Chronicstress-induced Epinephrine Promotes Glycolysis Via LDHA In Breast Cancer

1.BackgroundAmong females,breast cancer is the most commonly diagnosed cancer and the leading cause of cancer death.Breast cancer patients(and survivors)use to deal with important challenges daily,such as coping with stress and depression in order to improve treatment effectiveness.Chronic stress actives the sympathetic nervous system and then affect the immune,endocrine,metabolic and other functions of the system.A substantial body of literature reported that chronic stress facilitates the ability of proliferation and invasion result in promoting cancer progression and initiation.Chronic stress activates the hypothalamic-pituitary-adrenal axis,excites the sympathetic nervous system,and promotes the secretion of downstream hormones,thereby affecting the proliferation,metastasis and recurrence of tumors.Therefore,targeting the hormones secreted by chronic stress can provide a new treatment for tumor patients.Abnormal metabolism of tumor cells has become one of the important markers to distinguish them from normal cells.In contrast to normal differentiated cells,cancer cells prefer to glycolysis under aerobic condition to provide energy for cells themselves,which is known as Warburg effectLactate dehydrogenase A(LDHA)executes the final step of the Warburg effect by converting pyruvate to lactate.Deregulation of LDHA has been reported in a number of malignancies,inhibition of LDHA reduces malignant transformation and delays tumor formation,indicating an important role for LDHA in tumor initiation and progression.As might be predicted,LDHA consistently elevates‘stemness'properties of CSCs and enhances spheroid formation in hepatocellular cancer.This suggests that targeting cancer cell glycolysis,especially its key metabolic enzymes,may be a potential approach for cancer patients.2.Methods(1)(1)Mass spectrometry was employed to detect the metabolites of breast cancer cells treated with PBS and Epi;(2)Treated breast cancer cells with PBS and Epi and detected the oxygen consumption rate and extracellular acidification rate of cells by seahorse assay;(3)Glucose metabolism related kits were employed to examine the glucose uptake,lactic acid production and ATP production in cells treated with PBS and Epi.(2)(1)Western blot analysis was employed to compare the expressions of glucose metabolism related proteins in breast cancer cells after treatment with PBS and epinephrine;(2)Treated breast cancer cells with low or high glucose medium and detect glucose metabolism related proteins with Western blot;(3)Transient knockdown of the glucose metabolism related proteins in breast cancer cells,examining the expression of MYC;(4)Western blot analysis was employed to detect the glucose metabolism related proteins in breast cancer cells after treatment with LDHA activity inhibitor;(5)Treated breast cancer cells with lactic acid and examined the relevant protein levels by Western blot.(3)(1)Immunohistochemical assay was employed to detect LDHA in breast cancer patients;(2)Western blot was employed to detect LDHA in breast cancer patients between tumor and adjacent tissues;(3)Q-PCR was employed to detect LDHA in adherent culture and sphere formation of breast cancer cells.Survival analysis the correlation between the epinephrine and LDHA in breast cancer patients.(4)(1)MDA-MB-231 cells were stably infected with LDHA that was cloned into the pEGFP-C1 vector and screen the drugs targeting LDHA in the US drug library;(2)MTT assay was employed to examine the cytotoxicity of LDHA targeted drugs.(5)(1)Treated breast cancer cells with vitamin C or epinephrine and examined the glucose metabolism related proteinsby Western blot;(2)Lactic acid production kit was employed to detect the lactic acid in breast cancer cells after treatment with vitamin C or epinephrine;(3)Subcutaneously inject female NOD/SCID mice with 1×10~6 breast cancer cells(n=5),comparing tumor volume of different groups after a 30-day treatment.3.Results(1)(1)Metabolites of breast cancer cells are increased after treated with epinephrine;(2)Epinephrine-treated cells exhibited an increased extracellular acidation rate(ECAR)and decreased oxygen consumption rate(OCR).Epinephrine-treated cells increased glucose and lactate levels and decreased cellular ATP compared to control cells.(2)(1)Both HK2 and LDHA increased in response to epinephrine in breast cancer cells;(2)High glucose consistently triggered HK2 and LDHA expression and stimulated MYC in breast cancer cells;(3)Silencing LDHA significantly reversed induction of MYC by epinephrine,while silencing HK2 displayed no change on the effect of epinephrine;(4)The LDHA inhibitor sodium oxamate(Oxa)inhibited MYC expression in a dose-dependent manner.(5)The actic acid could inhibit the protein expression of MYC in breast cancer cells.(3)(1)Immunohistochemical staining(IHC)results revealed that high serum epinephrine was positively associated with high LDHA protein expression;(2)Breast cancer tissues displayed higher LDHA when compared to adjacent normal tissues;(3)Q-PCR analysis showed that MDA-MB-231-sphere-enriched cells displayed higher LDHA expression compared to those in MDA-MB-231-2D cells;(4)Patients with high serum epinephrine and high LDHA protein expression exhibited lower overall survival(OS)rate and disease-free survival(DFS)rate compared to patients with low epinephrine and LDHA levels.(4)(1)The screening identified 18 compounds,including vitamin C,which lowered fluorescence of EGFP-LDHA;(2)Vitamin C has no effect on cell viability at the experimental doses and time courses employed.(5)(1)Vitamin C attenuated the epinephrine-induced increase in LDHA in breast cancer cells;(2)Vitamin C suppressed lactate production in both the absence and presence of epinephrine;(3)Knockdown of LDHA showed a significant reduction in tumor volume compared to the control group.The treatment of Vitamin C had similar effect as knockdown of LDHA.4.Conclusion(1)Chronic stress-induced epinephrine promotes breast cancer glycolysis;(2)Chronic stress-induced epinephrine increases LDHA expression;(3)Breast cancer patient under chronic stress express higher LDHA;(4)Vitamin C inhibits chronic stress-induced epinephrine via LDHA.