Jiangbin Ye

Quantitative flux analysis reveals folate-dependent NADPH production

ATP is the dominant energy source in animals for mechanical and electrical work (for example, muscle contraction or neuronal firing). For chemical work, there is an equally important role for NADPH, which powers redox defence and reductive biosynthesis. The most direct route to produce NADPH from glucose is the oxidative pentose phosphate pathway, with malic enzyme sometimes also important. Although the relative contribution of glycolysis and oxidative phosphorylation to ATP production has been extensively analysed, similar analysis of NADPH metabolism has been lacking. Here we demonstrate the ability to directly track, by liquid chromatography–mass spectrometry, the passage of deuterium from labelled substrates into NADPH, and combine this approach with carbon labelling and mathematical modelling to measure NADPH fluxes. In proliferating cells, the largest contributor to cytosolic NADPH is the oxidative pentose phosphate pathway. Surprisingly, a nearly comparable contribution comes from serine-driven one-carbon metabolism, in which oxidation of methylene tetrahydrofolate to 10-formyl-tetrahydrofolate is coupled to reduction of NADP+ to NADPH. Moreover, tracing of mitochondrial one-carbon metabolism revealed complete oxidation of 10-formyl-tetrahydrofolate to make NADPH. As folate metabolism has not previously been considered an NADPH producer, confirmation of its functional significance was undertaken through knockdown of methylenetetrahydrofolate dehydrogenase (MTHFD) genes. Depletion of either the cytosolic or mitochondrial MTHFD isozyme resulted in decreased cellular NADPH/NADP+ and reduced/oxidized glutathione ratios (GSH/GSSG) and increased cell sensitivity to oxidative stress. Thus, although the importance of folate metabolism for proliferating cells has been long recognized and attributed to its function of producing one-carbon units for nucleic acid synthesis, another crucial function of this pathway is generating reducing power.

The GCN2-ATF4 pathway is critical for tumour cell survival and proliferation in response to nutrient deprivation

The transcription factor ATF4 regulates the expression of genes involved in amino acid metabolism, redox homeostasis and ER stress responses, and it is overexpressed in human solid tumours, suggesting that it has an important function in tumour progression. Here, we report that inhibition of ATF4 expression blocked proliferation and survival of transformed cells, despite an initial activation of cytoprotective macroautophagy. Knockdown of ATF4 significantly reduced the levels of asparagine synthetase (ASNS) and overexpression of ASNS or supplementation of asparagine in trans, reversed the proliferation block and increased survival in ATF4 knockdown cells. Both amino acid and glucose deprivation, stresses found in solid tumours, activated the upstream eukaryotic initiation factor 2α (eIF2α) kinase GCN2 to upregulate ATF4 target genes involved in amino acid synthesis and transport. GCN2 activation/overexpression and increased phospho‐eIF2α were observed in human and mouse tumours compared with normal tissues and abrogation of ATF4 or GCN2 expression significantly inhibited tumour growth in vivo. We conclude that the GCN2‐eIF2α‐ATF4 pathway is critical for maintaining metabolic homeostasis in tumour cells, making it a novel and attractive target for anti‐tumour approaches.

Pyruvate kinase M2 promotes de novo serine synthesis to sustain mTORC1 activity and cell proliferation

Despite the fact that most cancer cells display high glycolytic activity, cancer cells selectively express the less active M2 isoform of pyruvate kinase (PKM2). Here we demonstrate that PKM2 expression makes a critical regulatory contribution to the serine synthetic pathway. In the absence of serine, an allosteric activator of PKM2, glycolytic efflux to lactate is significantly reduced in PKM2-expressing cells. This inhibition of PKM2 results in the accumulation of glycolytic intermediates that feed into serine synthesis. As a consequence, PKM2-expressing cells can maintain mammalian target of rapamycin complex 1 activity and proliferate in serine-depleted medium, but PKM1-expressing cells cannot. Cellular detection of serine depletion depends on general control nonderepressible 2 kinase-activating transcription factor 4 (GCN2-ATF4) pathway activation and results in increased expression of enzymes required for serine synthesis from the accumulating glycolytic precursors. These findings suggest that tumor cells use serine-dependent regulation of PKM2 and GCN2 to modulate the flux of glycolytic intermediates in support of cell proliferation.

PERK promotes cancer cell proliferation and tumor growth by limiting oxidative DNA damage

To proliferate and expand in an environment with limited nutrients, cancer cells co-opt cellular regulatory pathways that facilitate adaptation and thereby maintain tumor growth and survival potential. The endoplasmic reticulum (ER) is uniquely positioned to sense nutrient deprivation stress and subsequently engage signaling pathways that promote adaptive strategies. As such, components of the ER stress-signaling pathway represent potential antineoplastic targets. However, recent investigations into the role of the ER resident protein kinase, RNA-dependent protein kinase (PKR)-like ER kinase (PERK) have paradoxically suggested both pro- and anti-tumorigenic properties. We have used animal models of mammary carcinoma to interrogate the contribution of PERK in the neoplastic process. The ablation of PERK in tumor cells resulted in impaired regeneration of intracellular antioxidants and accumulation of reactive oxygen species triggering oxidative DNA damage. Ultimately, PERK deficiency impeded progression through the cell cycle because of the activation of the DNA damage checkpoint. Our data reveal that PERK-dependent signaling is used during both tumor initiation and expansion to maintain redox homeostasis, thereby facilitating tumor growth.

Preferential Cytotoxicity of Bortezomib toward Hypoxic Tumor Cells via Overactivation of Endoplasmic Reticulum Stress Pathways

Hypoxia is a dynamic feature of the tumor microenvironment that contributes to drug resistance and cancer progression. We previously showed that components of the unfolded protein response (UPR), elicited by endoplasmic reticulum (ER) stress, are also activated by hypoxia in vitro and in vivo animal and human patient tumors. Here, we report that ER stressors, such as thapsigargin or the clinically used proteasome inhibitor bortezomib, exhibit significantly higher cytotoxicity toward hypoxic compared with normoxic tumor cells, which is accompanied by enhanced activation of UPR effectors in vitro and UPR reporter activity in vivo. Treatment of cells with the translation inhibitor cycloheximide, which relieves ER load, ameliorated this enhanced cytotoxicity, indicating that the increased cytotoxicity is ER stress-dependent. The mode of cell death was cell type-dependent, because DLD1 colorectal carcinoma cells exhibited enhanced apoptosis, whereas HeLa cervical carcinoma cells activated autophagy, blocked apoptosis, and eventually led to necrosis. Pharmacologic or genetic ablation of autophagy increased the levels of apoptosis. These results show that hypoxic tumor cells, which are generally more resistant to genotoxic agents, are hypersensitive to proteasome inhibitors and suggest that combining bortezomib with therapies that target the normoxic fraction of human tumors can lead to more effective tumor control.

Serine Catabolism Regulates Mitochondrial Redox Control during Hypoxia

UNLABELLED The de novo synthesis of the nonessential amino acid serine is often upregulated in cancer. In this study, we demonstrate that the serine catabolic enzyme, mitochondrial serine hydroxymethyltransferase (SHMT2), is induced when MYC-transformed cells are subjected to hypoxia. In mitochondria, SHMT2 can initiate the degradation of serine to CO2 and NH4+, resulting in net production of NADPH from NADP+. Knockdown of SHMT2 in MYC-dependent cells reduced cellular NADPH:NADP+ ratio, increased cellular reactive oxygen species, and triggered hypoxia-induced cell death. In vivo, SHMT2 suppression led to impaired tumor growth. In MYC-amplified neuroblastoma patient samples, there was a significant correlation between SHMT2 and hypoxia-inducible factor-1 α (HIF1α), and SHMT2 expression correlated with unfavorable patient prognosis. Together, these data demonstrate that mitochondrial serine catabolism supports tumor growth by maintaining mitochondrial redox balance and cell survival. SIGNIFICANCE In this study, we demonstrate that the mitochondrial enzyme SHMT2 is induced upon hypoxic stress and is critical for maintaining NADPH production and redox balance to support tumor cell survival and growth.

Induction of sarcomas by mutant IDH2

More than 50% of patients with chondrosarcomas exhibit gain-of-function mutations in either isocitrate dehydrogenase 1 (IDH1) or IDH2. In this study, we performed genome-wide CpG methylation sequencing of chondrosarcoma biopsies and found that IDH mutations were associated with DNA hypermethylation at CpG islands but not other genomic regions. Regions of CpG island hypermethylation were enriched for genes implicated in stem cell maintenance/differentiation and lineage specification. In murine 10T1/2 mesenchymal progenitor cells, expression of mutant IDH2 led to DNA hypermethylation and an impairment in differentiation that could be reversed by treatment with DNA-hypomethylating agents. Introduction of mutant IDH2 also induced loss of contact inhibition and generated undifferentiated sarcomas in vivo. The oncogenic potential of mutant IDH2 correlated with the ability to produce 2-hydroxyglutarate. Together, these data demonstrate that neomorphic IDH2 mutations can be oncogenic in mesenchymal cells.

ATF4, an ER Stress and Hypoxia-Inducible Transcription Factor and its Potential Role in Hypoxia Tolerance and Tumorigenesis

Hypoxia/anoxia promotes tumor aggressiveness and negatively impacts tumor response to therapy. Coordinate regulation of HIF-dependent and HIF-independent pathways has been shown to contribute to cellular adaptation to hypoxic stress, and to couple macromolecular synthesis rates to reduced energy availability. An important component of this type of adaptation is the activation of the endoplasmic reticulum kinase PERK by acute or prolonged hypoxia. Activated PERK subsequently induces phosphorylation of the translation initiation factor eIF2alpha and translational upregulation of the transcription factor ATF4. ATF4 is a basic leucine-zipper (bZip) transcription factor, which regulates amino acid metabolism, cellular redox state, and anti-stress responses. ATF4 expression can be regulated at transcriptional, translational, and post-translational levels. The functional activation of ATF4 under hypoxia and the overexpression of ATF4 in hypoxic areas of clinical samples of human tumors suggest that ATF4 plays a role in tumor hypoxic adaptation. Here we summarize recent findings regarding the regulation of ATF4 in transformed cells, clinical tumor samples and tumor models, and speculate on its potential role in tumor progression and chemoresistance.

GCN2 sustains mTORC1 suppression upon amino acid deprivation by inducing Sestrin2

Mammalian cells possess two amino acid-sensing kinases: general control nonderepressible 2 (GCN2) and mechanistic target of rapamycin complex 1 (mTORC1). Their combined effects orchestrate cellular adaptation to amino acid levels, but how their activities are coordinated remains poorly understood. Here, we demonstrate an important link between GCN2 and mTORC1 signaling. Upon deprivation of various amino acids, activated GCN2 up-regulates ATF4 to induce expression of the stress response protein Sestrin2, which is required to sustain repression of mTORC1 by blocking its lysosomal localization. Moreover, Sestrin2 induction is necessary for cell survival during glutamine deprivation, indicating that Sestrin2 is a critical effector of GCN2 signaling that regulates amino acid homeostasis through mTORC1 suppression.

Modulation of CCAAT/enhancer binding protein homologous protein (CHOP)-dependent DR5 expression by nelfinavir sensitizes glioblastoma multiforme cells to tumor necrosis factor-related apoptosis-inducing ligand (TRAIL).

Human glioblastoma multiforme cells demonstrate varying levels of sensitivity to tumor necrosis factor-related apoptosis-inducing ligand (TRAIL)-induced apoptosis. Endoplasmic reticulum (ER) stress has been shown to trigger cell death through apoptosis. We therefore pursued a strategy of integrating clinically relevant investigational agents that cooperate mechanistically through the regulation of ER stress and apoptosis pathways. Nelfinavir belongs to the protease inhibitor class of drugs currently used to treat patients with HIV and is in clinical trials as an anti-tumor agent. We found that Nelfinavir treatment led to ER stress-induced up-regulation of the DR5 receptor. This transactivation was mediated by the transcription factor CCAAT/enhancer binding protein homologous protein (CHOP). We also determined that ER stress-induced ATF4 up-regulation was responsible for modulation of CHOP. In contrast, DR4 receptor expression was unchanged by Nelfinavir treatment. Combining Nelfinavir with TRAIL led to a significantly enhanced level of apoptosis that was abrogated by siRNA silencing of DR5. We provide evidence that Nelfinavir-induced ER stress modulates DR5 expression in human glioblastoma multiforme cells and can enhance TRAIL efficacy. These studies provide a potential mechanistic rationale for the use of the Food and Drug Administration-approved agent Nelfinavir in combination with DR5 agonists to induce apoptosis in human malignancies.