Nuclear factor erythroid like NFE
Nuclear factor erythroid 2-like-2 (NFE2L2; hereafter NRF2) plays a crucial role in the basal and inducible expressions of multiple cytoprotective genes in response to electrophilic and oxidative stress . The cytosolic actin-binding protein Kelch-like ECH-associated protein 1 (KEAP1) primarily regulates NRF2 activity through the Culilin3-based E3 ligase-dependent degradation . In the presence of reactive oxygen species (ROS) or electrophiles, NRF2-KEAP1 binding is disrupted through modification of cysteine residues of KEAP1 protein, and thus free NRF2 protein accumulates in nucleus, which leads to the transactivation of antioxidant response element (ARE)-bearing cytoprotective genes [23,24]. However, high levels of NRF2 has been beneficial to cancer Candesartan by eliminating excess ROS, which are derived from uncontrolled energy production in cancer cells, and by facilitating tumor growth and anticancer drug metabolism [, , , ]. Moreover, NRF2 overactivation has been recognized as a factor inducing metabolic reprogramming of cancers, including the activation of the pentose phosphate pathway (PPP) and glutaminolysis .
We previously demonstrated that NRF2-silenced colon cancer cells failed to accumulate HIF-1α under hypoxic conditions, and thus tumor angiogenesis was blocked by NRF2-inhibition . In a subsequent study, we identified that miR-181c elevation leads to the reductions in mitochondrial O2 consumption rate and ATP production in NRF2-silenced cancer cells by inhibiting mitochondrial function. As a molecular event, miR-181c directly represses the level of the mitochondria-encoded cytochrome c oxidase (MT-CO1), a catalytic subunit of the mitochondrial complex IV . On the basis of these results, we hypothesized that miR-181c elevation might be a molecular link between NRF2-silencing and HIF-1α dysregulation in cancer cells. To test this idea, we compared levels of hypoxia-induced HIF-1α accumulation in breast cancer cells following the silencing of NRF2 or the overexpression of miR-181c. In addition, we examined the effect of NRF2-silencing and miR-181c on the changes in hypoxic metabolic pathways regulated by HIF-1α.
Materials and methods
Discussion Because NRF2 and HIF-1α are critical factors for sensing O2 and its related ROS, the mechanism of how NRF2 and HIF-1α are co-regulated in hypoxic conditions is intriguing. Our current study shows that NRF2-silencing suppresses hypoxia-inducible HIF-1α accumulation in breast cancer cells, and thereby inhibits the HIF-1α-mediated metabolic adaptation, including glycolysis activation, PPP facilitation, and autophagy stimulation, which eventually impairs the viability of NRF2-silenced cancer cells in hypoxic environment. Notably, we demonstrate that the NRF2-silencing effect on HIF-1α is mediated by miR-181c elevation. Breast cancer cells with miR-181c overexpression exhibited similar behaviors upon hypoxia: HIF-1α accumulation was attenuated and the levels of glycolysis enzymes were suppressed. Moreover, the inhibitory effect of NRF2-silencing on HIF-1α was blocked by treatment with a miR181c inhibitor. These results suggest a strong correlation between NRF2 and HIF-1α in the adaptive regulation of metabolic pathways, and further imply beneficial effects of NRF2-silencing on HIF-1α-mediated metabolic adaptation under hypoxic tumor environment. The role of miR-181c in mitochondria was first demonstrated in a study using cardiac cells from rats . In this study, miR-181c was identified as a mitochondria-localized miRNA, and was shown to increase O2 consumption and MMP by targeting MT-CO1 of the electron transport chain complex IV. Our previous study identified miR-181c as one of the miRNAs induced following NRF2-silencing in both HT29 and HCT116 colon cancer cells . However, unlike a study by Das et al. , we found that miR-181c-mediated electron transport chain dysfunction led to a decrease in MMP, mitochondrial respiration rate, and ATP production in normoxic cancer cells. As these changes could be partly compensated for by the activation of adenosine monophosphate (AMP)-activated protein kinase-a (AMPKα) signaling and consequent adaptive metabolic pathways to maintain energy homeostasis, NRF2-silenced cancer cells are vulnerable to AMPKα inhibition, which suggests the potential for combined inhibition of NRF2 and AMPKα to overcome adaptive behaviors of cancer cells . In addition to the role in normoxic cancer metabolism, our current study elucidated a novel role of miR-181c in hypoxic breast cancer cells. MiR-181c suppressed HIF-1α stabilization under hypoxic conditions and blocked the HIF-1α-mediated adaptive metabolic changes in glycolysis and autophagy. In particular, the inhibitory effect of miR-181c on HIF-1α accumulation can be attributed to the O2 redistribution effect which has been well-described in nitric oxide (NO)-treated cells [38,39]. When mitochondrial respiration is repressed by NO under hypoxic conditions, O2 is redistributed to non-respiratory targets such as PHD, allowing PHD to retain its enzymatic activity for HIF-1α hydroxylation, resulting in the inhibition of HIF-1α signaling. Indeed, HIF-1α accumulation was recovered in anoxic (0.1% O2) NRF2-silenced cancer cells  and miR-181c overexpressing breast cancer cells (data not shown). Several recent studies have indicated a role of miR-181c as a tumor suppressor. MiR-181c inhibited metastasis and migration in glioblastoma  and its overexpression in myelocytic leukemia cells suppressed chemoresistance . Reduced miR-181c expression has been associated with gastric cancer in patients . Together with these reports, our study suggests the beneficial effects of miR-181c for the control of the HIF-1α-mediated adaptive cancer behaviors in the hypoxic tumor microenvironment.