Nuclear factor erythroid like NFE L hereafter NRF plays a

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 [23]. The cytosolic actin-binding protein Kelch-like ECH-associated protein 1 (KEAP1) primarily regulates NRF2 activity through the Culilin3-based E3 ligase-dependent degradation [24]. 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 BI-D1870 receptor by eliminating excess ROS, which are derived from uncontrolled energy production in cancer cells, and by facilitating tumor growth and anticancer drug metabolism [[25], [26], [27], [28]]. 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 [28]. 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 [29]. 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 [30]. 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
Results
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 [37]. 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 [30]. However, unlike a study by Das et al. [37], 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 [30]. 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 [29] 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 [40] and its overexpression in myelocytic leukemia cells suppressed chemoresistance [41]. Reduced miR-181c expression has been associated with gastric cancer in patients [42]. 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.