Genetic Target Modulation Employing CRISPR/Cas9 Identifies Glyoxalase 1 as a Novel Molecular Determinant of Invasion and Metastasis in A375 Human Malignant Melanoma Cells In Vitro and In Vivo

摘要

Metabolic reprogramming is a molecular hallmark of cancer. Recently, we have reported the overexpression of glyoxalase 1 (encoded by ), 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 in melanomagenesis remains largely unexplored. Here, using genetic target modulation, we report the identification of as a novel molecular determinant of invasion and metastasis in malignant melanoma. First, A375 human malignant melanoma cells with deletion (A375- _KO) were engineered using CRISPR/Cas9, and genetic rescue clones were generated by stable transfection of KO clones employing a CMV-driven construct (A375- _R). After confirming target modulation at the mRNA and protein levels (RT-qPCR, immunodetection, enzymatic activity), phenotypic characterization indicated that deletion of does not impact proliferative capacity while causing significant sensitization to methylglyoxal-, chemotherapy-, and starvation-induced cytotoxic stress. Employing differential gene expression array analysis (A375- _KO versus A375- _WT), pronounced modulation of epithelial mesenchymal transition (EMT)-related genes [upregulated: , , ; (downregulated): and (up to tenfold; < 0.05)] was observed—all of which are consistent with EMT suppression as a result of deletion. Importantly, these expression changes were largely reversed upon genetic rescue employing A375- _R cells. Differential expression of as a function of 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- _KO cells, reversed again in genetic rescue clones. Strikingly, in a SCID mouse metastasis model, lung tumor burden imposed by A375- _KO cells was strongly attenuated as compared to A375- _WT cells. Taken together, these prototype data provide evidence in support of a novel function of in melanoma cell invasiveness and metastasis, and ongoing investigations explore the function and therapeutic potential of as a novel melanoma target.