mapping-metabolism-monitoring-lactate-dehydrogenase-activity-directly

Mapping Metabolism: Monitoring Lactate Dehydrogenase Activity Directly in Tissue

Mapping enzymatic activity in space and time is critical for understanding the molecular basis of cell behavior in normal tissue and disease. In situ ...

David Geffen School of Medicine

UCLA

Altered cellular metabolism is a defining feature of cancer [1]. The best studied metabolic phenotype of cancer is aerobic glycolysis--also known as the Warburg effect--characterized by increased metabolism of glucose to lactate in the presence of sufficient oxygen. Interest in the Warburg effect has escalated in recent years due to the proven utility of FDG-PET for imaging tumors in cancer patients and growing evidence that mutations in oncogenes and tumor suppressor genes directly impact metabolism. The goals of this review are to provide an organized snapshot of the current understanding of regulatory mechanisms important for Warburg effect and its role in tumor biology. Since several reviews have covered aspects of this topic in recent years, we focus on newest contributions to the field and reference other reviews where appropriate.

New aspects of the Warburg effect in cancer cell biology.

Differences in global levels of histone acetylation occur in normal and cancer cells, although the reason why cells regulate these levels has been unclear. Here we demonstrate a role for histone acetylation in regulating intracellular pH (pH(i)). As pH(i) decreases, histones are globally deacetylated by histone deacetylases (HDACs), and the released acetate anions are coexported with protons out of the cell by monocarboxylate transporters (MCTs), preventing further reductions in pH(i). Conversely, global histone acetylation increases as pH(i) rises, such as when resting cells are induced to proliferate. Inhibition of HDACs or MCTs decreases acetate export and lowers pH(i), particularly compromising pH(i) maintenance in acidic environments. Global deacetylation at low pH is reflected at a genomic level by decreased abundance and extensive redistribution of acetylation throughout the genome. Thus, acetylation of chromatin functions as a rheostat to regulate pH(i) with important implications for mechanism of action and therapeutic use of HDAC inhibitors.

Histone acetylation regulates intracellular pH.

Unlike many cancers that exhibit glycolytic metabolism, high-grade liposarcomas often exhibit low 2[18F]fluoro-2-deoxy-D-glucose uptake by positron emission tomography (PET), despite rapid tumor growth. Here, we used liquid chromatography tandem mass spectrometry to identify carbon sources taken up by liposarcoma cell lines derived from xenograft tumors in patients. Interestingly, we found that liposarcoma cell lines consume nucleosides from culture media, suggesting nucleoside salvage pathway activity. The nucleoside salvage pathway is dependent on deoxycytidine kinase (dCK) and can be imaged in vivo by PET with 1-(2'-deoxy-2'-[18F]fluoroarabinofuranosyl) cytosine (FAC). We found that liposarcoma cell lines and xenograft tumors exhibit dCK activity and dCK-dependent FAC uptake in vitro and in vivo. In addition, liposarcoma cell lines and xenograft tumors are sensitive to treatment with the nucleoside analogue prodrug gemcitabine, and gemcitabine sensitivity is dependent on dCK expression. Elevated dCK activity is evident in 7 of 68 clinical liposarcoma samples analyzed. These data suggest that a subpopulation of liposarcoma patients have tumors with nucleoside salvage pathway activity that can be identified noninvasively using [18F]-FAC-PET and targeted using gemcitabine.

Metabolomics strategy reveals subpopulation of liposarcomas sensitive to gemcitabine treatment.

Liposarcoma is a type of soft tissue sarcoma that exhibits poor survival and a high recurrence rate. Treatment is generally limited to surgery and radiation, which emphasizes the need for better understanding of this disease. Because very few in vivo and in vitro models can reproducibly recapitulate the human disease, we generated several xenograft models from surgically resected human dedifferentiated liposarcoma. All xenografts recapitulated morphological and gene expression characteristics of the patient tumors after continuous in vivo passages. Importantly, xenograftability was directly correlated with disease-specific survival of liposarcoma patients. Thus, the ability for the tumor of a patient to engraft may help identify those patients who will benefit from more aggressive treatment regimens. Gene expression analyses highlighted the association between xenograftability and a unique gene expression signature, including down-regulated PTEN tumor-suppressor gene expression and a progenitor-like phenotype. When treated with the PI3K/AKT/mTOR pathway inhibitor rapamycin alone or in combination with the multikinase inhibitor sorafenib, all xenografts responded with increased lipid content and a more differentiated gene expression profile. These human xenograft models may facilitate liposarcoma research and accelerate the generation of readily translatable preclinical data that could ultimately influence patient care.

Novel dedifferentiated liposarcoma xenograft models reveal PTEN down-regulation as a malignant signature and response to PI3K pathway inhibition.

The sterol regulatory element-binding proteins (SREBP) are key transcriptional regulators of lipid metabolism and cellular growth. It has been proposed that SREBP signaling regulates cellular growth through its ability to drive lipid biosynthesis. Unexpectedly, we find that loss of SREBP activity inhibits cancer cell growth and viability by uncoupling fatty acid synthesis from desaturation. Integrated lipid profiling and metabolic flux analysis revealed that cancer cells with attenuated SREBP activity maintain long-chain saturated fatty acid synthesis, while losing fatty acid desaturation capacity. We traced this defect to the uncoupling of fatty acid synthase activity from stearoyl-CoA desaturase 1 (SCD1)-mediated desaturation. This deficiency in desaturation drives an imbalance between the saturated and monounsaturated fatty acid pools resulting in severe lipotoxicity. Importantly, replenishing the monounsaturated fatty acid pool restored growth to SREBP-inhibited cells. These studies highlight the importance of fatty acid desaturation in cancer growth and provide a novel mechanistic explanation for the role of SREBPs in cancer metabolism.

An essential requirement for the SCAP/SREBP signaling axis to protect cancer cells from lipotoxicity.

Alternative splicing contributes to diverse aspects of cancer pathogenesis including altered cellular metabolism, but the specificity of the process or its consequences are not well understood. We characterized genome-wide alternative splicing induced by the activating EGFRvIII mutation in glioblastoma (GBM). EGFRvIII upregulates the heterogeneous nuclear ribonucleoprotein (hnRNP) A1 splicing factor, promoting glycolytic gene expression and conferring significantly shorter survival in patients. HnRNPA1 promotes splicing of a transcript encoding the Myc-interacting partner Max, generating Delta Max, an enhancer of Myc-dependent transformation. Delta Max, but not full-length Max, rescues Myc-dependent glycolytic gene expression upon induced EGFRvIII loss, and correlates with hnRNPA1 expression and downstream Myc-dependent gene transcription in patients. Finally, Delta Max is shown to promote glioma cell proliferation inÂ vitro and augment EGFRvIII expressing GBM growth inÂ vivo. These results demonstrate an important role for alternative splicing in GBM and identify Delta Max as aÂ mediator of Myc-dependent tumor cell metabolism.

EGFR Mutation-Induced Alternative Splicing of Max Contributes to Growth of Glycolytic Tumors in Brain Cancer.

&Eacute;pissage alternatif contribue &agrave; divers aspects de la pathogen&egrave;se du cancer, y compris l&#39;alt&eacute;ration du m&eacute;tabolisme cellulaire, mais la sp&eacute;cificit&eacute; du processus ou ses cons&eacute;quences ne sont pas bien comprises. Nous avons caract&eacute;ris&eacute; le g&eacute;nome &eacute;pissage alternatif induit par la mutation activatrice de EGFRvIII dans le glioblastome (GBM). EGFRvIII r&eacute;gule la ribonucl&eacute;oprot&eacute;ines nucl&eacute;aires h&eacute;t&eacute;rog&egrave;nes (hnRNP) A1 &eacute;pissage facteur, qui favorise l&#39;expression des g&egrave;nes glycolytique et conf&eacute;rant une survie significativement plus courte chez les patients &agrave; la hausse. HnRNPA1 promeut l&#39;&eacute;pissage d&#39;un transcrit codant le partenaire d&#39;interaction Myc Max, g&eacute;n&eacute;rant Delta Max, un activateur de la transformation de Myc-d&eacute;pendante. Delta Max, mais pas de pleine longueur Max, sauve l&#39;expression des g&egrave;nes glycolytique Myc d&eacute;pendante sur la perte de EGFRvIII induite et est en corr&eacute;lation avec l&#39;expression de hnRNPA1 et de la transcription du g&egrave;ne Myc d&eacute;pendante en aval chez les patients. Enfin, Delta Max est montr&eacute; &agrave; favoriser la prolif&eacute;ration de cellules de gliome in vitro et augmenter l&#39;EGFRvIII exprimant la croissance GBM in vivo. Ces r&eacute;sultats d&eacute;montrent un r&ocirc;le important pour l&#39;&eacute;pissage alternatif dans GBM et identifient le Delta Max comme m&eacute;diateur de m&eacute;tabolisme des cellules tumorales Myc-d&eacute;pendante.

EGFR Mutation induite par l&#39;&eacute;pissage alternatif de Max contribue &agrave; la croissance des tumeurs glycolytiques dans le cancer du cerveau.

The tetracycline antibiotics are widely used in biomedical research as mediators of inducible gene expression systems. Despite many known effects of tetracyclines on mammalian cells-including inhibition of the mitochondrial ribosome-there have been few reports on potential off-target effects at concentrations commonly used in inducible systems. Here, we report that in human cell lines, commonly used concentrations of doxycycline change gene expression patterns and concomitantly shift metabolism towards a more glycolytic phenotype, evidenced by increased lactate secretion and reduced oxygen consumption. We also show that these concentrations are sufficient to slow proliferation. These findings suggest that researchers using doxycycline in inducible expression systems should design appropriate controls to account for potential confounding effects of the drug on cellular metabolism.

Doxycycline alters metabolism and proliferation of human cell lines.

Virus infections trigger metabolic changes in host cells that support the bioenergetic and biosynthetic demands of viral replication. Although recent studies have characterized virus-induced changes in host cell metabolism (Munger et al., 2008; Terry et al., 2012), the molecular mechanisms by which viruses reprogram cellular metabolism have remained elusive. Here, we show that the gene product of adenovirus E4ORF1 is necessary for adenovirus-induced upregulation of host cell glucose metabolism and sufficient to promote enhanced glycolysis in cultured epithelial cells by activation of MYC. E4ORF1 localizes to the nucleus, binds to MYC, and enhances MYC binding to glycolytic target genes, resulting in elevated expression of specific glycolytic enzymes. E4ORF1 activation of MYC promotes increased nucleotide biosynthesis from glucose intermediates and enables optimal adenovirus replication in primary lung epithelial cells. Our findings show how a viral protein exploits host cell machinery to reprogram cellular metabolism and promote optimal progeny virion generation.

Adenovirus E4ORF1-induced MYC activation promotes host cell anabolic glucose metabolism and virus replication.

To colonize the liver, colon cancer metastases must overcome hypoxia and other metabolic stress. Loo et al. now show that metastatic cells achieve this by decreasing miR-483 and miR-551a expression, which derepresses creatine kinase expression and allows energy to be captured from extracellular ATP through generation and import of phosphocreatine.

The metabolic milieu of metastases.

We discovered recently that the central metabolite α-ketoglutarate (α-KG) extends the lifespan of C. elegans through inhibition of ATP synthase and TOR signaling. Here we find, unexpectedly, that (R)-2-hydroxyglutarate ((R)-2HG), an oncometabolite that interferes with various α-KG-mediated processes, similarly extends worm lifespan. (R)-2HG accumulates in human cancers carrying neomorphic mutations in the isocitrate dehydrogenase (IDH) 1 and 2 genes. We show that, like α-KG, both (R)-2HG and (S)-2HG bind and inhibit ATP synthase and inhibit mTOR signaling. These effects are mirrored in IDH1 mutant cells, suggesting a growth-suppressive function of (R)-2HG. Consistently, inhibition of ATP synthase by 2-HG or α-KG in glioblastoma cells is sufficient for growth arrest and tumor cell killing under conditions of glucose limitation, e.g., when ketone bodies (instead of glucose) are supplied for energy. These findings inform therapeutic strategies and open avenues for investigating the roles of 2-HG and metabolites in biology and disease.

2-Hydroxyglutarate Inhibits ATP Synthase and mTOR Signaling.

Viruses rewire host cell glucose and glutamine metabolism to meet the bioenergetic and biosynthetic demands of viral propagation. However, the mechanism by which viruses reprogram glutamine metabolism and the metabolic fate of glutamine during adenovirus infection have remained elusive. Here, we show MYC activation is necessary for adenovirus-induced upregulation of host cell glutamine utilization and increased expression of glutamine transporters and glutamine catabolism enzymes. Adenovirus-induced MYC activation promotes increased glutamine uptake, increased use of glutamine in reductive carboxylation and increased use of glutamine in generating hexosamine pathway intermediates and specific amino acids. We identify glutaminase (GLS) as a critical enzyme for optimal adenovirus replication and demonstrate that GLS inhibition decreases replication of adenovirus, herpes simplex virus 1 and influenza A in cultured primary cells. Our findings show that adenovirus-induced reprogramming of glutamine metabolism through MYC activation promotes optimal progeny virion generation, and suggest that GLS inhibitors may be useful therapeutically to reduce replication of diverse viruses. 

MYC-induced reprogramming of glutamine catabolism supports optimal virus replication.

Monocarboxylate transporter 1 (MCT1) inhibition is thought to block tumor growth through disruption of lactate transport and glycolysis. Here, we show MCT1 inhibition impairs proliferation of glycolytic breast cancer cells co-expressing MCT1 and MCT4 via disruption of pyruvate rather than lactate export. MCT1 expression is elevated in glycolytic breast tumors, and high MCT1 expression predicts poor prognosis in breast and lung cancer patients. Acute MCT1 inhibition reduces pyruvate export but does not consistently alter lactate transport or glycolytic flux in breast cancer cells that co-express MCT1 and MCT4. Despite the lack of glycolysis impairment, MCT1 loss-of-function decreases breast cancer cell proliferation and blocks growth of mammary fat pad xenograft tumors. Our data suggest MCT1 expression is elevated in glycolytic cancers to promote pyruvate export that when inhibited, enhances oxidative metabolism and reduces proliferation. This study presents an alternative molecular consequence of MCT1 inhibitors, further supporting their use as anti-cancer therapeutics. 

MCT1 Modulates Cancer Cell Pyruvate Export and Growth of Tumors that Co-express MCT1 and MCT4.

Cellular amino acid uptake is critical for mTOR complex 1 (mTORC1) activation and cell proliferation. However, the regulation of amino acid uptake is not well-understood. Here we describe a role for asparagine as an amino acid exchange factor: intracellular asparagine exchanges with extracellular amino acids. Through asparagine synthetase knockdown and altering of media asparagine concentrations, we show that intracellular asparagine levels regulate uptake of amino acids, especially serine, arginine and histidine. Through its exchange factor role, asparagine regulates mTORC1 activity and protein synthesis. In addition, we show that asparagine regulation of serine uptake influences serine metabolism and nucleotide synthesis, suggesting that asparagine is involved in coordinating protein and nucleotide synthesis. Finally, we show that maintenance of intracellular asparagine levels is critical for cancer cell growth. Collectively, our results indicate that asparagine is an important regulator of cancer cell amino acid homeostasis, anabolic metabolism and proliferation. 

Asparagine promotes cancer cell proliferation through use as an amino acid exchange factor.

The rate of glycolytic metabolism changes during differentiation of human embryonic stem cells (hESCs) and reprogramming of somatic cells to pluripotency. However, the functional contribution of glycolytic metabolism to the pluripotent state is unclear. Here we show that naive hESCs exhibit increased glycolytic flux, MYC transcriptional activity, and nuclear N-MYC localization relative to primed hESCs. This status is consistent with the inner cell mass of human blastocysts, where MYC transcriptional activity is higher than in primed hESCs and nuclear N-MYC levels are elevated. Reduction of glycolysis decreases self-renewal of naive hESCs and feeder-free primed hESCs, but not primed hESCs grown in feeder-supported conditions. Reduction of glycolysis in feeder-free primed hESCs also enhances neural specification. These findings reveal associations between glycolytic metabolism and human naive pluripotency and differences in the metabolism of feeder-/feeder-free cultured hESCs. They may also suggest methods for regulating self-renewal and initial cell fate specification of hESCs.

Glycolytic Metabolism Plays a Functional Role in Regulating Human Pluripotent Stem Cell State.

Cancer cells exhibit increased use of nutrients, including glucose and glutamine, to support the bioenergetic and biosynthetic demands of proliferation. We tested the small-molecule inhibitor of glutaminase CB-839 in combination with erlotinib on epidermal growth factor receptor (EGFR) mutant non-small cell lung cancer (NSCLC) as a therapeutic strategy to simultaneously impair cancer glucose and glutamine utilization and thereby suppress tumor growth. Here, we show that CB-839 cooperates with erlotinib to drive energetic stress and activate the AMP-activated protein kinase (AMPK) pathway in EGFR (del19) lung tumors. Tumor cells undergo metabolic crisis and cell death, resulting in rapid tumor regression in vivo in mouse NSCLC xenografts. Consistently, positron emission tomography (PET) imaging with F-fluoro-2-deoxyglucose (F-FDG) and C-glutamine (C-Gln) of xenografts indicated reduced glucose and glutamine uptake in tumors following treatment with CB-839 + erlotinib. Therefore, PET imaging with F-FDG and C-Gln tracers can be used to non-invasively measure metabolic response to CB-839 and erlotinib combination therapy.

Targeted Inhibition of EGFR and Glutaminase Induces Metabolic Crisis in EGFR Mutant Lung Cancer.

Copy number alteration (CNA) profiling of human tumors has revealed recurrent patterns of DNA amplifications and deletions across diverse cancer types. These patterns are suggestive of conserved selection pressures during tumor evolution but cannot be fully explained by known oncogenes and tumor suppressor genes. Using a pan-cancer analysis of CNA data from patient tumors and experimental systems, here we show that principal component analysis-defined CNA signatures are predictive of glycolytic phenotypes, including F-fluorodeoxy-glucose (FDG) avidity of patient tumors, and increased proliferation. The primary CNA signature is enriched for p53 mutations and is associated with glycolysis through coordinate amplification of glycolytic genes and other cancer-linked metabolic enzymes. A pan-cancer and cross-species comparison of CNAs highlighted 26 consistently altered DNA regions, containing 11 enzymes in the glycolysis pathway in addition to known cancer-driving genes. Furthermore, exogenous expression of hexokinase and enolase enzymes in an experimental immortalization system altered the subsequent copy number status of the corresponding endogenous loci, supporting the hypothesis that these metabolic genes act as drivers within the conserved CNA amplification regions. Taken together, these results demonstrate that metabolic stress acts as a selective pressure underlying the recurrent CNAs observed in human tumors, and further cast genomic instability as an enabling event in tumorigenesis and metabolic evolution.

Recurrent patterns of DNA copy number alterations in tumors reflect metabolic selection pressures.

Mutations in cancer reprogram amino acid metabolism to drive tumor growth, but the molecular mechanisms are not well understood. Using an unbiased proteomic screen, we identified mTORC2 as a critical regulator of amino acid metabolism in cancer via phosphorylation of the cystine-glutamate antiporter xCT. mTORC2 phosphorylates serine 26 at the cytosolic N terminus of xCT, inhibiting its activity. Genetic inhibition of mTORC2, or pharmacologic inhibition of the mammalian target of rapamycin (mTOR) kinase, promotes glutamate secretion, cystine uptake, and incorporation into glutathione, linking growth factor receptor signaling with amino acid uptake and utilization. These results identify an unanticipated mechanism regulating amino acid metabolism in cancer, enabling tumor cells to adapt to changing environmental conditions.

mTORC2 Regulates Amino Acid Metabolism in Cancer by Phosphorylation of the Cystine-Glutamate Antiporter xCT.

Altered metabolism is a hallmark of cancer growth, forming the conceptual basis for development of metabolic therapies as cancer treatments. We performed in vivo metabolic profiling and molecular analysis of lung squamous cell carcinoma (SCC) to identify metabolic nodes for therapeutic targeting. Lung SCCs adapt to chronic mTOR inhibition and suppression of glycolysis through the GSK3α/β signaling pathway, which upregulates glutaminolysis. Phospho-GSK3α/β protein levels are predictive of response to single-therapy mTOR inhibition while combinatorial treatment with the glutaminase inhibitor CB-839 effectively overcomes therapy resistance. In addition, we identified a conserved metabolic signature in a broad spectrum of hypermetabolic human tumors that may be predictive of patient outcome and response to combined metabolic therapies targeting mTOR and glutaminase.

Article	Year
New aspects of the Warburg effect in cancer cell biology. Seminars in cell & developmental biology\| PubMed ID: 22406683	2012
Histone acetylation regulates intracellular pH. Molecular cell\| PubMed ID: 23201122	2013
Metabolomics strategy reveals subpopulation of liposarcomas sensitive to gemcitabine treatment. Cancer discovery\| PubMed ID: 23230188	2012
Novel dedifferentiated liposarcoma xenograft models reveal PTEN down-regulation as a malignant signature and response to PI3K pathway inhibition. The American journal of pathology\| PubMed ID: 23416162	2013
An essential requirement for the SCAP/SREBP signaling axis to protect cancer cells from lipotoxicity. Cancer research\| PubMed ID: 23440422	2013
EGFR Mutation-Induced Alternative Splicing of Max Contributes to Growth of Glycolytic Tumors in Brain Cancer. Cell metabolism\| PubMed ID: 23707073	2013
Doxycycline alters metabolism and proliferation of human cell lines. PloS one\| PubMed ID: 23741339	2013
Adenovirus E4ORF1-induced MYC activation promotes host cell anabolic glucose metabolism and virus replication. Cell metabolism\| PubMed ID: 24703700	2014
The metabolic milieu of metastases. Cell\| PubMed ID: 25635452	2015
2-Hydroxyglutarate Inhibits ATP Synthase and mTOR Signaling. Cell metabolism\| PubMed ID: 26190651	2015
MYC-induced reprogramming of glutamine catabolism supports optimal virus replication. Nature communications\| PubMed ID: 26561297	2015
MCT1 Modulates Cancer Cell Pyruvate Export and Growth of Tumors that Co-express MCT1 and MCT4. Cell reports\| PubMed ID: 26876179	2016
Asparagine promotes cancer cell proliferation through use as an amino acid exchange factor. Nature communications\| PubMed ID: 27126896	2016
Glycolytic Metabolism Plays a Functional Role in Regulating Human Pluripotent Stem Cell State. Cell stem cell\| PubMed ID: 27618217	2016
Targeted Inhibition of EGFR and Glutaminase Induces Metabolic Crisis in EGFR Mutant Lung Cancer. Cell reports\| PubMed ID: 28099841	2017
Recurrent patterns of DNA copy number alterations in tumors reflect metabolic selection pressures. Molecular systems biology\| PubMed ID: 28202506	2017
mTORC2 Regulates Amino Acid Metabolism in Cancer by Phosphorylation of the Cystine-Glutamate Antiporter xCT. Molecular cell\| PubMed ID: 28648777	2017
The GSK3 Signaling Axis Regulates Adaptive Glutamine Metabolism in Lung Squamous Cell Carcinoma. Cancer cell\| PubMed ID: 29763624	2018

Heather R. Christofk

Biography

JoVE Journal Publications

Other Publications