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

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.


Introduction
A majority of skin cancer-related deaths is caused by malignant melanoma, a tumor originating from neural crest-derived melanocytes, and the exploration of novel molecular strategies for improved detection and treatment of melanoma is of substantial clinical significance [1][2][3]. Oncometabolic adaptation is now recognized as a hallmark of melanomagenesis, and recent research has focused on metabolic rewiring involved in proliferative and metastatic dysregulation characteristic of BRAF V600E -driven and BRAF kinase inhibitor-resistant melanoma [4,5]. Glyoxalase 1 (encoded by GLO1; NM_006708) is a glutathione-dependent enzyme involved in the detoxification of the reactive glycolytic byproduct methylglyoxal (MG) based on formation of (R)-S-lactoyl-glutathione from MG and reduced glutathione [6,7]. Cumulative evidence supports a crucial role of GLO1 expression in maintaining oncometabolic adaptations as observed in the context of tumor-associated aerobic glycolysis, commonly referred to as 'the Warburg effect', facilitating survival under hypoxic conditions and enabling escape from energy crisis and apoptosis [8][9][10]. Substantiating a role of GLO1 in metabolic reprogramming, cumulative research has focused on the emerging role of MG [and (R)-S-lactoylglutathione] as cellular oncometabolites, involved in tumorigenesis-associated proliferative control, redox dysregulation, epigenetic recoding, and regulation of EMT, cellular functions that have been attributed to posttranslational MG-adduction of specific target proteins including histones [8,9,[11][12][13][14][15][16]. Importantly, numerous malignancies (including those of the breast, colon, liver, lung, prostate, skin, stomach, and thyroid) have now been associated with a causative role of GLO1 dysregulation, and beyond serving as a prognostic factor of patient survival, development of pharmacological and genetic strategies targeting cancer cells through GLO1 modulation has attracted significant attention [10,[16][17][18][19][20][21][22][23][24].
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].
Here, in order to examine the consequences of genomic deletion of GLO1 in human melanoma cells more comprehensively, we have performed NanoString™ gene expression profiling (using the nCounter™ 'PanCancer Progression' Panel) complemented by phenotypic analysis performed in vitro and in vivo. We report for the first time that genomic GLO1 deletion modulates TXNIP expression, glucose metabolism, and redox homeostasis while accelerating human A375 malignant melanoma tumor growth. 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. S1). Briefly, double strand breaks were generated on both sides of exon 2 (chromosome 6, positions: 38, 687, 313 bp; 38,685,738 bp) with guide CRISPR RNAs (5 ′ -ACCCTCATGGACCAATCAGT-3 ′ and 5 ′ -TGATCATAGGTGTATACGA G-3 ′ ). Parental cells were transfected with Cas9 protein, crRNAs, and trans-activating crRNA (Integrated DNA Technologies, San Diego, CA) using the Lipofectamine RNAiMAX reagent (Thermo Fisher Scientific, Waltham, MA). Next, single cells were deposited in 96-well plates and once single cell colonies expanded (after approximately three weeks), individual clones were screened by PCR. Clones that were negative for a sequence inside the targeted deletion and negative for the undeleted chromosomal sequences but positive for ligation-junction fragment were scored as potentially homozygous for GLO1 exon 2 deletion. Absence of GLO1 expression was confirmed by single RT-qPCR, immunoblot, and enzymatic activity assays. GLO1 rescue expression construct: CMV-driven GLO1 reexpression (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. After 24 h transfection, cells were passaged at 1:10 dilution into fresh growth medium. Selection antibiotic, neomycin (500 μg/ml), was added to the growth media 24 h later to select for single-cell colonies. Individual single-cell colonies were then expanded, and cells were tested for GLO1 mRNA and protein expression.

Chemicals
NanoString nCounter TM gene expression analysis: The Nano-String nCounter™ technology is a direct digital detection system enabling multiplexed quantification of gene expression (740 target plus 30 housekeeping genes) that occurs without amplification using fluorescent probes that bind directly to chosen mRNA targets [27,28]. First, total mRNA was prepared using RNeasy Mini kit (Qiagen, Valencia, CA) and then 100 ng used for NanoString nCounter™ analysis (using the 'PanCancer Progression Panel', NanoString Technologies, Seattle, WA) comparing gene expression between wildtype human A375 malignant melanoma cells and the isogenic GLO1_KO [B40] variant. Total mRNA was hybridized with the 'PanCancer Progression' code set at 65 • C overnight. Further purification and binding of the hybridized probes to the optical cartridge was performed on the nCounter™ Prep Station, and finally the cartridge was scanned on the nCounter™ Digital Analyzer. RCC files were then imported into nSolver4.0 software (NanoString Technologies) and checked for data quality using default QC settings; all samples passed data quality QC. All samples were normalized using the geometric mean of the housekeeper genes. Expression ratios were calculated by dividing the mean values of all samples in one experimental group (GLO1_KO) by the mean values of all samples in the reference group (GLO1_WT). For 'pathway score analysis' each sample's gene expression profile was then condensed into a small set of pathway scores using nCounter™ Advanced Analysis software (version 2.0.115). Pathway scores were fit using the first principal component of each gene set's data, oriented such that each pathway score has positive weights for at least half its genes. Numerical pathway score represents average fold expression change (log2 scale) for all genes associated with the specific pathway, with positive scores indicating enhancement and negative scores indicating attenuation. A 'covariate plot' displays selected pathway scores against the covariate chosen (i.e. GLO1 genotype).
Comparative human Oxidative Stress Plus RT 2 Profiler™ gene expression array analysis: Total mRNA from cultured cells (200,000 in 35 mm dish format) was prepared using the RNeasy Mini kit (Qiagen) following our published standard procedures [26,29]. Reverse transcription was then performed using the RT 2 First Strand kit (Qiagen) from 500 ng total RNA. For gene expression array analysis, the human Oxidative Stress Plus RT 2 Profiler™ technology (Qiagen) assessing expression of 84 redox regulatory genes was used as published before [26]. Quantitative PCR was run using the following conditions: 95 • C (10 min), followed by 40 cycles at 95 • C (15 s) alternating with 60 • C (1 min) (Applied Biosystems, Carlsbad, CA). Gene-specific products were normalized to a group of 5 housekeeping genes (ACTB, B2M, GAPDH, HPRT1, RPLP0) and quantified using the comparative ΔΔCt method (ABI Prism 7500 sequence detection system user guide).
MMP9 ELISA: Quantikine™ MMP9 ELISA (PDMP900; R&D Systems, Minneapolis, MN) was used to measure the 92 kDa Pro-and 82 kDa active forms in cell culture supernatants of parental A375 cells and their GLO1 isogenic variants. Briefly, supernatants were collected, and samples prepared by removing particulates by centrifugation. Then, 100 μL of standards, controls, and 100-fold diluted supernatant samples were added to 100 μL of assay diluent in MMP9 pre-coated wells followed by ELISA procedure as specified by the manufacturer. Colorimetric analysis (450 nm) was performed using a BioTek Synergy 2 Reader (BioTek, Winooski, VT). Results were normalized to total sample protein using the Pierce™ BCA Protein Assay Kit (Thermo Fisher Scientific, Waltham, MA, USA) [26].
Cell proliferation assay and population doubling time: Cells (1,000 per 35 mm dish) were seeded followed by culture in fresh growth medium (72 h). Viable cells (as determined by trypan blue exclusion assay) were counted (d0 and d3) using a Cell Viability Analyzer (Beckman Coulter, Fullerton, CA, USA). Population doubling time (DT) was then calculated as follows: DT = T x ln2/ln (X e /X b ) [T is the incubation time (72 h), X b is the cell number at the beginning of the incubation time, and X e is the cell number at the end of the incubation time] [26].
Cell cycle analysis: Cells were harvested by trypsinization, resus- M-phase quantification by phospho-histone H3 (Ser10) flow cytometry: 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)] using a rabbit derived Alexa-488 conjugated antibody (Cell Signaling, Inc., Danvers, MA, USA) according to the manufacture's protocol. p-H3(Ser10)-positive cells in Mphase were expressed in percent of total gated cells [32].
Cellular oxygen consumption and extracellular acidification rates: Oxygen consumption rate (OCR) and extracellular acidification rate (ECAR) were measured using the XF e 96 Analyzer (Agilent, Santa Clara, CA) according to manufacturer's instructions as published by us before [36]. Briefly, 30,000 cells per well were seeded in the XF tissue culture plate in normal growth media. The cartridge was hydrated by adding 200 μL of calibrant solution to each well and incubated overnight in a 37 • C non-CO 2 incubator. After cartridge calibration, the cell culture plate was processed followed by data collection. For OCR, cells were washed with XF Cell MitoStress Test Assay Media containing unbuffered DMEM (4.5 g/L glucose; 1 mM sodium pyruvate; pH 7.4) using the XF Prep Station, followed by incubation (1 h) in the prep-station non-CO 2 incubator. Port A of the cartridge was then loaded with 25 μL of the mitochondrial uncoupler FCCP (1 μM).
Luminescent ATP assay: Cells were seeded at 50,000 cells/dish on 35-mm dishes. After 24 h, cells were harvested and counted, and ATP content per 10,000 cells was determined using the CellTiter-Glo™ luminescent assay (Promega, Madison, WI, USA) in 96 well format according to the manufacturer's instructions. Data are normalized to ATP content in untreated cells [26,32].
Detection of intracellular oxidative stress: Induction of intracellular oxidative stress by MG was analyzed by flow cytometry using 2 ′ ,7 ′dichlorodihydrofluorescein diacetate (DCFH-DA) as a sensitive nonfluorescent precursor dye according to a published standard procedure [26,37]. Cells were treated with MG (500 μM, 6 h), followed by DCFH-DA loading. Cells were incubated for 60 min in the dark (37 • C, 5% CO 2 ) with culture medium containing DCFH-DA (5 μg/ml). Cells were then harvested and analyzed immediately by flow cytometry.
Determination of reduced cellular glutathione content: Intracellular reduced glutathione was measured using the GSH-Glo™ Glutathione assay kit (Promega; San Luis Obispo, CA) [26,30,37]. Cells were seeded at 100,000 cells/dish on 35 mm dishes. After 24 h, cells were harvested by trypsinization and then counted using a Coulter counter. Cells were washed in PBS, and 10,000 cells/well (50 μL) were transferred onto a 96-well plate. GSH-Glo reagent (50 μL) containing luciferin-NT and glutathione-S-transferase was then added followed by 30 min incubation. After addition of luciferin detection reagent to each well (100 μL) and 15 min incubation luminescence reading was performed using a BioTek Synergy 2 Reader (BioTek, Winooski, VT).
Transwell invasion assay: Matrigel-coated 8 μm pore size translucent 24-well plate transwell chambers (BD Biosciences, San Jose, CA) were used to evaluate the invasion potential of A375 cells following a published standard procedure [26]. Briefly, 600 μL of normal growth medium (10% FBS) was added to the bottom of each well and a total of 2.5 × 10 4 cells resuspended in 250 μL of invasion buffer (normal growth medium; 0.5% FBS; 0.1% BSA) were seeded on top. After 24 h incubation at 37 • C, 5% CO 2 , non-invading cells were removed by wiping the upper side of the membrane, and invading cells fixed with methanol and stained with crystal violet (Sigma-Aldrich, St. Louis, MO, USA). The number of invading cells was quantified by counting 10 random fields per filter at 400 × magnification.
Anchorage-independent growth on soft agar: Agar (3% in sterile water) was autoclaved, kept at 48 • C on a water bath and diluted using normal growth media (0.6% final concentration), and then poured on a 6-well plate and solidified at room temperature. After solidification of the bottom layer, a top layer was prepared (0.3% final agar concentration containing 20,000 cells). The plate was then incubated for 14 days in a cell culture incubator. During this period, cells were fed with normal growth media. At the end of the experiment, plates were fixed and stained with crystal violet (0.005%). Individual colonies (larger than 70 μm) were counted using light microscopy [38].
The mice (n = 5 per group) were obtained from the University of Arizona Cancer Center SCID house colony at the age of 9 weeks with an average weight of 20 g. At the end of the experiment (21 d post injection), lungs were evaluated for presence and number of metastases, and tissues fixed in NBF. This study was performed in accordance with the recommendations of the National Institutes of Health (University of Arizona Institutional Animal Care and Use Committee; mouse protocol number: IACUC 17-298) [26].
Human A375 melanoma SCID mouse xenograft model: A SCID mouse colony was developed at the University of Arizona using original SCID (C⋅B-17/IcrACCSCID) obtained from Taconic (Germantown, New York). The mice were housed in microisolator cages (Allentown Caging Equipment Company, Allentown, N.J.) and maintained under specific pathogen-free conditions. The mice received NIH-31 irradiated pellets (Tekland Premier, Madison, WI) and autoclaved water. Animal facilities are approved by the Association for the Assessment and Accreditation of Laboratory Animal Care International and in accordance with United States Department of Agriculture, Department of Health and Human Services, and NIH regulations. A375 melanoma cells (1 × 10 6 /100 μL HBSS) were injected subcutaneously (lower right flank) on day 0 (n = 10 per genotype). Tumor growth curves were generated by monitoring average tumor volumes (mm 3 ) until day 28 after cell injection followed by tumor collection. All procedures were completed in accordance with the University of Arizona Institutional Animal Care and Use Committee (IACUC) protocol (IACUC 17-298). Specific tumor growth rate (SGR) was determined between day 6 (last time point of equal average tumor growth between groups) and day 28 (end of experiment with final tumor measurement) as follows: SGR = ln (V 2 /V 1 )/(t 2 -t 1 ), where V 1 and V 2 are the tumor volumes at day 6 (t 1 ) and day 28 (t 2 ), respectively [39].
Statistical analysis: Unless stated differently, data sets were analyzed employing analysis of variance (ANOVA) with Tukey's posthoc test using the Prism 8.4.3 software (Prism Software Corp., Irvine, CA); in respective bar graphs (analyzing more than two groups), means without a common letter differ (p < 0.05). For bar graphs comparing two groups only, statistical significance was calculated employing the Student's two-tailed t-test, utilizing Excel (Microsoft™, Redmond, WA). The level of statistical significance was marked as follows: *p < 0.05. Experiments involved at least nine individual replicates per data point, except for gene expression array analysis performed with three independent biological replicates analyzed in triplicate format. For NanoString nCounter™ expression profiling, individual samples were run in triplicate format of biological replicates, and data analysis was performed using the nSolver analysis software (4.0). For p-value adjustment (Benjamini-Yekutieli False Discovery Rate; p value threshold: 0.05) nCounter™ Advanced Analysis software (version 2.0.115) was used. Nonparametric data analysis of murine experimentation was performed using the Mann-Whitney test. Differences between groups were considered significant at *p < 0.05.

Results
NanoString™ gene expression and cancer pathway analysis identifies pronounced TXNIP upregulation as a consequence of CRISPR/ Cas9-based GLO1 deletion in human A375 malignant melanoma cells.
Using nCounter™ Advanced Analysis software, 'pathway score' profiling identified a number of gene expression networks, characterized by differential expression patterns as a function of GLO1_KO status (Fig. 1c). Among these, four out of six networks were characterized by upregulation of TXNIP [encoding 'thioredoxin-interacting protein'; also known as VDUP1 (Vitamin D3 up-regulated protein 1)], representing the most pronounced gene expression change elicited by GLO1 deletion: (i) 'regulation of metabolism', (ii) 'cellular growth', (iii) 'cell cycle', and (iv) 'metastasis suppression' (Fig. 1d). In addition, two additional pathways ('EMT to metastasis' and 'metastasis response', attenuated in response to GLO1_KO) were identified by score analysis, an observation in concordance with our previously published study that documented impaired A375 melanoma cell invasion and metastasis observable upon GLO1 deletion [26].
Therefore, our further investigations focused on networks characterized by upregulation of TXNIP, representing the most pronounced gene expression change associated with GLO1 deletion as identified by NanoString nCounter™ analysis.
Genomic GLO1 deletion upregulates TXNIP expression in human DU145 prostate carcinoma and A375 melanoma cells associated with attenuation of glucose uptake and metabolism in GLO1_KO cells.
In addition to upregulation of TXNIP expression, NanoString nCounter™ analysis identified other gene expression changes contributing to 'regulation of metabolism' pathway score alteration in GLO1_KO melanoma cells, i.e. downregulation of LDHA (encoding lactate dehydrogenase A) and SLC2A1 (GLUT1 encoding glucose transporter 1) (Fig. 1c and d and 2a). TXNIP expression changes (mRNA and protein levels) were also confirmed by single RT-qPCR and immunoblot analyses (A375 GLO1_WT versus GLO1_KO clones [B40 and C2]; Fig. 2b and c).
Next, we tested if TXNIP upregulation observable in A375 GLO1_KO melanoma cells is also detectable in CRISPR/Cas9-engineered DU145 GLO1_KO prostate carcinoma cells characterized by absence of GLO1 expression confirmed at the mRNA and protein levels (Fig. 2d,e and supplemental Fig. S1). Indeed, pronounced upregulation of TXNIP expression occurred in DU145 GLO1_KO clones [A16 and A29] as measured at the mRNA and protein levels ( Fig. 2d and e). We also tested if two pharmacological interventions associated with a direct increase in cellular MG levels were able to mimic effects of GLO1_KO status on TXNIP expression in A375 cells (Fig. 2f). Strikingly, treatment with MG, the oncometabolite inactivated by GLO1 enzymatic turnover, as well as treatment with TLSC702, a pharmacological GLO1 inhibitor elevating cellular MG levels through blockade of MG detoxification, caused significant upregulation of TXNIP expression as detected by RT-qPCR [21,40]. Taken together, this evidence (obtained from genetic and pharmacological target modulation) suggests that TXNIP expression is responsive to GLO1 expression status and that GLO1 might control TXNIP through regulation of cellular MG levels.
Next, we explored the possibility that genetic elimination of GLO1 in A375 malignant melanoma cells might be associated with pronounced changes affecting glucose metabolism (Fig. 3a-g). To this end, we designed experiments based on the established involvement of GLO1 in glycolysis-associated MG-detoxification and the critical role of the tumor suppressor TXNIP as a key modulator of cellular glucose uptake and energy metabolism [41][42][43]. First, a limited number of glucose-related signature metabolites was profiled using quantitative UPLC-MRM/MS analysis of cultured human A375 cells compared to an isogenic GLO1_KO [B40] variant (Fig. 3a). Pronounced changes Cellular glucose uptake was then assessed employing a flow cytometric assay that measures uptake of the fluorescent glucose probe 2-NBDG (Fig. 3b). Consistent with the glucose metabolite profile (glucose-6-phosphate) and downregulation of SLC2A1 (GLUT1) described above [Figs. 2a and 3a], GLO1_KO status was associated with  Fig. 3b}. Strikingly, oxygen consumption rates (OCR) as determined by metabolic flux analysis were increased significantly in GLO1_KO cells (Fig. 3c). Specifically, under conditions of chemical uncoupling (using FCCP), maximum mitochondrial oxygen consumption was increased by almost 15% in KO as compared to WT cells, and a small yet significant increase of basal oxygen consumption by approximately 5% was detectable in KO cells. Concordantly, KO cells displayed an approximately 30% reduction in extracellular acidification rate (ECAR) as compared to the isogenic wildtype control (Fig. 3d). At the same time, cellular ATP levels remained unchanged in KO as compared to WT cells (Fig. 3e). These observations concerning glucose uptake and metabolite profile (Fig. 3a  For all bar graph depictions, quantitative data analysis employed ANOVA with Tukey's post hoc test; means without a common letter differ from each other (p < 0.05). For bar graphs comparing two groups only, statistical significance was calculated employing the Student's two-tailed t-test (*p < 0.05). and b), oxygen consumption (Fig. 3c), glycolytic acidification (Fig. 3d), and ATP levels (Fig. 3e) suggest that in GLO1_KO cells an attenuation of glucose utilization is counterbalanced by increased mitochondrial oxygen consumption maintaining ATP levels, an observation consistent with metabolic reprogramming, a hypothesis to be tested by more detailed mechanistic experiments.
Next, guided by the observation that glucose uptake and metabolism through HBP were attenuated in GLO1_KO cells ( Fig. 3a and b), expression of SLC2A1 together with GFAT1 and GFAT2 (encoding the HBP key enzymes glutamine-fructose-6-phosphate transaminase 1 and 2, respectively) was assessed by RT-qPCR (Fig. 3f). Indeed, downregulation of GFAT1, GFAT2, and SLC2A1 mRNA expression was observable in GLO1_KO cells [B40 and C2] (displaying the characteristic absence of detectable GLO1 with unchanged GLO2 mRNA levels). Immunoblot analysis confirmed this observation at the protein level (Fig. 3g).
Finally, since tumor glucose metabolism (as associated with high flux through glycolysis and the hexosamine pathway) has recently been shown to modulate expression of specific genes relevant to cancer cell immune evasion, we focused on expression of PDL1, an important endogenous immunosuppressive mediator expressed by melanoma cells (including cultured A375; Fig. 3f and g) [44][45][46][47][48]. Indeed, we observed that PDL1 expression (GLO1_WT versus GLO1_KO [B40 and C2]) was attenuated as a consequence of GLO1_KO status, both at the mRNA and protein levels, a remarkable observation given the importance of PD-L1 as a crucial target for clinically relevant melanoma immunotherapeutic intervention.
Genomic deletion of GLO1 alters oxidative stress response gene expression in human A375 malignant melanoma cells.
GLO1_KO clones were also profiled for alteration of redox stress response gene expression, an analysis guided by the established role of TXNIP as an antagonistic modulator of thioredoxin function and key regulator of cellular redox homeostasis [49,50]. To this end, since only a limited number of redox-directed genes (including TXNIP) was interrogated by our NanoString nCounter™ 'PanCancer Progression' Panel, comparative human Oxidative Stress Plus RT 2 Profiler™ gene expression array analysis was performed with GLO1_WT and GLO1_KO [B40 and C2] cells (Fig. 4a-d). Pronounced modulation of redox-related gene expression as a function of GLO1 deletion was detected, shown by volcano and heat map depiction (Fig. 4a-b). In both GLO1_KO clones, out of 84 genes, 17 displayed upregulation and 13 downregulation by more than two-fold (Fig. 4c). Between KO clones, gene-specific changes (relative to WT control) occurred with comparable magnitude, 12 of which are featured in bar graph format (Fig. 4d).

PRDX1, PRDX3).
Also, displaying consistent overexpression as a function of GLO1_KO status, pronounced upregulation of SRXN1 (encoding sulfiredoxin, an enzyme involved in reductive regeneration of hyper-oxidized peroxiredoxins) was observed (Fig. 4b-d). Likewise, in GLO1_KO clones, expression of numerous key antioxidant factors involved in glutathione metabolism (i.e. synthesis, reductive regeneration, and electrophilic detoxification) was upregulated, including GSS (encoding the glutathione biosynthesis enzyme glutathione synthetase), GSR (encoding glutathione reductase), and GSTZ1 (encoding the glutathione-dependent enzyme glutathione-S-transferase zeta) (Fig. 4b-d). Numerous other genes encoding a broad range of antioxidant enzymes was upregulated as a consequence of GLO1 deletion including CAT (encoding catalase), SOD3 (encoding superoxide dismutase 3), and HMOX1 (encoding heme oxygenase 1). Of note, our oxidative stress gene expression array analysis also revealed that GLO1_KO status was associated with significant upregulation of PTGS1 (encoding the pro-inflammatory enzyme cyclooxygenase-1).
Next, overall cellular oxidative stress was monitored by flow cytometric detection of 2 ′ ,7 ′ -dihydrodichlorofluorescein-diacetate (DHDCF) oxidation (Fig. 4e). Remarkably, no changes were observable between GLO1_WT and KO cells (not exposed to external electrophilic stressors), even though MG exposure upregulated ROS levels in KO cells to a higher extent than that observable in wildtype cells, an observation documented previously [26]. Consistently, detection of reduced cellular glutathione revealed no changes observable between untreated WT and KO clones, not challenged by exposure to electrophilic stressors (Fig. 4f). Additional evidence in support of a regulatory function of GLO1 expression in A375 melanoma cell redox homeostasis was derived from the use of a rescue clone generated by CMV-driven GLO1 re-expression in KO cells (A375-GLO1_R), an experimental tool used by us before [26] (supplemental Fig. S2). Our molecular analysis indicated that rescue expression of GLO1 [associated with restoration of GLO1 mRNA levels and GLO1 enzymatic activity (supplemental Fig. S2a,b)] reversed TXNIP mRNA upregulation (supplemental Fig. S2c). Moreover, rescue expression of GLO1 reversed MG-induced oxidative stress (supplemental Fig. S2d) and redox-related stress response gene expression (TXNRD2, GSTZ1, SRXN1, HMOX1; supplemental Fig. S2e), all of which are associated with GLO1 deletion in A375 melanoma cells.
Taken together, these data indicate that GLO1 deletion causes a pronounced alteration of redox regulatory gene expression in A375 melanoma cells characterized by upregulated expression of a broad array of specific redox regulatory factors [including TXNIP ( Fig. 2a and  b), TXNRD2, PRDX2, PRDX5, SRXN1, GSS, GSR, GSTZ1, CAT, SOD3 . For all bar graph depictions, quantitative data analysis employed ANOVA with Tukey's post hoc test; means without a common letter differ from each other (p < 0.05). For bar graphs comparing two groups only, statistical significance was calculated employing the Student's two-tailed t-test (*p < 0.05). . (For interpretation of the references to colour in this figure legend, the reader is referred to the Web version of this article.) ( Fig. 4)]. These changes might serve compensatory antioxidant functions maintaining redox homeostasis in the absence of GLO1.
Genomic GLO1 deletion shortens population doubling time with increased M-phase cell cycle population and anchorage-independent growth, while limiting A375 melanoma cell invasive potential and metastasis.
Next, guided by NanoString nCounter™ analysis we focused on expression changes consistent with the observed alteration of 'cell cycle regulation' and 'cellular growth' pathway scores observable upon GLO1 deletion (Fig. 1c and d and 5a). Remarkably, among the most pronounced gene expression changes identified by NanoString™ analysis, many are associated with dysregulated proliferation and cell cycle control during melanomagenesis, including upregulated: CD24 (9.6fold), IL1A (9.3-fold), CXCL8 (5.2-fold), FSTL1 (4.0-fold), RRAS (2.4fold); and downregulated: CDKN1A (2.4-fold) [32,[51][52][53][54][55]. For example, recent research indicates a crucial role of CD24 as a cancer stem-like cell marker and driver of anchorage-independent growth and tumorigenesis in cutaneous melanoma [55]. Likewise, upregulation of CXCL8 and IL1A expression has earlier been shown to be involved in increased melanoma cell proliferation, clonogenicity, and tumorigenesis, and an inverse relationship between expression of IL1A and CDKN1A has been established in a panel of cultured malignant melanoma cells (including A375 cells) [52,53]. Given the importance of CDKN1A encoding the cyclin-dependent kinase inhibitor p21 Cip1 , a crucial inhibitor of cell cycle progression, we further confirmed downregulation of CDKN1A expression [as already indicated by NanoString™ analysis (Figs. 1b-5a)] by RT-qPCR and immunoblot analysis (Fig. 5b).
Subsequent cell cycle analysis indicated that cells lacking GLO1 expression displayed a significant increase in S-and G2/M-phase subpopulations (by approximately 5%, each), concomitant with an approximately 10% decrease in the G1-phase subpopulation (Fig. 5c, top  panels and bottom, left panels). Further analysis based on flow cytometric detection of the M-phase marker phospho-histone H3 [pH3 (Ser10)] indicated an almost 40% increase in cells transiting through Mphase (out of the total G2/M population) (Fig. 5d). Importantly, phenotypic analysis revealed that A375 cells lacking For bar graphs comparing two groups only, statistical significance was calculated employing the Student's two-tailed t-test (*p < 0.05). Nonparametric data analysis of murine experimentation was performed using the Mann-Whitney test (*p < 0.05). (For interpretation of the references to colour in this figure legend, the reader is referred to the Web version of this article.) Fig. 5c, bottom, right panel), a finding consistent with the observed cell cycle alterations, shifting cells towards S-and M-phase (Fig. 5c). In addition, these cells displayed an increased capacity for anchorageindependent growth as confirmed by 3D soft agar colony formation assay documenting an up to 20-fold increase in colony number in GLO1_KO clones (Fig. 5e). Taken together, these data indicate that loss of GLO1 expression is associated with gene expression changes and phenotypic alterations consistent with shortened population doubling time, accelerated cell cycle progression, and increased anchorageindependent growth.
Finally, an invasion assay conducted together with ELISA-based determination of extracellular MMP9 levels largely reiterated our published observation that GLO1_KO status is associated with significant suppression of melanoma cell invasiveness and MMP9 production (Fig. 6c) [26]. Strikingly, a tail vain injection metastasis model using SCID mice indicated that genetic deletion of GLO1 significantly attenuates lung metastasis of A375 melanoma cells (Fig. 6d).
Genomic deletion of GLO1 accelerates tumor growth in a SCID mouse xenograft model of human A375 malignant melanoma.
Next, the impact of genetic GLO1 status on A375 melanoma tumorigenicity was examined in a relevant SCID mouse model assessing tumor growth after subcutaneous injection (28 d period; Fig. 7a Fig. 7a and b).
Next, comparative immunohistochemical analysis of tumor specimens was performed (Fig. 7b and c). First, differential GLO1 expression status was confirmed at the protein level, and expression of antigens related to proliferation and cell cycle control (p21, Ki67) was consistent with the observed growth rates. Likewise, differential expression of tumor antigens representing metabolic and redox control (TXNIP, SLC2A1, GFPT1) as well as immune evasion (PD-L1) was observed as a function of GLO1 genotype (Fig. 7c). Concordantly, RT-qPCR analysis of tumor-derived mRNA confirmed critical expression changes [including CDKN1A, TXNIP, SLC2A1, GFPT1, GFPT2, MMP9, PDL1] already observed at the cell culture level (Fig. 7d). Taken together, these observations obtained in a murine xenograft model suggest that genomic GLO1 deletion accelerates tumor growth in a SCID mouse xenograft model of human A375 malignant melanoma. . For all bar graph depictions, quantitative data analysis employed ANOVA with Tukey's post hoc test; means without a common letter differ from each other (p < 0.05). For bar graphs comparing two groups only, statistical significance was calculated employing the Student's two-tailed t-test (*p < 0.05). Nonparametric data analysis of murine experimentation was performed using the Mann-Whitney test (*p < 0.05).

Discussion
Cumulative evidence supports a role of GLO1 expression in tumorigenesis that may involve various mechanisms including alteration of cellular energy and redox homeostasis, defense against electrophilic carbonyl and chemotherapeutic stress, and epigenetic control of histone adduction and gene expression [8,9,[11][12][13][14][15][16]. Specifically, an oncometabolic function of the glycolytic byproduct MG, regulated by the glyoxalase detoxification system, has been substantiated by numerous lines of investigation, and a double-edged, hormetic role of MG, serving pro-proliferative and tumorigenic functions at low concentrations while displaying cytotoxic, anti-proliferative, and tumor-suppressive activities at higher concentrations, has been demonstrated [6,8,11,13,16,24,64].
Here, employing NanoString™-based expression profiling and pathway analysis followed by phenotypic characterization, we have generated novel experimental evidence suggesting that CRISPR/Cas9based deletion of GLO1 is accompanied by gene expression changes involved in (i) attenuation of melanoma cell glucose uptake and energy metabolism, (ii) alteration of redox homeostasis, (iii) acceleration of cell cycle progression and proliferation, (iv) impairment of invasiveness and metastasis, and (v) potentiation of tumor growth in a murine xenograft model.
Importantly, in human melanoma, TXNIP expression has been shown to inhibit melanoma cell invasiveness without causing an attenuation of proliferative capacity, and B16F10 melanoma cells transfected with TXNIP displayed decreased lung metastasis in C57BL/6 mice resulted in decreased, and TXNIP has now been identified as a miR-regulated metastasis suppressor predicting melanoma patient survival [65][66][67][68].
Given the important role of TXNIP as a negative regulator of melanomagenesis, it will be important to elucidate the specific molecular mechanism of GLO1-dependent TXNIP regulation as supported here for the first time by genetic and pharmacological evidence. In this context, it is tempting to speculate that modulation of cellular MG, now recognized as an important glycolysis-derived oncometabolite involved in various aspects of tumorigenesis, might determine TXNIP responsiveness to genomic GLO1 deletion. Indeed, it will be interesting to examine if control of TXNIP expression occurs downstream of the GLO1 metabolite MG, an established electrophilic signaling molecule impacting various molecular targets (including NRF2 and miRs) that are known to regulate TXNIP mRNA levels [6,66,[69][70][71]. Our observation that TXNIP upregulation occurred in response to GLO1 inhibition by TLSC702 and also in response to treatment with the GLO1 substrate MG supports this hypothesis (Fig. 2f). Moreover, TXNIP upregulation was also observable in DU145 prostate carcinoma cells with genomic GLO1 deletion (Fig. 2d  and e). Obviously, more detailed mechanistic studies (performed in a wider range of diverse melanoma cell lines) are required to explore molecular basis and causative role of GLO1 in the modulation of melanoma cell gene expression (including TXNIP) identified by NanoString nCounter™ analysis (Fig. 1).
Remarkably, we were able to demonstrate an attenuation of cellular glucose uptake as a consequence of GLO1 deletion (Fig. 3b), observable together with downregulation of SLC2A1 (Figs. 2a and 3f). In contrast, even though cellular ATP levels were not modulated as a function of GLO1 expression, we observed that OCR was increased in GLO1_KO cells with concomitant ECAR reduction as substantiated by Seahorse metabolism analysis, indicative of increased mitochondrial respiration compensating for reduced glucose uptake and glycolytic flux, a hypothesis to be tested by future experiments (Fig. 3c-e). Indeed, alteration of glucose metabolism in GLO1_KO cells was evidenced by depletion of key metabolites essential to glycolysis (Glc-6P) and the hexosamine biosynthesis pathway (HBP: GlcN-6P, GlcNAc-6P, UDP-GlcNAc), accompanied by an increase in PPP intermediates (R-5P, Ru-5P) (Fig. 3e). Consistent with a lowering of HBP-related glucose metabolites as result of GLO1 deletion, we also observed the downregulated expression of hexosamine pacemaker enzymes (encoded by GFAT1 and GFAT2; Fig. 3f and g). Interestingly, HBP inhibition using a small molecule pharmacological GFAT1-inhibitor has recently been identified as a promising cancer drug target blocking metastasis and PD-L1-mediated immune evasion through alteration of extracellular protein glycosylation downstream of UDP-GlcNAc depletion [47]. Future research will aim at elucidating the molecular mechanisms connecting GLO1 expression status, and modulation of cellular MG metabolism with TXNIP-dependent control of glucose metabolism (glycolysis, HBP, PPP), a pathway that might offer novel therapeutic approaches targeting oncometabolism. Indeed, we observed that PDL1 expression, associated with oncometabolic alterations in various malignancies including malignant melanoma, was suppressed in cultured GLO1_KO melanoma cells and tumors ( Fig. 3f and g and Fig. 7c and d), opening the possibility that PDL1 expression, crucial to tumor cell immune evasion, might be amenable to therapeutic modulation by genetic or pharmacological GLO1 antagonism [48].
Following our key observation of pronounced TXNIP upregulation as a result of GLO1 deletion (Figs. 1 and 2a-c), our subsequent redoxfocused gene expression array analysis revealed that upregulated expression of genes controlling glutathione, i.e. biosynthesis (SLC7A11, GSS), regeneration (GSR), and glutathione-dependent detoxification (GPX1, GPX4, GSTZ1, GSTP1) ( Fig. 4a-d). Moreover, as a consequence of GLO1 deletion, alteration of the thioredoxin-related antioxidant network (TXN, TXNRD1, TXNRD2, PRDX1, PRDX2, PRDX3, PRDX5, SRXN1) together with upregulated expression of other major antioxidant defense factors including catalase (CAT) and extracellular superoxide dismutase 3 (SOD3) were observed. These pronounced expression changes [together with detection of elevated levels of PPP intermediates (Fig. 3a)] might be indicative of an adaptational change in redox homeostasis as a result of GLO1 deletion and TXNIP upregulation, consistent with the observation that no significant alteration of baseline cellular oxidative stress and glutathione levels were detectable in cells unless challenged by external MG (Fig. 4e and f). Further evidence in support of GLO1 control of TXNIP and redox response gene expression was obtained from the observation that rescue expression of GLO1 reversed TXNIP upregulation, redox-related stress response gene expression (TXNRD2, GSTZ1, SRXN1, HMOX1), and MG-induced oxidative stress, all of which are associated with GLO1 deletion in A375 melanoma cells (supplemental Fig. S2).
Guided by NanoString™ pathway score analysis, we also focused on the phenotypic assessment of proliferation, cell cycle progression, and clonogenicity as a function of GLO1_KO status (Fig. 5). Indeed, our cell cycle and proliferation analysis indicated an increase in cell populations in S-and M-phase, a decrease in population doubling time, and an increase in anchorage-independent growth/clonogenicity. These phenotypic characteristics are consistent with gene expression changes observable in GLO1_KO cells (including CD24, CXCL8, IL1A), all of which have been shown before to upregulate melanoma cell proliferative capacity, anchorage-independent growth, and tumorigenicity [32,52,53,55]. Likewise, NanoString™ pathway score analysis indicated downregulation of CDKN1A [encoding p21 (CIP1/WAF1), the cyclin dependent kinase inhibitor and suppressor of cell cycle progression], confirmed by us independently by RT-qPCR and immunoblot analysis.
In striking contrast to impaired metastatic potential and consistent with increased proliferative capacity and cell cycle alterations observed in vitro (Fig. 5), tumor growth rate of GLO1_KO as compared to wildtype cells was accelerated significantly (Fig. 7). Interestingly, immunohistochemical analysis confirmed the sustained maintenance of key expression changes associated with GLO1_KO status (upregulated: TXNIP; downregulated: GLUT1, GFAT1, GFAT2, CDKN1A) together with upregulation of the proliferation marker Ki-67. Also, tumor MMP9 expression was downregulated, an observation consistent with inhibition of EMT and metastatic potential, as documented and discussed before [22,26]. Strikingly, as already observed in cell culture ( Fig. 3f and g), downregulation of PDL1 expression was maintained in tumors lacking GLO1 expression, an observation confirmed at the mRNA and protein levels ( Fig. 7c and d).
Taken together, these data indicate that GLO1 deletion from A375 melanoma cells is associated with pronounced acceleration of in vivo tumorigenicity that, paradoxically, occurs with attenuation of metastatic potential. This striking phenotype is consistent with our Nano-String nCounter™ gene expression profiling revealing alterations that impact energy metabolism, redox homeostasis, proliferative and cell cycle control, and EMT/metastatic potential. Interestingly, the development of metastatic melanoma has been shown to require the dynamic shift of malignant cells between proliferative and invasive phenotypes, and it is therefore possible that decreased metastatic potential downstream of GLO1 elimination is associated with increased proliferative capacity as observed by us (Figs. 5-7) [73][74][75]. Specifically, cumulative evidence suggests that melanoma aggressiveness originates from an intrinsic plasticity referred to as 'phenotype switching' driving tumor progression, allowing the dynamic, reversible, and seemingly paradoxical transformation of malignant cells between a hyper-proliferative/hypo-invasive to a hypo-prolifera tive/hyper-invasive stage [73][74][75]. Thus, it might be hypothesized that this molecular switch is amenable to modulation by GLO1 elimination or pharmacological intervention as observed here for the first time. In this context, it also seems worth mentioning that our in vivo experiments were conducted in immunosuppressed SCID mice, producing seemingly opposing tumor-relevant outcomes, i.e. (i) attenuated lung metastasis and (ii) accelerated tumor growth (Figs. 6 and 7, respectively). Thus, in order to reconcile these findings and to define the potential therapeutic value of GLO1 antagonism, our ongoing experiments aim at examining the possibility that GLO1-inhibition (potentially associated with enhanced tumorigenic potential) might be counterbalanced by increased tumor immunogenicity (downstream of downregulated PDL1 expression) and attenuation of metastasis in immunocompetent host systems.
Future research has to elucidate the specific molecular basis underlying GLO1 control of melanoma cell tumorigenicity and invasiveness observed here in A375 cells, using a more comprehensive panel of melanoma cells representative of the human disease and elucidating the mechanistic involvement of novel targets shown here for the first time to be responsive to genetic GLO1 modulation (including TXNIP, GFAT1, and PDL1). Importantly, given the documented efficacy of small molecule pharmacological inhibitors, our observations suggest feasibility of therapeutic intervention targeting GLO1 control of glucose utilization, redox homeostasis, cell proliferation, metastasis, and immune evasion, a hypothesis to be explored by future experimentation.

Declaration of competing interest
All authors declare that there are no conflicts of interest to disclose.