Identification of glucose-independent metabolic pathways associated with anti-proliferative effect of metformin, their coordinate derangement with cMyc downregulation and reversibility in liver cancer cells

Several studies indicated anti-cancer effects of metformin in liver cancer. This was attributed to the activation of LKB-AMPK axis, which is associated with anti-hyperglycaemic effect and cytotoxicity. However, despite lack of evidence on cytotoxic effect of physiological metformin concentrations and ability of cancer cells to survive under glucose-deprivation, no study has examined the glucose-independent effect of non-cytotoxic metformin or metabolic reprogramming associated with it. In addition, no study has ever been conducted on reversibility of anti-cancer effects of metformin. Here, the dose-dependent effects of metformin on HepG2 cells were examined in presence and absence of glucose. The longitudinal evolution of metabolome was analyzed along with gene and protein expression as well as their correlations with and reversibility of cellular phenotype and metabolic signatures. Metformin concentrations up to 2.5mM were found to be non-cytotoxic but anti-proliferative irrespective of presence of glucose. Apart from mitochondrial impairment, derangement of fatty acid desaturation, one-carbon, glutathione and polyamine metabolism were associated with non-cytotoxic metformin treatment irrespective of glucose supplementation. Depletion of pantothenic acid, downregulation of essential amino acid uptake, metabolism and purine salvage were identified as novel glucose-independent effects of metformin. These were significantly correlated with cMyc expression and reduction in proliferation. Rescue experiments established reversibility upon metformin withdrawal and tight association between proliferation, metabotype and cMyc expression. Taken together, derangement of novel glucose-independent metabolic pathways and concomitant cMyc downregulation co-ordinately contribute to anti-proliferative effect of metformin even at non-cytotoxic concentrations, which is reversible and may influence its therapeutic utility. ### Competing Interest Statement The authors have declared no competing interest. * ACC1 : Acetyl CoA carboxylase alpha aka ACACA ACC2 : Acetyl CoA carboxylase beta aka ACACB ADA : Adenosine deaminase ADK : Adenosine kinase AHCYL1 : S adenosylhomocysteine hydrolase like protein AMD1 : Adenosylmethionine decarboxylase AMPK : AMP-activated protein kinase ANOVA : Analysis of Variance APRT : Adenine phosphoribosyltransferase BCAA : Branched-chain amino acids BCAT1 : Branched chain amino acid transaminase BrdU : Bromodeoxyuridine cMyc : Cellular myelocytomatosis oncogene CoA : Coenzyme A DMEM : Dulbecco’s Modified Eagle Medium EAA : Essential Amino Acids GCLC : Glutamate Cysteine Ligase Catalytic Subunit GCLM : Glutamate Cysteine Ligase Modifier Subunit GLS1 : Glutaminase GLS2 : Glutaminase GOT1 : Glutamic oxaloacetic transaminase 1 cytosolic GOT2 : Glutamic oxaloacetic transaminase 2 mitochondrial GPT1 : Glutamic pyruvic transaminase GPT2 : Glutamic pyruvic transaminase HPRT1 : Hypoxanthine phosphoribosyltransferase 1 LACC 1 : Laccase Domain Containing LAT1 : L type amino acid transporter 1 aka SLC7A5 LKB : Liver Kinase B aka STK11. MAT2A : Methionine adenosyltransferase II alpha MTAP : Methylthioadenosine Phosphorylase MTR : 5 methyltetrahydrofolate homocysteine methyltransferase NAFLD : Non-alcoholic fatty liver disease NEAA : Non-essential Amino Acids ODC1 : Ornithine decarboxylase PAOX : Polyamine Oxidase SHMT1 : Serine hydroxymethyltransferase 1 SHMT2 : Serine hydroxymethyltransferase 2 SLC16A1 : Solute Carrier Family 16 Member 1 SLC16A10 : Solute Carrier Family 16 Member 10 SLC16A3 : Solute Carrier Family 16 Member 3 SLC1A5 : Solute Carrier Family 1 Member 5 aka ASCT2 Glutamine Transporter SLC2A1 : Solute carrier family 2 facilitated glucose transporter member 1 aka GLUT1 SLC2A3 : Solute carrier family 2 facilitated glucose transporter member 3 aka GLUT3 SLC3A2 : Solute carrier family 3 member 2 aka CD98 SLC43A1 : Solute Carrier Family 43 Member1 SLC43A2 : Solute Carrier Family 43 Member 2 SLC7A1 : Solute Carrier Family 7 Member 1 SLC7A11 : Solute Carrier Family 7 Member 11 SMOX : Spermine oxidase SMS : Spermine synthase SRM : Spermidine synthase SSAT1 : Spermidine /spermine N1 acetyltransferase 1 aka SAT1 TCA : Tricarboxylic acid cycle XTT : Methoxynitrosulfophenyl-tetrazolium carboxanilide.