Oxygen availability and metabolic reprogramming in cancer

The HIF hydroxylase system

The HIF system itself has been reviewed extensively elsewhere (1, 2). Initially identified as a transcriptional regulator bound to a hypoxia-response element (HRE) of the erythropoietin (EPO) gene to promote EPO production, it readily became apparent that this pathway operated much more widely, and it is currently recognized as a key modulator of the transcriptional response to hypoxic stress. Briefly, HIFs are heterodimeric transcription factors consisting of α and β subunits. Three HIFα isoforms exist in the mammalian genome (1α, 2α, and 3α), of which HIF1α and HIF2α are the best characterized. HIFα subunits heterodimerize with stable HIF1β (or ARNT), recognize, and then bind to HREs ((G/A)CGTG) throughout the genome to regulate downstream gene expression. Hypoxia-inducible behavior is conferred by the HIFα subunits, the protein abundance and transcriptional activity of which are regulated by O₂-dependent prolyl and asparaginyl hydroxylation. In well-oxygenated environments, HIFα subunits are hydroxylated by prolyl hydroxylases (PHDs) and factor-inhibiting HIF (FIH), and in turn they are targeted for proteosomal degradation by an E3 ubiquitin ligase, the von Hippel-Lindau protein (pVHL) complex. In hypoxic conditions, PHD and FIH activities are diminished, and HIFα proteins are stabilized, which consequently induces transcription of thousands of genes supplying adaptive functions, such as vascular endothelial growth factor (VEGF), carbonic anhydrase IX (CAIX), EPO, and others.

HIF regulation of glucose metabolism

HIF regulation of glutamine metabolism

Glutamine is the most abundant amino acid in the circulation and is an essential precursor for the synthesis of proteins, fatty acids, nucleotides, and many other important molecules (19). Glutamine metabolism has been reviewed extensively (20) and is briefly outlined here. Upon cellular entry via transporters such as SLC1A5, glutamine is converted by glutaminases to generate glutamate. Glutamate plays a variety of important roles in eukaryotic cells (Fig. 1A). For example, it is required for glutathione synthesis, the major cellular antioxidant, and is also the source of amino groups for other non-essential amino acids such as proline, alanine, aspartate, serine, and glycine. In addition, glutamate can be converted to α-ketoglutarate (α-KG), which is either oxidized by α-ketoglutarate dehydrogenase (αKGDH) to succinate and enters a “forward” tricarboxylic acid (TCA) cycle to generate ATP or reductively carboxylated by isocitrate dehydrogenase (cytosolic IDH1 or mitochondrial IDH2) to produce isocitrate and citrate by a “reverse” TCA cycle.

Under hypoxia, Wise et al. (21) demonstrated that glutamine becomes a major source of citrate through reductive carboxylation via wild-type IDH2 activity in the glioblastoma cell line SF188. Consequently, hypoxic cells are unable to proliferate when they are either glutamine-starved or rendered IDH2-deficient. This metabolic reprogramming might be partly through HIF, as constitutive HIF activation recapitulates the preferential reductive metabolic pathway even in normoxic conditions (21). In addition, IDH1 is also essential for HIF-mediated production of citrate at 1% O₂, and citrate produced via IDH1-dependent reductive carboxylation is utilized for de novo lipogenesis, required for maintaining cell growth in hypoxic conditions (22–24). This shift appears to be due to proteolysis of the E1 subunit of the αKGDH complex, named “oxoglutarate dehydrogenase 2” (OGDH2) (25). Sun and Denko (25) found that the E3 ubiquitin ligase SIAH2 destabilized OGHD2 in hypoxia in an HIF-dependent manner, which results in diminished αKGDH activity and decreased glutamine oxidation. In addition, a ubiquitination-resistant OGDH2 336KA mutant impedes tumor growth in vivo, accompanied by a reduction in glutamine incorporation into lipids (25), highlighting this pathway as an essential mechanism to rewire glutamine fate in HIF-stabilized tumor cells. Finally, in VHL^−/− ccRCC cells, glutamine is also utilized to generate aspartate for de novo pyrimidine biosynthesis via reductive carboxylation and glutathione for redox balance (26). When glutaminase is inhibited, nucleoside depletion and increased ROS levels lead to DNA replication stress, which sensitizes cells to poly(ADP-ribose) polymerase inhibition in vitro and in vivo. Taken together, these studies exemplify alternative fates of glutamine in an HIF-dependent manner to support cancer cell proliferation and viability under hypoxic stress or as a consequence of genetic mutations.

HIF regulation of lipid synthesis

Lipid synthesis provides essential building blocks and signaling molecules for tumor growth. Thus, despite a high-energy cost, de novo lipogenesis is clearly increased in most cancer cells (27–29). In this process, acetyl-CoA and NADPH together generate free fatty acid chains through stepwise enzymatic reactions via acetyl-CoA carboxylase (ACC) and fatty-acid synthase (FASN) (Fig. 1A). These fatty acids are then: 1) used to generate phospholipids, the basic units of lipid bilayers making up cell and organelle membranes; 2) incorporated into neutral lipids, such as triglycerides and cholesterol esters, for storage; 3) utilized to produce essential signaling molecules, such as sphingosine 1-phosphate, lysophosphatidic acid, diglyceride, and inositol 1,4,5-trisphosphate, which control inflammation, cell migration, and survival in cancer; and 4) employed to modify proteins, among others.

In hypoxic conditions, glucose-derived pyruvate entry into the TCA cycle is inhibited, as well as subsequent citrate and acetyl-CoA production. Therefore, cells must shift to alternative carbon sources to generate acetyl-CoA for fatty acid synthesis. These sources include citrate synthesis from reductive carboxylation of glutamate (reviewed above) and acetyl-CoA production through metabolizing acetate via cytoplasmic acetyl-CoA synthetase (ACSS2) (30). Indeed, based on ¹³C-tracing and mass spectrometry, cells cultured in O₂-replete conditions produce more than 90% acetyl-CoA from glucose and glutamine-derived carbon. However, at 1% O₂, acetate instead appears to be the primary hypoxia-induced contributor to acetyl-CoA in multiple cell lines (31). In addition, hypoxia enhances ACSS2 expression, increasing the use of acetate to sustain lipid biomass production and imparting a competitive growth advantage under microenvironmental stress (32). Exactly how ACSS2 is up-regulated in hypoxia remains unclear, although it has been suggested that sterol regulatory element-binding protein 2 (SREBP2) mainly controls ACSS2 expression, whereas HIF signaling enhances the up-regulation of ACSS2 by SREBP2 (32).

Multiple genes involved in lipid metabolism have been reported to be strongly induced under hypoxia. One example is the regulation of SREBP1 via phosphorylation of AKT followed by HIF1α activation (33). Of note, SREBP1 is a major transcriptional regulator of lipid metabolic genes, such as FASN. Moreover, some lipid metabolic genes are direct HIF transcriptional targets. For example, in hypoxic breast cancer MCF-7 cells and glioblastoma U87 cells, induction of fatty acid-binding protein 3 (FABP3), FABP7, and adipose differentiation-related protein (ADRP, also known as perilipin2, PLIN2) is an HIF1α-dependent but HIF-2α-independent event. These proteins are essential for lipid droplet (LD) formation, which helps to maintain ROS levels in these cells (34). Furthermore, PLIN2 up-regulation in ccRCC is HIF2α-dependent, which promotes lipid storage and contributes to “clear cell” phenotypes (35). Interestingly, PLIN2-mediated LD formation maintains integrity of the ER, suppressing cytotoxic ER stress responses that otherwise result from elevated protein synthetic activity characteristic of ccRCC cells. Therefore, PLIN2 depletion results in apoptosis and further sensitizes these cells to ER stress, identifying it as a targetable vulnerability created by HIF2α/PLIN2 signaling in this common renal malignancy.

HIF and redox stress

Because of its role as a potent electron acceptor, O₂ has the tendency to form highly reactive oxygen species. Mitochondrial O₂ metabolism is the dominant source of superoxide production via the mitochondrial electron transport chain (ETC). Several studies found HIFα subunits can be stabilized by mitochondrial ROS (36–39). Reciprocally, suppressing the production of ROS by ETC seems to be a fundamental function of HIF.

Under hypoxic conditions, HIF actively regulates mitochondrial function and subsequent ROS production through multiple mechanisms. First, HIF1α uncouples glycolysis and oxidative mitochondrial metabolism by transcriptionally up-regulating pyruvate dehydrogenase kinase 1 (PDK1) (40, 41). PDK1 phosphorylates and inhibits the function of mitochondrial pyruvate dehydrogenase (PDH), which transforms pyruvate into acetyl-CoA, subsequently used in the TCA cycle to carry out cellular respiration (Fig. 1A). Enforced PDK1 expression in hypoxic HIF1α-null cells attenuates ROS generation and protects these cells from hypoxia-induced apoptosis (40). At the same time, accumulating pyruvate is metabolized by lactate dehydrogenase A (LDHA) to generate lactate, which is then exported from the cell by MCT4, as discussed previously. Importantly, both LDHA and MCT4 are also HIF1α targets (42). Taken together, HIF1α-dependent transcriptional regulation coordinately impedes the entry of substrates for mitochondrial respiration and toxic ROS production. Second, HIF1α attenuates mitochondrial function by down-regulating several ETC components. For example, components of cytochrome c oxidase (COX; complex IV), the final ETC enzyme, are regulated in an HIF1α-dependent manner (43). HIF1α reciprocally manipulates COX4 subunit expression by activating transcription of the gene encoding COX4-2, while at the same time enhancing COX4-1 protein degradation by increasing the abundance of LON, a mitochondrial protease (43). The replacement of COX4-1 with COX4-2 allows improved adaption to hypoxia, with reduced ROS production. In addition, HIF1α increases mitochondrial NDUFA4L2 (NADH dehydrogenase 1α subcomplex, 4-like 2), which attenuates mitochondrial oxygen consumption through reduced complex I activity, limiting intracellular ROS production under low O₂ conditions (44). Furthermore, HIF1α suppresses succinate dehydrogenase subunit B, a component of mitochondrial complex II (45), but whether this contributes to ROS level regulation is unclear. Finally, HIF influences mitochondrial function through effects at the whole-organelle level. In ccRCC cells lacking VHL, HIF1α negatively regulates c-Myc activity and subsequent mitochondrial biogenesis, O₂ consumption, and ROS production (46). Moreover, the HIF target BNIP3 contributes to reduced mitochondrial mass through increased mitophagy (47). Taken together, these adaptive pathways underlying HIF activation attenuate ROS production and provide protective mechanisms for cancer cell growth. Whether they are “Achilles heels” and how we can utilize them against cancer are under investigation.

HIF, miR-210, and metabolism

The microRNAs (miRNAs) are small, non-coding RNA molecules ∼22 nucleotides in length that are evolutionarily conserved (48). Targeting most protein-coding transcripts, miRNAs are involved in nearly all physiological and pathological processes, including cancer cell metabolism. In recent years, more than 50 hypoxia-regulated miRNAs have been identified. Among them, miR-210 exhibits a robust and consistent up-regulation under hypoxia in virtually all cell types through an HIF1α-dependent regulation (49) and influences intracellular metabolism in both normal and malignant cells. For example, first, miR-210 represses the iron–sulfur cluster assembly proteins (ISCU1 in cytosol and ISCU2 in mitochondria) (50), which promote the assembly of [4Fe-4S] and [2Fe-2S] iron–sulfur clusters, which are incorporated into enzymes responsible for mitochondrial respiration, such as those in ETC complexes I, II, and III (51). Therefore, miR-210 expression decreases activity of complex I, oxygen consumption, and ATP generation (50). Moreover, an miR-210 antagonist decreases clonogenic survival in hypoxic MCF7 and HeLa cells (52). In both cases, miR-210 suppression increases ROS production. Second, the transcript coding for one of ETC components, SDHD, subunit D of succinate dehydrogenase complex, is a bona fide miR-210 target. SDHD knockdown mimics miR-210 mediated mitochondrial dysfunction. In addition, SDHD targeting also seems to stabilize HIF1α, which creates a positive feedback loop (53). Taken together, miR-210 serves as an important mediator shifting mitochondrial oxidative phosphorylation to glycolysis downstream of HIF. Whether other miRNAs are involved in more metabolic processes needs to be further investigated.

l-2-Hydroxyglutarate (l-2HG) production and function

With the discovery of oncogenic mutations in isocitrate dehydrogenase enzymes (IDH1 and IDH2), the enzymatic product d-2HG is broadly appreciated as an oncometabolite that inhibits αKG and a large family of αKG-dependent dioxygenases, including histone demethylases and methylcytosine dioxygenases of the TET family, causing epigenetic dysregulation and a block in cellular differentiation (54). Although intensive efforts have been made to investigate the cellular functions of IDH-mutant derived d-2HG, and a selective, potent inhibitor of mutant IDH2, AG-221, was shown to be effective in IDH2 mutation-positive acute myeloid leukemia (55, 56), its mirror-image enantiomer l-2HG is less understood. Interestingly, recent studies found that hypoxia dramatically induces production of l-2HG in both normal and malignant cells (57, 58).

In different types of cancer cell lines, hypoxia substantially increases 2HG production 5–25-fold, detailed analysis specifies a selective accumulation of the l-enantiomer of 2HG in these cells (57). Surprisingly, this reaction is not through IDH1/2, but LDHA and malate dehydrogenase (MDH1 and MDH2) via their “promiscuous” catalytic activity (57). Further study confirmed these results by showing that purified LDH and MDH enzymes catalyze stereospecific reduction of αKG to l-2HG and that acidic reaction conditions dramatically enhance LDH- and MDH-mediated production of l-2HG in vitro and in cells. Mechanistically, hypoxia-induced acidic pH enhances LDHA-mediated reduction of αKG by driving equilibrium toward a protonated form of αKG that binds more stably to the LDHA enzyme (59). In normal cells, including human pulmonary arterial endothelial and smooth muscle cells, hypoxia also directs the production of l-2HG (58), highlighting it as a fundamental mechanism for hypoxia adaptation. Functionally, acid-enhanced production of l-2HG leads to stabilization of HIF1α in normoxic conditions (59). In addition, like d-2HG, l-2HG also inhibits the Jumonji family histone lysine demethylase KDM4C, resulting in aberrant accumulation of trimethylated histone 3 lysine 9 (H3K9me3) (57). Interestingly, a recent report demonstrated that tumor hypoxia reduces TET enzyme activity, which is independent of hypoxia-associated alterations in TET expression, proliferation, metabolism, HIF activity, or ROS production, and it is dependent directly on O₂ shortage (60). This effect results in hypermethylation at gene promoters (including those suppressing glycolysis), which accounts for up to half of the hypermethylation events in cells. However, whether and how much hypoxia-mediated l-2HG plays a role in this process needs to be investigated in more detail. Furthermore, mitochondrial l-2HG is oxidized and converted back to αKG by l-2HG dehydrogenase (L2HGDH) (61), and αKG and l-2HG form a redox couple linked to NADH/NAD⁺ and FADH₂/FAD (Fig. 1B). Therefore, 2HG may provide cells with a reservoir of reducing equivalents, and the αKG-2HG exchange maintains cellular redox balance. Consistent with this idea, a recent study in hematopoietic stem cells reported that loss of the mitochondrial complex III subunit Rieske iron–sulfur protein results in impaired mitochondrial respiration, increased NADH/NAD⁺ ratio, and increased 2HG levels, accompanied with DNA and histone hypermethylation (62). Because L2HGDH activity is dependent on active complex III (63), loss of complex III activity both increases NADH/NAD⁺ and impairs L2HGDH activity to favor 2HG production.

How HIF proteins contribute to l-2HG production seems to be context-dependent. In lung fibroblasts, whereas VHL knockdown and HIFα stabilization increase total 2HG production in normoxia, HIF1α depletion also elevates l-2HG in hypoxia, indicating HIF1α is sufficient but not necessary for l-2HG accumulation in these cells (58). Consistent with this, in SF188 cancer cells, HIF1A knockdown in hypoxia does not decrease but slightly increases l-2HG levels (57). In addition, HIF1α enhances hypoxia-induced l-2HG production in VHL-deficient RCC4 cells, as HIF1α depletion partly reverses hypoxia-mediated l-2HG increments (57). However, in murine CD8⁺ T lymphocytes, Hif1α^−/− abolished 2HG accumulation under hypoxia, and l-2HG constitutes more than 90% of the 2HG pool, suggesting l-2HG production is dependent on HIF1α in this case (64).

Alternative effect of hypoxia on lipid metabolism

O₂ deficiency affects lipid metabolism not only through HIF-dependent pathways, as discussed above, but also via direct effects on enzymes involved in this process. Stearoyl-CoA desaturase-1 (SCD1) is a key enzyme catalyzing the rate-limiting step in the formation of monounsaturated fatty acids. This process conducts electron flow from NADPH to the terminal electron acceptor molecular O₂, rendering O₂ a critical factor in the formation of double bonds in stearoyl-CoA. Indeed, Kamphorst et al. (65) found that consistent with impaired SCD1 activity, hypoxic cancer cells display decreased C18:1 production, as well as the C18:1/C18:0 ratios. Furthermore, SCD1 enzymatic activity is required to maintain cell viability in Tsc2^−/− mouse embryonic fibroblasts with O₂ deprivation (66). This is accompanied by elevated toxic ER stress, which might be caused by an unmet requirement of ER membrane quality in the conditions of increased protein synthesis by dysregulated mTORC1. This mechanism has been confirmed in multiple systems by demonstrating that SCD1 inhibition increases ER stress in vitro and in vivo (67, 68) and can be employed as a strategy to treat cancers.