The Expression Profile Of ACOT7 In Breast Cancer And Mechanism Of Its Role On Promoting Proliferation And Metastasis Via PGC1α-mediated Regulation Of Oxidative Phosphorylation

Background:Currently,breast cancer(BC)has surpassed lung cancer to become the most common malignant tumor worldwide.There are 2.26 million new cases of BC each year,accounting for 11.7%of all cancers.With the improvement of early screening and treatment means of BC,the five-year survival rate of BC has reached about 80%,but the recurrence and metastasis of BC are still the main causes of death of BC patients.Therefore,it is critical to investigate the molecular mechanisms of BC recurrence and metastasis and identify key targets in BC recurrence and metastasis.Metabolic remodeling is one of the most important features of tumors.Tumor cells adaptively reprogram their metabolism to provide sufficient energy and materials for biomacromolecule synthesis.In 1920s,Otto Warburg first discovered that tumor cells still tend to generate energy through glycolysis,regardless of oxygen availability,which led to the traditional consumption that oxidative phosphorylation(OXPHOS)might be inhibited in tumor cells.However,several studies have shown that OXPHOS is up-regulated in some cancers,including BC,pancreatic ductal adenocarcinoma,leukemia and lymphoma.Therefore,inhibition of OXPHOS may serve as a potential anti-tumor strategy.The aim of this study is to explore the biological basis and upstream mechanisms of dysregulated metabolism in BC cells,which may provide potential therapeutic targets for the precision treatment of BC.Metabolic remodeling of tumor is often accompanied by alterations in the expression of metabolic enzymes.In addition to catalyzing intracellular metabolic reactions,metabolic enzymes also participate in regulating gene expression,cell cycle,DNA damage repair,cell antioxidant capacity,proliferation,survival,apoptosis and tumor microenvironment,further expanding the metabolic dependence of tumor cells,and endowing tumor cells with the ability to adapt to different environmental stimuli.Therefore,exploring the role of these metabolic enzymes in the tumorigenesis and development,as well as the prospects of these metabolic enzymes as new targets for anti-tumor therapy,not only provide new molecular targets for individualized cancer therapy,but also have important guiding significance for the development of drugs targeting tumor metabolism.Acyl-CoA thioesterases play important roles in lipid metabolism,which are responsible for maintaining the ratio of activated fatty acids to free fatty acids and the content of CoA in cells.Acyl-CoA thioesterase 7(ACOT7)is highly expressed in the cytoplasm and abundant in brain tissue and testis.ACOT7 plays an important role in the prevention of neurotoxicity by regulating fatty acid metabolism within neurons.At present,ACOT7 has been found to be upregulated in ovarian cancer,prostate cancer,colon cancer,hepatocellular carcinoma and other cancers.Some studies have shown that ACOT7 promotes the proliferation of BC and lung cancer by regulating PKCζ-p53-p21 pathway.ACOT7 can be transcriptionally activated by KLF13 to promote the progression of hepatocellular carcinoma.However,the role of ACOT7 in the malignant progression of BC are still unclear.In this study,we used a series of in vivo and in vitro functional experiments to explore the function and possible mechanisms of ACOT7 in BC.Methods:1.GEPIA database was used to verify the expression of acyl-CoA thioesterases in BC.2.Quantitative PCR was used to detect the expression levels of ACOT7 mRNA in BC tissues and adjacent non-tumor tissues.3.Immunohistochemistry was used to detect the expression level of ACOT7 protein in BC tissues.4.CCK8 and EdU experiments were used to explore the effect of ACOT7 gene on the proliferative ability of BC cells in vitro.5.Transwell and scratch experiments were used to evaluate the role of ACOT7 gene on the migrative and invasive ability of BC cells.6.Seahorse analysis,ATP level kit and mitochondrial complex I activity kit were used to detect the regulatory effect of ACOT7 gene on mitochondrial function and cell metabolism in BC cells.7.DCFH-DA and Mitosox Red probes were performed to evaluate intracellular ROS and mitochondrial ROS levels,respectively.JC-1 method was performed to evaluate the mitochondrial membrane potential of cells.8.Western blot and immunofluorescence were used to confirm the effect of ACOT7 gene on the expression of PGC1α and its downstream genes.9.PGC1α was knocked down in ACOT7-overexpressing cells,and its effects on the proliferation,migration and invasion of BC cells were detected by EdU and Transwell experiments.10.PGC1α was knocked down in ACOT7-overexpressing cells,and its effects on oxygen consumption rate,ATP level and mitochondrial complex I activity of BC cells were detected by Seahorse analysis,ATP level kit and mitochondrial complex I activity kit.11.Statistical analysis:SPSS(Version 19)and GraphPad(Version 8)were used for statistical analysis.Results:1.GEPIA database was used to detect the expression of acyl-CoA thioesterases in BC,and found that ACOT7 was significantly overexpressed in BC.2.ACOT7 was upregulated in BC tissues compared with non-tumorous adjacent tissues verified by quantitative PCR(p=0.009).The mRNA level of ACOT7 in BC tissues with lymph node metastasis was significantly higher than that in BC tissues without lymph node metastasis(p=0.037).The mRNA level in BC patients with tumor size≥3cm was significantly higher than that in BC patients with tumor size<3cm(p<0.001).3.The expression of ACOT7 protein was positively associated with tumor size(p=0.032),lymph node metastasis(p=0.022)and Ki-67 index(p=0.027),but not with other clinicopathological parameters.4.Proliferation,migration and invasion-related function experiments indicated that ACOT7 overexpression could enhance the proliferative,invasive and migrative capabilities of BC cells.On the contrary,ACOT7 knockdown could inhibit the proliferative,invasive and migrative capabilities of BC cells.Subcutaneous tumorigenesis assay showed that ACOT7 overexpression could promote the growth of subcutaneous tumor(p=0.036)and increase the tumor weight(p=0.003).5.Gene set enrichment analysis of BC data in the TGGA database showed that ACOT7 was highly enriched in OXPHOS-related pathways.Seahorse analysis was used to detect mitochondrial respiration,and it was found that ACOT7 overexpression could significantly improve the oxygen consumption rates of BC cells.Further application of ATP level kit and mitochondrial complex I activity detection kit confirmed that ACOT7 overexpression increased ATP level and activity of mitochondrial complex I in BC cells.6.Tests of DCFH-DA and Mitosox Red probes indicated that ACOT7 knockdown significantly elevated the level of intracellular ROS and mitochondrial ROS.JC-1 method was used to test the mitochondrial membrane potential of tumor cells,and it was found that ACOT7 knockdown could significantly reduce the mitochondrial membrane potential.7.Western blot and immunofluorescence indicated that knockdown of ACOT7 downregulated the expression levels of PGC1α and its downstream genes,and overexpression of ACOT7 upregulated the expression levels of PGC1α and its downstream genes.The expression level of ACOT7 was significantly correlated with the expression level of PGC1α and NRF1 by immunohistochemistry in our BC tissue microarrays.8.We further knocked down PGC1α in ACOT7-overexpressing cells and found that PGC1α knockdown could reverse the promoting effects of ACOT7 on the proliferation,migration and invasion of BC cells.And PGC1α knockdown in ACOT7overexpressing cells could reverse the promoting effects of ACOT7 on the oxygen consumption rates,ATP level and mitochondrial complex I activity.Conclusion:1.ACOT7 mRNA was significantly overexpressed in BC tissues than in adjacent non-cancer tissues.In the verification of BC tissue samples in our tissue microarrays,analysis of pathological characteristics showed that high expression of ACOT7 protein was positively correlated with tumor size,lymph node metastasis and Ki67 index.With the increase of ACOT7 mRNA level,the overall survival and distant metastasis-free survival of BC patients decreased.Therefore,ACOT7 may serve as a potential prognostic biomarker for BC.2.ACOT7 promoted the proliferation,migration and invasion of BC cells in vivo and vitro.3.ACOT7 upregulated oxygen consumption rate,ATP production and mitochondrial respiration chain complex I activity in BC cells.4.Mechanistically,ACOT7 upregulated PGC1α expression to promote oxygen consumption rates of BC cells and enhance tumor proliferation,migration and invasion.