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

Abstract

Metabolic reprogramming is a molecular hallmark of cancer. Recently, we have reported the overexpression of glyoxalase 1 (encoded by GLO1), 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 GLO1 in melanomagenesis remains largely unexplored. Here, using genetic target modulation, we report the identification of GLO1 as a novel molecular determinant of invasion and metastasis in malignant melanoma. First, A375 human malignant melanoma cells with GLO1 deletion (A375-GLO1_KO) were engineered using CRISPR/Cas9, and genetic rescue clones were generated by stable transfection of KO clones employing a CMV-driven GLO1 construct (A375-GLO1_R). After confirming GLO1 target modulation at the mRNA and protein levels (RT-qPCR, immunodetection, enzymatic activity), phenotypic characterization indicated that deletion of GLO1 does not impact proliferative capacity while causing significant sensitization to methylglyoxal-, chemotherapy-, and starvation-induced cytotoxic stress. Employing differential gene expression array analysis (A375-GLO1_KO versus A375-GLO1_WT), pronounced modulation of epithelial mesenchymal transition (EMT)-related genes [upregulated: CDH1, OCLN, IL1RN, PDGFRB, SNAI3; (downregulated): BMP1, CDH2, CTNNB1, FN1, FTH1, FZD7, MELTF, MMP2, MMP9, MYC, PTGS2, SNAI2, TFRC, TWIST1, VIM, WNT5A, ZEB1, and ZEB2 (up to tenfold; p < 0.05)] was observed—all of which are consistent with EMT suppression as a result of GLO1 deletion. Importantly, these expression changes were largely reversed upon genetic rescue employing A375-GLO1_R cells. Differential expression of MMP9 as a function of GLO1 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-GLO1_KO cells, reversed again in genetic rescue clones. Strikingly, in a SCID mouse metastasis model, lung tumor burden imposed by A375-GLO1_KO cells was strongly attenuated as compared to A375-GLO1_WT cells. Taken together, these prototype data provide evidence in support of a novel function of GLO1 in melanoma cell invasiveness and metastasis, and ongoing investigations explore the function and therapeutic potential of GLO1 as a novel melanoma target.

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

“…Previously, using cell culture models (comparing primary melanocytes and malignant melanoma lines) as well as patient samples in tissue microarray format, we have documented that GLO1 is overexpressed during melanoma progression [ 25 ]. Moreover, employing CRISPR/Cas 9-based GLO1 deletion and rescue expression, we have documented a novel role of GLO1 as a molecular determinant of invasion and metastasis observable in experimental human malignant melanoma in vitro and in vivo , a finding consistent with independent evidence indicating GLO1 -control of prostate carcinoma cell EMT and metastatic behavior [ 22 , 26 ].…”

“…CRISPR/Cas9-based engineering of GLO1_ KO A375 malignant melanoma and GLO1_ KO DU145 prostate carcinoma cells: Homozygous GLO1 gene knock-out in human malignant A375 melanoma cells was performed using genetic engineering as published before [ 26 ]. Likewise, DU145 prostate carcinoma GLO1 _KO cells were engineered using a similar approach (supplemental data; Fig.…”

“…GLO1 rescue expression construct: CMV-driven GLO1 re-expression (A375- GLO1 _R) in a KO clone (A375 GLO1 _KO [B40]) was performed and validated following our previously published methodology [ 26 ]. Briefly, stable A375- GLO1 rescue cells (A375- GLO1 _R) were generated by overnight incubation of A375- GLO1 _KO cells with DNA (4 μg)-Lipofectamine® 2000 (10 μg) complexes at 37 °C.…”

Glyoxalase System in the Progression of Skin Aging and Skin Malignancies

“…As an adaptive response to elevated MGO stress, cancer cells are known to overexpress glyoxalase enzymes. Glo1 is also overexpressed in both melanoma [ 38 , 39 , 40 ] and non-melanoma cells [ 41 ]. As glyoxalase activity is increased in tumor cells, glyoxalase has become a potential therapeutic target in tumorigenesis.…”

“…Similarly, Jandova et al also reported that silencing of Glo1 inhibits A375 melanoma cell migration and invasion by modulating epithelial mesenchymal transition-related genes, and this expression was reversed by the re-expression of Glo1 in Glo1 knockout A375 cells. In addition, the tumor migration in SCID (Severe combined immunodeficient) mice induced by A375-Glo1 knockout cells was relatively lower than that in wild-type A375 cells [ 39 ]. These studies strongly suggest that Glo1 plays a major role in melanoma tumor invasion and migration.…”