The Pro-Oncogenic Sphingolipid-Metabolizing Enzyme β-Galactosylceramidase Modulates the Proteomic Landscape in BRAF(V600E)-Mutated Human Melanoma Cells

Abstract

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.

Proteomic Analysis Highlights the Impact of the Sphingolipid Metabolizing Enzyme β-Galactosylceramidase on Mitochondrial Plasticity in Human Melanoma

“…Such relationship was confirmed by the analysis of the proteomic data obtained by LC-MS/MS on human melanoma A2058 and A375 cell lines that had been engineered to stably overexpress human GALC by lentiviral infection [ 15 ]. GALC overexpression results in an increased tumorigenic potential in these cells and in significant changes in their proteomic landscape, leading to the modulation of the expression of proteins involved in various aspects of melanoma progression, including endoplasmic reticulum responses, metastasis, and immune escape [ 15 , 16 ]. Here, we focused our attention on a set of 98 proteins whose cellular levels were significantly downregulated in GALC -overexpressing cells when compared to control cells.…”

“…GALC is a lysosomal sphingolipid-metabolizing enzyme that catalyzes the removal of galactose from terminal β-galactose-containing sphingolipids, including β-galactosylceramide [ 11 , 12 ]. Previous observations have shown that GALC may exert pro-oncogenic functions in human melanoma [ 13 , 15 ]. The present work extends these observations and indicates that GALC exerts a significant impact on melanoma mitochondrial plasticity.…”

“…GALC-overexpressing A2058-upGALC and A375-upGALC melanoma cells and the corresponding control mock cells have been described in a previous publication [ 15 ]. In this present work, we investigated the proteomic data obtained by LC-MS/MS analysis of the extracts of the A2058 and A357 mock and GALC-overexpressing cells [ 16 ].…”

“…The analysis of this list of genes on different platforms identified various enriched Gene Ontology (GO) terms and Kyoto Encyclopedia of Genes and Genomes (KEGG) pathways related to mitochondrial proteins and function. Accordingly, by taking advantage of previous proteomic data obtained in our laboratory by liquid chromatography–tandem mass spectrometry (LC-MS/MS) analysis [ 15 , 16 ], we identified a set of 98 proteins whose expression is downregulated at protein and Mrna levels in GALC overexpressing A2058 and A375 human melanoma cells (upGALC cells) harboring the BRAF (V600E) mutation. Among them, a STRING-defined cluster of 42 downregulated proteins was associated with GO and KEGG categorizations related to mitochondrion and energetic metabolism.…”

Dataset: Impact of β-Galactosylceramidase Overexpression on the Protein Profile of Braf(V600E) Mutated Melanoma Cells

“…However, approximately 50% of human melanomas are characterized by the BRAF (V600E) tumor driver mutation [21][22][23][24][25][26], which represents a major target in melanoma therapy [27][28][29][30]. These observations prompted us to assess the role of GALC in human melanoma in the presence of a BRAF-mutated background [31]. To this purpose, liquid chromatography-tandem mass spectrometry (LC-MS/MS) was used to investigate the impact of GALC overexpression on the proteomic profile of BRAF (V600E)-mutated A2058 and A375 human melanoma cells.…”