Uveal Melanoma Cell Line Proliferation Is Inhibited by Ricolinostat, a Histone Deacetylase Inhibitor

Uveal melanoma (UM) is the most common adult eye cancer. UM originates in the iris, ciliary body or choroid (collectively known as the uvea), in the middle layer of the eye. This first or primary UM is treated by targeting cancer cells using ocular radiation implants or by surgical removal of the eye. However, when UM spreads to the liver and other parts of the body, patients have a poor survival prognosis. Unfortunately, there are no effective treatment options for UM that has spread. Our aim is to help identify effective treatments for UM. In our study, we identified that the drug ACY-1215 prevents the growth of cells derived from UM in the eye and a UM that spread to the liver. Our pre-clinical study uncovered a potential treatment approach for advanced UM. Metastatic uveal melanoma (MUM) is characterized by poor patient survival. Unfortunately, current treatment options demonstrate limited benefits. In this study, we evaluate the efficacy of ACY-1215, a histone deacetylase inhibitor (HDACi), to attenuate growth of primary ocular UM cell lines and, in particular, a liver MUM cell line in vitro and in vivo, and elucidate the underlying molecular mechanisms. A significant (p = 0.0001) dose-dependent reduction in surviving clones of the primary ocular UM cells, Mel270, was observed upon treatment with increasing doses of ACY-1215. Treatment of OMM2.5 MUM cells with ACY-1215 resulted in a significant (p = 0.0001), dose-dependent reduction in cell survival and proliferation in vitro, and in vivo attenuation of primary OMM2.5 xenografts in zebrafish larvae. Furthermore, flow cytometry revealed that ACY-1215 significantly arrested the OMM2.5 cell cycle in S phase (p = 0.0001) following 24 h of treatment, and significant apoptosis was triggered in a time- and dose-dependent manner (p < 0.0001). Additionally, ACY-1215 treatment resulted in a significant reduction in OMM2.5 p-ERK expression levels. Through proteome profiling, the attenuation of the microphthalmia-associated transcription factor (MITF) signaling pathway was linked to the observed anti-cancer effects of ACY-1215. In agreement, pharmacological inhibition of MITF signaling with ML329 significantly reduced OMM2.5 cell survival and viability in vitro (p = 0.0001) and reduced OMM2.5 cells in vivo (p = 0.0006). Our findings provide evidence that ACY-1215 and ML329 are efficacious against growth and survival of OMM2.5 MUM cells

1,4-dihydroxy quininib activates ferroptosis pathways in metastatic uveal melanoma and reveals a novel prognostic biomarker signature

Uveal melanoma (UM) is an ocular cancer, with propensity for lethal liver metastases. When metastatic UM (MUM) occurs, as few as 8% of patients survive beyond two years. Efficacious treatments for MUM are urgently needed. 1,4-dihydroxy quininib, a cysteinyl leukotriene receptor 1 (CysLT1) antagonist, alters UM cancer hallmarks in vitro, ex vivo and in vivo. Here, we investigated the 1,4-dihydroxy quininib mechanism of action and its translational potential in MUM. Proteomic profiling of OMM2.5 cells identified proteins differentially expressed after 1,4-dihydroxy quininib treatment. Glutathione peroxidase 4 (GPX4), glutamate-cysteine ligase modifier subunit (GCLM), heme oxygenase 1 (HO-1) and 4 hydroxynonenal (4-HNE) expression were assessed by immunoblots. Biliverdin, glutathione and lipid hydroperoxide were measured biochemically. Association between the expression of a specific ferroptosis signature and UM patient survival was performed using public databases. Our data revealed that 1,4-dihydroxy quininib modulates the expression of ferroptosis markers in OMM2.5 cells. Biochemical assays validated that GPX4, biliverdin, GCLM, glutathione and lipid hydroperoxide were significantly altered. HO-1 and 4-HNE levels were significantly increased in MUM tumor explants from orthotopic patient-derived xenografts (OPDX). Expression of genes inhibiting ferroptosis is significantly increased in UM patients with chromosome 3 monosomy. We identified IFerr, a novel ferroptosis signature correlating with UM patient survival. Altogether, we demontrated that in MUM cells and tissues, 1,4-dihydroxy quininib modulates key markers that induce ferroptosis, a relatively new type of cell death driven by iron-dependent peroxidation of phospholipids. Furthermore, we showed that high expression of specific genes inhibiting ferroptosis is associated with a worse UM prognosis, thus, the IFerr signature is a potential prognosticator for which patients develop MUM. All in all, ferroptosis has potential as a clinical biomarker and therapeutic target for MUM

Zebrafish as a model for dissecting the in vivo roles of Collagen VI

My PhD project was focused on using Danio rerio (zebrafish) as an in vivo model to dissect the expression and function of Collagen VI (COL6), a key extracellular matrix component found in several tissues and involved in a range of physiological and pathological processes. Mutations of COL6 genes in humans can cause different forms of inherited muscular diseases, including Bethlem myopathy and Ullrich congenital muscular dystrophy. A number of animal models were generated for COL6-related myopathies, displaying a spectrum of phenotypes. In particular, studies carried out in COL6 knockout (Col6a1–/–) mice provided a valuable tool for throwing light on the in vivo roles of COL6. Indeed, those studies demonstrated that COL6 exerts several cytoprotective functions, which span from counteracting apoptosis and oxidative damage, to the regulation of cell differentiation and autophagy. Despite this, no study until now ever assessed in detail which functions COL6 plays during development and in regulating intracellular signalling pathways. To gain further insights into these aspects, we decided to exploit zebrafish, a model that has been widely used for studies of vertebrate development and gene function. Indeed, thanks to its transparency and rapid development, zebrafish represents a powerful tool to visualize the expression pattern of a gene in the whole organism. In addition, the availability of transgenic biosensor lines makes zebrafish an ideal tool for the in vivo study of signaling pathways. During the first part of my PhD, I characterized in detail the spatio-temporal expression pattern and distribution of COL6 in zebrafish embryos, larvae and adults. The results I obtained demonstrate that zebrafish COL6 genes exhibit features that are very similar to those found in their mammalian orthologs. In particular, by exploiting a new antibody generated against zebrafish COL6, I was able to characterize precisely the expression pattern of this distinctive ECM protein during zebrafish development and adult life. These data are of major relevance since they provide the basis for functional studies in this animal model, and they have been collected in a manuscript entitled “Spatio-temporal expression and distribution of collagen VI during zebrafish development”, which is currently under revision. In the second part of my PhD, I carried out a number of functional studies exploiting a novel zebrafish col6a1 null line generated through CRISPR/Cas9 site-specific mutagenesis. The ablation of COL6 in this zebrafish model allowed me to identify neuromuscular defects and distinctive alterations in the three-dimensional architecture of craniofacial cartilages. In addition, knockout of COL6 affected Wnt signaling during embryogenesis, thus pointing at a potential role of COL6 in regulating this major signalling pathway. Altogether, the zebrafish col6a1 null line represents a valuable tool to unveil previously unknown links between molecular and signaling defects and COL6 deficiency. Indeed, the data reported in this thesis underline how zebrafish represents a very useful in vivo model for increasing our understanding of the role of this major ECM protein in different physiological and pathological contexts, both during development and adult life. Moreover, the zebrafish col6a1 null line represents a suitable tool for future drug testing aimed at finding novel treatments for COL6-related diseases.ll mio progetto di dottorato si è basato sull'utilizzo del modello animale Danio rerio (zebrafish) al fine di analizzare l'espressione e la funzione del collagene VI (COL6), un componente chiave della matrice extracellulare presente in vari tessuti e coinvolto in numerosi processi fisiologici e patologici. Mutazioni a carico dei geni codificanti per le catene del COL6 sono note causare diverse forme di malattie muscolari ereditarie nell'uomo, tra cui la miopatia di Bethlem e la distrofia muscolare congenita di Ullrich. Diversi modelli animali sono stati generati al fine di studiare queste miopatie. In particolare, gli studi condotti su topi knockout per il COL6 (Col6a1–/–) hanno permesso di mettere in luce alcuni dei ruoli chiave che questa proteina esercita in vivo. Tali lavori hanno infatti dimostrato che il COL6 svolge importanti funzioni citoprotettive, che vanno dal contrastare l'apoptosi e i danni ossidativi alla regolazione del differenziamento e dell'autofagia. Ciò nonostante, nessuno studio ha finora mai indagato a fondo quali siano le funzioni del COL6 durante lo sviluppo embrionale e nella regolazione delle vie di segnalazione intracellulari. Pertanto, allo scopo di ottenere ulteriori conoscenze su questi aspetti, abbiamo deciso di utilizzare lo zebrafish, un modello ampiamente impiegato negli studi sullo sviluppo dei vertebrati e sulla funzione genica. Grazie infatti alla sua trasparenza e al suo rapido sviluppo, lo zebrafish rappresenta un ottimo modello per visualizzare il pattern di espressione di un gene nell'intero organismo. Inoltre, la disponibilità di numerose linee transgeniche reporter rende lo zebrafish uno strumento ideale per lo studio in vivo delle vie di segnalazione intracellulare. Durante la prima parte del mio lavoro di dottorato, ho analizzato in dettaglio il pattern di espressione spazio-temporale e la distribuzione delle diverse catene del COL6 in embrioni, larve e adulti di zebrafish. I dati ottenuti dimostrano che i geni COL6 presentano un pattern di espressione nello zebrafish molto simile a quella dei rispettivi ortologhi in mammifero. In particolare, sfruttando un nuovo anticorpo che riconosce in maniera specifica il COL6 di zebrafish, ho potuto caratterizzare in dettaglio la distribuzione della proteina in questo organismo, sia durante lo sviluppo embrionale che in età adulta. Tali dati sono di notevole interesse, in quanto forniscono una base essenziale per poter effettuare degli studi funzionali in questo modello animale. I risultati ottenuti sono stati quindi raccolti in un lavoro intitolato "Spatio-temporal expression and distribution of collagen VI during zebrafish development", il quale è attualmente in fase di revisione. Nella seconda parte del mio dottorato ho svolto studi funzionali sfruttando una nuova linea di zebrafish priva di COL6, generata nel laboratorio attraverso la tecnologia di mutagenesi sito-specifica CRISPR/Cas9. In tale linea ho individuato numerosi difetti neuromuscolari e peculiari alterazioni nella struttura tridimensionale delle cartilagini craniofacciali. Inoltre, ho identificato che l’assenza di COL6 causa alterazioni nella via di segnale di Wnt, suggerendo un ruolo del COL6 nel regolare questa via di segnalazione intracellulare. Complessivamente, i dati ottenuti dimostrano che la linea di zebrafish non esprimente il COL6 rappresenta un valido modello in vivo per svelare connessioni ancora poco conosciute tra carenza di COL6 e deficit molecolari e di vie di segnalazione. Le evidenze riportate in questa tesi sottolineano infatti come lo zebrafish sia estremamente utile nello studio del ruolo di questa proteina di matrice in diversi contesti fisiologici e patologici, sia durante lo sviluppo che nella vita adulta. Inoltre, il modello zebrafish COL6 knockout rappresenta lo strumento ideale per effettuare studi farmacologici volti ad identificare nuovi trattamenti per le malattie associate ad alterazioni del COL6

Spatio-temporal expression and distribution of collagen VI during zebrafish development

International audienceCollagen VI (ColVI) is an extracellular matrix (ECM) protein involved in a range of physiological and pathological conditions. Zebrafish (Danio rerio) is a powerful model organism for studying vertebrate development and for in vivo analysis of tissue patterning. Here, we performed a thorough characterization of ColVI gene and protein expression in zebrafish during development and adult life. Bioinformatics analyses confirmed that zebrafish genome contains single genes encoding for α1(VI), α2(VI) and α3(VI) ColVI chains and duplicated genes encoding for α4(VI) chains. At 1 day post-fertilization (dpf) ColVI transcripts are expressed in myotomes, pectoral fin buds and developing epidermis, while from 2 dpf abundant transcript levels are present in myosepta, pectoral fins, axial vasculature, gut and craniofacial cartilage elements. Using newly generated polyclonal antibodies against zebrafish α1(VI) protein, we found that ColVI deposition in adult fish delineates distinct domains in the ECM of several organs, including cartilage, eye, skin, spleen and skeletal muscle. Altogether, these data provide the first detailed characterization of ColVI expression and ECM deposition in zebrafish, thus paving the way for further functional studies in this species

Collagen VI ablation in zebrafish causes neuromuscular defects during developmental and adult stages

Collagen VI (COL6) is an extracellular matrix protein exerting multiple functions in different tissues. In humans, mutations of COL6 genes cause rare inherited congenital disorders, primarily affecting skeletal muscles and collectively known as COL6-related myopathies, for which no cure is available yet. In order to get insights into the pathogenic mechanisms underlying COL6-related diseases, diverse animal models were produced. However, the roles exerted by COL6 during embryogenesis remain largely unknown. Here, we generated the first zebrafish COL6 knockout line through CRISPR/Cas9 site-specific mutagenesis of the col6a1 gene. Phenotypic characterization during embryonic and larval development revealed that lack of COL6 leads to neuromuscular defects and motor dysfunctions, together with distinctive alterations in the three-dimensional architecture of craniofacial cartilages. These phenotypic features were maintained in adult col6a1 null fish, which displayed defective muscle organization and impaired swimming capabilities. Moreover, col6a1 null fish showed autophagy defects and organelle abnormalities at both embryonic and adult stages, thus recapitulating the main features of patients affected by COL6-related myopathies. Mechanistically, lack of COL6 led to increased BMP signaling, and direct inhibition of BMP activity ameliorated the locomotor activity of col6a1 null embryos. Finally, treatment with salbutamol, a beta(2)-adrenergic receptor agonist, elicited a significant amelioration of the neuromuscular and motility defects of col6a1 null fish embryos. Altogether, these findings indicate that this newly generated zebrafish col6a1 null line is a valuable in vivo tool to model COL6-related myopathies and suitable for drug screenings aimed at addressing the quest for effective therapeutic strategies for these disorders. (C) 2022 Published by Elsevier B.V

Ergolide mediates anti-cancer effects on metastatic uveal melanoma cells and modulates their cellular and extracellular vesicle proteomes

&lt;p&gt; Background: Uveal melanoma is a poor prognosis cancer. Ergolide, a sesquiterpene lactone isolated from Inula Brittanica, exerts anti-cancer properties. The objective of this study was to 1) evaluate whether ergolide reduced metastatic uveal melanoma (MUM) cell survival/viability in vitro and in vivo; and 2) to understand the molecular mechanism of ergolide action.&lt;/p&gt;&lt;p&gt; Methods: Ergolide bioactivity was screened via long-term proliferation assay in UM/MUM cells and in zebrafish MUM xenograft models. Mass spectrometry profiled proteins modulated by ergolide within whole cell or extracellular vesicle (EVs) lysates of the OMM2.5 MUM cell line. Protein expression was analyzed by immunoblots and correlation analyses to UM patient survival used The Cancer Genome Atlas (TCGA) data.&lt;/p&gt;&lt;p&gt; Results: Ergolide treatment resulted in significant, dose-dependent reductions (48.5 to 99.9%; p&lt;0.0001) in OMM2.5 cell survival in vitro and of normalized primary zebrafish xenograft fluorescence (56%; p&lt;0.0001) in vivo, compared to vehicle controls. Proteome-profiling of ergolide-treated OMM2.5 cells, identified 5023 proteins, with 52 and 55 proteins significantly altered at 4 and 24 hours, respectively ( p&lt;0.05; fold-change &gt;1.2). Immunoblotting of heme oxygenase 1 (HMOX1) and growth/differentiation factor 15 (GDF15) corroborated the proteomic data. Additional proteomics of EVs isolated from OMM2.5 cells treated with ergolide, detected 2931 proteins. There was a large overlap with EV proteins annotated within the Vesiclepedia compendium. Within the differentially expressed proteins, the proteasomal pathway was primarily altered. Interestingly, BRCA2 and CDKN1A Interacting Protein (BCCIP) and Chitinase Domain Containing 1 (CHID1), were the only proteins significantly differentially expressed by ergolide in both the OMM2.5 cellular and EV isolates and they displayed inverse differential expression in the cells versus the EVs.&lt;/p&gt;&lt;p&gt; Conclusions: Ergolide is a novel, promising anti-proliferative agent for UM/MUM. Proteomic profiling of OMM2.5 cellular/EV lysates identified candidate pathways elucidating the action of ergolide and putative biomarkers of UM, that require further examination.&lt;/p&gt

Ergolide mediates anti-cancer effects on metastatic uveal melanoma cells and modulates their cellular and extracellular vesicle proteomes

&lt;p&gt;The most common form of adult eye cancer is uveal melanoma (UM). Once UM cancer cells spread to organs in the rest of the body, metastatic UM (MUM), there is a poor prognosis for patients with only one approved drug treatment. Hence, it is vital to better understand the cellular and extracellular proteins that regulate UM pathology in order to uncover biomarkers of disease and therapeutic targets. In this original study, we demonstrate a compound called ergolide is capable of severely reducing the metabolic activity and growth of UM cancer cells, grown as isolated monolayers. Ergolide was also able to reduce the growth of human MUM cells growing as tumors in transplanted zebrafish larvae. We identify that ergolide alters specific proteins found in the human UM cells. These proteins once analyzed in detail offer opportunities to understand how new treatment strategies can be developed for UM.&lt;/p&gt