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), PPARγ coactivator 1α (PGC-1α), 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. Common features across diverse mammalian tumors include the rapid glycolytic activity, greater expression of lactate dehydrogenases (LDH) and subsequent overproduction of lactic acid, occurring regardless of the presence of oxygen (Warburg effect) (1,2). This unique trait of cancer has been studied for decades, where researchers have geared off original focus from its role in metabolism to its role in acidification of the tumor microenvironment (TME), a formidable driving force for tumor growth, metastasis, aggressiveness, chemo/radiation resistance and loss of immune surveillance (3-8). This loss of immune surveillance 469 This article is freely accessible online.

Targeting the pH Paradigm at the Bedside: A Practical Approach

“…Further research is needed to determine what comes first, the Warburg effect or the pH paradigm. However, after the seminal studies by the groups headed by Pouyssegur [12][13][14][15], and Reshkin [5,[16][17][18][19][20][21], evidence hints towards the second option, based on: (a) The finding of increased intracellular alkalinity at the very beginning of malignization [5]. (b) The need for a high intracellular pH for the increased activity of glycolytic enzymes [22].…”

“…(c) The persistence of the pH paradigm, even in conditions in which lactic acid cannot be produced. (d) The Warburg effect (aerobic glycolysis) is not indispensable for tumor survival and progression [13]. (e) Hypoxia is involved in both the Warburg effect and pH inversion.…”

Metabolic Response to the Mitochondrial Toxin 1-Methyl-4-phenylpyridinium (MPP+) in LDH-A/B Double-knockout LS174T Colon Cancer Cells

“…Interestingly, we have been studying the consequences of LDHA inhibitors for many years in diverse tumor cells and repeatedly failed to see any adverse effects on the production of lactic acid or tumor cell energy survival systems, with the only possible consequence being a reduction in cell proliferation (11)(12)(13). In collaboration with Dr. Jacques Pouyssegurs laboratory (Nice, France), our recent work further defined transcriptomic changes in colon cancer cells which were genetically engineered to knockout both LDHA/B genes (14), and in doing so, constructed a cell line lacking transcripts required to synthesize numerous LDH isoforms (15). That work was crucial in demonstrating the need to simultaneously down-regulate LDH-A and LDH-B genes in order to shut off the production of lactic acid in cancer cells.…”

“…That work was crucial in demonstrating the need to simultaneously down-regulate LDH-A and LDH-B genes in order to shut off the production of lactic acid in cancer cells. However, even with this, cancer cells remain immortal, experience slower doubling rates, and exhibit heightened mitochondrial OXPHOS capacity (15).…”

“…In this study we explore the metabolic consequences of using a well-known multi-complex mitochondrial complex I, IV inhibitor,1-methyl-4-phenylpyridinium (MPP+) in LDH-A/B DKO colon cancer cells which are devoid of the metabolic apparatus to carry out anaerobic glycolytic SLP (15).…”