Background: The poor response to chemotherapy and the brief response to vemurafenib in metastatic melanoma patients, make the identification of new therapeutic approaches an urgent need. Interestingly the increased expression and activity of the Aurora kinase B during melanoma progression suggests it as a promising therapeutic target. Methods: The efficacy of the Aurora B kinase inhibitor barasertib-HQPA was evaluated in BRAF mutated cells, sensitive and made resistant to vemurafenib after chronic exposure to the drug, and in BRAF wild type cells. The drug effectiveness has been evaluated as cell growth inhibition, cell cycle progression and cell migration. In addition, cellular effectors of drug resistance and response were investigated. Results: The characterization of the effectors responsible for the resistance to vemurafenib evidenced the increased expression of MITF or the activation of Erk1/2 and p-38 kinases in the newly established cell lines with a phenotype resistant to vemurafenib. The sensitivity of cells to barasertib-HQPA was irrespective of BRAF mutational status. Barasertib-HQPA induced the mitotic catastrophe, ultimately causing apoptosis and necrosis of cells, inhibited cell migration and strongly affected the glycolytic metabolism of cells inducing the release of lactate. In association i) with vemurafenib the gain in effectiveness was found only in BRAF(V600K) cells while ii) with nab-paclitaxel, the combination was more effective than each drug alone in all cells. Conclusions: These findings suggest barasertib as a new therapeutic agent and as enhancer of chemotherapy in metastatic melanoma treatment.

Background: Metastatic melanoma represents the most deadly form of skin cancer. The poor response to chemotherapy and the brief response to the anti-BRAF vemurafenib in selected population of patients, make the identification of new therapeutic approaches an urgent need. Our goal is the evaluation of the efficacy of barasertib, an aurora B kinase inhibitor impairing cytokinesis, in both mutated and non-mutated melanoma cell lines. Materials and methods: Panel of melanoma cells: BRAFV600E mutated cells (MBA72 and Hmel1), the same cell in which the resistance to vemurafenib was induced by chronic exposure to it (MBA72R and Hmel1R) and BRAF wt (HBL and LND1). Cells were characterized for vemurafenib and barasertib effectiveness on cell growth by MTT assay after 3 and 6 days of continuous exposure. Cell cycle was determined by flowcytometry and migration was evaluated by wound-healing assay. Results: Cells with BRAFV600E mutation are sensitive to vemurafenib conversely, those with BRAF wt and the resistant ones showed an IC50 of at least 10 folds higher. 3days-barasertib exposure strongly reduced cell growth (30-60% at 30 and 300nM, respectively) in all cell lines; when the drug was given together with vemurafenib, no gain in effectiveness was evident. Prolonged exposure to barasertib (6 days) showed a progressive increase of effectiveness particularly in cells BRAF wt. The analysis of cell death mechanisms involved in determining the effectiveness of barasertib and vemurafenib showed that the first drug induced both apoptosis and necrosis conversely, the latter mainly apoptosis. Cell cycle analysis demonstrated that barasertib induced an increase in cell size and in polyploidia, suggesting also the mitotic catastrophe as a further cell death mechanism. Moreover, the anti-metastatic behaviour of this agent has been evaluated in function of drug concentration and time exposure. Preliminary results showed a strong reduction of cell migration after drug exposure. Conclusions: The sensitivity of melanoma cells to barasertib is irrespective to BRAF mutational status; however, cells BRAF wt show an higher nuclear modification. In conclusion, our results suggest barasertib as a novel therapeutic approach in melanoma treatment irrespective of BRAFV600E mutation.

Melanogenesis is mostly studied in melanocytes and melanoma cells, but much less is known about other pigment cell systems. Liver, spleen, kidney, and other organs of lower vertebrates harbour a visceral pigment cell system with an embryonic origin that differs from that of melanocytes distinct embryonic origin. In teleosts, melanin-containing cells occur in the reticulo-endothelial system and are mainly in the kidney and spleen. The Atlantic salmon (Salmo salar L.) is an ichthyic breeding species of considerable economic importance. The accumulation of pigments in salmon visceral organs and musculature adversely affects the quality of fish products and is a problem for the aquaculture industry. With the aim to reveal novel functions and behaviour of the salmonid extracutaneous pigment system, we investigated aspects of the melanogenic systems in the tissues of Atlantic salmon, as well as in SHK-1 cells, which is a long-term cell line derived from macrophages of the Atlantic salmon head-kidney. We demonstrate that a melanogenic system is present in SHK-1 cells, head-kidney, and spleen tissues. As teleosts lack lymph nodes and Peyer’s patches, the head-kidney and spleen are regarded as the most important secondary lymphoid organs. The detection of tyrosinase activity in lymphoid organs indicates that a link exists between the extracutaneous pigmentary system and the immune system in salmon.

We isolated two novel cell lines from different types of sporadic human malignant melanoma: the hmel1 line was obtained from a melanoma skin metastasis and the hmel9 cell line from a primary superficial spreading melanoma. The karyotype and pigmentation parameters were assessed in these cell lines. Cytogenetic analysis in early stages of culture revealed that both cell lines had chromosome instability and simultaneous growth of heteroploid subpopulations. The molecular analysis of some genes involved in melanoma showed that both cell lines harbor BRAF mutations. The unpigmented hmel1 and the pigmented hmel9 lines were found to express the tyrosinase gene. The tyrosine hydroxylase activity was detectable only in hmel9 cells and practically absent in the hmel1 cell line. This activity was found to be correlated with the relative tyrosinase protein amount in both melanoma cell lines. The biological behaviour in the two melanoma cell lines, derived from two different types of melanoma lesions displaying distinct clinical and histopathological features, confirms the heterogeneous characteristics of sporadic melanoma. Similarities and/or differences between cell lines extracted from different melanoma cases could be useful in the future for diagnostic, prognostic and therapeutic purposes.

Melanomas of the oral cavity are extremely rare. Their rarity and their independence on exposure to UV radiation make them particularly interesting. The authors analyzed an oral multiphasic melanoma composed by a nodular nonpigmented ulcerated central region, a nodular ulcerated pigmented area, a pigmented nonulcerated region, and an area similar to a dysplastic nevus. They determined the expression of some genes involved in the differentiation and cellular transformation in morphologically different regions of melanoma. All these areas were also analyzed by electron microscopy. The various regions composing the melanoma expressed genes involved in melanogenesis and melanoma progression in a different manner. Electron microscopy observation of ultrathin sections of each region evidenced ultrastructural differences, being the cellular architecture more compromised in the most aggressive parts of the neoplasm. This pilot study identified morphological, molecular, and ultrastructural differences that characterize each region of the multiphasic melanoma.

PP-7 PGC1 alpha expression and correlation with BRAF mutational status I. Maida1 , A. Ferretta1 , T. Cocco1 , P. Zanna1 , R. Labarile1 , S. Guida3 , C. Grieco1 , L. Porcelli2 , A. Azzariti2 , S. Tommasi2 , A. Albano2 , S. Strippoli2 , M. Guida2 , R. Filotico3 , G. Guida1 1 Department of Basic Medical Sciences, Neurosciences and Sense Organs, School of Medicine, University of Bari, Bari, Italy; 2 Clinical Experimental Oncology Laboratory and Medical Oncology Department, National Cancer Institute, Bari, Bari, Italy; 3 Unit of Dermatology and Venereology, University of Bari, P.O. “A. Perrino”, Brindisi, Italy In this study we present the behaviour of the mitochondrial master regulator gene PGC1alpha in novel sporadic melanoma cell lines. The HBL and LND1 cell lines, wild type for BRAF, highly express PGC1alpha while hmel1, hmel9, hmel11, Mba72, M3, presenting BRAF mutations at the V600 residue, show a down regulation of this gene. Mutations in homo and/or heterozigosis of V600 in BRAF protein kinase always correspond to a down regulation of PGC1alpha as described for the cell lines A375, SKmel28, SKmel2, RPM7951, WM115 in Vasquez et al., 2013. MITF expression levels, measured by western blotting in our melanoma cell lines, were more abundant in HBL and LND1 cell lines with respect to the other cell lines harbouring BRAF mutations. There is a direct correspondence between PGC1alpha and MITF levels: higher levels of PGC1alpha are associated with an enhanced MITF quantity. The analysis of cAMP levels in our melanoma cell lines showed a similar trend, being higher in wt BRAF cell lines compared to the other cell lines. Our data confirm the key role of BRAF mutations, MITF and cAMP levels in melanoma biology, suggesting a very important association with the transcriptional co-activator PGC1alpha, involved in energy metabolism, in mitochondrial biogenesis but also in various physiological stimuli which are reprogrammed in melanoma cells. This factor, also involved in regulating angiogenetic mechanisms, could be an interesting gene for target therapy but also it could provide an important diagnostic role.

The differentiation, proliferation and survival of melanocytes and melanoma cells are controlled at various levels. Recently, the importance of translational regulation in promoting and sustaining tumorigenesis is being increasingly recognized. The expression of certain translational factors transiently increases in normal cells in response to growth factors and is constitutively upregulated in tumor cells. The initiation factor IF2 is a central regulator of translation, and is the target of the main pathways of translational control. Activation of eIF2 kinases in response to stress or tumoral transformation has been reported; increased phosphorylation of eIF2 alpha has been correlated with a metastatic phenotype in some kinds of tumors. Phosphorylated ERK levels can be used as marker of cell growth since they are higher in the actively proliferating cells. We investigated whether phosphorylated eIF2-alpha and phosphorylated ERK levels are reliable markers of tumorigenic potential in different melanoma cell lines, and to get insight into the mechanisms whereby eIF2-alpha phosphorylation may modulate cellular transformation. In our melanoma cell lines, showed different levels of phosphorylated eIF2alpha and phosphorylated ERK, which were higher in the cell lines derived from metastatic tumours as compared to primary melanoma cell lines. We showed that after treatment with a MEK inhibitor, ERK phosphorylation was inhibited while eIF2alpha phosphorylation was enhanced in all melanoma cell lines. Moreover Western blotting and confocal analyses revealed that phosphorylated eIF2alpha was localized in the nucleus of melanoma cell lines, supporting a possible relationship between phosphorylation levels and subcellular localization of phosphorylated eIF2 alpha and cell proliferation in malignant melanoma.

Introduzione: Il melanoma maligno è oggi considerato una patologia emergente a causa dell’aumento costante dell'incidenza e dell’assenza di terapie efficaci nel trattamento dei pazienti con melanoma metastatico. Esistono inoltre sempre maggiori evidenze in letteratura relative alla complessità dei meccanismi molecolari legati all'insorgenza e alla progressione della malattia. Nel melanoma familiare è ormai certo che mutazioni dei geni CDK4 ed CDKN2A giocano un ruolo fondamentale. Nel melanoma sporadico invece la letteratura non fornisce ancora certezze nonostante questo tipo di melanoma risulti essere più rappresentato nella popolazione. Obiettivi: Nonostante l'eterogeneità dell'assetto genetico, nei pazienti affetti da melanoma si mira all’individuazione di eventuali correlazioni tra le vie cellulari coinvolte e la biologia della malattia al fine di fornire informazioni di supporto nella messa punto di nuove target therapy. È stato condotto uno studio di genotipizzazione di alcuni dei geni coinvolti nella suscettibilità al melanoma e di caratterizzazione di alcune vie di regolazione della melanogenesi, della proliferazione cellulare e della adesione cellulare (MC1R, RAS/RAF/MAPK, βcatenine) in tre nuove linee cellulari di melanoma sporadico (hmel1, hmel9, hmel11) isolate nei nostri laboratori. I risultati ottenuti sono stati confrontati con quelli ottenuti su linee cellulari di controllo (HBL, LND1). Risultati: L’attività della tirosinasi, enzima chiave della melanogenesi, nelle linee cellulari isolate da melanoma sporadico, correla solo parzialmente con il livello di espressione del gene della tirosinasi e non correla col genotipo MC1R e la densità del suddetto recettore a livello della membrana plasmatica. Le nuove linee cellulari non presentano mutazioni di MC1R nonostante la trasduzione del segnale ad esso correlata risulti compromessa. Questo dato ci suggerisce che un signalling difettoso può essere più frequente di quanto ipotizzabile in relazione al genotipo MC1R. Nelle stesse linee, il gene BRAF risulta invece mutato al residuo V600 con conseguente fosforilazione e attivazione costitutiva di ERK. Le linee cellulari di controllo sono wild type (wt) per i geni MC1R e BRAF. Questo dato è a sostegno dell’ipotesi di un coinvolgimento del pathway di RAS/RAF/MAPK nella genesi del melanoma. Sono stati inoltre analizzati i livelli e la localizzazione subcellulare delle βcatenine. Esse presentano una diversa localizzazione nelle linee cellulari provenienti da melanoma sporadico (membrana/citoplasma), rispetto alle linee di melanoma controllo (nucleo/citoplasma). Questo suggerisce un diverso ruolo di tali proteine nell’adesione cellula-cellula o nella proliferazione delle diverse linee cellulari. Lo studio da noi condotto ha inoltre evidenziato l’assenza di correlazione tra il livello e la distribuzione delle βcatenine e il trattamento con NDP-MSH e il pathway di RAS / RAF / MAPK. Studi di sequenziamento delle βcatenine hanno poi consentito di riscontrare mutazioni nelle linee cellulari HBL, LND1 e hmel1 che sono estratte da metastasi cutanee di melanoma. Nella linea hmel1 la mutazione porta alla sostituzione di Trp383 con Cys383. Questa mutazione non è mai stata descritta in letteratura. Conclusioni: Le nuove linee cellulari presentano una compromissione del pathway del cAMP pur esprimendo un MC1R wt. Tale alterazione è stata precedentemente descritta in linee cellulari con attivazione costitutiva del pathway RAS/RAF/MAPK. I nostri risultati confermano l’associazione tra mutazione di BRAF e disaccoppiamento funzionale di MC1R con la produzione di cAMP. Secondo quanto riportato in letteratura, le βcatenine hanno un ruolo importante nel determinare l’aggressività del melanoma. Il sequenziamento delle βcatenine ha consentito di individuare la mutazione Trp383 → Cys383 che sembrerebbe ridurre l’affinità delle βcatenine per APC, compromettendo l'assemblaggio del

Background: Metabolic reprogramming is commonly found in cancer but it is poorly understood in melanoma. Recent works [1,2] provided new insights concerning molecular mechanisms involved in mitochondrial biogenesis of melanoma. This work aims to find possible correlations between pathways involved in the onset and progression of the disease in order to provide supporting information in this field. In particular we studied the behaviour of the mitochondrial master regulator gene PGC1alpha in novel sporadic melanoma cell lines and its relations with BRAF mutational status. Materials and methods: We studied new cell lines extracted from sporadic metastatic melanomas (hmel1, M3, Mba72) and primary melanomas (hmel9, hmel11), genotyped for genes involved in melanoma development compared to control melanoma cell lines (HBL, LND1) wt for MC1R and BRAF genes. Hmel1, hmel9 and hmel11 have already been described in Zanna et al., 2011 [3] and Zanna et al., 2013 [4]. We evaluated PGC1a levels and some of its mitochondrial target genes and the mitochondrial respiratory capacity, the amount of ROS, and the lactate level. We related these data to BRAF mutational stauts and analyzed MITF and cAMP levels. Results: The HBL and LND1 cell lines, wt for BRAF, highly express PGC1alpha while hmel1, hmel9, hmel11, Mba72, M3, presenting BRAF mutations at the V600 residue, show a downregulation of this gene. MITF expression levels were more abundant in HBL and LND1 cell lines with respect to the other cell lines harbouring BRAF mutations. There is a direct correspondence between PGC1alpha and MITF levels: higher levels of PGC1alpha are associated with an enhanced MITF quantity. The analysis of cAMP levels in our melanoma cell lines showed a similar trend, being higher in wt BRAF cell lines compared to the other cell lines. Conclusions: Our data confirm the key role of BRAF mutations, MITF and cAMP levels in melanoma biology, suggesting a very important association with the transcriptional co-activator PGC1alpha, involved in energy metabolism and in mitochondrial biogenesis but also in various physiological stimuli that are reprogrammed in melanoma cells. These data support the divergent pathways hypothesis for melanoma, which may require a reappraisal of targeted cancer prevention and target therapeutic activities. References 1. Haq R, et al: Cancer Cell 2013, 23:302-315. 2. Vasquez F, et al: Cancer Cell 2013, 23:287-301. 3. Zanna P, et al: J Biol Regul Homeost Agents 2011, 25:239-247. 4. Zanna P, et al: J Biol Regul Homeost Agents 2013, 27:131-141.

Malignant transformation and tumorigenesis include a complex series of cellular and biomecular events which are not yet completely known. Emerging evidence supports the fundamental role of translational control mechanisms in the modulation of cell functions. Cell growth and proliferation depend on protein synthesis that is regulated, in part, by translational initiation factors. These factors transiently increase in normal cells in response to growth factors and are constitutively elevated in transformed cells. In particular, the eIF2 translation factor seems to be involved in the abnormal regulation of protein synthesis that leads to a tumorigenic phenotype. Recent works suggest the translation factor eIF2 to be involved in the pathogenesis and/or tumour progression in some kind of tumours such as bronchioloaveolar carcinoma, non-Hodgkin lymphomas, breast tumour, brain tumour, gastrointestinal carcinomas, melanoma. Moreover other data indicate that the pERK levels could influence the RAS/RAF/MAPK pathway modifying the cell proliferation parameters. In this study we have analysed the phosphorylated eIF2-alpha and ERK levels in different melanoma cell lines derived from metastatic or primary cutaneous melanoma lesions, as well as in a line from the primary skin lesion of a patient with metastatic melanoma. Our results show higher levels of phosphorylated eIF2alpha and ERK in the metastatic cell lines (including the line from the primary lesion in the patient with distant metastases) as compared to the primary melanoma cell lines. In such cell lines, the gene expression of proteins involved in cell cycle regulation (i.e., cyclin D1 and p53) was also examined. The results of our study suggest the relationship between p-eIF2 alpha and tumoral progression in the malignant melanoma. Further analyses could allow to identify phosphorylated eIF2 as a possible prognostic marker.

Premessa Il gene MC1R (melanocortin 1 receptor) è noto per il suo ruolo chiave nella regolazione della pigmentazione ed a tutt’oggi è discusso il suo coinvolgimento nella suscettibilità al melanoma (1). Materiali e metodi Al fine di valutare l’associazione tra varianti del gene MC1R e il rischio di melanoma in Puglia (di cui attualmente non sono ancora disponibili dati), abbiamo condotto uno studio caso-controllo che ha coinvolto 101 soggetti affetti da melanoma e 102 soggetti sani. Risultati Abbiamo osservato 42 polimorfismi di MC1R, incluse nuove varianti non precedentemente descritte in letteratura. La variante più frequente nei casi e nei controlli è la Val60Leu. Un incremento del rischio di melanoma è stato riscontrato in individui portatori di due o più varianti del recettore (odds ratio (OR) 2.336, 95% intervallo di confidenza (CI) 1.163 - 4.691), rispetto ai soggetti wild-type o portatori di una singola mutazione. In particolare, quando una di queste varianti è RHC il rischio di sviluppare melanoma aumenta (OR 3.122, 95% CI 1.080 - 9.023). A tal proposito, la combinazione di polimorfismi multipli di MC1R più rappresentata è la Met92Val e Thr314Thr. Abbiamo inoltre analizzato la possibile relazione tra melanoma e altri fattori di rischio: significativo risulta il ruolo di un numero di nevi >50 e il fototipo I e II. Conclusioni I nostri risultati supportano il ruolo dei polimorfismi di MC1R nella suscettibilità al melanoma in Puglia. Di particolare interesse è la frequenza della variante Val60Leu nella popolazione pugliese, in linea con i risultati della popolazione del Sud dell’Europa (2). Bibliografia 1. Raimondi S et al. – Journal of Cancer 2008; 122: 2753-60 2. Martínez-Cadenas C et al. – Mol Biol Evol 2013 2013 Oct 10. [Epub ahead of print]