| Summary: | Metabolic reprogramming is a molecular hallmark of cancer. Recently, we have reported the overexpression of glyoxalase 1 (encoded by <i>GLO1</i>), a glutathione-dependent enzyme involved in detoxification of the reactive glycolytic byproduct methylglyoxal, in human malignant melanoma cell culture models and clinical samples. However, the specific role of <i>GLO1</i> in melanomagenesis remains largely unexplored. Here, using genetic target modulation, we report the identification of <i>GLO1</i> as a novel molecular determinant of invasion and metastasis in malignant melanoma. First, A375 human malignant melanoma cells with <i>GLO1 </i>deletion (A375-<i>GLO1</i>_KO) were engineered using CRISPR/Cas9, and genetic rescue clones were generated by stable transfection of KO clones employing a CMV-driven <i>GLO1</i> construct (A375-<i>GLO1</i>_R). After confirming <i>GLO1</i> target modulation at the mRNA and protein levels (RT-qPCR, immunodetection, enzymatic activity), phenotypic characterization indicated that deletion of <i>GLO1</i> does not impact proliferative capacity while causing significant sensitization to methylglyoxal-, chemotherapy-, and starvation-induced cytotoxic stress. Employing differential gene expression array analysis (A375-<i>GLO1</i>_KO versus A375-<i>GLO1</i>_WT), pronounced modulation of epithelial–mesenchymal transition (EMT)-related genes [upregulated: <i>CDH1</i>, <i>OCLN</i>, <i>IL1RN, PDGFRB, SNAI3</i>; (downregulated): <i>BMP1, CDH2, CTNNB1, FN1, FTH1, FZD7, MELTF, MMP2, MMP9, MYC, PTGS2, SNAI2, TFRC, TWIST1, VIM, WNT5A, ZEB1, </i>and <i>ZEB2</i> (up to tenfold; <i>p</i> < 0.05)] was observed—all of which are consistent with EMT suppression as a result of <i>GLO1</i> deletion. Importantly, these expression changes were largely reversed upon genetic rescue employing A375-<i>GLO1</i>_R cells. Differential expression of <i>MMP9</i> as a function of <i>GLO1</i> status was further substantiated by enzymatic activity and ELISA analysis; phenotypic assessment revealed the pronounced attenuation of morphological potential, transwell migration, and matrigel 3D-invasion capacity displayed by A375-<i>GLO1</i>_KO cells, reversed again in genetic rescue clones. Strikingly, in a SCID mouse metastasis model, lung tumor burden imposed by A375-<i>GLO1</i>_KO cells was strongly attenuated as compared to A375-<i>GLO1</i>_WT cells. Taken together, these prototype data provide evidence in support of a novel function of <i>GLO1</i> in melanoma cell invasiveness and metastasis, and ongoing investigations explore the function and therapeutic potential of <i>GLO1</i> as a novel melanoma target.
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