Mitochondrial malic enzyme 2 promotes breast cancer metastasis via stabilizing HIF-1α under hypoxia

Objective: α-ketoglutarate(α-KG) is the substrate to hydroxylate collagen and hypoxia-inducible factor-1α(HIF-1α), which are important for cancer metastasis. Previous studies have shown that the upregulation of collagen prolyl 4-hydroxylase in breast cancer cells stabilizes the expression of HIF-1α by depleting α-KG levels. We hypothesized that mitochondrial malic enzyme 2(ME2) might also affect HIF-1α expression via modulating α-KG levels in breast cancer cells.Methods: We evaluated ME2 protein expression in 100 breast cancer patients using immunohistochemistry and correlated with clinicopathological indicators. The effect of ME2 knockout on cancer metastasis was evaluated using an orthotopic breast cancer model. The effect of ME2 knockout or knockdown on the levels of α-KG and HIF-1α proteins in breast cancer cell lines was determined both in vitro and in vivo.Results: ME2 was found to be upregulated in the human breast cancerous tissues compared with the matched precancerous tissues(P<0.001). The elevated expression of ME2 was associated with a poor prognosis(P=0.019).ME2 upregulation was also related to lymph node metastasis(P=0.016), pathological staging(P=0.033), and vascular cancer embolus(P=0.014). Also, ME2 knockout significantly inhibited lung metastasis in vivo. In the tumors formed by ME2 knockout cells, the levels of α-KG were significantly increased and collagen hydroxylation level did not change significantly but HIF-1α protein expression was significantly decreased, compared to the control samples. In cell culture, cells with ME2 knockout or knockdown demonstrated significantly higher α-KG levels but significantly lower HIF-1α protein expression than control cells under hypoxia. Exogenous malate and α-KG exerted similar effect on HIF-1α in breast cancer cells to ME2 knockout or knockdown. Additionally,treatment with malate significantly decreased 4 T1 breast cancer lung metastasis. ME2 expression was associated with HIF-1α levels in human breast cancer samples(P=0.008).Conclusions: Our results provide evidence that upregulation of ME2 is associated with a poor prognosis of breast cancer patients and propose a mechanistic understanding of a link between ME2 and breast cancer metastasis.

Lactate dehydrogenases amplify reactive oxygen species in cancer cells in response to oxidative stimuli

Previous studies demonstrated that superoxide could initiate and amplify LDH-catalyzed hydrogen peroxide production in aqueous phase,but its physiological relevance is unknown.Here we showed that LDHA and LDHB both exhibited hydrogen peroxide-producing activity,which was significantly enhanced by the superoxide generated from the isolated mitochondria from HeLa cells and patients'cholangiocarcinoma specimen.After LDHA or LDHB were knocked out,hydrogen peroxide produced by Hela or 4T1 cancer cells were significantly reduced.Re-expression of LDHA in LDHA-knockout HeLa cells partially restored hydrogen peroxide production.In HeLa and 4T1 cells,LDHA or LDHB knockout or LDH inhibitor FX11 significantly decreased ROS induction by modulators of the mitochondrial electron transfer chain(antimycin,oligomycin,rotenone),hypoxia,and pharmacological ROS inducers piperlogumine(PL)and phenethyl isothiocyanate(PEITC).Moreover,the tumors formed by LDHA or LDHB knockout HeLa or 4T1 cells exhibited a significantly less oxidative state than those formed by control cells.Collectively,we provide a mechanistic understanding of a link between LDH and cellular hydrogen peroxide production or oxidative stress in cancer cells in vitro and in vivo.