Metabolic adaptation is an important survival

Metabolic adaptation is an important survival strategy for cancer cells within the hypoxic tumor environment. Under the condition of O2 limitation, metabolic pathways shift from energy-efficient oxidative phosphorylation to the anaerobic glycolysis pathway for the purpose of ATP generation [13,43]. This change is achieved by the upregulation of the HIF-1α-mediated glycolysis-involved GLUT1, HK2 and PDK1, an inhibitor of the conversion of pyruvate to Voreloxin Hydrochloride [43]. At the same time, the formation of lactic acid from pyruvate is facilitated by the HIF-1α-mediated upregulation of LDHA, which contributes to the acidification of the tumor microenvironment [44]. In our results, all of these hypoxia-induced metabolic changes were suppressed by NRF2-silencing. The HIF-1α-inducible expressions of GLUT1, HK2, PDK1, and LDHA were low in NRF2-silenced breast cancer cells and, accordantly, levels of hypoxia-inducible glycolysis metabolites such as G6P, F16P, GAP, and lactate were diminished by NRF2-silencing. As a result of PDK1 elevation, levels of the TCA metabolites citric acid and isocitric acid were substantially reduced following hypoxia, which provides evidence that the TCA cycle is inhibited under hypoxic conditions. However, the elevation of α-KG levels in control cells might imply the contribution of the glutaminolysis pathway in the TCA cycle [28,45]. However, NRF2-silenced cells did not show α-KG elevation. In line with the inhibition of glycolysis pathway, NRF2-silenced MCF-7 cells exhibited lower levels of HIF-1α-induced ATP and ADP and cell viability under hypoxic conditions. Similar to NRF2-silenced MCF-7, overexpressing miR-181c caused suppression of hypoxia-induced glycolysis enzymes and cell viability. In addition to the glycolysis pathway, activation of the PPP is another core feature of hypoxic cancer cells to provide necessary cellular building blocks [46,47]. The HIF-1α-mediated activation of the PPP provides precursors for nucleotide synthesis, which aids rapid proliferation of cancer cells under energy-limited hypoxic conditions. PPP activation additionally enhances NADPH levels and maintains redox homeostasis by providing reducing power to antioxidant enzymes such as GSH reductase [48]. Our metabolome analysis revealed that hypoxia-inducible levels of Ru5P, R5P, and S7P were low in NRF2-silenced breast cancer cells, indicating the suppression of the PPP. In agreement with this, hypoxic levels of PPP-derived nucleotide precursors were lower in NRF2-silenced breast cancer cells than in control cells. These results indicate that the supply of building blocks can be restricted by NRF2 inhibition in hypoxic cancer cells, which is supported by reduced cell viability following hypoxic incubation in NRF2-silenced cells. Levels of NADPH were similarly diminished under hypoxic conditions in both cell lines, implying the extensive consumption of NADPH in hypoxic conditions. It is noteworthy that NRF2 can upregulate multiple metabolic genes, which can lead to metabolic shift in normoxic condition. Expression of PPP enzymes such as G6P dehydrogenase (G6PD), 6-phosphogluconate dehydrogenase (PGD), and transketolase (TKT) was directly regulated by NRF2 in lung cancer cells, and NRF2 overexpression redirected cell metabolism to PPP-mediated purine nucleotide synthesis [28]. In another study, NRF2-mediated regulation of PPP genes was demonstrated to be an indirect event: NRF2 signaling diminished the expression of miR-1 and miR-206, which are inhibitor miRNAs for PPP enzymes such as G6PD and TKT and this, in turn, led to an elevation in PPP metabolism [49]. Additionally, lung adenocarcinoma with simultaneous mutations in Keap1 and Kras showed an increased glutamine utilization for energy production [45]. NRF2 also facilitates the metabolism from glucose to serine by indirectly regulating its biosynthetic enzymes [50]. These results indicate that NRF2 is positively involved in facilitation of glucose metabolism toward PPP and serine synthesis, and enhancement of glutaminolysis, which all together promotes tumor proliferation in normoxic condition. Therefore, NRF2 silencing inhibited PPP metabolism and consequently suppressed tumor cell proliferation by restricting purine nucleotides supply [45,49], which implies the beneficial effects of NRF2 inhibition in both normoxic and hypoxic tumor environments.