Study On The Function And Molecular Mechanism Of Golgi Body Protein ACBD3 In Regulating Breast Cancer Stem Cells

1 Purposes:To study the biological function of ACBD3(Acyl-CoA binding domain containing 3)in breast cancer stem cells,to explore the molecular mechanism of ACBD3 regulating breast cancer stem cells,and to identify the correlation between ABCD3 and clinicopathological stage,laying a new theoretical foundation for the research and development of new drugs targeting breast cancer stem cells.2 Background:The leading cause of cancer-related deaths in women is breast cancer.The existence of cancer stem cells,so that breast cancer patients often metastasis and recurrence,leading to the death of patients.To explore the molecular mechanism of the regulation of cancer stem cells,and then to study the specific expression profile and functional characteristics of cancer stem cells will be beneficial to develop new targeted drugs that specifically kill cancer stem cells in the near future.ACBD3(also known as GCP60,GOLPH1,PAP7)is a golgi protein involved in steroid metabolism,apoptosis,iron balance,embryonic development,and cycle control in normal cells.ACBD3 is involved in PKARIA-mediated tumorigenesis.In addition,ACBD3 expression was 3.8 times higher in non-responders than in responders of lung cancer patients treated with geffetinib.ACBD3 regulates the asymmetric division of neural progenitors.However,there have been no studies on the regulation of ACBD3 on breast cancer stem cells.3 Methods:Firstly,in order to analyze the expression of ACBD3 mRNA in breast Cancer patients,this study used The Cancer Genome Atlas(TCGA)public database to analyze the expression level of ACBD3 mRNA in 1099 cases.Realtime q-PCR and Western blotting were used to examine the mRNA transcription and protein translation levels of ACBD3 in breast cancer cells and paired clinical tissue samples.Then,the association between ACBD3 expression and cancer stem cell related genes in TCGA public database was analyzed by gene enrichment software.The correlation between ACBD3 and the characteristics of cancer stem cells was verified by real-time q-PCR,Western blotting,flow cytometry,mammary balloon formation and mouse in situ tumor formation.Finally,immunofluorescence staining was used to detect the changes of ACBD3 on the sublocalization of ?-Catenin cells,and the correlation between ACBD3 and the key protein of Wnt signaling pathway in the paraffin section samples of breast cancer was explored by immunohistochemistry.4 Results:First,mRNA levels of ACBD3 in breast cancer are significantly upregulated in Luminal A,Luminal B,HER2,nomal-like and high metastatic Basal-like types,while their expression is low in normal tissues.ACBD3 expression was significantly increased in stage III and IV patients compared with patients with early breast cancer.Secondly,TCGA public data analysis shows that the expression level of ACBD3 is positively correlated with cell proliferation-related genes and cell cycle regulating genes.The balloon formation experiment of breast cancer showed that the up-regulated expression of ACBD3 promoted the balloon formation of breast cancer cells.Finally,GSEA analysis found that ACBD3 can enrich the genes related to Wnt/?-Catenin signaling pathway.Confocal immunofluorescence staining showed that the up-regulated expression of ACBD3 enhanced the entry of ?-Catenin into the nucleus and activated the Wnt/?-Catenin signal transduction pathway.Immunohistochemical results of clinical breast cancer samples showed that when the expression of ACBD3 was up-regulated,the nuclear entry of ?-Catenin was increased,while when the expression of ACBD3 was low,the result was opposite.5 Conclusions:In summary,the mRNA transcription and protein translation of ACBD3 in breast cancer cells and clinical samples are up-regulated,which indicates a poor prognosis.ACBD3 can enhance the ball-forming ability of breast cancer cells and the tumorigenic size in mice,and promote the characteristics of breast cancer stem cells.The molecular mechanism may be to activate the Wnt/?-Catenin signaling pathway by increasing the entry of ?-Catenin protein into the nucleus,thereby increasing tumor growth.In this study,a new ACBD3-dependent mechanism for regulating the function of breast cancer stem cells was found,which may provide a new target for targeting breast cancer stem cells.