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 opposing ways as a consequence of GLO1 elimination.

乙二醛酶 1（由GLO1编码）是一种谷胱甘肽依赖性酶，可对糖酵解副产物甲基乙二醛 (MG) 进行解毒，MG 是一种参与代谢重编程的癌代谢物。最近，我们已经证明GLO1在人类恶性黑色素瘤细胞和患者肿瘤中过表达，并证实了GLO1作为黑色素瘤侵袭和转移的分子决定因素的新作用。在这里，使用 NanoString™ 基因表达谱（nCounter™ 'PanCancer Progression Panel'），我们报告了人类 A375 恶性黑色素瘤细胞中基于 CRISPR/Cas 9 的GLO1缺失改变了葡萄糖代谢和氧化还原稳态，同时加速了肿瘤发生。Nanostring™ 分析确定了 TXNIP（编码硫氧还蛋白相互作用蛋白），细胞能量代谢和氧化还原稳态的主要调节剂，显示响应GLO1消除的最明显的表达变化，通过 RT-qPCR 和免疫印迹分析证实。TXNIP在缺乏GLO1的 CRISPR/Cas9 工程化的 DU145 前列腺癌细胞中也上调，并且用 MG 或药理学 GLO1 抑制剂 (TLSC702) 处理模拟 GLO1_KO状态，表明GLO1通过调节 MG控制TXNIP表达。GLO1 _KO 状态的特征是 ( i ) 氧化应激反应基因表达改变，( ii）通过下调基因表达（GLUT1、GFAT1、GFAT2、LDHA）和相关关键代谢物（6-磷酸葡萄糖、UDP-N-乙酰氨基葡萄糖）的减少和 ( iii ) 免疫检查点调节 ( PDL1 ) 减弱葡萄糖摄取和代谢）。虽然证实了我们之前的发现，即GLO1缺失通过调节 EMT 相关基因（例如TGFBI、MMP9、ANGPTL4、TLR4、SERPINF1）来限制侵袭和转移，但我们观察到GLO1_KO黑色素瘤细胞的群体倍增时间缩短，细胞周期随着 M 期群体的增加而改变，并且不依赖于贴壁的生长增强，这是表达分析支持的表型（CXCL8、CD24、IL1A、CDKN1A）。一致地，在 SCID 小鼠黑色素瘤异种移植模型中可以观察到GLO1_KO肿瘤的加速生长速度，伴随着TXNIP过表达和代谢重编程，这表明 A375 黑色素瘤肿瘤的生长和转移可能因GLO1消除而以相反的方式失调。