Marco Presta

Background/Objectives: Mitochondrial plasticity, reflected by the capacity of cancer cells to shift between oxidative phosphorylation and glycolytic metabolism in response to genetic alterations and changes in the tumor microenvironment, represents a key feature of melanoma progression. Sphingolipids contribute to the metabolic adaptations that support tumor development. Previous studies have demonstrated that β-galactosylceramidase (GALC), a lysosomal enzyme involved in sphingolipid metabolism, alters lipid composition and promotes a pro-oncogenic phenotype in melanoma cells. Here, we investigated the impact of GALC knockout (KO) on mitochondrial energy metabolism in human melanoma. Methods: CRISPR/Cas9-mediated gene editing was used in BRAF^(V600E) mutated A2058 human melanoma cells to generate nine GALC KO clones and three control clones. Targeted analyses of the mitochondrial sphingolipid profile, transcriptomic profiling, and mitochondrial structural and functional assessments, including the Seahorse Mito-Stress Test, were performed in GALC KO and control cell clones. Results: GALC loss induced sphingolipid-mediated reprogramming of mitochondrial metabolism in the absence of major structural alterations. This metabolic phenotype was characterized by bioenergetic insufficiency, potentially resulting from a sphingolipid-driven impairment of mitochondrial respiratory chain function. Conclusions: Overall, our findings indicate that GALC loss suppresses mitochondrial metabolism through sphingolipid-mediated mechanisms and provides new insights into the potential exploitation of mitochondrial metabolic vulnerabilities for the development of therapeutic strategies targeting melanoma.

Mitochondrial plasticity, characterized by the dynamic balance between glycolysis and oxidative phos-phorylation in response to genetic and microenvironmental changes, is a hallmark of melanoma progression. Sphingolipids play a significant role in various aspects of cancer cell biology, including metabolic reprogramming. Previous observations had shown that the lysosomal sphingolipid-metabolizing enzyme β-galactosylceramidase (GALC) rewires the lipid profile of mouse melanoma cells, exerting pro-oncogenic functions, gene silencing leading to a decreased oncogenic activity in murine and human melanoma cells. Here, we have focused on the mitochondrial sphingolipid composition and energetic metabolism in GALC knockout (KO) A2058 human melanoma cells. Targeted analysis of the mitochondrial sphingolipid profile, transcriptomic data, and mitochondrial structural and functional studies indicate that GALC loss drives a sphingolipid-mediated reprogramming of mitochondrial metabolism in absence of major structural alterations, characterized by bioenergetic insufficiency possibly due to ceramide- and sphingomyelin-driven impairment of respiratory chain function. Overall, these data indicate that GALC KO leads to a sphin-golipid-driven mitochondrial metabolic suppression and may provide novel information for the development of efficacious approaches in mitochondrial targeting melanoma therapies.

Gremlin‐1 is a secreted antagonist of bone morphogenetic protein (BMP) signalling, highly expressed in various malignant tumours and is associated with poor prognosis. In addition to its established pro‐angiogenic activity, its potential role in endothelial differentiation in tumour contexts remains incompletely defined. Here, we investigated whether gremlin‐1 contributes to tumour vascularization by promoting both endothelial differentiation and vascular recruitment. The expression of gremlin‐1 in murine embryonic stem cells (ESCs) promoted tumoroid development enriched in mesodermal and endothelial lineages in vitro, as indicated by the upregulation of lineage‐specific markers and the presence of CD31‐positive vascular‐like networks. In vivo, gremlin‐1‐expressing ESCs generated larger teratomas with pronounced stromal expansion and increased vascularization, while retaining multilineage differentiation capacity. Using the chick chorioallantoic membrane (CAM) assay to discriminate donor‐derived vasculogenesis from host‐driven angiogenesis, we observed that gremlin‐1‐expressing grafts exhibited enhanced growth and vascularization. Species‐specific endothelial labelling revealed the presence of both ESC‐derived and host‐derived endothelial cells within vascular structure. Collectively, our findings identify gremlin‐1 as a regulator of tissue vascularization that integrates intrinsic endothelial differentiation with extrinsic angiogenic responses, a mechanism potentially relevant to vascular remodelling in several pathological conditions including tumour growth.

Krabbe disease (KD) is an autosomal recessive sphingolipidosis due to mutations of the GALC gene encoding for the lysosomal β-galactosylceramidase (GALC) that removes β-galactose from β-galactosylceramide, β-lactosylceramide (LacCer) and the neurotoxic metabolite β-galactosylsphingosine (psychosine). At present, the accumulation of psychosine is thought to be the main cause of demyelination, neurodegeneration and neuroinflammation that characterize the early infantile KD with a 1.5–2-year median survival. Currently, the standard of care of KD is haematopoietic stem cell transplantation which, however, improves the lifespan of Krabbe patients only when performed before symptoms appear. Thus, a better understanding of the pathogenesis of KD is required for the development of more efficacious therapeutic approaches. This largely depends upon the availability of novel suitable animal models of the disease. Zebrafish (Danio rerio) represents a useful platform for the study of the mechanisms responsible for human hereditary diseases, including sphingolipidoses, and for the identification of new therapeutics. Two co-orthologues of human GALC have been identified in zebrafish, named galca and galcb. Here, we generated a mutant zebrafish line for each of the two co-orthologues by CRISPR/Cas9 genome editing. Galcb knockout (KO), but not galca KO, exerts a dramatic decrease of total GALC activity both in zebrafish embryos and in the brain of adult mutants. At 3–4 months post-fertilization, galcb KO zebrafish showed impaired locomotion and reduced lifespan. Gene expression analysis, immunohistochemistry, spectral confocal reflectance and transmission electron microscopy showed the presence of demyelination, neuroinflammation and neurodegeneration in the brain of galcb KO mutants. Notably, double galca/galcb KO did not cause a further worsening of the disease when compared with galcb KO mutants. Finally, targeted lipidomic analysis demonstrated a dramatic accumulation of the bioactive sphingolipid LacCer in the brain of both galcb KO and double galca/galcb KO mutants with a modest increase of psychosine levels. Accordingly, activation of LacCer-related signalling occurs in the brain of galcb KO animals. Furthermore, intraventricular injection of LacCer upregulates the expression of various proinflammatory markers and increase mpeg1-positive macrophage infiltration in the brain of 5 dpf zebrafish embryos. In conclusion, galcb KO zebrafish recapitulates several pathological features of KD and is characterized by the accumulation of the bioactive LacCer. This model sheds new light on a possible role of LacCer as a neuroinflammatory/neurodegenerative metabolite in KD with implications for the development of novel therapeutic strategies.

Mitochondrial plasticity, marked by a dynamism between glycolysis and oxidative phosphorylation due to adaptation to genetic and microenvironmental alterations, represents a characteristic feature of melanoma progression. Sphingolipids play a significant role in various aspects of cancer cell biology, including metabolic reprogramming. Previous observations have shown that the lysosomal sphingolipid-metabolizing enzyme β-galactosylceramidase (GALC) exerts pro-oncogenic functions in melanoma. Here, mining the cBioPortal for a Cancer Genomics data base identified the top 200 nuclear-encoded genes whose expression is negatively correlated with GALC expression in human melanoma. Their categorization indicated a significant enrichment in Gene Ontology terms and KEGG pathways related to mitochondrial proteins and function. In parallel, proteomic analysis by LC-MS/MS of two GALC overexpressing human melanoma cell lines identified 98 downregulated proteins when compared to control mock cells. Such downregulation was confirmed at a transcriptional level by a Gene Set Enrichment Analysis of the genome-wide expression profiling data obtained from the same cells. Among the GALC downregulated proteins, we identified a cluster of 42 proteins significantly associated with GO and KEGG categorizations related to mitochondrion and energetic metabolism. Overall, our data indicate that changes in GALC expression may exert a significant impact on mitochondrial plasticity in human melanoma cells.

β-Galactosylceramidase (GALC) is a lysosomal enzyme involved in sphingolipid metabolism by removing β-galactosyl moieties from β-galactosyl ceramide and β-galactosyl sphingosine. Previous observations have shown that GALC exerts a pro-oncogenic activity in human melanoma. Here, the impact of GALC overexpression on the proteomic landscape of BRAF-mutated A2058 and A375 human melanoma cell lines was investigated by liquid chromatography–tandem mass spectrometry analysis of the cell extracts. The results indicate that GALC overexpression causes the upregulation/downregulation of 172/99 proteins in GALC-transduced cells when compared to control cells. Gene ontology categorization of up/down-regulated proteins indicates that GALC may modulate the protein landscape in BRAF-mutated melanoma cells by affecting various biological processes, including RNA metabolism, cell organelle fate, and intracellular redox status. Overall, these data provide further insights into the pro-oncogenic functions of the sphingolipid metabolizing enzyme GALC in human melanoma.

β-Galactosylceramidase (GALC) is a lysosomal enzyme involved in sphingolipid metabolism by removing β-galactosyl moieties from β-galactosylceramide and β-galactosylsphingosine. Previous observations have shown that GALC may exert pro-oncogenic functions in melanoma and Galc silencing, leading to decreased oncogenic activity in murine B16 melanoma cells. The tumor-driving BRAF(V600E) mutation is present in approximately 50% of human melanomas and represents a major therapeutic target. However, such mutation is missing in melanoma B16 cells. Thus, to assess the impact of GALC in human melanoma in a more relevant BRAF-mutated background, we investigated the effect of GALC overexpression on the proteomic landscape of A2058 and A375 human melanoma cells harboring the BRAF(V600E) mutation. The results obtained by liquid chromatography-tandem mass spectrometry (LC-MS/MS) demonstrate that significant differences exist in the protein landscape expressed under identical cell culture conditions by A2058 and A375 human melanoma cells, both harboring the same BRAF(V600E)-activating mutation. GALC overexpression resulted in a stronger impact on the proteomic profile of A375 cells when compared to A2058 cells (261 upregulated and 184 downregulated proteins versus 36 and 14 proteins for the two cell types, respectively). Among them, 25 proteins appeared to be upregulated in both A2058-upGALC and A375-upGALC cells, whereas two proteins were significantly downregulated in both GALC-overexpressing cell types. These proteins appear to be involved in melanoma biology, tumor invasion and metastatic dissemination, tumor immune escape, mitochondrial antioxidant activity, endoplasmic reticulum stress responses, autophagy, and/or apoptosis. Notably, analysis of the expression of the corresponding genes in human skin cutaneous melanoma samples (TCGA, Firehose Legacy) using the cBioPortal for Cancer Genomics platform demonstrated a positive correlation between GALC expression and the expression levels of 14 out of the 27 genes investigated, thus supporting the proteomic findings. Overall, these data indicate for the first time that the expression of the lysosomal sphingolipid-metabolizing enzyme GALC may exert a pro-oncogenic impact on the proteomic landscape in BRAF-mutated human melanoma.