Abstract 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.

Suppressor/enhancer of Lin-12-like (SEL1L) is a component of the endoplasmic reticulum-associated degradation (ERAD) pathway which is part of the unfolded protein response (UPR). SEL1L may exert pro-tumorigenic or oncosuppressive functions in different tumor types, but its role in human cutaneous malignant melanoma (cMM) remains largely unexplored. Here, in silico analysis revealed that SEL1L is upregulated in cMM compared to normal skin. In addition, SEL1L expression in cMM positively correlated with Clark level at diagnosis, higher expression being associated with poorer patient survival. SEL1L co-expression with ERAD- and UPR-related genes, along with Gene Ontology enrichment analysis, supported its role in ER-stress responses in cMM. Immunohistochemistry and quantitative digital morphometric analysis of SEL1L protein levels in 60 human samples of benign melanocytic tumors and cMMs at different Breslow T category showed that SEL1L levels in cMM progressively increase in association with T category, tumor thickness, and the presence of ulceration. In addition, total and tumor-associated endothelial SEL1L expression correlates with microvessel density in the corresponding tumor specimens. In keeping with a non-redundant role of SEL1L in cMM, grafting of SEL1L-silenced A2058 human melanoma cells in immunodeficient mice yielded tumors with reduced cell proliferation, enhanced apoptosis, and increased necrosis, accompanied by heightened hypoxia and reduced vascularization. Accordingly, SEL1L-silenced cells showed upregulation of the angiosuppressive thrombospondin-encoding genes THSP1 and THSP2 and a reduced angiogenic potential in an endothelial sprouting assay in vitro and in the chick embryo chorionallantoic membrane assay in vivo. Together, these findings indicate that SEL1L may exert a pro-tumorigenic/pro-angiogenic function in human melanoma and pave the way to further studies aimed at investigating its potential as a therapeutic target in cMM.

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.

PURPOSE:TMED10 is involved in unconventional protein secretion and ER-Golgi trafficking. TMED10 may exert protumorigenic or oncosuppressive functions in different tumor types, but its role in human cutaneous melanoma has never been explored. Here, TMED10 expression has been investigated in human benign melanocytic tumors and cutaneous malignant melanoma (cMM). METHODS:This study utilized in silico analysis on various publicly available web platforms to investigate TMED10 gene expression in normal skin and human melanoma at different Clark levels, and immunohistochemistry and digital morphometric analysis of TMED10 protein levels in 60 human samples of benign melanocytic tumors and cMMs at different Breslow T category. RESULTS:In silico analysis revealed that TMED10 is upregulated in cMM compared to normal skin. TMED10 expression in cMM positively correlated with Clark level at diagnosis, and higher expression was associated with the BRAFV600E mutation and poorer patient survival. TMED10 co-expression with TMED2, TMED8, HSP90AA1 and HSP90B1, along with Gene Ontology enrichment analysis, supported its role in ER-Golgi trafficking and unconventional protein secretion in human melanoma. Digital morphometric analysis of immunohistochemical investigation of 60 human specimens confirmed a significant increase in TMED10 protein levels with Breslow thickness-based T category in tumor cells and tumor-associated blood vessels. At variance, TMED10 immunoreactivity in infiltrating lymphocytes was reduced in T3 and T4 cMM. CONCLUSION:These findings point to a pro-tumorigenic function of TMED10 in human cMM and pave the way to further studies aimed at identifying its contribution to tumor progression, neovascularization, and immune escape, and its potential as a therapeutic target in cMM.

Adrenocortical carcinoma (ACC) is a rare endocrine neoplastic disease that originates from the cortical cortex of adrenal gland. Unfortunately, after complete resection of the tumor, ACC relapses either locally or with distant metastasis in about 74 % of patients and for these patients the therapeutic options are still severely limited. In this study we demonstrate that the system composed by the fibroblast growth factors (FGFs) and their receptors (FGFRs) might represent a promising therapeutic target for ACC. Indeed, human ACC specimens and cell lines express FGF ligands and FGF receptors and show FGFR activation, suggesting the presence of an autocrine FGF/FGFR loop of stimulation able to sustain ACC growth. Accordingly, inhibition of FGFR activation by TK inhibitors (erdafitinib and infigratinib) or FGF trapping (by NSC12) significantly hampered ACC growth and survival in vitro. Importantly, oral administration of erdafitinib strongly affected tumor growth in vivo by reducing tumor cell proliferation/survival and tumor angiogenesis. Mechanistically, FGF/FGFR inhibition in ACC cells strongly decreased the levels of the oncoprotein c-Myc and induced oxidative stress and DNA damage, leading to reduced tumor cell proliferation and increased tumor cell apoptosis. Altogether these results demonstrate for the first time the impact of FGF/FGFR blockade on ACC cell growth and survival both in vitro and in vivo. This study may set the rationale to start clinical trials investigating the therapeutic potential of FDA approved FGFR-TK inhibitors for the treatment of aggressive ACC.

Hyalocytes are macrophage-like cells residing in the eye vitreous cortex. Even though hyalocytes have been firstly described in the mid-Nineteenth century, they have been poorly explored. Recent researches highlighted hyalocyte involvement in both physiological and pathological processes of the vitreoretinal interface. Nonetheless, the majority of works involving hyalocyte cultures were carried out in animals, while fewer studies were performed on humans because their isolation requires vitrectomy. The aim of this study was to differentiate human induced pluripotent stem cells (iPSCs) into hyalocytes as a non-invasive method to continuously obtain cells. iPSCs were first differentiated into hematopoietic stem/progenitor cells (HSPCs) and then into macrophages. Macrophages were either left untreated (NT) or treated with ascorbic acid (AA) alone or combined with bFGF and/or TGF-β1. Additionally, macrophages were cultured in the presence of a pool of vitreous bodies from vitrectomies. Cells were analyzed for morphology and then for gene and protein expression through qRT-PCR, immunofluorescence, Western Blot, and flow cytometry. Similar to cells treated with the vitreous body, macrophages treated with AA alone or in combination with bFGF exhibited a more elongated shape compared to NT or cells treated with TGF-β1. Additionally, these treatments resulted in gene expression downregulation for S100A4, S100A10, S100B, and CX3CR1, while upregulating COL6A1, HLA-DRA, and CD74. At the protein level, S100B, CD14, and CD49d were downregulated with all treatments, while collagen VI and HLA-DR were upregulated. This work demonstrates that hyalocytes can be differentiated by treatment of iPSC-derived macrophages with ascorbic acid for a period of 21 days.

Vascular endothelial growth factor (VEGF) is a key player in the development and progression of several diseases, most notably cancer and retinal disorders. Over the last twenty years, VEGF has emerged as a significant therapeutic target for these conditions. This study reports the isolation and characterization of a fully synthetic, humanized, affinity-matured single-domain antibody fragment (VHH) designed to target VEGF. Our approach involved a two-step strategy to construct a phage-displayed library: first, diversifying the CDR3 region of the NbBCII10FGLA nanobody and then diversifying the CDR1 and CDR2 regions. The selection process involved three rounds of biopanning against VEGF isoform 121. The resulting antibody fragment, NbH1, showed a four-fold increase in affinity for human VEGF compared to the original VHH. Significantly, this enhanced affinity did not compromise the solubility or thermal stability of NbH1. Molecular modeling indicated that NbH1 recognizes a unique epitope on VEGF. Functional assays, including competitive and conventional ELISAs, demonstrated that NbH1 effectively disrupts the interaction between VEGF and its main receptor VEGFR2, and with bevacizumab, making it particularly promising for therapeutic applications. This novel molecule demonstrates cross-species activity and does not interfere with VEGFR1 binding. Furthermore, NbH1 inhibits VEGF-induced phosphorylation of VEGFR2 in human endothelial cells and hampers their sprouting in a 3D spheroid angiogenesis assay, highlighting its potential for treating pathological angiogenesis in vivo.

AbstractAmong blood cancers, multiple myeloma (MM) represents the second most common neoplasm and is characterized by the accumulation and proliferation of monoclonal plasma cells within the bone marrow. Despite the last few decades being characterized by the development of different therapeutic strategies against MM, at present such disease is still considered incurable. Although MM is highly heterogeneous in terms of genetic and molecular subtypes, about 67% of MM cases are associated with abnormal activity of the transcription factor c-Myc, which has so far revealed a protein extremely difficult to target. We have recently demonstrated that activation of fibroblast growth factor (FGF) signaling protects MM cells from oxidative stress-induced apoptosis by stabilizing the oncoprotein c-Myc. Accordingly, secretion of FGF ligands and autocrine activation of FGF receptors (FGFR) is observed in MM cells and FGFR3 genomic alterations represent some 15–20% MM cases and are associated with poor outcome. Thus, FGF/FGFR blockade may represent a promising strategy to indirectly target c-Myc in MM. On this basis, the present review aims at providing an overview of recently explored connections between the FGF/FGFR system and c-Myc oncoprotein, sustaining the therapeutic potential of targeting the FGF/FGFR/c-Myc axis in MM by using inhibitors targeting FGF ligands or FGF receptors. Importantly, the provided findings may represent the rationale for using FDA approved FGFR TK inhibitors (i.e. Pemigatinib, Futibatinib, Erdafitinib) for the treatment of MM patients presenting with an aberrant activation of this axis.

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.

Krabbe disease is a sphingolipidosis characterized by the genetic deficiency of the acid hydrolase β-galactosylceramidase (GALC). Most of the studies concerning the biological role of GALC performed on Krabbe patients and Galc-deficient twitcher mice (an authentic animal model of the disease) indicate that the pathogenesis of this disorder is the consequence of the accumulation of the neurotoxic GALC substrate β-galactosylsphingosine (psychosine), ignoring the possibility that this enzyme may exert a wider biological impact. Indeed, limited information is available about the effect of GALC downregulation on the cell lipidome in adult and developing organisms. The teleost zebrafish (Danio rerio) has emerged as a useful platform to model human genetic diseases, including sphingolipidoses, and two GALC co-orthologs have been identified in zebrafish (galca and galcb). Here, we investigated the effect of the competitive and irreversible GALC inhibitor β-galactose-cyclophellitol (GCP) on the lipid profile of zebrafish embryos. Molecular modelling indicates that GCP can be sequestered in the catalytic site of the enzyme and covalently binds human GALC, and the zebrafish Galca and Galcb proteins in a similar manner. Accordingly, GCP inhibits the β-galactosylceramide hydrolase activity of zebrafish in vitro and in vivo, leading to significant alterations of the lipidome of zebrafish embryos. These results indicate that the lack of GALC activity deeply affects the lipidome during the early stages of embryonic development, and thereby provide insights into the pathogenesis of Krabbe disease.

Fibroblast growth factors (FGFs) act as proangiogenic and mitogenic cytokines in several cancers, including multiple myeloma (MM). Indeed, corrupted FGF autocrine and paracrine secretion induces an aberrant activation of the FGF receptor (FGFR) signaling sustaining cancer cell spreading and resistance to pharmacological treatments. Thus, FGF traps may represent a promising anti-cancer strategy to hamper the ligand-dependent activation of the FGF/FGFR system. We previously identified NSC12 as the first orally available small molecule FGF trap able to inhibit the growth and progression of several FGF-dependent tumor models. NSC12 is a pregnenolone derivative carrying a 1,1-bis-trifluoromethyl-1,3-propanediol chain in position 17 of the steroid nucleus. Investigation of structure-activity relationships (SARs) provided more potent and specific NSC12 steroid derivatives and highlighted that the C17-side chain is pivotal for the FGF trap activity. Here, a scaffold hopping approach allowed to obtain two FGF trap compounds (22 and 57) devoid of the steroid nucleus and able to efficiently bind FGF2 and to inhibit FGFR activation in MM cells. Accordingly, these compounds exert a potent anti-tumor activity on MM cell lines both in vitro and in vivo and on MM patient-derived primary cells, strongly affecting the survival of both proteasome-inhibitor sensitive and resistant MM cells. These results propose a new therapeutic option for relapsed/refractory MM patients and set the bases for the development of novel FGF traps prone to chemical diversification to be used in the clinic for the treatment of those tumors in which the FGF/FGFR system plays a pivotal role, including MM.

Sig1R promotes hERG- and AKT-dependent FDM-induced cell spreading, VEGF secretion and endothelial transmigration

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.

Single file with supplemental figures, methods, tables, legends: Figure S1: Effects of FGF blockade in MM; Figure S2: FGF inhibition induces oxidative stress-mediated apoptosis in MM cells; Figure S3: Mitochondrial oxidative stress is responsible for the antitumor activity exerted in vivo by FGF inhibition; Figure S4: Oxidative stress-induced apoptosis following FGF inhibition is c-Myc-dependent; Figure S5: Western blot analysis for caspase 3 activation in PBMCs compared to NSC12-sensitive patient-derived MM cells treated with NSC12 for 12 hours; Figure S6: Effects of FGF inhibition in BTZ-resistant KMS-11 cells; Figure S7: High doses of BTZ hamper basal and NSC12-induced c-Myc degradation; Table S1: List of Log2 2-fold down- or upregulated genes in KMS-11 cells after 6 and 12 hours of NSC12 6μM treatment; Table S2: Patient-derived MM cells treated with NSC12; Reagent table.

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