Acid Sphingomyelinase Downregulation Enhances Mitochondrial Fusion and Promotes Oxidative Metabolism in a Mouse Model of Melanoma.

Melanoma is the most severe type of skin cancer. Its unique and heterogeneous metabolism, relying on both glycolysis and oxidative phosphorylation, allows it to adapt to disparate conditions. Mitochondrial function is strictly interconnected with mitochondrial dynamics and both are fundamental in tumour progression and metastasis. The malignant phenotype of melanoma is also regulated by the expression levels of the enzyme acid sphingomyelinase (A-SMase). By modulating at transcriptional level A-SMase in the melanoma cell line B16-F1 cells, we assessed the effect of enzyme downregulation on mitochondrial dynamics and function. Our results demonstrate that A-SMase influences mitochondrial morphology by affecting the expression of mitofusin 1 and OPA1. The enhanced expression of the two mitochondrial fusion proteins, observed when A-SMase is expressed at low levels, correlates with the increase of mitochondrial function via the stimulation of the genes PGC-1alpha and TFAM, two genes that preside over mitochondrial biogenesis. Thus, the reduction of A-SMase expression, observed in malignant melanomas, may determine their metastatic behaviour through the stimulation of mitochondrial fusion, activity and biogenesis, conferring a metabolic advantage to melanoma cells.


Introduction
Whereas the majority of malignant tumours rely on enhanced glycolysis for energy supply (i.e., the Warburg effect) [1], melanoma, the most aggressive form of skin cancer, has a unique metabolism, orchestrated by its environment and specific signalling mutations [2][3][4][5]. Although melanoma cells can rely on anaerobic metabolism, oxidative phosphorylation (OxPhos) also plays a role, which becomes critical in defined subsets of melanomas [6][7][8]. Increasing evidence indicates that mitochondrial respiration contributes to transformation, development of drug resistance and metastasis in melanomas, thus defining a pleiotropic role of mitochondria in tumourigenesis [9][10][11][12][13]. In normal and cancer cells, mitochondria exist in a dynamic network resulting from the interplay between fission and fusion events [14,15], governed by nutrient levels and energy demands [16]. In fragmented mitochondria, when fission exceeds fusion, oxidative metabolism is reduced and glycolytic intermediates are preserved for a highly activated glycolysis to provide fuel for cell proliferation [17,18]. In contrast, when mitochondrial fusion prevails, the outcome is an extension in the mitochondrial network, an event that provides specific metabolic advantages to cells under high energy needs, such as metastatic cells. The metabolic shift towards a more pronounced aerobic activity [18,19] also accounts for tumour resistance to drugs such as B-RAF inhibitors [20][21][22]. In mammalian cells, the primary players in mitochondrial morphology are mitofusin1 and mitofusin2 (Mfn1, Mfn2), and optic atrophy 1 (OPA1), which are essential for outer and inner mitochondrial membrane fusion, respectively, and dynamin-related protein 1 (Drp1), which is essential for the process of fission [14,15,19,23].
The malignant phenotype of melanoma, in terms of tumour progression and metastatic capacity, is also regulated by sphingolipids [24,25], in particular those originating from the activity of the lysosomal hydrolase acid sphingomyelinase (A-SMase), an enzyme responsible for the conversion of sphingomyelin to ceramide, a well-known pro-apoptotic molecule in both normal and cancer cells [26]. In melanoma cells, sphingolipid signalling is deregulated so as to prevent the accumulation of ceramide and protect cells from apoptosis [27,28]. One of the mechanism adopted by melanoma to promote cell proliferation and resistance to apoptosis is the downregulation of A-SMase expression and activity during tumour progression [29][30][31]. Although the role of mitochondria and A-SMase in melanoma share some similarities, and the localisation of A-SMase to mitochondria has been suggested [32], whether regulation by A-SMase of mitochondria explains its function in melanoma has never been studied.
Here, we report on a pivotal role of A-SMase in determining mitochondrial morphology and bioenergetics in a mouse model of melanoma cells. The reduction of A-SMase expression, reported to prompt progression of melanomas and influence their metastatic behaviour [31], leads to an increase of mitochondrial fusion, activity and biogenesis, thus conferring a selective metabolic advantage to melanoma cells.

Cell Models
The cell clones used in the in vivo experiments were generated from the murine melanoma cell line B16-F1 of the American Type Culture Collection. As described previously [31], the B16-W6_pSIL10 clone was generated from a subclone of the parental cell line B16-F1 (i.e., B16-W6) by transfecting the cells with the plasmid pSilencer4.1-CMV (Invitrogen-Life Technologies, Monza, Italy) containing a shRNA sequence responsible for A-SMase silencing. The control clone B16-pSILscr was generated by transfecting B16-W6 cells with the plasmid pSilencer4.1-CMV containing a shRNA scrambled sequence. The B16-B9 clone was also used as control of naturally occurring down-regulation of A-SMase.

Animal Handling and Allograft Tumour Model
Female C57BL/6 mice (6-8 weeks old) were purchased from Charles River Laboratories (Calco, Italy), housed in a regulated environment (23 ± 1 • C, 50% ± 5% humidity) with a 12 h light/dark cycle, and provided with food and water ad libitum. On day 0, mice were injected sub-cutaneously with a tumourigenic dose of 2.5 × 10 4 melanoma cells in the lower-right flank [29,30,33,34]. Before the injection, melanoma cells were controlled for their levels of expression of A-SMase. Tumour growth was monitored every 2-3 days by means of a caliper and the volume was calculated (length × width 2/2). Mice were sacrificed when the tumour size reached ca. 500 mm 3 volume; the tumour was then collected for transmission electron microscopy analyses. All studies were conducted in accordance with the Italian law on animal care N • 116/1992 and the European Communities Council Directive EEC/609/86. The experimental protocols (01/11) were approved by the Ethics Committee of the University of Milan. All efforts were made to reduce both animal suffering and the number of animals used.

Transmission Electron Microscopy
Samples collected from melanoma transplants were reduced into smaller blocks and fixed overnight at 4 • C in a solution containing 2% formaldehyde and 2% glutaraldehyde in 0.1 M sodium cacodylate buffer, pH 7.3. Specimens were washed in cacodylate buffer and postfixed at 0 • C for 1.5 h in 2% osmium tetroxide. The samples were washed in distilled water, stained en bloc in 2% aqueous uranyl acetate, dehydrated through an ascending series of ethanol, and embedded in Epon Araldite resin. For ultrastructural observations, at least 5 ultra-thin sections (60-90 nm) were obtained from each tumour. Sections were collected on 100-mesh grids, counterstained with lead citrate, and photographed (magnification 2500×) with an EM 10 electron microscope (Carl Zeiss, Oberkochen, Germany). Micrographs were scanned in a flat-bed scanner and images were merged.
For each melanoma transplant, at least 40,000 µm 2 of each section area was analysed (Image ProPlus 6.0 software, Media Cybernetics, Bethesda, MD). Software was used to manually trace mitochondrial length, mitochondrial area and cytoplasm area of cells.

Immunofluorescence and Mitochondria Morphometric Analysis
B16-F1_siASM and B16-F1_scr cells, for a set of experiments, and B16-W6 and B16-B9 cells, for another, cultured in 120-mm coverslips were fixed in 4% paraformaldehyde in 0.1 M phosphate buffer (PB), pH 7.4, for 10 min. Samples were then washed in PB and pre-incubated for 30 min at room temperature with 5% bovine serum albumin (BSA; Life Technologies, Monza, Italy) and 10% of normal goat serum (Life Technologies) in PB containing 0.1% Triton X-100. Subsequently, samples were stained overnight at 4 • C with mouse anti-cyclophilin f (#ab110324; Abcam, Cambridge, UK) primary antibody at a dilution of 1:200 in PB containing 0.1% Triton X-100. Cells were then stained for 1 h at room temperature with the appropriate Alexa Fluor secondary antibody with or without the cytopainter phalloidin-ifluor 555 (#ab176756; Abcam) that binds to F-actin filaments (cytoskeleton detection). Finally, samples were cover-slipped in a ProLong Gold Antifade Mountant with or without DAPI (Life Technologies). Images were acquired with a Zeiss LSM 710 confocal microscope (Carl Zeiss) [35,36].
To quantify mitochondrial morphology, an ImageJ macro was used [37]. In brief, acquired images were converted to binary and the signal from cyclophylin (green) was optimised to a threshold allowing resolution of individual mitochondria. The macro "analyze particles" was applied to detect mitochondrial outlines and quantify both mitochondrial size and interconnectivity for each mitochondrion. The mean area/perimeter ratio was used as an index of mitochondrial interconnectivity where high values indicate mitochondria with many physical interactions, while low values are indicative of single mitochondria. For the analysis of mitochondrial branching, the binary image was converted to a skeleton by using the "skeletonize" plugin. Finally, the length of each branch and the number of branches were determined by using the "analyze skeleton" plugin [38].

Quantitative Real Time-PCR (qPCR)
The analysis of mRNA expression was performed as previously described [31,33,35,39,40]. Briefly, total RNA from cells was extracted with the PureZol RNA Isolation Reagent (Bio-Rad, Hercules, CA, USA), according to the manufacturer's protocol. First-strand cDNA was generated from 1 µg of total RNA using the iScript Reverse Transcription Supermix (Bio-Rad, Hercules, CA, USA). A set of primer pairs (Eurofins Genomics, Milan, Italy) was designed to hybridise to unique regions of the appropriate gene sequence (Table 1). qPCR was performed using the SsoAdvanced Universal SYBR Green Supermix and the CFX96 Touch Real-Time PCR Detection System (Bio-Rad, Hercules, CA, USA). The fold change was determined relative to the control after normalisation to internal standards (actin, rpl32, 36b4) through the use of the formula 2 −∆∆CT . Mitochondrial DNA (mtDNA) was quantified as previously described [41,42]. Total DNA was isolated from cells using the QIAamp DNA mini kit (Qiagen, Milano, Italy) according to the protocol provided by the manufacturer. The mtDNA content was measured by quantitative PCR, normalising the quantity of a not-polymorphic mtDNA with a single copy nuclear gene (RNAse P). Table 1. List of primers designed for PCR.

Mitochondria Respiratory Rate
Mitochondria respiratory rates were measured into the O2K oxygraph chambers (Oroboros Instruments, Innsbruck, Austria) at 37 • C in the respiration medium MiR06 (0.5 mM EGTA, 3 mM MgCl2, 60 mM K-lactobionate, 20 mM taurine, 10 mM KH2PO4 20 mM Hepes, 110 mM sucrose and 1 g/L bovine serum albumin fatty acid-free, 280 U/mL catalase (pH 7.1)) [37,41,42,47]. Basal respiration was observed when the signal of oxygen consumption was stable, while the leak respiration was induced after the addition of oligomycin (0.5 µM). Stepwise titration (0.5 µM each step) of the uncoupler carbonyl cyanide-4-(trifluoromethoxy) phenylhydrazone (FCCP) induced the progressive release of the proton gradient until maximal respiration was achieved. The residual oxygen consumption was evaluated by blocking mitochondrial respiration with the addition of 0.5 µM rotenone and 2.5 µM antimycin A (AA), and this value was subtracted by each steady-state. Coupling efficiency was the ratio: (Basal Respiration -Leak respiration)/Basal respiration.

Mitochondrial Membrane Potential Analysis
Mitochondrial potential was measured by cell staining with 500 nM of the sensitive fluorescent dye, TMRM (Sigma-Aldrich, Saint Louis, MO, USA), as previously described [33,48,49]. Fluorescence was analysed by a Gallios Flow Cytometer (Beckman-Coulter, Brea, CA, USA) and the software FCS Express 4 (De Novo System, Portland, OR, USA).

Statistical Analysis
Statistical significance of differences between the groups was evaluated using Student's t-test (single comparisons) or one-way ANOVA, followed by the Newman-Keuls post-test (multiple comparisons). When data were not normally distributed and the variance between the samples differed significantly, the Mann-Whitney test or the Wilconox test was used. Data belonging from different experiments were represented and averaged in the same graph. The GraphPad Prism software package (Graph Software, San Diego, CA, USA) was used. The results were expressed as means ± SEM of the indicated n values.

A-SMase Expression Determines Mitochondrial Morphology
Depending on the cell type and physiological conditions, mitochondria can be present either as numerous morphologically distinct small organelles, or they can form large interconnected networks [14,15]. To assess the relationship between A-SMase expression and mitochondrial morphology, we used transmission electron microscopy to analyse the shape of the mitochondria of explanted murine melanoma allografts. To this end, mice were injected subcutaneously with B16-W6_pSIL10 cells, a cell clone obtained by constitutively knocking-down A-SMase in B16-F1 cells [29][30][31]. B16-pSILscr cells transfected with the scrambled vector were used as a control. By ultrastructural analysis we found that control tumours presented mostly rounded and small mitochondria, while elongated and more tubular mitochondria accumulated in B16-W6_pSIL10 melanomas ( Figure 1A). The measurement of the mitochondria mean length and area confirmed that these organelles were significantly longer and larger in the absence of A-SMase ( Figure 1B). The effect of A-SMase expression levels on mitochondrial shape was also confirmed in allograft melanomas derived from the B16-B9 clone, in which A-SMase is expressed naturally at low levels. In these tumours, mitochondria appear round, rather elongated in shape, and with a larger area (Supplementary Figure  S1A).

A-SMase Expression Regulates Mitochondrial Elongation through Mfn1 and OPA1
Given our initial observation, we aimed to determine whether the differences in mitochondrial size observed in explanted tumours ( Figure 1A,B) depended on A-SMase expression and, if so, the

A-SMase Expression Regulates Mitochondrial Elongation through Mfn1 and OPA1
Given our initial observation, we aimed to determine whether the differences in mitochondrial size observed in explanted tumours ( Figure 1A,B) depended on A-SMase expression and, if so, the mechanism behind this event. To this end, we analysed in vitro the effect of A-SMase silencing on mitochondrial morphology by transiently transfecting B16-F1 cells with a siRNA specific for A-SMase (B16-F1_siASM cells) (Figure 2A) [31]. We found that the downregulation of A-SMase resulted in an increased percentage of cells with elongated mitochondria which were characterised by augmented interconnectivity, number of branches and branch length compared to scrambled control (B16-F1_scr) ( Figure 2B,C). These results are in line with those obtained in the two clones derived from B16-F1 cells expressing A-SMase at low (B19-B9) and high levels (B16-W6). B19-B9 cells displayed a mitochondrial network with elongated mitochondria, similar to that observed in B16-F1_siASM cells, while B16-W6 showed more rounded mitochondria (Supplementary Figure S1B). All these data confirm further that A-SMase expression affects mitochondrial morphology.
The balance of mitochondrial fusion and fission dictates the morphology, abundance, function and spatial distribution of mitochondria. Therefore, we analysed the expression of the players of mitochondrial fusion, i.e., Mfn1, Mfn2 and OPA1 and fission i.e., Drp1 [14,15,19,23]. We found that the expression of Mfn1 and OPA1 at both the mRNA and protein level increased significantly in B16-F1_siASM cells, while no differences were observed for the mRNA of Mfn2 and Drp1 ( Figure 3A,B). On the contrary, the analysis of Mnf1 and OPA1 in a clone overexpressing A-SMase (B16_B1A) showed that the increase of A-SMase expression induced a reduction of the two markers of mitochondrial fusion (Supplementary Figure S1C).
To better understand this mechanism, we investigated whether the microphtalmia-associated transcription factor (Mitf), a key target of A-SMase action in melanoma [31], was involved in mitochondrial dynamics and their changes. As shown in Figure 3C, we found increased levels of Mitf mRNA in B16-F1_siASM cells when compared to B16-F1_scr cells ( Figure 3C), further confirming its dependency on A-SMase expression. Notably, the silencing of Mitf ( Figure 3D) in B16-F1 cells induced a significant inhibition of Mfn1 and OPA1 mRNA expression ( Figure 3D), therefore acting, as expected, in an opposite manner to A-SMase. To further explore the Mitf-dependency in the context of the A-SMase pathway, we silenced A-SMase and Mitf together in B16-F1 cells (B16-F1_siASM/siMitf) (Supplementary Figure S2A,B), finding that silencing Mitf completely abolished the effect of A-SMase down-regulation on Mfn1 and OPA1 expression ( Figure 3D).
Taken together, these data indicate that low levels of A-SMase expression, as reported to occur in melanoma [31], are a key determinant of mitochondrial morphology, acting by increasing mitochondrial fusion through Mitf upregulation.

A-SMase Downregulation Improves Mitochondrial Function
Mitochondrial dynamics are regulated by cellular bioenergetic demands. Mitochondria are the major source of ATP and metabolites necessary to fulfill the bioenergetics and biosynthetic requirements of cells [50,51]. Thus, we evaluated whether the changes in mitochondrial morphology following A-SMase silencing might affect mitochondrial activity.
In B16-F1_siASM cells, we found an increased production of ATP via OxPhos, sustained by an improved mitochondrial coupling efficiency ( Figure 4A). This event depends on the activation of the respiratory chain and not on upstream biochemical pathways. Indeed, no differences were observed in the expression of genes encoding for proteins involved in glycolysis and Krebs cycles, and no modifications were detected in the production of glycolytic ATP ( Figure 4B,C). No changes were noticed in the mitochondrial membrane potential of B16-F1_siASM compared to B16-F1_scr, measured by loading the cells with the potentiometric probe tetramethylrhodamine methyl ester (TMRM) [33] ( Figure 4D).
A-SMase silencing reduces the sensitivity to Cisplatin of B16 melanoma cells [29]. Therefore, we assessed whether mitochondria could be involved in melanoma cell response to the chemotherapeutic drug. Cisplatin (10 µg/mL for 16 h) enhanced OxPhos ATP production and mitochondrial membrane potential in B16-F1_siASM cells compared to B16-F1_scr ( Figure 4E,F). This indicates that A-SMase has a role in the cytotoxic response to Cisplatin, and that this goes through the activation of mitochondrial pathways.

A-SMase Downregulation Improves Mitochondrial Function
Mitochondrial dynamics are regulated by cellular bioenergetic demands. Mitochondria are the major source of ATP and metabolites necessary to fulfill the bioenergetics and biosynthetic requirements of cells [50,51]. Thus, we evaluated whether the changes in mitochondrial morphology following A-SMase silencing might affect mitochondrial activity.
In B16-F1_siASM cells, we found an increased production of ATP via OxPhos, sustained by an improved mitochondrial coupling efficiency ( Figure 4A). This event depends on the activation of the respiratory chain and not on upstream biochemical pathways. Indeed, no differences were observed in the expression of genes encoding for proteins involved in glycolysis and Krebs cycles, and no modifications were detected in the production of glycolytic ATP ( Figure 4B,C). No changes were noticed in the mitochondrial membrane potential of B16-F1_siASM compared to B16-F1_scr, measured by loading the cells with the potentiometric probe tetramethylrhodamine methyl ester (TMRM) [33] (Figure 4D).
A-SMase silencing reduces the sensitivity to Cisplatin of B16 melanoma cells [29]. Therefore, we assessed whether mitochondria could be involved in melanoma cell response to the chemotherapeutic drug. Cisplatin (10 µg/mL for 16 h) enhanced OxPhos ATP production and mitochondrial membrane potential in B16-F1_siASM cells compared to B16-F1_scr ( Figure 4E,F). This indicates that A-SMase has a role in the cytotoxic response to Cisplatin, and that this goes through the activation of mitochondrial pathways.

A-SMase Downregulation Increases Mitochondrial Biogenesis
The increase of mitochondrial mass by mitochondrial biogenesis is a mechanism that promotes metastasis and resistance to chemotherapy in different cancers [9,[52][53][54].
The silencing of A-SMase did not lead to changes in mitochondrial content, measured via the analysis of mitochondrial DNA (mtDNA) in B16-F1_scr and B16-F1_siASM cells ( Figure 5A). This datum was confirmed by the expression of genes encoding different electron transport chain subunits, namely, COX subunit I and IV (COX I and COX IV), cytochrome b and c (CYT B and CYT C), and ATPase ( Figure 5B). However, when we analysed the expression of the major genes involved in the mitochondrial biogenesis machinery, we found in A-SMase silenced cells a higher level of mRNA of the peroxisome proliferator activated receptor-gamma co-activator 1 alpha (PGC-1alpha), as well as one of its downstream targets, the mitochondrial transcription factor A (TFAM) ( Figure 5C). Of note, Mitf silencing alone or in combination with the downregulation of A-SMase in B16-F1 cells reduces the expression of both PGC-1alpha and TFAM ( Figure 5D), indicating that ASMase levels affect the mitochondrial biogenesis system through the regulation of Mitf.

Discussion
The therapeutic potential of targeting sphingolipid metabolism in melanoma, a tumour for which a resolutive cure is still lacking and with a high risk of local or disseminated recurrence after surgical excision of the primary tumour [55], has been previously demonstrated [24,27,[56][57][58]. Notably, some studies showed the involvement of A-SMase in critical phases of melanoma development such as proliferation, migration, the ability to metastasise, and the response to chemotherapy [29][30][31][59][60][61][62][63].
In the current study, we evaluated for the first time the role of A-SMase in the regulation of mitochondrial function and morphology in a mouse model of melanoma cells. We discovered that the A-SMase expression level is correlated with mitochondrial elongation, which is concurrent with an increased oxidative phosphorylation and the activation of the biogenesis machinery.
We found that A-SMase impacts mitochondrial elongation by regulating the expression of mitochondrial fusion proteins. A-SMase silencing promoted mitochondrial fusion by enhancing the expression of Mfn-1 and OPA1. A recent study showed that expression levels of the proteins involved in mitochondrial fusion and fission differ among melanoma cells [19]. In particular, this study revealed that inhibiting mitochondrial fusion in human melanoma cells significantly decreases oxygen consumption rate, thus indicating that oxidative phosphorylation depends on mitochondrial fusion in melanoma cells.

Discussion
The therapeutic potential of targeting sphingolipid metabolism in melanoma, a tumour for which a resolutive cure is still lacking and with a high risk of local or disseminated recurrence after surgical excision of the primary tumour [55], has been previously demonstrated [24,27,[56][57][58]. Notably, some studies showed the involvement of A-SMase in critical phases of melanoma development such as proliferation, migration, the ability to metastasise, and the response to chemotherapy [29][30][31][59][60][61][62][63].
In the current study, we evaluated for the first time the role of A-SMase in the regulation of mitochondrial function and morphology in a mouse model of melanoma cells. We discovered that the A-SMase expression level is correlated with mitochondrial elongation, which is concurrent with an increased oxidative phosphorylation and the activation of the biogenesis machinery.
We found that A-SMase impacts mitochondrial elongation by regulating the expression of mitochondrial fusion proteins. A-SMase silencing promoted mitochondrial fusion by enhancing the expression of Mfn-1 and OPA1. A recent study showed that expression levels of the proteins involved in mitochondrial fusion and fission differ among melanoma cells [19]. In particular, this study revealed that inhibiting mitochondrial fusion in human melanoma cells significantly decreases oxygen consumption rate, thus indicating that oxidative phosphorylation depends on mitochondrial fusion in melanoma cells.
High expression of Mfn1, Mfn2 and OPA1 has also been linked to cancer cell proliferation, survival and invasion, while their inhibition blocks cell growth and triggers apoptosis of different cancer cells. Conversely, mitochondrial fission occurs during apoptosis and seems important for progression of the apoptotic pathway [17,[64][65][66][67].
Mitochondrial dynamics and function are tightly interconnected; in order to satisfy the metabolic requirements of the cell, mitochondria constantly divide, elongate and connect with each other to form tubular networks or fragmented granules [17,68]. The analysis of melanoma cell lines and patient samples indicated that in some subsets of melanoma OxPhos plays a critical role [6][7][8], as between 35-50% of them show a "High-OxPhos" phenotype, characterised by increased mitochondrial respiration. This phenotype is, first and foremost, dictated by PGC-1alpha. The high expression of PGC-1alpha correlates with increased expression of mitochondrial transcriptional factors and mitochondrial fusion and fission mediators [13], together with a decreased overall survival in patients with stage III melanoma [7]. The "High-OxPhos" phenotype of melanoma cells corroborates what we observed in A-SMase-silenced B16-F1 cells, in which we found an increased production of OxPhos ATP and the activation of the mitochondrial biogenesis machinery. Notably, we did not notice differences between the control and the silenced cells in the content of mitochondrial DNA, a proxy for mitochondrial mass. However, the mitochondrial mass is determined by the balance of organelle biogenesis and degradation. We already demonstrated that autophagy in melanoma cells relies upon A-SMase expression levels and that low levels of A-SMase increase the autophagic process [29,69]. The fact that A-SMase deficiency enhances mitochondrial biogenesis machinery but fails to increment mitochondrial content may conceivably be through the activation of autophagy.
A-SMase signalling in melanoma involves the transcription factor Mitf [31]. A-SMase promotes the proteasomal degradation of Mitf, such that its levels increase whenever the levels of A-SMase are reduced, such as during melanoma development [31]. Here, we demonstrate that A-SMase regulates Mitf also at transcriptional levels, with the downregulation of Mitf resulting in the inhibition of Mfn-1 and OPA1 mRNA. The effect of Mitf on the expression of the two proteins may occur through a direct interaction with their promoters, or may involve the activation/inhibition of other molecular players. The analysis of the promoters of Mfn1 and OPA1 (Mus musculus), carried out by using the EPD database (https://epd.epfl.ch//index.php) [70], showed multiple putative consensus sequences for Mitf with both genes ( Mitf controls the expression of genes crucial for melanoma cell differentiation, proliferation, and survival [71,72]. The functional roles of Mitf have now been broadened by the identification of this transcription factor, together with PGC-1alpha, as a marker for melanoma subtypes that depend on mitochondrial OxPhos [8]. Melanomas present heterogeneous metabolic and energetic states, which are defined by the levels of expression of PGC-1alpha. PGC-1alpha-negative melanoma cells have a reduced bioenergetic capacity but high rates of glycolysis consistent with a glycolytic "Warburg" state. Conversely, PGC-1alpha-positive cells show elevated rates of mitochondrial oxidative metabolism [7]. Mitf expression sustains the transcription of PGC-1alpha in melanoma cells [7]. In our cell model, A-SMase downregulation induced the increase of Mitf and the "High OxPhos" phenotype; the silencing of the transcription factor correlated with the downregulation of PGC-1alpha and of its downstream target TFAM, whose expression has been correlated recently with glucose consumption and ATP production in melanoma cells [73]. In summary, our study demonstrates that A-SMase downregulation modulates the following: (i) mitochondrial morphology, by enhancing the expression of Mfn1 and OPA1; and (ii), mitochondrial biogenesis and function, through the stimulation of PGC-1alpha and TFAM. Moreover, we demonstrate that A-SMase acts on the two above biological events by modulating the expression of the mitochondrial fusion machinery and biogenesis in a Mitf-dependent manner. Our findings, therefore, expand the understanding of A-SMase in melanomas and provide new insights into its diverse roles in shaping the melanoma phenotype.