International Journal of Molecular Sciences

Article Metastatic Melanoma Progression Is Associated with Endothelial Nitric Oxide Synthase Uncoupling Induced by Loss of eNOS:BH4 Stoichiometry

Fabiana Henriques Machado de Melo 1,2,*, Diego Assis Gonçalves 3,4 , Ricardo Xisto de Sousa 5, Marcelo Yudi Icimoto 6 , Denise de Castro Fernandes 7, Francisco R. M. Laurindo 7 and Miriam Galvonas Jasiulionis 1,*

1 Pharmacology Department, Universidade Federal de São Paulo, São Paulo 05508-090, Brazil 2 Department of Pharmacology, Institute of Biomedical Science, University of São Paulo, São Paulo 05508-060, Brazil 3 Micro-Imuno-Parasitology Department, Universidade Federal de São Paulo, São Paulo 05508-090, Brazil; [email protected] 4 Parasitology Department, Microbiology and Immunology, Federal University of Juiz de Fora, Juiz de Fora 36036-900, Brazil 5 Department of Physiological Sciences, Santa Casa de São Paulo School of Medical Sciences, São Paulo 01221-020, Brazil; [email protected] 6 Biophysics Department, Universidade Federal de São Paulo, São Paulo 05508-090, Brazil; [email protected] 7 Vascular Biology Laboratory, Heart Institute (InCor), University of São Paulo School of Medicine,


São Paulo 05508-060, Brazil; [email protected] (D.d.C.F.); [email protected] (F.R.M.L.)
 * Correspondence: [email protected] or [email protected] (F.H.M.d.M.); [email protected] or [email protected] (M.G.J.) Citation: Melo, F.H.M.d.; Gonçalves, D.A.; Sousa, R.X.d.; Icimoto, M.Y.; Fernandes, D.d.C.; Laurindo, F.R.M.; Abstract: Melanoma is the most aggressive type of skin cancer due to its high capability of developing Jasiulionis, M.G. Metastatic metastasis and acquiring chemoresistance. Altered redox homeostasis induced by increased reactive Melanoma Progression Is Associated oxygen species is associated with melanomagenesis through modulation of redox signaling pathways. −• with Endothelial Nitric Oxide Dysfunctional endothelial nitric oxide synthase (eNOS) produces superoxide anion (O2 ) and Synthase Uncoupling Induced by contributes to the establishment of a pro-oxidant environment in melanoma. Although decreased Loss of eNOS:BH4 Stoichiometry. Int. tetrahydrobiopterin (BH4) bioavailability is associated with eNOS uncoupling in endothelial and J. Mol. Sci. 2021, 22, 9556. https:// human melanoma cells, in the present work we show that eNOS uncoupling in metastatic melanoma doi.org/10.3390/ijms22179556 cells expressing the genes from de novo biopterin synthesis pathway Gch1, Pts, and Spr, and high BH4 concentration and BH4:BH2 ratio. Western blot analysis showed increased expression of Nos3, Academic Editor: Lucie Germain altering the stoichiometry balance between eNOS and BH4, contributing to NOS uncoupling. Both treatment with L-sepiapterin and eNOS downregulation induced increased nitric oxide (NO) and Received: 28 June 2021 • Accepted: 17 August 2021 decreased O2 levels, triggering NOS coupling and reducing cell growth and resistance to anoikis Published: 3 September 2021 and dacarbazine chemotherapy. Moreover, restoration of eNOS activity impaired tumor growth in vivo. Finally, NOS3 expression was found to be increased in human metastatic melanoma samples

Publisher’s Note: MDPI stays neutral compared with the primary site. eNOS dysfunction may be an important mechanism supporting with regard to jurisdictional claims in metastatic melanoma growth and hence a potential target for therapy. published maps and institutional affil- iations. Keywords: metastatic melanoma; eNOS uncoupling; tetrahydrobiopterin; L-sepiapterin; eNOS:BH4 stoichiometry

Copyright: © 2021 by the authors. Licensee MDPI, Basel, Switzerland. 1. Introduction This article is an open access article Cutaneous melanoma originates from melanocytes, neural-crest-derived pigment- distributed under the terms and producing cells located in the epidermis. Even though the development of targeted therapy conditions of the Creative Commons and immunotherapy has improved the overall survival of melanoma patients, its incidence Attribution (CC BY) license (https:// continues to rise around the world, caused by increased life expectancy and ultraviolet creativecommons.org/licenses/by/ radiation exposure [1–4]. Although melanoma occurrence is low compared to other types 4.0/).

Int. J. Mol. Sci. 2021, 22, 9556. https://doi.org/10.3390/ijms22179556 https://www.mdpi.com/journal/ijms Int. J. Mol. Sci. 2021, 22, 9556 2 of 20

of cutaneous malignancies, it is responsible for approximately 75% of all skin-cancer-related deaths, which is associated with its chemoresistance to available therapeutic approaches and high ability to metastasize. Different mechanisms associated with melanoma pathogenesis have already been described. Data from our group and others have shown that the establishment of a pro- oxidant intracellular milieu contributes to melanocyte malignant transformation through modulation of signaling pathways that favor proliferation and cell survival, and inhibit apoptosis, as was shown for other skin cancers [5–9]. The contribution of DNA and protein damage in the carcinogenesis induced by UV-derived ROS is also well established in non-melanoma skin cancers [10,11]. It was shown that the activation of RAS/RAC1/ERK signaling pathway in melanocytes submitted to a chronic stress condition is associated with oncogenesis, through regulation of global DNA methylation and DNMT1 expression. Moreover, melanocyte malignant transformation was severely impaired in the presence of −• a superoxide anion (O2 ) scavenger, showing the involvement of altered redox activity in melanomagenesis [7]. Dysfunctional mitochondria and increased activity of NADPH oxidase contribute to the disturbance of redox homeostasis in melanoma cells by raising reactive oxygen species (ROS) levels [12–14]. The involvement of nitric oxide synthases (NOSs) in redox landscape alteration has also been described in tumor cells [6,9]. NOSs are homodimeric enzymes that catalyze nitric oxide (NO) synthesis from L- arginine and molecular oxygen. The NOS reductase domain has homology with the cytochrome P450 enzyme, generating electrons flowing from the NADPH through the flavin domains FAD and FMN, and then transferred to the heme group of the oxidase domain of the other monomer, where L-arginine oxidation occurs. Integrated mechanisms, including activation by calcium–calmodulin, post-translational modifications as phosphorylation and glutathionylation, and substrate and cofactor availability, regulate NOS activity [15–17]. A critical aspect of NOS function is the bioavailability of the tetrahydrobiopterin cofac- tor BH4. BH4 is an unstable molecule that is easily oxidated to 7-8-dihydrobiopterin (BH2), which binds to the active enzyme site with the same affinity and is not able to catalyze NO formation. Consequently, reduction in BH4 concentration or BH4:BH2 ratio results in enzyme destabilization and dimer conformation disruption, inducing NOS dysfunction and reducing NO production. This state is referred to as uncoupling because NADPH oxidation and reduction of molecular oxygen are uncoupled from arginine hydroxylation and NO formation. However, the electron transfer from NADPH through the flavins −• domains to molecular oxygen is not inhibited, resulting in the generation of O2 [18,19]. Moreover, it was shown that in endothelial cells, not only the levels of BH4 and BH2, but also eNOS:BH4 stoichiometry determines eNOS function [20,21]. Increased expression of the three isoforms of NOS has been described in numerous tumor cells and has been related to carcinogenesis [9,22–24]. In melanoma, increased NOS2 expression was correlated with worse prognosis and poor survival [25,26]. Other authors have demonstrated elevated expression of eNOS and nNOS [22,27] in melanoma tissues. However, these studies do not always address if the enzyme is functional and if it −• is producing NO or O2 . −• Previous data from our group suggested that eNOS uncoupling is a source of O2 in melanocytes submitted to anchorage blockade, since L-NAME, an eNOS inhibitor, and −• L-sepiapterin, a BH4 precursor, decreased O2 and increased NO levels [5,6]. The involve- ment of eNOS uncoupling in melanocyte malignant transformation was also shown, since L-NAME abrogated melanocyte transformation induced by sustained stress conditions. −• Besides that, inhibition of O2 production by L-sepiapterin treatment rendered metastatic melanoma cells less resistant to anoikis and more sensitive to carboplatin chemotherapy [6], showing the participation of NOS uncoupling in capabilities acquired during melanoma progression. To investigate the contribution of uncoupling NOS and the mechanisms associated with its dysfunction in melanoma development, a murine melanoma model, comprising non-tumorigenic melan-a melanocytes, pre-malignant 4C melanocytes, non-metastatic Int. J. Mol. Sci. 2021, 22, x FOR PEER REVIEW 3 of 20

carboplatin chemotherapy [6], showing the participation of NOS uncoupling in capabilities acquired during melanoma progression. To investigate the contribution of uncoupling NOS and the mechanisms associated Int. J. Mol. Sci. 2021, 22, 9556 3 of 20 with its dysfunction in melanoma development, a murine melanoma model, comprising non‐tumorigenic melan‐a melanocytes, pre‐malignant 4C melanocytes, non‐metastatic 4C11− melanoma cells, and metastatic 4C11+ melanoma cells were used [28]. Based on the results4C11− heremelanoma presented, cells, we and suggested metastatic that 4C11+ altered melanoma intracellular cells were eNOS:BH4 used [28 stoichiometry]. Based on the is aresults key determinant here presented, of eNOS we suggested uncoupling that in altered metastatic intracellular melanoma eNOS:BH4 cells and stoichiometry is associated withis a key melanoma determinant aggressiveness. of eNOS uncoupling in metastatic melanoma cells and is associated with melanoma aggressiveness. 2. Results 2.1.2. Results Increased Expression of De Novo Tetrahydrobiopterin Biosynthesis Enzymes during Melanoma2.1. Increased Progression Expression of de Novo Tetrahydrobiopterin Biosynthesis Enzymes during Melanoma Progression Our group has previously showed decreased NO and increased O2−• levels in cell Our group has previously showed decreased NO and increased O −• levels in cell lines lines corresponding to distinct stages of melanoma progression. In metastatic2 melanoma corresponding to distinct stages of melanoma progression. In metastatic melanoma cells, L- cells, L‐sepiapterin treatment reversed this pattern, inducing NO production and−• sepiapterin treatment reversed this pattern, inducing NO production and suppressing O2 , suppressing O2−•, suggesting BH4 restoration and NOS coupling [6]. Intracellular BH4 suggesting BH4 restoration and NOS coupling [6]. Intracellular BH4 amount is mainly con- amount is mainly controlled by the activity of the de novo pathway comprising the trolled by the activity of the de novo pathway comprising the enzymes GTP cyclohydrolase enzymes GTP cyclohydrolase 1 (GTPCH1), the rate‐limiting enzyme in BH4 biosynthesis; 1 (GTPCH1), the rate-limiting enzyme in BH4 biosynthesis; 6-pyruvoyltetrahydrobiopterin 6synthase‐pyruvoyltetrahydrobiopterin (PTPS); and sepiapterin synthase reductase (PTPS); (SR) and [19]. sepiapterin To elucidate reductase the mechanisms (SR) [19]. un-To elucidatederlying NOSthe mechanisms uncoupling, underlying the expression NOS of uncoupling, the genes encoding the expression these three of the enzymes genes wasencoding analyzed these by three real-time enzymes qPCR was in melan-aanalyzed melanocytes, by real‐time 4CqPCR pre-malignant in melan‐a melanocytes, 4C4C11 pre− non-metastatic‐malignant melanocytes, melanoma cells,4C11 and− non 4C11+‐metastatic metastatic melanoma melanoma cells, cells. Surprisingly,and 4C11+ metastaticthe expression melanoma of Gch1 cells.was Surprisingly, found to be increased the expression in 4C11 of −Gch1and was 4C11+ found tumorigenic to be increased cells whenin 4C11 compared− and 4C11+ with tumorigenic melan-a melanocytes cells when and compared 4C pre-malignant with melan melanocytes‐a melanocytes(Figure and1A), 4C whilepre‐malignantPts and SprmelanocytesmRNA levels (Figure were 1A), augmented while Pts and only Spr in mRNA the metastatic levels were 4C11+ augmented cell line (Figureonly in1 theB,C). metastatic In endothelial 4C11+ cells cell andline hepatocytes,(Figure 1B,C). it wasIn endothelial demonstrated cells that and Gch1hepatocytes,activity, itand was consequently demonstrated BH4 that synthesis, Gch1 activity, is allosterically and consequently regulated BH4 by the synthesis, enzyme is GTP allosterically feedback regulatedregulator, GFRP.by the Interestingly,enzyme GTP Gchfrfeedbackexpression regulator, was GFRP. found Interestingly, increased in 4C Gchfr pre-malignant expression wasmelanocytes found increased and 4C11 in− melanoma4C pre‐malignant cells; however, melanocytes it is reducedand 4C11 in− 4C11+melanoma metastatic cells; however,melanoma it cellsis reduced when comparedin 4C11+ metastatic with melan-a melanoma melanocytes cells when (Figure compared1D). Data with sets melan from‐a TCGAmelanocytes showed (Figure increased 1D). DataGCH1 setsexpression from TCGA along showed with increased melanoma GCH1 progression expression (n = along 248) (Figurewith melanoma S3A) and progression decreased expression (n = 248) (Figure of GCHFR S3A)in and melanoma decreased (n =expression 461) compared of GCHFR with innormal melanoma skin (n (n = = 558) 461) (Figure compared S3D) with [29]. normal In a study skin published (n = 558) (Figure by Talantov S3D) and[29]. colleagues, In a study publishedhigh PTS (Figure by Talantov S3B) and andSPR colleagues,levels (Figure high PTS S3C) (Figure were observed S3B) and inSPR cutaneous levels (Figure melanoma S3C) (nwere = 45) observed compared in cutaneous with benign melanoma nevi (n = 18),(n = corroborating 45) compared the with data benign found nevi in our (n melanoma = 18), cor‐ roboratingmodel [30] the (GSE3189). data found in our melanoma model [30] (GSE3189).

Figure 1. Cont.

Int. J. Mol. Sci. 2021Int. J., 22 Mol., x FOR Sci. 2021 PEER, 22 REVIEW, 9556 4 of 20 4 of 20

Figure 1. ExpressionFigure 1. ofExpression de novo tetrahydrobiopterin of de novo tetrahydrobiopterin synthesis pathway synthesis along pathway with melanoma along with pro melanoma‐ pro- gression. Relativegression. mRNA Relative levels of mRNA the enzymes levels of from the de enzymes novo tetrahydrobiopterin from de novo tetrahydrobiopterin via (A) Gch1, (B) via (A) Gch1, Pts, (C) Spr, and(B) (PtsD) ,(GfrpC) wereSpr, anddetermined (D) Gfrp inwere melan determined‐a melanocytes in melan-a (Ma), 4C melanocytes pre‐malignant (Ma), melano 4C pre-malignant‐ cytes, 4C11− non‐metastatic melanoma cells, and 4C11+ metastatic melanoma cells by real‐time melanocytes, 4C11− non-metastatic melanoma cells, and 4C11+ metastatic melanoma cells by real- qPCR using specific primers. β‐actin gene was used as an internal control. (E) Tetrahydrobiopterin time qPCR using specific primers. β-actin gene was used as an internal control. (E) Tetrahydro- biosynthesis pathways. In the de novo pathway, tetrahydrobiopterin (BH4) is synthesized by the sequential actionbiopterin of three biosynthesis enzymes from pathways. guanosine In the triphosphate de novo pathway, (GTP). tetrahydrobiopterin The first step is catalyzed (BH4) is synthesized by the reactionby‐limiting the sequential enzyme, action GTP ofcyclohydrolase three enzymes 1 (GTPCH1), from guanosine which triphosphate converts GTP (GTP). into 7,8 The‐ first step is dihydroneopterincatalyzed triphosphate, by the reaction-limiting which is converted enzyme, by the GTP action cyclohydrolase of the enzyme 1 (GTPCH1), pyroviltetrahydro which‐ converts GTP biopterin synthaseinto 7,8-dihydroneopterin (PTPS) into 6‐pyrovil triphosphate, tetrahydrobiopterin. which is The converted last reaction by the is action catalyzed of the by enzyme the pyrovilte- enzyme sepiapterintrahydrobiopterin reductase (SR) synthase which (PTPS) produces into BH4 6-pyrovil from tetrahydrobiopterin. 6‐pyrovil tetrahydrobiopterin. The last reaction BH4 is catalyzed can be spontaneouslyby the enzyme oxidized sepiapterin to dihydrobiopterin reductase (SR) (BH2). which To produces restore BH4, BH4 BH2 from is 6-pyrovil reduced tetrahydrobiopterin.by the enzyme dihydrofolateBH4 can bereductase spontaneously (DHFR) oxidized in the salvage to dihydrobiopterin pathway. BH4 (BH2).can also To be restore converted BH4, to BH2 qui‐ is reduced by nonoid‐BH2 (qBH2), an unstable molecule that is easily converted to BH2, being reduced to BH4 by the enzyme dihydrofolate reductase (DHFR) in the salvage pathway. BH4 can also be converted the enzyme dihydropteridine reductase (DHPR). The SR enzyme also reduces exogenous se‐ to quinonoid-BH2 (qBH2), an unstable molecule that is easily converted to BH2, being reduced to piapterin to BH2, which is reduced by DHFR to BH4. GTP feedback regulator (GRFR) binds to GTPCH1 andBH4 regulates by the BH4 enzyme synthesis dihydropteridine in a feedback reductase mechanism. (DHPR). Values The are SR reported enzyme alsoin the reduces bar exogenous graphs and expressedsepiapterin as the to BH2,means which ± S.D. is The reduced experiments by DHFR were to performed BH4. GTP in feedback triplicate regulator and p values (GRFR) binds to were based onGTPCH1 the one‐way and ANOVA regulates test BH4 followed synthesis by inBonferroni a feedback post mechanism.‐test; *, p < 0.05; Values **, p are< 0.01; reported ***, in the bar p < 0.001. graphs and expressed as the means ± S.D. The experiments were performed in triplicate and p values were based on the one-way ANOVA test followed by Bonferroni post-test; *, p < 0.05; **, p < 0.01; 2.2. Tetrahydrobiopterin***, p < 0.001. Concentration Is Elevated in Melanoma Cells In endothelial cells, increased Gch1 expression is associated with elevated BH4 levels 2.2. Tetrahydrobiopterin Concentration Is Elevated in Melanoma Cells [31,32]. Since increased expression of all the enzymes from the de novo synthesis pathway was observed in Inmelanoma endothelial cells, cells, the increasedconcentrationGch1 ofexpression biopterins iswas associated analyzed with by HPLC. elevated BH4 lev- BH4 and biopterinsels [31,32 concentration]. Since increased was augmented expression along of all with the melanoma enzymes from progression, the de novo be‐ synthesis pathway was observed in melanoma cells, the concentration of biopterins was analyzed ing higher in melanoma cells (Figure 2A,D), which corroborates the increased activity of by HPLC. BH4 and biopterins concentration was augmented along with melanoma pro- the de novo BH4 synthesis cycle. However, BH2 amount did not change in all cells evalu‐ gression, being higher in melanoma cells (Figure2A,D), which corroborates the increased ated (Figure 2B). BH4:BH2 ratio was higher in 4C pre‐malignant melanocytes and in 4C11− activity of the de novo BH4 synthesis cycle. However, BH2 amount did not change in all e 4C11+ melanoma cells when compared to melanocytes (Figure 2C). cells evaluated (Figure2B). BH4:BH2 ratio was higher in 4C pre-malignant melanocytes and in 4C11− e 4C11+ melanoma cells when compared to melanocytes (Figure2C).

Int.Int. J. J. Mol. Mol. Sci. Sci. 20212021,, 2222,, x 9556 FOR PEER REVIEW 5 5of of 20 20

Int. J. Mol. Sci. 2021, 22, x FOR PEER REVIEW 5 of 20

FigureFigure 2. 2. TetrahydrobiopterinTetrahydrobiopterin metabolism metabolism is is altered altered along with melanoma progression. ( (AA)) The concentrationconcentrationFigure 2. Tetrahydrobiopterin of of BH4, BH4, ( (BB)) BH2, BH2, metabolism ( (CC)) BH4:BH2 BH4:BH2 is alteredratio, ratio, and and along ( (DD )with) total total melanoma biopterin progression.in in melan melan-a‐a melanocytes (A) The concentration of BH4, (B) BH2, (C) BH4:BH2 ratio, and (D) total biopterin in melan‐a melanocytes (Ma),(Ma), 4C 4C pre pre-malignant‐malignant melanocytes, 4C11−− nonnon-metastatic‐metastatic melanoma melanoma cells, cells, and 4C11+ metastatic (Ma), 4C pre‐malignant melanocytes, 4C11− non‐metastatic melanoma cells, and 4C11+ metastatic melanomamelanoma cells were evaluatedevaluated byby HPLC. HPLC. Values Values are are reported reported in in the the bar bar graphs graphs and and expressed expressed as the as themelanoma means ± cells S.D. wereThe experimentsevaluated by wereHPLC. performed Values are in reported triplicate in and the pbar values graphs were and based expressed on the as one ‐ means ± S.D. The experiments were performed in triplicate and p values were based on the one-way waythe meansANOVA ± S.D. test The followed experiments by Bonferroni were performed post‐test; in triplicate*, p < 0.05; and **, pp values< 0.01; were ***, p based < 0.001. on the one‐ ANOVAway ANOVA test followed test followed by Bonferroni by Bonferroni post-test; post‐test; *, p *,< p 0.05; < 0.05; **, **,p < p 0.01;< 0.01; ***, ***,p p< < 0.001. 0.001. 2.3. Increased Expression of Endothelial Nitric Oxide Synthase in Metastatic Melanoma Cells 2.3.2.3. Increased Expression Expression of of Endothelial Endothelial Nitric Nitric Oxide Oxide Synthase Synthase in inMetastatic Metastatic Melanoma Melanoma Cells Cells It has been described that eNOS uncoupling can be caused paradoxically by the over‐ ItIt has has been been described described that that eNOS eNOS uncoupling uncoupling can be can caused be caused paradoxically paradoxically by the over by‐ the expressionoverexpressionexpression ofof NOS3 of NOS3 in endothelialendothelialin endothelial cells cells [20]. [20]. cells Since Since [20]. decreased Sincedecreased decreased BH4 BH4 levels levels BH4 or levels BH4:BH2or BH4:BH2 or BH4:BH2 ratio ratio wereratiowere not werenot observedobserved not observed in melanoma in melanoma cells cells and and cellsincreased increased and increasedexpression expression expressionof ofNos3 Nos3 has has been of beenNos3 observed observedhas been inobservedin melan melan‐‐aa in melanocytesmelanocytes melan-a melanocytes submittedsubmitted submittedto to sustained sustained to stress sustainedstress condition, condition, stress which condition, which resulted resulted which in mela in resulted mela‐ ‐ nocyteinnocyte melanocyte malignant malignant malignant transformation transformation [5] [5] and and gave gave [5] rise and rise to gave to the the cells rise cells used to used the in cells thisin this study, used study, in we this weeval study, eval‐ ‐ uatedweuated evaluated eNOS eNOS expressionexpression eNOS expression inin thesethese melanoma inmelanoma these melanoma cell cell lines lines by cell by real linesreal‐time‐time by qPCR. real-time qPCR. Increased Increased qPCR. expres Increased expres‐ ‐ sionexpressionsion ofof Nos3Nos3 of waswasNos3 foundwas foundinin 4C11+4C11+ in 4C11+metastatic metastatic metastatic melanoma melanoma melanoma cells cells (Figure (Figure cells (Figure3), 3),suggesting suggesting3), suggesting that that eNOSthateNOS eNOS uncoupling uncoupling uncoupling could could bebe aa resultresult be a result of of loss loss of of of loss eNOS:BH4 eNOS:BH4 of eNOS:BH4 stoichiometry. stoichiometry. stoichiometry.

FigureFigure 3.3. Increased expression expression of of endothelial endothelial nitric nitric oxide oxide synthase synthase in metastatic in metastatic melanoma melanoma cells. cells. FigureRelative 3. endothelialIncreased expressionnitric oxide ofsynthase endothelial mRNA nitric expression oxide wassynthase analyzed in metastatic in melan‐a melanomamelanocytes cells. Relative endothelial nitric oxide synthase mRNA expression was analyzed in melan-a melanocytes Relative(Ma), 4C endothelial pre‐malignant nitric melanocytes, oxide synthase 4C11 mRNA− non‐metastatic expression melanoma was analyzed cells, and in melan 4C11+‐ ametastatic melanocytes − (Ma),(Ma),melanoma 4C 4C pre pre-malignant cells‐malignant by real‐time melanocytes, qPCR using 4C11 specific− nonnon-metastatic primers.‐metastatic Values melanoma melanoma are reported cells, cells, in theand bar 4C11+ graphs metastatic and melanomamelanomaexpressed ascells cells the by bymeans real real-time‐ ±time S.D. qPCR qPCRThe experiments using using specific specific were primers. primers. performed Values Values in triplicate are are reported reported and p valuesin the bar barwere graphs graphs based and and expressedexpressedon the one as as‐way the the ANOVAmeans means ±± S.D.testS.D. followed The The experiments experiments by Bonferroni were were postperformed performed‐test; ***, in in p triplicate triplicate< 0.001. and and pp valuesvalues were were based based onon the the one one-way‐way ANOVA ANOVA test followed by Bonferroni post post-test;‐test; ***, p < 0.001.

Int. J. J. Mol. Sci. Sci. 20212021,, 2222,, x 9556 FOR PEER REVIEW 6 6of of 20

2.4.2.4. Downregulation Downregulation of of eNOS eNOS Protein Protein Restores Its Function ToTo investigate if the overexpression of Nos3 in 4C11+ metastatic melanoma cells was associatedassociated with with its its dysfunction and and the altered redox status observed in these cells, Nos3 was knocked down in 4C11+ melanoma melanoma cells cells using an eNOS shRNA. A scramble control (TRC1)(TRC1) or eNOS shRNA was introduced in 4C11+ cells and two clones (#1 and #2) with decreaseddecreased Nos3Nos3 expressionexpression were were established as as evaluated by by RT RT-qPCR‐qPCR (Figure 44A)A) andand Western blotblot (Figure (Figure4 B).4B). The The results results suggest suggest that that downregulation downregulation of eNOS of eNOS protein protein restores re‐ −•−• storesits function, its function, since since we observed we observed increased increased NO NO (Figure (Figure4C) 4C) and and decreased decreased O O2 2 levels (Figure(Figure 44D)D) afterafter eNOSeNOS silencing.silencing.

Figure 4. Endothelial nitric oxide synthase downregulation induced increased nitric oxide and reduced superoxide anion levels. Knockdown Knockdown of of eNOS eNOS was was evaluated evaluated by by (A) ( ART) RT-qPCR‐qPCR and and (B) (WesternB) Western blotting blotting in wild in wild-type‐type 4C11+ 4C11+ metastatic metastatic mel‐ anomamelanoma cells cells (WT) (WT) and after and transduction after transduction with viral with particles viral particles containing containing two different two different shRNA sequences shRNA sequences for eNOS for (she eNOSNOS #1 and #2) or control non‐target shRNA (TRC1). (C) Superoxide anion and (D) nitric oxide amount was analyzed using (sheNOS #1 and #2) or control non-target shRNA (TRC1). (C) Superoxide anion and (D) nitric oxide amount was analyzed DHE or DAF, respectively, by flow cytometry. Values are reported in the bar graphs and expressed as the means ± S.D. using DHE or DAF, respectively, by flow cytometry. Values are reported in the bar graphs and expressed as the means The experiments were performed in triplicate and p values were based on the one‐way ANOVA test followed by Bonfer‐ ± roniS.D. post The‐test; experiments **, p < 0.01; were ***, p performed < 0.001. in triplicate and p values were based on the one-way ANOVA test followed by Bonferroni post-test; **, p < 0.01; ***, p < 0.001. 2.5. Decreased Expression and Coupling of eNOS in the Presence of a Superoxide Scavenger 2.5. Decreased Expression and Coupling of eNOS in the Presence of a Superoxide Scavenger Elevated ROS concentration induced the expression of eNOS, as shown at both the transcriptionalElevated ROS and translational concentration levels induced in endothelial the expression cells [33,34]. of eNOS, Previous as shown data at from both our the grouptranscriptional showed andthat translational O2−• depletion levels impaired in endothelial melan cells‐a melanocyte [33,34]. Previous malignant data fromtransfor our‐ −• mationgroup showed induced that by Osequential2 depletion cycles impaired of anchorage melan-a blockade, melanocyte giving malignant rise to transformationcells—namely Mn3,induced Mn4, by and sequential Mn5—with cycles a of senescent anchorage‐like blockade, phenotype giving and rise low to cells—namelyproliferation rate Mn3, [7]. Mn4, To determineand Mn5—with if oxidative a senescent-like stress during phenotype melanoma and progression low proliferation contributes rate [7 to]. Toincreased determine Nos3 if expressionoxidative stress found during in 4C11+ melanoma metastatic progression melanoma contributes cells, its expression to increased wasNos3 evaluatedexpression in Mn3found and in 4C11+Mn5 cells. metastatic We found melanoma decreased cells, Nos3 its expression expression in was Mn3 evaluated and Mn5 in cells Mn3 compared and Mn5 tocells. 4C11+ We metastatic found decreased melanomaNos3 cellsexpression (Figure S1A). in Mn3 Moreover, and Mn5 eNOS cells activity compared seems to 4C11+ to be metastatic melanoma cells (Figure S1A). Moreover, eNOS activity seems to be restored, restored, since O2−• is reduced (Figure S1B) and NO amount elevated (Figure S1C) in Mn3 and Mn5 in comparison with 4C11+ melanoma cells.

Int. J. Mol. Sci. 2021, 22, 9556 7 of 20

Int. J. Mol. Sci. 2021, 22, x FOR PEER REVIEW 7 of 20

−• since O2 is reduced (Figure S1B) and NO amount elevated (Figure S1C) in Mn3 and Mn5 2.6.in comparison L‐Sepiapterin with Supplementation 4C11+ melanoma Decreases cells. Superoxide Anion and Increases Nitric Oxide Levels2.6. L-Sepiapterin Supplementation Decreases Superoxide Anion and Increases Nitric Oxide Levels ItIt was was reported reported that that administration administration of BH4 of BH4 in mice in mice overexpressing overexpressing eNOS eNOS in vivoin pre vivo‐ ventedprevented eNOS eNOS uncoupling uncoupling by byrestoring restoring NO NO production production [20,35]. [20,35 ].To To evaluate evaluate if if increased increased intracellularintracellular BH4 cancan restorerestore eNOS eNOS function function in 4C11+in 4C11+ metastatic metastatic melanoma melanoma cells, cells, they they were weretreated treated or not or with not 40withµM 40 L-sepiapterin μM L‐sepiapterin for two for hours. two Inhours. the presence In the presence of L-sepiapterin, of L‐se‐ piapterin, decreased−• O2−• levels were observed, as demonstrated by flow cytometer (Fig‐ decreased O2 levels were observed, as demonstrated by flow cytometer (Figure5A) and ureHPLC 5A) analysesand HPLC (Figure analyses5B). (Figure In addition, 5B). In NO addition, was increased NO was after increased L-sepiapterin after L‐sepiapterin treatment treatmentas demonstrated as demonstrated by flow cytometer by flow cytometer analyses (Figure analyses5C) (Figure and by NO5C) analyzerand by NO(Figure analyzer5D). (FigureThese results 5D). These corroborate results corroborate with previous with data previous from our data laboratory, from our wherelaboratory, a cell where line iso- a celllated line from isolated a murine from a metastatic murine metastatic melanoma melanoma (Tm5 lineage) (Tm5 lineage) showed showed the same the pattern same pat [6‐]. ternL-sepiapterin [6]. L‐sepiapterin treatment treatment was efficient, was efficient, since both since increased both increased intracellular intracellular BH4 and BH4 biopterin and biopterinconcentration concentration (Figure S2A,D) (Figure and S2A,D) BH4:BH2 and BH4:BH2 ratio were ratio observed were observed (Figure S2C). (Figure S2C).

FigureFigure 5. 5. L‐sepiapterinL-sepiapterin treatment treatment restored restored endothelial endothelial nitric nitric oxide oxide synthase synthase function. function. 4C11+ meta 4C11+‐ staticmetastatic melanoma melanoma cells were cells treated were treated or not (non or not‐treated) (non-treated) with 40 μ withM L 40‐sepiapterinµM L-sepiapterin (L‐SEP) for (L-SEP) two hours,for two and hours, superoxide and superoxide anion levels anion were levels analyzed were using analyzed (A) DHE using by (A HPLC) DHE or by (B HPLC) flow cytometry, or (B) flow and nitric oxide amount were evaluated by (C) NO analyzer or by (D) flow cytometry using DAF. cytometry, and nitric oxide amount were evaluated by (C) NO analyzer or by (D) flow cytometry Values are reported in the bar graphs and expressed as the means ± S.D. The experiments were using DAF. Values are reported in the bar graphs and expressed as the means ± S.D. The experiments performed in triplicate and p values were based on the one‐way ANOVA test followed by Bonfer‐ roniwere post performed‐test; ***, inp < triplicate 0.001. and p values were based on the one-way ANOVA test followed by Bonferroni post-test; ***, p < 0.001.

2.7.2.7. Restoration Restoration of of eNOS eNOS Function Function Reduced Reduced Cell Cell Survival Survival of of Metastatic Metastatic Melanoma Melanoma Cells Cells SinceSince reduced reduced Nos3Nos3 expression,expression, as as well well as as L L-sepiapterin‐sepiapterin treatment treatment in in 4C11+ 4C11+ metastatic metastatic melanomamelanoma cells, induced restorationrestoration of of its its activity activity by by increasing increasing NO NO synthesis synthesis and and decreas- de‐ creasing−• O2−•, cell survival was evaluated. Downregulation of eNOS decreased cell viabil‐ ing O2 , cell survival was evaluated. Downregulation of eNOS decreased cell viability ityof of 4C11+ 4C11+ metastatic metastatic melanoma melanoma cells cells compared compared to to wild wild typetype andand scramble control cells cells (Figure(Figure 6A)6A) for for 72 72 and and 96 96 h hand and colony colony formation formation in vitroin vitro (Figure(Figure 6B).6 eNOSB). eNOS silencing silencing also conferredalso conferred anoikisanoikis sensitivitysensitivity to 4C11+ to 4C11+ metastatic metastatic melanoma melanoma cells when cells compared when compared to scram to‐ bledscrambled control control cells (Figure cells (Figure 6C). 6C).

Int. J. J. Mol. Sci. Sci. 20212021,, 2222,, x 9556 FOR PEER REVIEW 8 8of of 20

FigureFigure 6. 6. EndothelialEndothelial nitric nitric oxide oxide synthase synthase knockdown knockdown reduced reduced melanoma melanoma cell cellgrowth growth in vitro.in vitro (A). Cell(A) Cellviability,viability, (B) clonogenic (B) clonogenic capability, capability, and (C) and anoikis (C) resistanceanoikis resistance were analyzed were analyzedin 4C11+ metastatic in 4C11+ melanomametastatic cells melanoma WT, TRC1, cells she WT,NOS TRC1,#1, and she NOSsheNO#1,S#2. and Cell she NOviabilityS#2. Cellwas evaluated viability was for 24, evaluated 48, 72, and 96 h by MTT. The clonogenic capability was determined by estimating clone formation after 9 for 24, 48, 72, and 96 h by MTT. The clonogenic capability was determined by estimating clone forma- days of cell culture and anoikis resistance by maintaining melanoma cells in suspension for 96 h and tion after 9 days of cell culture and anoikis resistance by maintaining melanoma cells in suspension evaluating viable cells by MTT. Values are reported in the bar graphs and expressed as the means ± S.D.for 96 The h andexperiments evaluating were viable performed cells by in MTT. triplicate Values and are p values reported were in based the bar on graphs the one and‐way expressed ANOVA testas the followed means by± S.D.Bonferroni The experiments post‐test or were on the performed two‐way in ANOVA triplicate test and followedp values by were Bonferroni based on post the‐ test;one-way ***, p ANOVA < 0.001. test followed by Bonferroni post-test or on the two-way ANOVA test followed by Bonferroni post-test; ***, p < 0.001. 2.8. Downregulation of eNOS Attenuated Metastatic Melanoma Growth In Vivo 2.8. Downregulation of eNOS Attenuated Metastatic Melanoma Growth In Vivo Since we observed decreased cell viability, colony formation, and anoikis resistance Since we observed decreased cell viability, colony formation, and anoikis resistance in in 4C11+ metastatic melanoma cells silenced for eNOS, in vivo tumor growth was ana‐ 4C11+ metastatic melanoma cells silenced for eNOS, in vivo tumor growth was analyzed. lyzed. Metastatic melanoma 4C11+ cells‐wild type, scramble, and silenced for eNOS were Metastatic melanoma 4C11+ cells-wild type, scramble, and silenced for eNOS were inoc- inoculated in the subcutaneous flank of female C57BL/6 mice, and tumor development ulated in the subcutaneous flank of female C57BL/6 mice, and tumor development was was observed. Tumors of all groups appeared after 15 days; however, the tumor develop‐ observed. Tumors of all groups appeared after 15 days; however, the tumor development ment of 4C11+ metastatic melanoma cells silenced for eNOS was mitigated, as observed of 4C11+ metastatic melanoma cells silenced for eNOS was mitigated, as observed after after21 days 21 dayswhen when the animals the animals were were sacrificed sacrificed and the and tumor the tumor mass measured.mass measured. Decreased Decreased eNOS eNOSexpression expression caused caused a significant a significant reduction reduction in both in tumor both tumor volume volume and weight and weight (Figure (Figure7A,B). 7A,B).

Int.Int. J.J. Mol.Mol. Sci.Sci. 20212021,, 2222,, 9556x FOR PEER REVIEW 99 of 2020

FigureFigure 7.7. InIn vivovivo melanomamelanoma growthgrowth waswas impairedimpaired byby endothelialendothelial nitricnitric oxideoxide synthasesynthase downregu-downregu‐ lation.lation. 4C11+ metastatic metastatic melanoma melanoma cells cells were were subcutaneously subcutaneously inoculated inoculated into into C57BL/6 C57BL/6 mice mice and and (A) (tumorA) tumor weightweight and and (B) volume (B) volume were were determined. determined. WT: WT: wild wild type type 4C11+ 4C11+ metastatic metastatic melanoma melanoma cell line; cell line;TRC1: TRC1: control control non‐ non-targettarget shRNA; shRNA; sheNOS sheNOS#1 and#1 andwhe wheNOSNOS#2: clones#2: clones silenced silenced for eNOS. for eNOS. Values Values are reported in the bar graphs and expressed as the means ± S.D. The experiments were performed in are reported in the bar graphs and expressed as the means ± S.D. The experiments were performed in triplicate and p values were based on the one‐way ANOVA test followed by the Bonferroni post‐ triplicate and p values were based on the one-way ANOVA test followed by the Bonferroni post-test test or two‐way ANOVA test followed by Bonferroni post‐test; *, p < 0.05, ***, p < 0.001. or two-way ANOVA test followed by Bonferroni post-test; *, p < 0.05, ***, p < 0.001.

2.9.2.9. L-SepiapterinL‐Sepiapterin SupplementationSupplementation InhibitsInhibits thethe GrowthGrowth ofof MetastaticMetastatic MelanomaMelanoma CellsCells AA pro-oxidantpro‐oxidant microenvironment microenvironment sustained sustained by by eNOS eNOS uncoupling uncoupling seems seems to contributeto contrib‐ toute the to survival the survival of metastatic of metastatic melanoma melanoma cells, since cells, L-sepiapterin since L‐sepiapterin treatment treatment reduced reduced cell via- bilitycell viability at 24, 48, at 72, 24, and 48, 96 72, h and as shown 96 h as by shown MTT (Figure by MTT8A) (Figure and clonogenic 8A) and assay clonogenic(Figure assay8B). −• 2−• Decreased(Figure 8B). O 2Decreasedlevels inducedO levels by L-sepiapterininduced by L‐ supplementationsepiapterin supplementation also rendered also 4C11+ ren‐ metastaticdered 4C11+ melanoma metastatic cells melanoma less resistant cells tolessanoikis resistant(Figure to anoikis8C). Moreover, (Figure 8C). L-sepiapterin Moreover, in- L‐ creasedsepiapterin 4C11+ increased melanoma 4C11+ sensitivity melanoma to dacarbazine sensitivity chemotherapy, to dacarbazine as shown chemotherapy, in Figure8 D.as shown in Figure 8D.

Int. J. J. Mol. Sci. Sci. 20212021,, 2222,, x 9556 FOR PEER REVIEW 1010 of of 20

FigureFigure 8. LL-sepiapterin‐sepiapterin impaired impaired cell cell growth and improved sensitivity to apoptosis of metastatic melanomamelanoma cells. cells. 4C11+ 4C11+ metastatic metastatic melanoma melanoma cells cells were were treated treated (+) (+) or ornot not (non (non-treated)‐treated) (‐) (-)with with 40 μ40Mµ LM‐sepiapterin L-sepiapterin (L‐SEP) (L-SEP) and and (A) cell (A) cellviability, viability, (B) clonogenic (B) clonogenic capability, capability, (C) anoikis (C) anoikis resistance,resistance, and (andD) dacarbazine (D) dacarbazine (DAC) (DAC) treatment treatment sensitivity sensitivity were were evaluated. evaluated. WT: wild WT: wildtype type4C11+ 4C11+ metastatic metastatic mel‐ anomamelanoma cell cellline; line; TRC1: TRC1: control control non non-target‐target shRNA; shRNA; she sheNOSNOS#1#1 and and she sheNOSNOS#2:#2: clones clones silenced silenced for eNOS.eNOS. Values Values are reported in the barbar graphsgraphs andand expressedexpressed asas thethe meansmeans ±± S.D. The experiments were performed in triplicate and p values were based on the one one-way‐way ANOVA ANOVA test followed by the Bonferroni post‐test or on the two‐way ANOVA test followed by Bonferroni post‐test; *, p < 0.05; **, Bonferroni post-test or on the two-way ANOVA test followed by Bonferroni post-test; *, p < 0.05; p < 0.01; ***, p < 0.001. **, p < 0.01; ***, p < 0.001.

2.10.2.10. Increased Increased NOS3 NOS3 Expression Expression in Human Metastatic Compared with Primary Melanomas AnalysisAnalysis of of Riker gene array with 87 samples from Oncomine showed high NOS3 expressionexpression in in metastatic melanoma melanoma when when compared compared to to the the primary site (Figure 99A)A) [[36]36] (GSE7553).(GSE7553). Moreover,Moreover, analysis analysis from from TCGA TCGA showed showed increased increasedNOS3 NOS3expression expression along along with withmelanoma melanoma progression progression (Figure (Figure9B) [29 9B)]. [29].

Int. J. Mol. Sci. 2021, 22, 9556 11 of 20 Int. J. Mol. Sci. 2021, 22, x FOR PEER REVIEW 11 of 20

Figure 9. Increased endothelial nitric oxide synthase expression isis correlated with human melanoma aggressiveness. (A)) Melanoma metastasismetastasis (n(n == 40) expresses higher levelslevels ofof NOS3NOS3 comparedcompared toto thethe primaryprimary sitesite (n(n == 16). Microarray data by the Riker study was obtained from the Oncomine databasedatabase as indicated (GSE7553). (GSE7553). Boxes Boxes represent represent the the interquartile interquartile range range (25th–75th (25th–75th percentile). percentile). The The bars bars de‐ note the median; *p < −1 × 104. (B) Increased NOS3 expression along with melanoma progression (n denote the median; • p < −1 × 104.(B) Increased NOS3 expression along with melanoma progression = 48). Data regarding melanoma progression was obtained from gepia2. (n = 48). Data regarding melanoma progression was obtained from gepia2.

3. Discussion Previous datadata from from our our group group showed showed the establishmentthe establishment of a murine of a murine melanoma melanoma model wheremodel differentwhere different melanoma melanoma cell lines cell were lines obtained were obtained after submitting after submitting melan-a melan melanocytes‐a mela‐ tonocytes serial cyclesto serial of cycles anchorage of anchorage blockade, blockade, which was which associated was associated with oxidative with oxidative stress [28 ].stress The −• −• involvement[28]. The involvement of O2 in of the O regulation2 in the ofregulation signaling of pathways signaling and pathways epigenetic and mechanisms epigenetic associatedmechanisms with associated melanocyte with malignant melanocyte transformation malignant transformation was demonstrated was [5 ,demonstrated7]. Moreover, the[5,7]. participation Moreover, the of eNOSparticipation uncoupling of eNOS was uncoupling suggested was since suggested L-NAME since and L-sepiapterinL‐NAME and −• decreasedL‐sepiapterin O2 decreasedand increased O2−• and NO increased during adhesion NO during impediment. adhesion impediment. More importantly, More theim‐ treatmentportantly, ofthe melan-a treatment with of L-NAME melan‐a duringwith L‐ de-adhesionNAME during impaired de‐adhesion the acquisition impaired of the a ma- ac‐ lignantquisition phenotype, of a malignant reinforcing phenotype, the role reinforcing of uncoupled the role eNOS of uncoupled in melanoma eNOS development in melanoma [6]. developmentNOS function [6]. is regulated by several integrated mechanisms, with intracellular avail- abilityNOS of the function BH4 cofactor is regulated being by essential several integrated [18]. Intracellular mechanisms, BH4 concentrationwith intracellular is tightly avail‐ controlledability of the in differentBH4 cofactor ways being and is essential maintained [18]. by Intracellular de novo, salvage, BH4 concentration and recycle synthesis is tightly pathwayscontrolled (Figure in different1E) [ 37ways]. In and de novo is maintained BH4 biosynthesis, by de novo, BH4 salvage, is synthesized and recycle from synthesis GTP in a three-steppathways reaction(Figure 1E) as follows: [37]. In de GTPCH1 novo BH4 (EC biosynthesis, 3.5.4.16), encoded BH4 is by synthesizedGCH1 gene, from is followed GTP in bya three 6-pyruvoyl-tetrahydrobiopterin‐step reaction as follows: GTPCH1 synthase (EC PTPS 3.5.4.16), (EC 4.6.1.10) encoded and by sepiapterin GCH1 gene, reductase is fol‐ SRlowed (EC by 1.1.1.153). 6‐pyruvoyl In‐ endothelialtetrahydrobiopterin cells and synthase cardiomyocytes, PTPS (EC it 4.6.1.10) was shown and sepiapterin that GTPCH1 re‐ activityductase canSR (EC be regulated 1.1.1.153). transcriptionally In endothelial cells since andGCH1 cardiomyocytes,mRNA expression it was is shown correlated that withGTPCH1 protein activity content can andbe regulated BH4 production transcriptionally [32,38,39 since]. Surprisingly, GCH1 mRNA we expression found increased is cor‐ expressionrelated with of proteinGch1 mRNA content in and 4C11 BH4− and production 4C11+ melanoma [32,38,39]. cellsSurprisingly, when compared we found with in‐ melan-acreased expression melanocytes of andGch1 4C mRNA pre-malignant in 4C11− melanomaand 4C11+ cellsmelanoma (Figure cells1A). when Since compared we have previouslywith melan suggested‐a melanocytes that eNOS and 4C uncoupling pre‐malignant is associated melanoma with cells melanoma (Figure cell 1A). growth Since and we apoptosishave previously resistance suggested [6], we expectedthat eNOS to observeuncoupling decreased is associatedGch1 expression with melanoma and activity. cell However,growth and this apoptosis result corroborates resistance the[6], literature we expected data, to since observeGCH1 decreasedexpression Gch1 is increased expression in estrogen-receptor-negativeand activity. However, this breast result tumors corroborates and cells the present literature in the data, tumor since microenvironment, GCH1 expression like stromal fibroblasts and inflammatory and endothelial cells, being a significant predictor is increased in estrogen‐receptor‐negative breast tumors and cells present in the tumor of poor prognosis in patients [40]. Overexpression of GTPCH1 in glioblastoma is also asso- microenvironment, like stromal fibroblasts and inflammatory and endothelial cells, being ciated with tumor growth and higher glioma grade, recurrence, and worse survival [41]. a significant predictor of poor prognosis in patients [40]. Overexpression of GTPCH1 in Epidermal melanocytes already express GTPCH1 in vitro and in vivo and can synthesize glioblastoma is also associated with tumor growth and higher glioma grade, recurrence, BH4, which is involved in melanogenesis [42]. Furthermore, Gch1 mRNA expression is and worse survival [41]. Epidermal melanocytes already express GTPCH1 in vitro and in increased in the presence of high ROS concentration [43]. Therefore, these circumstances vivo and can synthesize BH4, which is involved in melanogenesis [42]. Furthermore, Gch1 could explain the Gch1 upregulation observed in melanoma cells. Pts and Spr mRNA mRNA expression is increased in the presence of high ROS concentration [43]. Therefore, expression were increased only in 4C11+ metastatic melanoma cells (Figure1B,C). Expres- these circumstances could explain the Gch1 upregulation observed in melanoma cells. Pts

Int. J. Mol. Sci. 2021, 22, 9556 12 of 20

sion of PTPS is augmented in the early phases of colorectal cancer and under hypoxia, and its induction in colon carcinoma cell lines was associated with tumor growth [44]. Moreover, PTPS downregulation decreased BH4 synthesis, demonstrating that altered ex- pression of other enzymes from the BH4 synthesis pathway may also affect its production. In our melanoma model, we observed up-regulation of genes associated with hypoxia response along with melanoma progression [45]. Therefore, increased Hif-1 expression in 4C11+ melanoma cells can be associated with Pts up-regulation. The overexpression of SPR mRNA was also found in neuroblastoma, breast cancer, and hepatocarcinoma cells [46–48]. It was demonstrated that SR is associated with tumor growth through different mechanisms, including interaction with ornithine decarboxylase, induction of FoxO3a/Bim signaling pathway, and oxidative stress. The mechanisms underlying the role of SR in melanoma development are under investigation. The de novo BH4 biosynthesis is tightly regulated by GFRP. By an allosteric mechanism, high BH4 concentration decreases GTPCH1 activity via interaction with GFRP, while L-phenylalanine stimulates BH4 produc- tion [49]. We observed increased Gchfr expression in 4C pre-malignant melanoma cells and 4C11− melanoma cells; however, in 4C11+ metastatic melanoma cells, Gchfr expression is reduced (Figure1D). Under oxidative stress conditions, Gchfr mRNA is also upregu- −• lated in endothelial cells and cardiomyocytes [43]. Since O2 is increased along with melanoma progression [6], the overexpression of Gchfr in 4C pre-malignant melanocytes and 4C11− non-metastatic melanoma cells could be associated with altered redox home- ostasis. However, decreased Gchfr expression observed in 4C11+ melanoma cells need more investigation. The functional GFRP/GTPCH1 axis has been described in epidermal melanocytes [42], suggesting that this feedback mechanism could be controlling BH4 syn- thesis in melanoma cells. However, it was shown in other systems that modifications in GFRP expression do not necessarily occur concomitantly with changes in GTPCH1 activity or BH4 synthesis [32]. Moreover, public data from Oncomine and TCGA showed that high GCH1 expression correlates with melanoma aggressiveness (Figure S3A), increased expression of PTS (Figure S3B) and SPR (Figure S3C), and decreased GCHFR expression (Figure S3D) in melanoma when compared to benign nevi. According to the increased expression of the Gch1 gene, BH4 and total biopterins concentration were increased in 4C11− and 4C11+ melanoma cells when compared to melanocytes (Figure2A,D), showing that in the tumor, as demonstrated in other cells, GTPCH1 activity and BH4 production are regulated at the transcriptional level. BH4 is easily oxidized in the presence of high levels of ROS; however, we did not observe changes in BH2 concentration throughout the devel- opment of melanoma (Figure2B), suggesting that the salvage and recycle pathways are functional, since these cells produce high levels of ROS [6]. Consequently, BH4:BH2 ratio was increased in 4C pre-malignant cells and 4C11− and 4C11+ melanoma cells (Figure2C). Although this information is not always related to cancer cells, it would be relevant to evaluate BH4:BH2 ratio, since the accumulation of BH2 could induce eNOS uncoupling by competing with BH4 for eNOS active site. As mentioned earlier, although BH2 binds to the active site of eNOS with the same affinity, it does not have the ability to catalyze the forma- tion of NO. Therefore, BH4:BH2 ratio in addition to the BH4 absolute molar concentration are key determinants of eNOS function [21,50,51]. Based on these results, it would be reasonable to think that NOS is not uncoupled in these cells. However, we have described −• a reduction in NO and an increase in O2 in Tm5 metastatic melanoma cells, which is in apparent contradiction with the results shown here. Moreover, L-sepiapterin treatment −• induced a switch in the redox status of melanoma cells, impairing O2 production and stimulating NO synthesis [6]. It was reported in vitro and in vivo that the stoichiometric relationship between eNOS and BH4 also determines eNOS activity. These authors demon- strated that Nos3 superexpression in the absence of GTPCH1 activity induces uncoupling, −• increasing O2 levels and reducing NO synthesis [20,21]. The mechanism underlying uncoupling is that the amount of BH4 is not enough to bind overexpressed eNOS. In human −• melanoma cells, the results also suggested that the imbalance between NO and O2 is caused by the alteration in BH4:eNOS stoichiometry and decreased BH4:BH2 ratio [9]. Int. J. Mol. Sci. 2021, 22, 9556 13 of 20

We demonstrated increased Nos3 mRNA expression in 4C11+ metastatic melanoma cells (Figure3), as observed during melanocyte anchorage blockade impediment [ 5]. Therefore, increased ROS levels observed in 4C11+ melanoma cells could be a result of a disturbance between BH4 amount and Nos3 expression. To evaluate this hypothesis, Nos3 mRNA expression was downregulated in 4C11+ melanoma cells and two clones were settled down, sheNOS#1 and sheNOS#2 (Figure4A,B). Decreased Nos3 expression restored eNOS −• function by impairing O2 production (Figure4C) and elevating NO levels (Figure4D), as shown previously in endothelial cells [20]. Upregulation of Nos3 expression has been associated with K-Ras-driven tumors, as pancreatic ductal adenocarcinoma and papillomas, since NO, through wild-type Ras S-nitrosylation, activates this signaling pathway, leading to tumor growth [52,53]. The participation of mutated RAS in melanoma development is well known [54,55]. There is no evidence that N-RAS mutations are caused by UV-induced DNA damage; however, RAS activation can be induced by increased ROS production, which is also a result of UV exposure [7,56]. In fact, we and others have shown that Ras is redox-sensitive, being associated with melanocyte malignant transformation and −• melanoma development. In the presence of MnTBAP, an O2 scavenger, Ras activity is decreased [7]. Therefore, increased Nos3 expression may lead to uncoupling, altering cellu- lar redox status, and overactivating the Ras transduction pathway, which, in turn, would contribute to melanomagenesis. Moreover, in B16F10 melanoma tumors from chronically stressed animals, Nos3 is overexpressed and contributes to tumor development [57]. It was shown that eNOS expression is increased in the presence of high ROS levels in endothelial cells [33,34]. Therefore, increased Nos3 expression could be a result of altered redox home- ostasis found in 4C11+ metastatic melanoma cells. Corroborating this idea, we observed −• decreased Nos3 expression (Figure S1A), reduced O2 (Figure S1B), and increased NO amount (Figure S1C) in clones obtained after submitting melan-a cells to sequential cycles of anchorage impediment in the presence of MnTBAP, a superoxide scavenger [7]. MnT- BAP treatment impaired an efficient malignant transformation, and one of the mechanisms underlying this phenomenon could be eNOS coupling. The restoration of NOS activity in 4C11+ melanoma cells was also observed after L-sepiapterin treatment, as indicated by −• decreased O2 (Figure5A,B) and increased NO (Figure5C,D), corroborating the hypothe- sis of eNOS uncoupling. Recovering redox homeostasis with L-sepiapterin treatment was already showed in vitro and in vivo, including in cancer models [6,58–60]. L-sepiapterin supplementation impaired chemically-induced murine colitis and colon cancer [59] and reduced breast tumor growth in vitro and in vivo [60] through the restoration of coupled NOS activity. Furthermore, BH4 and L-sepiapterin decreased human metastatic melanoma cell survival by inducing apoptosis [9]. In agreement with these studies, L-sepiapterin decreased 4C11+ cell viability (Figure8A) and clonogenicity capability (Figure8B). Fur- thermore, it also rendered 4C11+ melanoma cells more sensitive to anoikis (Figure8C) and dacarbazine treatment (Figure8D). Downregulation of Nos3 also decreased cell viability (Figure6A), clonogenicity capability (Figure6B), and anoikis resistance of 4C11+ melanoma cells (Figure6C). More important, reduced expression of eNOS in 4C11+ melanoma cells impaired tumor growth in vivo, as demonstrated by decreased tumor weight (Figure7A) and volume (Figure7B). Corroborating the role of eNOS in RAS-driven cancers, genetic ablation of NOS3 gene disrupted pancreatic carcinoma and papilloma development and improved mice median survival. These effects were also shown after oral administration of L-NAME [53]. Finally, Oncomine and TCGA analysis showed that NOS3 expression correlated with human melanoma progression (Figure9), reinforcing the participation of eNOS in melanoma development. As far as we know, this is the first time that increased eNOS expression has been related to metastatic melanoma. It has been reported that eNOS expression is increased in melanoma tissue when compared with melanocytic nevi; however, the melanoma grade was not discriminated [22]. It is important to note that −• Nos3 overexpression does not correlate with increased NO production, but instead O2 generation, which means that is fundamental to assess enzyme function to understand the real participation of any protein in pathological conditions. Int. J. Mol. Sci. 2021, 22, 9556 14 of 20

4. Materials and Methods 4.1. Cell Culture A murine melanocyte malignant transformation model was developed in our labo- ratory after submitting an immortalized but non-tumorigenic melan-a melanocytes to a sustained stress condition, giving rise to pre-malignant 4C melanocytes, non-metastatic 4C11− melanoma cells, and metastatic 4C11+ melanoma cells [7,28]. Melan-a cells were cultured at 37 ◦C in humidified 95% air/5% CO2 in RPMI pH 6.9 supplemented with 5% fetal bovine serum (Invitrogen, Scotland, UK), 200 nM 12-phorbol-13-myristate acetate (PMA; Calbiochem, Darmstadt, Germany), 100 U/mL penicillin, and 100 U/mL strepto- mycin (Invitrogen, Grand Island, NY, USA). Pre-malignant 4C melanocyte lineage and non-metastatic 4C11− and metastatic 4C11+ melanoma cell lines were cultured as melan-a cells, but in the absence of PMA. 4C11+ melanoma cells were treated or not with 40 µM −• L-sepiapterin (Cayman, Ann Arbor, MI, USA) for 2 hours for NO and O2 quantification; 96 h for BH4 quantification, MTT, and anoikis resistance assay; and 9 days for colony formation assay.

4.2. Stable Silencing of eNOS by shRNA eNOS was silenced by shRNA using MISSION® Lentiviral Transduction Particles from (Sigma-Aldrich, Saint Louis, MO, USA). Two shRNA sequences for eNOS (sheNOS#1, Clone ID: NM_008713.2-3844s1c1, and sheNOS1#2, Clone ID: NM_008713.2657s1c1) and a control plasmid containing a non-target sequence (non-Mammalian shRNA Control, TCR1, SHC002) were used. Lentiviral particles were produced from TRC1 and TRC1.5 libraries consisting of sequence-verified cloned into the pLKO-1-puro vector and were used to establish cell lines with stable eNOS knockdown or expressing the non-target shRNA sequence as a control. Metastatic melanoma 4C11+ cells were plated on a 24-well plate and maintained in complete medium for 24 h. Viral particles at MOI 1.0 (multiplicity of infection) were added to the cells, gently mixed, and incubated overnight. After this period, medium containing viral particles was replaced by a complete fresh medium for 24 h. In order to establish stable-silenced eNOS clones, puromycin-resistant cells were selected and submitted to clonal dilution.

4.3. Nitric Oxide Quantification NO concentration was evaluated after a gas-phase chemiluminescence reaction of NO with ozone by an NO analyzer (NOA 280; Sievers Instruments, Boulder, CO, USA). A standard curve was established with a set of serial dilutions (0.1–100 µM) of sodium nitrate. NO metabolite concentrations in samples cells were settled by comparison with a standard curve and expressed as micromoles per milligram of protein using the NOANalysis soft- ware (version 3.21; Sievers Instruments, Inc., Boulder, CO). NO levels were also measured using a non-fluorescent indicator for nitric oxide, 4-5-diaminofluorescein diacetate (DAF- 2DA, Molecular Probes, Eugene, OR, USA). Cells were incubated with 5 µM DAF-2DA at 37 ◦C for 30 min in the dark, rinsed with phosphate-buffered saline (PBS), and NO levels analyzed by flow cytometry (FACSCalibur; Becton-Dickinson, Franklin Lakes, NJ, USA) (excitation wavelength 495 nm; emission wavelength 515 nm).

4.4. Superoxide Anion Quantification The amount of superoxide anion was measured using dihydroethidium (DHE; Molec- −• ular Probes, Eugene, OR, USA), a non-fluorescent cell-permeable indicator for O2 . For flow cytometer analysis, cells were washed and incubated in PBS for 30 min at 37 ◦C, incubated with 25 µM DHE for 40 min at 37 ◦C in the dark, washed, and analyzed by flow cytometry (FACSCalibur; Becton–Dickinson, Franklin Lakes, NJ, USA) (excitation wavelength 480 nm; emission wavelength 567 nm). For high-performance liquid chro- matography (HPLC) evaluation, cells were washed three times with PBS and incubated in PBS containing 100 µM DTPA (diethylenetriamine pentaacetic acid) (Sigma, St. Louis, MO, USA) and 50 µM DHE for 30 min at 37 ◦C. After washing, acetonitrile extraction was Int. J. Mol. Sci. 2021, 22, 9556 15 of 20

performed and simultaneous fluorescent detection of 2-hydroxyethidium (2-E+OH) and ethidium was done. DHE-derived product 2-E+OH was expressed as ratios of generated 2-E+OH over consumed DHE (initial DHE concentration minus remaining DHE) [61].

4.5. Biopterin Quantification The concentrations of tetrahydrobiopterin (BH4), 7,8-dihydrobipterin (BH2), total biopterin, and BH4:BH2 ratio were analyzed by reversed-phase high-performance liquid chromatography as previously described [62]. Melan-a, 4C, 4C11− and 4C11+, and 4C11+ cells treated or not with 40 µM L-sepiapterin for 96 h were washed twice with cold PBS (pH 7.4). After centrifugation, cells were resuspended in 0.5 mL 0.1 M phosphoric acid contain- ing 5 mM dithioerythritol (DTT) and sonicated for 40 s, and 35 µL 2 M trichloroacetic acid (TCA) was added. The solution was centrifuged at 12,000× g for 1 min, and the supernatant was used immediately for the quantification of all biopterins. The total biopterin amount was measured following oxidation in acidic conditions, whereas BH2 quantification was conducted after its oxidation in alkaline conditions. BH4 was calculated from the difference between the amount of biopterin formed by oxidation in acidic conditions and the amount of biopterin formed by oxidation in alkaline conditions. For oxidation reaction under acidic conditions, 100 µL cell extract was mixed with 15 µL 0.2 M TCA and 15 µL 1% I2/2% KI in 0.2 M TCA. For oxidation under alkaline conditions, 100 µL cell extract were mixed with 15 µL 1 M NaOH and 15 µL 1% I2/2% KI in 3 M NaOH. The oxidation reaction was carried out for 1 h in the dark at room temperature. To inactivate the excess of iodine, 25 µL 0.114 M ascorbic acid was added and centrifuged at 4 ◦C for 12 min, and 100 µL of the supernatant was injected into an HPLC system (LCMS-2020, Shimadzu Co., Kyoto, Japan) on a C18 Vydac reversed-phase column (5 µm, 4.6 mm id × 205) and detected by fluorescence (lex = 350 nm; lem = 450 nm). Biopterins were eluted by an isocratic mobile phase solution (5% methanol and 7.5 mM sodium phosphate buffer, pH 6.35) at a flow rate of 1.0 mL/min. Data were collected and analyzed by LC solution software (Shimadzu Co., Kyoto, Japan) and normalized against protein concentration.

4.6. RNAm Expression Analysis RNA was isolated using Trizol (Invitrogen, Carlsbad, CA, USA) according to the manufacturer’s specifications. Equal amounts of each cDNA synthetized were quantified by real time-PCR in a Corbett Rotor-Gene 6000 Detection System version 1.7 using an SYBR green PCR master mix (Qiagen, Dusseldorf, German) and specific primers. Relative quantification (RQ) of the amplicons was calculated according to the 2-∆∆Cq method. Gene normalizing was performed using β-actin.

4.7. Western Blot Subconfluent cell cultures were trypsinized and washed in PBS, and whole-cell lysates were prepared using NP-40 lysis buffer (10% NP-40 in 100 mM NaCl and 50 mM Tris pH 7.4, containing 30 mM sodium pyrophosphate, 50 mM NaF, 1 mM NaVO4, 2 mg/mL aprotinin, 2 mg/mL leupeptin, 2 mg/mL pepstatin, and 1 mM PMSF), kept for 15 min on ice, followed by centrifugation at 13,000× g for 15 min at 4 ◦C. The supernatant was collected for protein concentration using a Bio-Rad protein assay dye reagent (Bio-Rad, Hercules, CA, USA). Equivalent amounts of protein (40 µg) were denatured in SDS sample buffer (240 mM Tris–HCl pH 6.8, 0.8% SDS, 200 mM beta-mercaptoethanol, 40% glycerol, and 0.02% bromophenol blue) for 5 min. Protein lysates were resolved by SDS-PAGE and transferred onto nitrocellulose membranes (Bio-Rad, Hercules, CA, USA). The specific antibody used was a rabbit anti-eNOS (Cell Signaling Technology Danver, MA, USA) followed by incubation with an anti-rabbit conjugated to peroxidase (Bio-Rad, Hercules, CA, USA). The signal was visualized by chemoluminescence using the Immobilon Forte Western HRP substrate (Merck KGaA, Darmstadt, Germany). The band intensities were quantified using Processing and Analysis in Java, ImageJ 1.38b (Wayne Rasband, National Institute of Health, USA, http://www.rsb.info.nih.gov/ij/, accessed on 27 June 2021). Int. J. Mol. Sci. 2021, 22, 9556 16 of 20

4.8. MTT Assay The viability of 4C11+ melanoma cells—wild type, non-target (TRC1), sheNOS#1, sheNOS#2, and 4C11+ cells, treated or not with 40 µM L-sepiapterin—was evaluated using the standard MTT assay (Calbiochem, Hesse, Germany). Cells were harvest with trypsin ◦ and cultured on 96-well plates at 37 C in a humidified atmosphere containing 5% CO2. Cell viability was analyzed every 96 h when 5 mg/mL MTT was added to the culture. Cells ◦ were kept in an incubator at 37 C and 5% CO2 with MTT for one hour. After the medium was withdrawn, 100 µL of isopropanol (Merck, Hesse, Germany) was added to all wells and incubated for 15 min. Absorbance was quantified on a spectrophotometer at 620 nm (Multiskan EX, Labsystem, Vantaa, Finland). Cell viability was also evaluated in cells treated or not with 500 µM dacarbazine and 500 µM dacarbazine plus 40 µM L-sepiapterin for 48 h.

4.9. Colony Formation Assay 4C11+ melanoma cells—wild type, non-target (TRC1), sheNOS#1, sheNOS#2, and 4C11+ cells, treated or not with 40 µM L-sepiapterin—were harvested with mild trypsin treatment, and two hundred cells were plated on 60 mm dishes. After nine days, the plates were washed in PBS, fixed in 3.7% (v/v) formaldehyde for 15 min, washed with PBS, stained with 1% Toluidine Blue in 1% sodium tetraborate (borax) for five minutes, and washed with water. For the quantification of surviving cells, the dye was solubilized in 1% SDS under agitation for one hour, and the absorbance at 620 nm was measured using a spectrophotometer.

4.10. Anoikis Resistance Assay To estimate anoikis resistance, 4C11+ melanoma cells—wild type, non-target (TRC1), sheNOS#1, sheNOS#2, and 4C11+, treated or not with 40 µM L-sepiapterin—were main- tained for 96 h in 100 mm2 dishes coated with 1% agarose. After this time, cells were collected, centrifuged, and seeded in a 96-well plate. Cell viability was assessed by MTT after cell adhesion.

4.11. Tumor Growth Assay Cells (2 × 105) were subcutaneously injected into the flanks of 6-to-10-week-old C57BL/6 female mice (6 animals per group). Animals were kept under 12 h daylight cycles without water or food restriction, and tumor growth was observed and measured every two days. On day 21, mice were sacrificed, and the tumor weight was determined. Tumor volume (mm3) was measured using the formula d2 × D/2, where d represents the minimum diameter and D the maximum diameter. All procedures involving animals were performed after approval from the Research Ethics Committee of the Universidade Federal de São Paulo, Brazil (Approval no. 0219/07).

4.12. Statistical Analysis Data analysis was performed for two groups by Student’s t-test, and for three or more groups by analysis of variance (factorial ANOVA) with Bonferroni post-test, using the Graphpad Prism 7.0® statistical software (GraphPad, San Diego, CA, USA). The significance level was established at p < 0.05.

5. Conclusions In conclusion, eNOS uncoupling is involved in the maintenance of a pro-oxidant milieu in melanoma cells, which in turn contributes to melanoma progression. Moreover, eNOS uncoupling in melanoma cells is a result of disrupted BH4:eNOS stoichiometry. Targeting eNOS in melanoma cells might be considered as a potential target therapy, and further studies in this field are warranted. Int. J. Mol. Sci. 2021, 22, 9556 17 of 20

Supplementary Materials: The following are available online at https://www.mdpi.com/article/10 .3390/ijms22179556/s1. Author Contributions: Conceived and designed the experiments: F.H.M.d.M. and M.G.J. Per- formed the experiments: F.H.M.d.M., D.A.G., R.X.d.S., and M.Y.I. Analyzed the data: F.H.M.d.M., D.A.G., D.d.C.F., F.R.M.L., and M.G.J. Contributed reagents/materials/analysis: F.H.M.d.M., D.d.C.F., F.R.M.L., and M.G.J. Wrote the paper: F.H.M.d.M. and M.G.J. All authors have read and agreed to the published version of the manuscript. Funding: This work had financial support from Fundação de Amparo à Pesquisa do Estado de São Paulo—FAPESP (2014/13663-0). FHMM had been supported by Capes and FAPESP (2017/04352-0), RXS by FAPESP (2017/10695-7), and DAG by CNPq. Institutional Review Board Statement: The study was conducted according to the guidelines of the Declaration of Helsinki, and approved by the Ethics Committee of Universidade Federal de São Paulo. Approval No. 0219/07. Informed Consent Statement: Not applicable. Data Availability Statement: Not applicable. Conflicts of Interest: The authors declare no conflict of interest.

Abbreviations NOS: nitric oxide synthase; NO, nitric oxide; ROS, reactive oxygen species; BH4, tetrahydrobiopterin; BH2, dihydrobiopterin; GTPCH1, GTP cyclohydrolase protein; Gch1, cyclohydrolase gene; Pts, 6- pyruvoyltetrahydrobiopterin synthase gene; PTPS, 6-pyruvoyltetrahydrobiopterin synthase protein; Spr, sepiapterin reductase gene; SR, sepiapterin reductase protein; Gchfr, GTP feedback regulator gene; GFRP, GTP feedback regulator protein.

References 1. Lo, J.A.; Fisher, D.E. The Melanoma Revolution: From UV Carcinogenesis to a New Era in Therapeutics. Science 2014, 346, 945–949. [CrossRef][PubMed] 2. Rastrelli, M.; Tropea, S.; Rossi, C.R.; Alaibac, M. Melanoma: Epidemiology, Risk Factors, Pathogenesis, Diagnosis and Classifica- tion. In Vivo 2014, 28, 1005–1011. [PubMed] 3. Curti, B.D.; Faries, M.B. Recent Advances in the Treatment of Melanoma. N. Engl. J. Med. 2021, 89, 73–74. [CrossRef] 4. Strashilov, S.; Yordanov, A. Aetiology and Pathogenesis of Cutaneous Melanoma: Current Concepts and Advances. Int. J. Mol. Sci. 2021, 22, 6395. [CrossRef] 5. Campos, A.C.E.; Molognoni, F.; Melo, F.H.M.; Galdieri, L.C.; Carneiro, C.R.W.; D’Almeida, V.; Correa, M.; Jasiulionis, M.G. Oxida- tive Stress Modulates DNA Methylation during Melanocyte Anchorage Blockade Associated with Malignant Transformation. Neoplasia 2007, 9, 1111–1121. [CrossRef] 6. Melo, F.H.M.; Molognoni, F.; Morais, A.S.; Toricelli, M.; Mouro, M.G.; Higa, E.M.S.; Lopes, J.D.; Jasiulionis, M.G. Endothelial Nitric Oxide Synthase Uncoupling as a Key Mediator of Melanocyte Malignant Transformation Associated with Sustained Stress Conditions. Free Radic. Biol. Med. 2011, 50, 1263–1273. [CrossRef] 7. Molognoni, F.; De Melo, F.H.M.; Da Silva, C.T.; Jasiulionis, M.G. Ras and Rac1, Frequently Mutated in Melanomas, Are Activated by Superoxide Anion, Modulate Dnmt1 Level and Are Causally Related to Melanocyte Malignant Transformation. PLoS ONE 2013, 8, e81937. [CrossRef] 8. Bisevac, J.P.; Djukic, M.; Stanojevic, I.; Stevanovic, I.; Mijuskovic, Z.; Djuric, A.; Gobeljic, B.; Banovic, T.; Vojvodic, D. Association between Oxidative Stress and Melanoma Progression. J. Med. Biochem. 2018, 37, 12–20. [CrossRef][PubMed] 9. Gonçalves, D.A.; Xisto, R.; Gonçalves, J.D.; da Silva, D.B.; Moura Soares, J.P.; Icimoto, M.Y.; Sant’Anna, C.; Gimenez, M.; de Angelis, K.; Llesuy, S.; et al. Imbalance between Nitric Oxide and Superoxide Anion Induced by Uncoupled Nitric Oxide Synthase Contributes to Human Melanoma Development. Int. J. Biochem. Cell Biol. 2019, 115, 105592. [CrossRef] 10. Alves-Fernandes, D.K.; Jasiulionis, M.G. The Role of SIRT1 on DNA Damage Response and Epigenetic Alterations in Cancer. Int. J. Mol. Sci. 2019, 20, 3153. [CrossRef] 11. Ng, C.Y.; Yen, H.; Hsiao, H.-Y.; Su, S.-C. Phytochemicals in Skin Cancer Prevention and Treatment: An Updated Review. Int. J. Mol. Sci. 2018, 19, 941. [CrossRef] 12. Meitzler, J.L.; Makhlouf, H.R.; Antony, S.; Wu, Y.; Butcher, D.; Jiang, G.; Juhasz, A.; Lu, J.; Dahan, I.; Jansen-Dürr, P.; et al. Decoding NADPH Oxidase 4 Expression in Human Tumors. Redox Biol. 2017, 13, 182–195. [CrossRef] Int. J. Mol. Sci. 2021, 22, 9556 18 of 20

13. Chong, S.J.F.; Lai, J.X.H.; Qu, J.; Hirpara, J.; Kang, J.; Swaminathan, K.; Loh, T.; Kumar, A.; Vali, S.; Abbasi, T.; et al. A Feedforward Relationship between Active Rac1 and Phosphorylated Bcl-2 Is Critical for Sustaining Bcl-2 Phosphorylation and Promoting Cancer Progression. Cancer Lett. 2019, 457, 151–167. [CrossRef][PubMed] 14. Aminzadeh-gohari, S.; Weber, D.D.; Catalano, L.; Feichtinger, R.G.; Kofler, B.; Lang, R. Targeting Mitochondria in Melanoma. Biomolecules 2020, 10, 1395. [CrossRef] 15. Peng, H.; Zhuang, Y.; Chen, Y.; Rizzo, A.N.; Chen, W. The Characteristics and Regulatory Mechanisms of Superoxide Generation from ENOS Reductase Domain. PLoS ONE 2015, 10, e0140365. [CrossRef][PubMed] 16. Yuyun, M.F.; Ng, L.L.; Ng, G.A. Endothelial Dysfunction, Endothelial Nitric Oxide Bioavailability, Tetrahydrobiopterin, and 5-Methyltetrahydrofolate in Cardiovascular Disease. Where Are We with Therapy? Microvasc. Res. 2018, 119, 7–12. [CrossRef] [PubMed] 17. Takata, T.; Araki, S.; Tsuchiya, Y.; Watanabe, Y. Oxidative Stress Orchestrates Mapk and Nitric-Oxide Synthase Signal. Int. J. Mol. Sci. 2020, 21, 8750. [CrossRef] 18. Alp, N.J.; Channon, K.M. Regulation of Endothelial Nitric Oxide Synthase by Tetrahydrobiopterin in Vascular Disease. Arterioscler. Thromb. Vasc. Biol. 2004, 24, 413–420. [CrossRef][PubMed] 19. Kim, H.K.; Han, J. Tetrahydrobiopterin in Energy Metabolism and Metabolic Diseases. Pharmacol. Res. 2020, 157, 104827. [CrossRef] 20. Bendall, J.K.; Alp, N.J.; Warrick, N.; Cai, S.; Adlam, D.; Rockett, K.; Yokoyama, M.; Kawashima, S.; Channon, K.M. Stoichiometric Relationships between Endothelial Tetrahydrobiopterin, Endothelial NO Synthese (ENOS) Activity, and ENOS Coupling in Vivo: Insights from Transgenic Mice with Endothelial-Targeted GTP Cyclohydrolase 1 and ENOS Overexpression. Circ. Res. 2005, 97, 864–871. [CrossRef] 21. Crabtree, M.J.; Tatham, A.L.; Al-Wakeel, Y.; Warrick, N.; Hale, A.B.; Cai, S.; Channon, K.M.; Alp, N.J. Quantitative Regulation of Intracellular Endothelial Nitric-Oxide Synthase (ENOS) Coupling by Both Tetrahydrobiopterin-ENOS Stoichiometry and Biopterin Redox Status Insights from Cells with TET-Regulated GTP Cyclohydrolasei Expression. J. Biol. Chem. 2009, 284, 1136–1144. [CrossRef] 22. Tu, Y.T.; Tao, J.; Liu, Y.Q.; Li, Y.; Huang, C.Z.; Zhang, X.B.; Lin, Y. Expression of Endothelial Nitric Oxide Synthase and Vascular Endothelial Growth Factor in Human Malignant Melanoma and Their Relation to Angiogenesis. Clin. Exp. Dermatol. 2006, 31, 413–418. [CrossRef] 23. Li, C.; Hu, Z.; Liu, Z.; Wang, L.E.; Gershenwald, J.E.; Lee, J.E.; Prieto, V.G.; Duvic, M.; Grimm, E.A.; Wei, Q. Polymorphisms of the Neuronal and Inducible Nitric Oxide Synthase Genes and the Risk of Cutaneous Melanoma: A Case-Control Study. Cancer 2007, 109, 1570–1578. [CrossRef] 24. Thomas, D.D.; Wink, D.A. NOS2 as an Emergent Player in Progression of Cancer. Antioxidants Redox Signal. 2017, 26, 963–965. [CrossRef][PubMed] 25. Ekmekcioglu, S.; Ellerhorst, J.; Smid, C.M.; Prieto, V.G.; Munsell, M.; Buzaid, A.C.; Grimm, E.A. Inducible Nitric Oxide Synthase and Nitrotyrosine in Human Metastatic Melanoma Tumors Correlate with Poor Survival. Clin. Cancer Res. 2000, 6, 4768–4775. [PubMed] 26. Ekmekcioglu, S.; Ellerhorst, J.A.; Prieto, V.G.; Johnson, M.M.; Broemeling, L.D.; Grimm, E.A. Tumor INOS Predicts Poor Survival for Stage III Melanoma Patients. Int. J. Cancer 2006, 119, 861–866. [CrossRef] 27. Ahmed, B.; Van Den Oord, J.J. Expression of the Neuronal Isoform of Nitric Oxide Synthase (NNOS) and Its Inhibitor, Protein Inhibitor of NNOS, in Pigment Cell Lesions of the Skin. Br. J. Dermatol. 1999, 141, 12–19. [CrossRef] 28. Oba-Shinjo, S.M.; Correa, M.; Ricca, T.I.; Molognoni, F.; Pinhal, M.A.; Neves, I.A.; Marie, S.K.; Sampaio, L.O.; Nader, H.B.; Chammas, R.; et al. Melanocyte Transformation Associated with Substrate Adhesion Impediment. Neoplasia 2006, 8, 231–241. [CrossRef][PubMed] 29. Tang, Z.; Kang, B.; Li, C.; Chen, T.; Zhang, Z. GEPIA2: An Enhanced Web Server for Large-Scale Expression Profiling and Interactive Analysis. Nucleic Acids Res. 2019, 47, W556. [CrossRef] 30. Talantov, D.; Mazumder, A.; Yu, J.X.; Briggs, T.; Jiang, Y.; Backus, J.; Atkins, D.; Wang, Y. Novel Genes Associated with Malignant Melanoma but Not Benign Melanocytic Lesions. Clin. Cancer Res. 2005, 11, 7234–7242. [CrossRef][PubMed] 31. Gesierich, A.; Niroomand, F.; Tiefenbacher, C.P. Role of Human GTP Cyclohydrolase I and Its Regulatory Protein in Tetrahydro- biopterin Metabolism. Basic Res. Cardiol. 2003, 98, 69–75. [CrossRef][PubMed] 32. Tatham, A.L.; Crabtree, M.J.; Warrick, N.; Cai, S.; Alp, N.J.; Channon, K.M. GTP Cyclohydrolase I Expression, Protein, and Activity Determine Intracellular Tetrahydrobiopterin Levels, Independent of GTP Cyclohydrolase Feedback Regulatory Protein Expression. J. Biol. Chem. 2009, 284, 13660–13668. [CrossRef] 33. Zhen, J.; Lu, H.; Wang, X.Q.; Vaziri, N.D.; Zhou, X.J. Upregulation of Endothelial and Inducible Nitric Oxide Synthase Expression by Reactive Oxygen Species. Am. J. Hypertens. 2008, 21, 28–34. [CrossRef] 34. Ji, X.W.; Lyu, H.J.; Zhou, G.H.; Wu, B.; Zhu, Y.Y.; Wu, T.H.; Zhang, F.; Jin, S.N.; Cho, K.W.; Wen, J.F. Physcion, a Tetra-Substituted 9,10-Anthraquinone, Prevents Homocysteine-Induced Endothelial Dysfunction by Activating Ca2+- and Akt-ENOS-NO Signaling Pathways. Phytomedicine 2021, 81, 153410. [CrossRef][PubMed] 35. Ozaki, M.; Kawashima, S.; Yamashita, T.; Hirase, T.; Namiki, M.; Inoue, N.; Hirata, K.; Yasui, H.; Sakurai, H.; Yoshida, Y.; et al. Overexpression of Endothelial Nitric Oxide Synthase Accelerates Atherosclerotic Lesion Formation in ApoE-Deficient Mice. J. Clin. Invest. 2002, 110, 331–340. [CrossRef][PubMed] Int. J. Mol. Sci. 2021, 22, 9556 19 of 20

36. Riker, A.I.; Enkemann, S.A.; Fodstad, O.; Liu, S.; Ren, S.; Morris, C.; Xi, Y.; Howell, P.; Metge, B.; Samant, R.S.; et al. The Gene Expression Profiles of Primary and Metastatic Melanoma Yields a Transition Point of Tumor Progression and Metastasis. BMC Med. Genom. 2008, 1, 13. [CrossRef][PubMed] 37. Thöny, B.; Auerbach, G.; Blau, N. Tetrahydrobiopterin Biosynthesis, Regeneration and Functions. Biochem. J. 2000, 347, 1–16. [CrossRef] 38. Katusic, Z.S.; Stelter, A.; Milstien, S. Cytokines Stimulate GTP Cyclohydrolase I Gene Expression in Cultured Human Umbilical Vein Endothelial Cells. Arterioscler. Thromb. Vasc. Biol. 1998, 18, 27–32. [CrossRef][PubMed] 39. Shimizu, S.; Hiroi, T.; Ishii, M.; Hagiwara, T.; Wajima, T.; Miyazaki, A.; Kiuchi, Y. Hydrogen Peroxide Stimulates Tetrahydro- biopterin Synthesis through Activation of the Jak2 Tyrosine Kinase Pathway in Vascular Endothelial Cells. Int. J. Biochem. Cell Biol. 2008, 40, 755–765. [CrossRef] 40. Chen, L.; Zeng, X.; Kleibeuker, E.; Buffa, F.; Barberis, A.; Leek, R.D.; Roxanis, I.; Zhang, W.; Worth, A.; Beech, J.S.; et al. Paracrine Effect of GTP Cyclohydrolase and Angiopoietin-1 Interaction in Stromal Fibroblasts on Tumor Tie2 Activation and Breast Cancer Growth. Oncotarget 2016, 7, 9353–9367. [CrossRef] 41. Tran, A.N.; Walker, K.; Harrison, D.G.; Chen, W.; Mobley, J.; Hocevar, L.; Hackney, J.R.; Sedaka, R.S.; Pollock, J.S.; Goldberg, M.S.; et al. Reactive Species Balance via GTP Cyclohydrolase i Regulates Glioblastoma Growth and Tumor Initiating Cell Maintenance. Neuro. Oncol. 2018, 20, 1055–1067. [CrossRef] 42. Chavan, B.; Gillbro, J.M.; Rokos, H.; Schallreuter, K.U. GTP Cyclohydrolase Feedback Regulatory Protein Controls Cofactor 6-Tetrahydrobiopterin Synthesis in the Cytosol and in the Nucleus of Epidermal Keratinocytes and Melanocytes. J. Investig. Dermatol. 2006, 126, 2481–2489. [CrossRef] 43. Kalivendi, S.; Hatakeyama, K.; Whitsett, J.; Konorev, E.; Kalyanaraman, B.; Vásquez-Vivar, J. Changes in Tetrahydrobiopterin Levels in Endothelial Cells and Adult Cardiomyocytes Induced by LPS and Hydrogen Peroxide—A Role for GFRP? Free Radic. Biol. Med. 2005, 38, 481–491. [CrossRef][PubMed] 44. Zhao, Q.; Zheng, K.; Ma, C.; Li, J.; Zhuo, L.; Huang, W.; Chen, T.; Jiang, Y. PTPS Facilitates Compartmentalized LTBP1 S-Nitrosylation and Promotes Tumor Growth under Hypoxia. Mol. Cell 2020, 77, 95.e5–107.e5. [CrossRef][PubMed] 45. De Souza, C.F.; Xander, P.; Monteiro, A.C.; dos Santos Silva, A.G.; da Silva, D.C.P.; Mai, S.; Bernardo, V.; Lopes, J.D.; Jasiulionis, M.G. Mining Gene Expression Signature for the Detection of Pre-Malignant Melanocytes and Early Melanomas with Risk for Metastasis. PLoS ONE 2012, 7, e44800. [CrossRef][PubMed] 46. Lange, I.; Geerts, D.; Feith, D.J.; Mocz, G.; Koster, J.; Bachmann, A.S. Novel Interaction of Ornithine Decarboxylase with Sepiapterin Reductase Regulates Neuroblastoma Cell Proliferation. J. Mol. Biol. 2014, 426, 332–346. [CrossRef] 47. Wu, Y.; Chen, P.; Sun, L.; Yuan, S.; Cheng, Z.; Lu, L.; Du, H.; Zhan, M. Sepiapterin Reductase: Characteristics and Role in Diseases. J. Cell. Mol. Med. 2020, 24, 9495–9506. [CrossRef] 48. Zhang, X.; Chen, Y.; Wang, K.; Tang, J.; Chen, Y.; Jin, G.; Liu, X. The Knockdown of the Sepiapterin Reductase Gene Suppresses the Proliferation of Breast Cancer by Inducing ROS-Mediated Apoptosis. Int. J. Clin. Exp. Pathol. 2020, 13, 2228–2239. 49. Harada, T.; Kagamiyama, H.; Hatakeyama, K. Feedback Regulation Mechanisms for the Control of GTP Cyclohydrolase I Activity. Science 1993, 260, 1507–1510. [CrossRef] 50.V ásquez-Vivar, J. Tetrahydrobiopterin, Superoxide and Vascular Dysfunction. Free Radic. Biol. Med. 2009, 47, 1108. [CrossRef] 51. Crabtree, M.J.; Smith, C.L.; Lam, G.; Goligorsky, M.S.; Gross, S.S. Ratio of 5,6,7,8-Tetrahydrobiopterin to 7,8-Dihydrobiopterin in Endothelial Cells Determines Glucose-Elicited Changes in NO vs. Superoxide Production by ENOS. Am. J. Physiol. Heart Circ. Physiol. 2008, 294.[CrossRef] 52. Lim, K.H.; Ancrile, B.B.; Kashatus, D.F.; Counter, C.M. Tumour Maintenance Is Mediated by ENOS. Nature 2008, 452, 646–649. [CrossRef] 53. Lampson, B.L.; Kendall, S.D.S.; Ancrile, B.B.; Morrison, M.M.; Shealy, M.J.; Barrientos, K.S.; Crowe, M.S.; Kashatus, D.F.; White, R.R.; Gurley, S.B.; et al. Targeting ENOS in Pancreatic Cancer. Cancer Res. 2012, 72, 4472–4482. [CrossRef][PubMed] 54. Hodis, E.; Watson, I.R.; Kryukov, G.V.; Arold, S.T.; Imielinski, M.; Theurillat, J.P.; Nickerson, E.; Auclair, D.; Li, L.; Place, C.; et al. A Landscape of Driver Mutations in Melanoma. Cell 2012, 150, 251–263. [CrossRef][PubMed] 55. Montor, W.R.; Salas, A.R.O.S.E.; de Melo, F.H.M. Receptor Tyrosine Kinases and Downstream Pathways as Druggable Targets for Cancer Treatment: The Current Arsenal of Inhibitors. Mol. Cancer 2018, 17, 55. [CrossRef][PubMed] 56. Paluncic, J.; Kovacevic, Z.; Jansson, P.J.; Kalinowski, D.; Merlot, A.M.; Huang, M.L.H.; Lok, H.C.; Sahni, S.; Lane, D.J.R.; Richardson, D.R. Roads to Melanoma: Key Pathways and Emerging Players in Melanoma Progression and Oncogenic Signaling. Biochim. Biophys. Acta Mol. Cell Res. 2016, 1863, 770–784. [CrossRef] 57. Barbieri, A.; Palma, G.; Rosati, A.; Giudice, A.; Falco, A.; Petrillo, A.; Petrillo, M.; Bimonte, S.; Di Benedetto, M.; Esposito, G.; et al. Role of Endothelial Nitric Oxide Synthase (ENOS) in Chronic Stress-Promoted Tumour Growth. J. Cell. Mol. Med. 2012, 16, 920–926. [CrossRef] 58. Teng, R.J.; Du, J.; Xu, H.; Bakhutashvili, I.; Eis, A.; Shi, Y.; Pritchard, K.A.; Konduri, G.G. Sepiapterin Improves Angiogenesis of Pulmonary Artery Endothelial Cells with in Utero Pulmonary Hypertension by Recoupling Endothelial Nitric Oxide Synthase. Am. J. Physiol. Lung Cell. Mol. Physiol. 2011, 301, 334–345. [CrossRef] 59. Cardnell, R.J.G.; Rabender, C.S.; Ross, G.R.; Guo, C.; Howlett, E.L.; Alam, A.; Wang, X.Y.; Akbarali, H.I.; Mikkelsen, R.B. Sepiapterin Ameliorates Chemically Induced Murine Colitis and Azoxymethane-Induced Colon Cancer. J. Pharmacol. Exp. Ther. 2013, 347, 117–125. [CrossRef] Int. J. Mol. Sci. 2021, 22, 9556 20 of 20

60. Rabender, C.S.; Alam, A.; Sundaresan, G.; Cardnell, R.J.; Yakovlev, V.A.; Mukhopadhyay, N.D.; Graves, P.; Zweit, J.; Mikkelsen, R.B. The Role of Nitric Oxide Synthase Uncoupling in Tumor Progression. Mol. Cancer Res. 2015, 13, 1034–1043. [CrossRef] 61. Fernandes, D.C.; Wosniak, J.; Pescatore, L.A.; Bertoline, M.A.; Liberman, M.; Laurindo, F.R.M.; Santos, C.X.C. Analysis of DHE-Derived Oxidation Products by HPLC in the Assessment of Superoxide Production and NADPH Oxidase Activity in Vascular Systems. Am. J. Physiol. Cell Physiol. 2007, 292.[CrossRef][PubMed] 62. Marinos, R.S.; Zhang, W.; Wu, G.; Kelly, K.A.; Meininger, C.J. Tetrahydrobiopterin Levels Regulate Endothelial Cell Proliferation. Am. J. Physiol. Hear. Circ. Physiol. 2001, 281, H482–H489. [CrossRef][PubMed]