| Objective:As a conserved evolutionary process,autophagyis the only mechanism to degrade large structures such as organelles and protein aggregates for cell to survive under stress.Recent studies indicated that autophagy has been playing paradoxical roles in cancer for it can prevent initiation of some cancers by keep the stability of none cancer cell,and also may support tumor growth by continuous energy supply.It is widely agreed that there is a huge difference of expression of autophagy related genes between tumor and adjacent tissue,and the expression difference is always correlated with clinicopathologic parameters including TNM stage,histologicalgrade and lymph node metastasis.Therefore,autophagy related genes are becoming increasingly recognized for their potentials in tumorigenesis and treatment,and prognostic marker.Breast cancer is the leading cause of death among women all around the world.The life style,diet structure,the way of procreation are all been through great changes as the rapid development of economy in China,the morbidity of breast cancer in our country is growing faster than ever.As a disease with high heterogeneity,Breast cancer is classified into luminal type,HER2 type and triple negative breast cancer(triple negative breast cancer)according to its biomarkers including estrogen receptor(ER),progesterone receptor(PR)and epidermal growth factor receptor 2(HER2).triple negative breast cancer,lacks of all 3 biomarkers,account for 15~20% breast cancer and the patients with triple negative breast cancer are the subgroup with the worst prognosis because it is highly recurrence many years after operation.Therefore,it is still urgent to find new prognostic markers and molecule targets to cure triple negative breast cancer patients.Atg7(Autophagy related gene 7),known as ubiquitin E1-like activating enzyme,is essential for the assembly and function in the expansion of autophagosomal membranes.Atg7-deficient mice impaired both constitutive and starvation-induced autophagy,leading to early death soon after birth due to respiratory distress.Atg7 is reported to play significant role in lung cancer,liver cancer,prostate cancer and it is a promising target for clinical therapy.According to some studies,Atg7 is overexpressed in breast cancer and variant in Atg7 rs8154(A>G)was significantly associated with breast cancer-specific survival,also,Atg7 silencing in MCF-7 cells enhances the efficacy and apoptotic effect of docetaxeland exhibit improved anticancer effects.These studies strongly suggested that Atg7 is a important molecule in breast cancer.In our research,we intend to illuminate the correlation between the Atg7’s expression and breast cancer patients’ clinicopathologic parameters,prognosis by immunohistochemical experiment.In addition,we seek to exam the function of Atg7 in breast cancer cell lines.This study aim to provide new potential target and prognostic marker for breast cancer clinical therapy.Methods:1.Kaplan-Meier Plotter data base were to used to analyze the correlation between Atg7 m RNA level and breast cancer patients’ overall suvival time after surgery,survival curve were also drew.90 patient(including all molecular types)were randomly picked and then immunohistochemical were perform to examine Atg7 protein expression.2.Multiple breast cancer cell lines including MCF-7,T47D(luminal),SKBR3(HER2)and MDA-MB-231,BT549(triple negative)were cultured and basal level of Atg7 expression were examined by western blot.3.A micro array containing 165 patients were used to perform immunohistochemical,Atg7 expression were correlated with patients’ clinicopathologic parameters including Ki-67 level,TNM stage,lymph node metastasisandhistologicalgrade,tumor size.42 surgically resected triple negative breast cancer patients were selected from First Affiliated Hospital of China Medical University from 2009 to 2013.Correlation between Atg7 expression and patients’ over survival time were analyzed.4.Atg7 were transfected into or knock down in breast cancer cell,then the migration,proliferation,epithelial-mesenchymal transition of Atg7 were examined by transwell,CCK8,western blot and the apoptosis level were examined by flow cytometry in order to test the function of Atg7 in breast cancer cells.5.The OCR and ECAR were measured using a Seahorse XFp analyzer.Tumor formation was performed by subcutaneous injection in nude mice with BT549 cells.Results:1.Kaplan-Meier Plotter data base indicated that among all types of breast cancer,higher Atg7 m RNA expression were correlated with better prognosis.In triple negative breast cancer however,higher Atg7 m RNA expression were correlated with worse prognosis.Immunohistochemical results of 90 patients showed that Atg7 expression in triple negative breast cancer tissues is obvious lower than non triple negative breast cancer and adjacent duct.2.Micro array results indicated that Atg7 expression is correlated with patients’ age,Ki-67 level,TNM stage,lymph node metastasis and histologicalgrade but unrelated to tumor size.Moreover,survival curve according to 42 patients’ Atg7 expression and overall survival time indicated that higher Atg7 is correlated with better prognosis.3.Result of western blot showed that Atg7 protein level is obviously lower in MDA-MB-231 and BT549 cells compared to MCF-7,T47D(luminal)and SKBR-3(Her-2)cells.Migration,proliferation,epithelial-mesenchymal transition of MDA-MB-231 and BT549 cells were inhibited and apoptosis level were upregulated after Atg7 transfection.Knock down of Atg7 in MCF-7 cell accelerated cell proliferation and migration but inhibited epithelial-mesenchymal transition progress,and overexpression of Atg7 in MCF-7 knock down cell rescued the results cause by Atg7 knock down.4.On OCR,the basal consumption rate and the spare respiratory capacity were both reduced by overexpressing Atg7.On ECAR,Atg7 led to decreased levels of lactate production in MDA-MB-231 and BT-549 cells.In addition,Oligomycin(an aerobic glycolysis accelerator)not FCCP(an oxidative phosphorylation activator)recovered Atg7-induced loss of N-cadherin.5.Tumors of Atg7 group in nude mice were significantly smaller than tumors of NC group after 35 days growing.Conclusion:1.Atg7 expression in triple negative breast cancer tissues is lower than non triple negative breast cancer and adjacent duct.2.Higher level of Atg7 expression was associated with prolonged prognosis in triple negative breast cancer patients.3.Atg7 inhibited migration,proliferation,epithelial-mesenchymal transition and accelerated cell apoptosis in triple negative breast cancer cells.4.Atg7 reduced mitochondrial metabolism by inhibiting both OCR and ECAR.Atg7 prevents EMT mainly through inhibiting aerobic glycolysis in TNBC cells. |
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