Genomic GLO1 deletion modulates TXNIP expression, glucose metabolism, and redox homeostasis while accelerating human A375 malignant melanoma tumor growth

Abstract

Glyoxalase 1 (encoded by GLO1 ) is a glutathione-dependent enzyme detoxifying the glycolytic byproduct methylglyoxal (MG), an oncometabolite involved in metabolic reprogramming. Recently, we have demonstrated that GLO1 is overexpressed in human malignant melanoma cells and patient tumors and substantiated a novel role of GLO1 as a molecular determinant of invasion and metastasis in melanoma. Here, employing NanoString™ gene expression profiling (nCounter™ ‘PanCancer Progression Panel’), we report that CRISPR/Cas 9-based GLO1 deletion from human A375 malignant melanoma cells alters glucose metabolism and redox homeostasis, observable together with acceleration of tumorigenesis. Nanostring™ analysis identified TXNIP (encoding thioredoxin-interacting protein), a master regulator of cellular energy metabolism and redox homeostasis, displaying the most pronounced expression change in response to GLO1 elimination, confirmed by RT-qPCR and immunoblot analysis. TXNIP was also upregulated in CRISPR/Cas9-engineered DU145 prostate carcinoma cells lacking GLO1 , and treatment with MG or a pharmacological GLO1 inhibitor (TLSC702) mimicked GLO1 _KO status, suggesting that GLO1 controls TXNIP expression through regulation of MG. GLO1 _KO status was characterized by ( i ) altered oxidative stress response gene expression, ( ii ) attenuation of glucose uptake and metabolism with downregulation of gene expression ( GLUT1 , GFAT1 , GFAT2 , LDHA ) and depletion of related key metabolites (glucose-6-phosphate, UDP-N-acetylglucosamine), and ( iii ) immune checkpoint modulation ( PDL1 ). While confirming our earlier finding that GLO1 deletion limits invasion and metastasis with modulation of EMT-related genes (e.g. TGFBI , MMP9 , ANGPTL4 , TLR4, SERPINF1 ) , we observed that GLO1_KO melanoma cells displayed a shortened population doubling time, cell cycle alteration with increased M-phase population, and enhanced anchorage-independent growth, a phenotype supported by expression analysis ( CXCL8 , CD24 , IL1A , CDKN1A ). Concordantly, an accelerated growth rate of GLO1 _KO tumors, accompanied by TXNIP overexpression and metabolic reprogramming, was observable in a SCID mouse melanoma xenograft model, demonstrating that A375 melanoma tumor growth and metastasis can be dysregulated in opp...

Deuterium Oxide (D2O) Induces Early Stress Response Gene Expression and Impairs Growth and Metastasis of Experimental Malignant Melanoma

“…The following cell lines were purchased from ATCC (Manassas, VA, USA): human malignant melanoma cells (A375 (CRL-1619), A375-Luc2 (containing BRAF V600E mutation; CRL-1619-LUC2), NRAS-mutant-A375-Luc2 (isogenic variant containing both BRAF V600E and NRAS Q61K mutations; CRL-1619IG-2-LUC2), and G361 (CRL-1424)), human pancreatic ductal adenocarcinoma cells (PANC-1-Luc2 (CRL-1469-LUC2), MIA PaCa-2 (CRL-1420), and Capan-2 (HTB-80)), and normal skin fibroblasts (Hs27 (CRL-1634)), cultured under standard conditions as specified by the manufacturer [ 48 , 49 , 50 ]. LOX-IMVI melanoma cells (SCC201) were purchased from Millipore Sigma.…”

“…After D 2 O (up to 90%; up to 6 days) treatment, viability of melanoma and pancreatic cancer cells was assessed by annexinV-FITC/propidium iodide (PI) dual staining followed by flow cytometric analysis as published before using an apoptosis detection kit according to the manufacturer’s specifications (APOAF, Sigma Aldrich, St. Louis, MO, USA) [ 48 , 49 ].…”

“…Using a rabbit derived Alexa-488 conjugated antibody (Cell Signaling), cells in M-phase were detected by bivariate flow cytometric determination of cellular DNA content (PI-staining) and histone H3 phosphorylated at Ser 10 (p-H3(Ser10)); p-H3(Ser10)-positive cells were expressed in percent of total gated cells as published before [ 49 , 50 ].…”

“…The human Stress and Toxicity Pathway Finder RT 2 Profiler TM technology (Qiagen) assessing expression of 84 stress response regulatory genes (normalized to a group of five housekeeping genes ( ACTB , B2M , GAPDH , HPRT1 , and RPLP0 )) was employed following our published procedures [ 49 , 53 ].…”

“…To evaluate the cellular invasion potential, a published standard procedure was followed [ 49 , 52 , 54 ]. Either normal growth medium (10% FBS; 0% D 2 O) or D 2 O supplemented medium (10% FBS; 27% D 2 O) was added to the bottom of each well and a total of 4 × 10 4 cells resuspended in invasion buffer (0.5% FBS; 0.1% BSA) were seeded on top.…”