Acid Sphingomyelinase Downregulation Enhances Mitochondrial Fusion and Promotes Oxidative Metabolism in a Mouse Model of Melanoma
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cells Article Acid Sphingomyelinase Downregulation Enhances Mitochondrial Fusion and Promotes Oxidative Metabolism in a Mouse Model of Melanoma 1, 1,2, 1, 3 Marco Coazzoli y, Alessandra Napoli y , Paulina Roux-Biejat y, Clara De Palma , Claudia Moscheni 1 , Elisabetta Catalani 4 , Silvia Zecchini 1, Vincenzo Conte 5 , Matteo Giovarelli 1, Sonia Caccia 1 , Patrizia Procacci 5 , Davide Cervia 4 , Emilio Clementi 1,6,* and Cristiana Perrotta 1,* 1 Department of Biomedical and Clinical Sciences “Luigi Sacco” (DIBIC), Università degli Studi di Milano, 20157 Milano, Italy; [email protected] (M.C.); [email protected] (A.N.); [email protected] (P.R.-B.); [email protected] (C.M.); [email protected] (S.Z.); [email protected] (M.G.); [email protected] (S.C.) 2 Unit of Clinical Pharmacology, University Hospital “Luigi Sacco”-ASST Fatebenefratelli Sacco, 20157 Milano, Italy 3 Department of Medical Biotechnology and Translational Medicine (BIOMETRA), Università degli Studi di Milano, 20129 Milano, Italy; [email protected] 4 Department for Innovation in Biological, Agro-food and Forest systems (DIBAF), Università degli Studi della Tuscia, 01100 Viterbo, Italy; [email protected] (E.C.); [email protected] (D.C.) 5 Department of Biomedical Sciences for Health (SCIBIS), Università degli Studi di Milano, 20133 Milano, Italy; [email protected] (V.C.); [email protected] (P.P.) 6 Scientific Institute IRCCS “Eugenio Medea”, 23842 Bosisio Parini, Italy * Correspondence: [email protected] (E.C.); [email protected] (C.P.) These authors contributed equally to this work. y Received: 13 February 2020; Accepted: 28 March 2020; Published: 31 March 2020 Abstract: 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. Keywords: melanoma; acid sphingomyelinase; mitochondrial dynamics; mitochondrial function 1. 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 [2–5]. Although melanoma cells can Cells 2020, 9, 848; doi:10.3390/cells9040848 www.mdpi.com/journal/cells Cells 2020, 9, 848 2 of 19 rely on anaerobic metabolism, oxidative phosphorylation (OxPhos) also plays a role, which becomes critical in defined subsets of melanomas [6–8]. 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 [9–13]. 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 [20–22]. 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 [29–31]. 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. 2. Materials and Methods 2.1. 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. For the in vitro experiments, we used B16-F1 cells transiently transfected with a A-SMase-specific (B16-F1_siASM: sense 50-GGCUACCGAGUUUACCAAAtt-30/antisense 50-UUUGGUAAACUCGG UAGCCag-30) or Mitf-specific (B16-F1_siMitf: sense: 50-GGACAAUCACAACUUGAUUtt-30/ antisense: 50-AAUCAAGUUGUGAUUGUCCtt-30) siRNA or a non-targeting siRNA (B16-F1_scr: sense 50-GCCTATGCTAATCGCAAATGTtt-30/antisense 50-ACAUUUGCGAUUAGCAUAGGCag-30), alone or in combination. Briefly, B16-F1 cells were seeded at 40% confluence and transfected when at 60% confluence with the siRNAs using Lipofectamine RNAiMAX transfection reagent according to the manufacturer’s protocol for 48 h. B16-B1A cells, constitutively transfected with a the pEF1/Myc Cells 2020, 9, 848 3 of 19 plasmid (Invitrogen-Life Technologies) containing the cDNA for A-SMase [30], were also used as a model of A-SMase overexpression. Cells were cultured in Iscove’s modified Dulbecco’s medium supplemented with 10% heat-inactivated foetal bovine serum (FBS), glutamine (200 mM), and penicillin/streptomycin (100 U/mL), and grown at 37 ◦C in a humidified atmosphere containing 5% CO2. 2.2. 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 2.5 104 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 mm3 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. 2.3. 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