disappeared after the use of bevacizumab.Immunohistochemistry showed an increase in CD31 density.3.The results of protein profile,Western blot,IP and ubiquitin showed that mitochondrial associated apoptosis inducing factor 2(AIFM2)was the deubiquitinated substrate of deubiquitin enzyme ATXN3,and ATXN3 could maintain the protein stability of AIFM2.In vitro studies have shown that the supernatant of breast cancer cells knocked down ATXN3 can inhibit the ring formation and migration of endothelial cells,while the overexpression of AIFM2 can reverse this process.4.The results of ELISA and immunohistochemistry showed that breast cancer patients with high expression of ATXN3 had higher expression of VEGF-A in serum and CD31 in tissue.Conclusionsour study revealed for the first time the role of ATXN3 in breast cancer angiogenesis,which maintains the protein stability of AIFM2 by deubiquitination,thus promoting breast cancer cells to secrete more angiogenic factors(VEGA-A)and induce angiogenesis.ATXN3 protein is positively correlated with angiogenic markers in serum(VEGF-A)and tissue(CD31),suggesting that the level of serum ATXN3 maybe used as an index for clinical diagnosis and prognosis.