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Whole-transcriptome Analysis of Fully Viable Energy Efficient Glycolytic-null Cancer Cells Established by Double Genetic Knockout of Lactate Dehydrogenase A/B or Glucose-6-Phosphate Isomerase.

ABSTRACT: BACKGROUND/AIM:Nearly all mammalian tumors of diverse tissues are believed to be dependent on fermentative glycolysis, marked by elevated production of lactic acid and expression of glycolytic enzymes, most notably lactic acid dehydrogenase (LDH). Therefore, there has been significant interest in developing chemotherapy drugs that selectively target various isoforms of the LDH enzyme. However, considerable questions remain as to the consequences of biological ablation of LDH or upstream targeting of the glycolytic pathway. MATERIALS AND METHODS:In this study, we explore the biochemical and whole transcriptomic effects of CRISPR-Cas9 gene knockout (KO) of lactate dehydrogenases A and B [LDHA/B double KO (DKO)] and glucose-6-phosphate isomerase (GPI KO) in the human colon cancer cell line LS174T, using Affymetrix 2.1 ST arrays. RESULTS:The metabolic biochemical profiles corroborate that relative to wild type (WT), LDHA/B DKO produced no lactic acid, (GPI KO) produced minimal lactic acid and both KOs displayed higher mitochondrial respiration, and minimal use of glucose with no loss of cell viability. These findings show a high biochemical energy efficiency as measured by ATP in glycolysis-null cells. Next, transcriptomic analysis conducted on 48,226 mRNA transcripts reflect 273 differentially expressed genes (DEGS) in the GPI KO clone set, 193 DEGS in the LDHA/B DKO clone set with 47 DEGs common to both KO clones. Glycolytic-null cells reflect up-regulation in gene transcripts typically associated with nutrient deprivation / fasting and possible use of fats for energy: thioredoxin interacting protein (TXNIP), mitochondrial 3-hydroxy-3-methylglutaryl-CoA synthase 2 (HMGCS2), PPAR? coactivator 1? (PGC-1?), and acetyl-CoA acyltransferase 2 (ACAA2). Other changes in non-ergometric transcripts in both KOs show losses in "stemness", WNT signaling pathway, chemo/radiation resistance, retinoic acid synthesis, drug detoxification, androgen/estrogen activation, and extracellular matrix reprogramming genes. CONCLUSION:These findings demonstrate that: 1) The "Warburg effect" is dispensable, 2) loss of the LDHAB gene is not only inconsequential to viability but fosters greater mitochondrial energy, and 3) drugs that target LDHA/B are likely to be ineffective without a plausible combination second drug target.

SUBMITTER: Mazzio E

PROVIDER: S-EPMC7472444 | biostudies-literature | 2020 Sep-Oct

REPOSITORIES: biostudies-literature

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Whole-transcriptome Analysis of Fully Viable Energy Efficient Glycolytic-null Cancer Cells Established by Double Genetic Knockout of Lactate Dehydrogenase A/B or Glucose-6-Phosphate Isomerase.

Mazzio Elizabeth E Badisa Ramesh R Mack Nzinga N Cassim Shamir S Zdralevic Masa M Pouyssegur Jacques J Soliman Karam F A KFA

Cancer genomics & proteomics 20200901 5

<h4>Background/aim</h4>Nearly all mammalian tumors of diverse tissues are believed to be dependent on fermentative glycolysis, marked by elevated production of lactic acid and expression of glycolytic enzymes, most notably lactic acid dehydrogenase (LDH). Therefore, there has been significant interest in developing chemotherapy drugs that selectively target various isoforms of the LDH enzyme. However, considerable questions remain as to the consequences of biological ablation of LDH or upstream ...[more]

PMID: 32859627

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Project description:For a long time cancer cells are known for increased uptake of glucose and its metabolization through glycolysis. Glyceraldehyde-3-phosphate dehydrogenase (GAPDH) is a key regulatory enzyme of this pathway and can produce ATP through oxidative level of phosphorylation. Previously, we reported that GAPDH purified from a variety of malignant tissues, but not from normal tissues, was strongly inactivated by a normal metabolite, methylglyoxal (MG). Molecular mechanism behind MG mediated GAPDH inhibition in cancer cells is not well understood.GAPDH was purified from Ehrlich ascites carcinoma (EAC) cells based on its enzymatic activity. GAPDH associated proteins in EAC cells and 3-methylcholanthrene (3MC) induced mouse tumor tissue were detected by mass spectrometry analysis and immunoprecipitation (IP) experiment, respectively. Interacting domains of GAPDH and its associated proteins were assessed by in silico molecular docking analysis. Mechanism of MG mediated GAPDH inactivation in cancer cells was evaluated by measuring enzyme activity, Circular dichroism (CD) spectroscopy, IP and mass spectrometry analyses.Here, we report that GAPDH is associated with glucose-6-phosphate isomerase (GPI) and pyruvate kinase M2 (PKM2) in Ehrlich ascites carcinoma (EAC) cells and also in 3-methylcholanthrene (3MC) induced mouse tumor tissue. Molecular docking analyses suggest C-terminal domain preference for the interaction between GAPDH and GPI. However, both C and N termini of PKM2 might be interacting with the C terminal domain of GAPDH. Expression of both PKM2 and GPI is increased in 3MC induced tumor compared with the normal tissue. In presence of 1 mM MG, association of GAPDH with PKM2 or GPI is not perturbed, but the enzymatic activity of GAPDH is reduced to 26.8 ± 5 % in 3MC induced tumor and 57.8 ± 2.3 % in EAC cells. Treatment of MG to purified GAPDH complex leads to glycation at R399 residue of PKM2 only, and changes the secondary structure of the protein complex.PKM2 may regulate the enzymatic activity of GAPDH. Increased enzymatic activity of GAPDH in tumor cells may be attributed to its association with PKM2 and GPI. Association of GAPDH with PKM2 and GPI could be a signature for cancer cells. Glycation at R399 of PKM2 and changes in the secondary structure of GAPDH complex could be one of the mechanisms by which GAPDH activity is inhibited in tumor cells by MG.

Project description:Study hypothesisDetailed structural comparisons of sperm-specific glyceraldehyde 3-phosphate dehydrogenase, spermatogenic (GAPDHS) and the somatic glyceraldehyde 3-phosphate dehydrogenase (GAPDH) isozyme should facilitate the identification of selective GAPDHS inhibitors for contraceptive development.Study findingThis study identified a small-molecule GAPDHS inhibitor with micromolar potency and >10-fold selectivity that exerts the expected inhibitory effects on sperm glycolysis and motility.What is known alreadyGlycolytic ATP production is required for sperm motility and male fertility in many mammalian species. Selective inhibition of GAPDHS, one of the glycolytic isozymes with restricted expression during spermatogenesis, is a potential strategy for the development of a non-hormonal contraceptive that directly blocks sperm function.Study design, samples/materials, methodsHomology modeling and x-ray crystallography were used to identify structural features that are conserved in GAPDHS orthologs in mouse and human sperm, but distinct from the GAPDH orthologs present in somatic tissues. We identified three binding pockets surrounding the substrate and cofactor in these isozymes and conducted a virtual screen to identify small-molecule compounds predicted to bind more tightly to GAPDHS than to GAPDH. Following the production of recombinant human and mouse GAPDHS, candidate compounds were tested in dose-response enzyme assays to identify inhibitors that blocked the activity of GAPDHS more effectively than GAPDH. The effects of a selective inhibitor on the motility of mouse and human sperm were monitored by computer-assisted sperm analysis, and sperm lactate production was measured to assess inhibition of glycolysis in the target cell.Main results and the role of chanceOur studies produced the first apoenzyme crystal structures for human and mouse GAPDHS and a 1.73 Å crystal structure for NAD(+)-bound human GAPDHS, facilitating the identification of unique structural features of this sperm isozyme. In dose-response assays T0501_7749 inhibited human GAPDHS with an IC50 of 1.2 μM compared with an IC50 of 38.5 μM for the somatic isozyme. This compound caused significant reductions in mouse sperm lactate production (P= 0.017 for 100 μM T0501_7749 versus control) and in the percentage of motile mouse and human sperm (P values from <0.05 to <0.0001, depending on incubation conditions).Limitations, reasons for cautionThe chemical properties of T0501_7749, including limited solubility and nonspecific protein binding, are not optimal for drug development.Wider implications of the findingsThis study provides proof-of-principle evidence that GAPDHS can be selectively inhibited, causing significant reductions in sperm glycolysis and motility. These results highlight the utility of structure-based drug design and support further exploration of GAPDHS, and perhaps other sperm-specific isozymes in the glycolytic pathway, as contraceptive targets.Large scale dataNone. Coordinates and data files for three GAPDHS crystal structures were deposited in the RCSB Protein Data Bank (http://www.rcsb.org).Study funding and competing interestsThis work was supported by grants from the National Institutes of Health (NIH), USA, including U01 HD060481 and cooperative agreement U54 HD35041 as part of the Specialized Cooperative Centers Program in Reproduction and Infertility Research from the Eunice Kennedy Shriver National Institute of Child Health and Human Development, and TW/HD00627 from the NIH Fogarty International Center. Additional support was provided by subproject CIG-05-109 from CICCR, a program of CONRAD, Eastern Virginia Medical School, USA. There are no conflicts of interest.

Dataset Information

Whole-transcriptome Analysis of Fully Viable Energy Efficient Glycolytic-null Cancer Cells Established by Double Genetic Knockout of Lactate Dehydrogenase A/B or Glucose-6-Phosphate Isomerase.

Publications

Whole-transcriptome Analysis of Fully Viable Energy Efficient Glycolytic-null Cancer Cells Established by Double Genetic Knockout of Lactate Dehydrogenase A/B or Glucose-6-Phosphate Isomerase.

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