The Expression Of Mitochondrial Malic Enzymes In Breast Cancer And Its Correlation With Metastasis And Prognosis

Background Worldwide,breast cancer is one of the most commonly diagnosed cancers and remains the leading cause of cancer-related death among females.As the hub of metabolism reprogramming,mitochondria are involved in the development of malignant tumors and contribute to the progression and metastasis of breast cancer.The mitochondrial malic enzymes(MEs)include two subtypes,ME2 and ME3.By recycling the tricarboxylic acid(TCA)cycle intermediate malate into TCA cycle carbon source pyruvate,MEs have a regulatory role in TCA flux.Meanwhile,the reaction is with a concomitant reduction of NAD(P)+ to NAD(P)H,which affect the redox homeostasis in mitochondrial.Mitochondrial MEs have been found to play multiple roles in different cancers,including lung cancer,pancreatic cancer and glioma,etc.However,the role of mitochondrial MEs in breast cancer has not been well characterized.The present study was undertaken to explore the effect of ME2 and ME3 expression on breast cancer and possible mechanism involved,and to provide a novel strategies for breast cancer targeting therapy through mitochondrial metabolic pathway.Methods The protein expression of ME2 and ME3 was evaluated by immunohistochemistry on breast cancer patients and correlated with clinicopathological indicators.The effect of ME2 on cancer metastasis was evaluated by an orthotopic breast cancer model.The function role of ME2 was further explored through determining the effect of ME2 on the TCA intermediates and redox homeostasis in high metastatic breast cancer cell lines(4T1 and MDA-MB-231)in vitro and in vivo.Results Part I ME2 promotes breast cancer metastasis via stabilizing HIF-1α 1.ME2 is overexpression in breast cancer tissues and associated with a poor prognosis High expression of ME2 was displayed in breast cancerous tissues(63.0%)compared to the matched precancerous tissues(33.3%).Meanwhile,high expression of ME2 had an inferior survival than the patients with low expression of ME2.2.ME2 is associated with metastasis-related indicators ME2 high expression was associated with lymph node metastasis,pathological staging,and vascular cancer embolus.In the subgroup analysis,high expression of ME2 exhibited more significant worse prognostic impact in the subgroup of patients with no lymph node metastasis and without vascular cancer embolus.In orthotopic breast cancer model,ME2 knockout significantly inhibited lung metastasis.3.ME2 promotes breast cancer metastasis via stabilizing HIF-1α In the tumors formed by ME2 knockout 4T1 cells,the level of TCA intermediates malate,α-ketoglutarate(α-KG)and citrate significantly increased.Meanwhile,ME2 knockout significant inhibited hypoxia-inducible factor 1-α(HIF-1α)protein level in mouse tumor tissue.In cell culture,ME2 knockout or knockdown cells also demonstrated a significantly higher α-KG level but significantly lower HIF-1α protein level than control cells under hypoxia.Meanwhile,the high expression of ME2 in human breast cancer is significantly correlated with the positive expression of HIF-1α.The present results indicated that ME2 was related to the expression of HIF-1α,and that ME2 might promote breast cancer metastasis via stabilizing HIF-1α.4.ME2 stabilizes HIF-1α by regulating α-KG The inhibition of HIF-1α expression induced by down-regulation of ME2 was accompanied by increased α-KG level both in vivo and in vitro.In cell culture,exogenous α-KG significantly reduced HIF-1α concentrations under hypoxia.With the prolonged hypoxic culture,ME2 knockout cells showed increasing α-KG and then maintained at a high level than control cells.The direct reaction of ME2 knockout or knockdown was the accumulation of malate.Likewise,exogenous malate also mimics the similar effect of ME2 inhibition.With the prolonged hypoxic culture,adding malate showed increasing α-KG gradually and then led to the instability of HIF-1α.Meanwhile,treatment with malate significantly decreased 4T1 breast cancer lung metastasis.In summary,ME2 stabilized HIF-1α expression by mediating malate /α-KG/ HIF-1α pathway under hypoxia.Part II ME3 inhibits breast cancer carcinogenesis and progression 1.ME3 is inversely associated with the progression from precancerous lesions to cancerous lesions The ME3 positive immunostaining rate was higher in normal breast tissues and decreased stepwise from normal(92.1%)to usual hyperplasia(91.1%),atypical hyperplasia(64.2%),carcinoma in situ(62.5%)and invasive carcinoma(45.5%).2.ME3 is inversely associated with the progression of breast cancer The decreasing tendency was observed for ME3 positive immunostaining rate from Tis(75.0%)through T1(62.5%)and T2(37.5%)to T3(33.3%).ME3 expression was also related with negative lymph node metastasis.Similarly,the decreasing tendency of ME3 positive immunostaining rate was also significant from stage 0(75.0%)through I(72.0%),II(44.4%)to III(24.1%).3.The correlation of ME3 expression and prognosis in breast cancerThe patients with negative expression of ME3 had worse outcome and ME3 was an independent risk factor for prognosis.Conclusions 1.ME2 is overexpressed in breast cancer and related to metastasis and poor prognosis in breast cancer patients.ME2 promotes breast cancer metastasis by mediating the regulation of malate/α-KG/HIF-1α pathway under hypoxia.Targeting ME2 or regulating its substrate malate is a promising target for the control of breast cancer metastasis.2.ME3 positive expression is decreased with the progression from precancerous to cancerous lesions.And ME3 positive expression is inversely correlated with the breast cancer TNM staging.It suggests that ME3 involved in or accompanied with breast cancer carcinogenesis and progression.ME3 may be a promising biomarker for better prognosis of breast cancer patients.