ROS as Regulators of Cellular Processes in Melanoma.

In this review, we examine the multiple roles of ROS in the pathogenesis of melanoma, focusing on signal transduction and regulation of gene expression. In recent years, different studies have analyzed the dual role of ROS in regulating the redox system, with both negative and positive consequences on human health, depending on cell concentration of these agents. High ROS levels can result from an altered balance between oxidant generation and intracellular antioxidant activity and can produce harmful effects. In contrast, low amounts of ROS are considered beneficial, since they trigger signaling pathways involved in physiological activities and programmed cell death, with protective effects against melanoma. Here, we examine these beneficial roles, which could have interesting implications in melanoma treatment.

The Streptococcus agalactiae cell wall-anchored protein PbsP mediates adhesion to and invasion of epithelial cells by exploiting the host vitronectin/αv integrin axis.

Binding of microbial pathogens to host vitronectin (Vtn) is a common theme in the pathogenesis of invasive infections. In this study, we characterized the role of Vtn in the invasion of mucosal epithelial cells by Streptococcus agalactiae (i.e. group B streptococcus or GBS), a frequent human pathogen. Moreover, we identified PbsP, a previously described plasminogen-binding protein of GBS, as a dual adhesin that can also interact with human Vtn through its streptococcal surface repeat (SSURE) domains. Deletion of the pbsP gene decreases both bacterial adhesion to Vtn-coated inert surfaces and the ability of GBS to interact with epithelial cells. Bacterial adherence to and invasion of epithelial cells were either inhibited or enhanced by cell pretreatment with, respectively, anti-Vtn antibodies or Vtn, confirming the role of Vtn as a GBS ligand on host cells. Finally, antibodies directed against the integrin αv subunit inhibited Vtn-dependent cell invasion by GBS. Collectively, these results indicate that Vtn acts as a bridge between the SSURE domains of PbsP on the GBS surface and host integrins to promote bacterial invasion of epithelial cells. Therefore, inhibition of interactions between PbsP and extracellular matrix components could represent a viable strategy to prevent colonization and invasive disease by GBS.

Genistein reduces proliferation of EP3-expressing melanoma cells through inhibition of PGE2-induced IL-8 expression.

Genistein, a natural isoflavone found in soybean products, is considered as a powerful anti-cancer agent, although the involved mechanisms are not fully understood. There is a growing body of evidence that, among the genes inhibited by genistein and responsible for cell cycle progression, invasion, metastasis, and angiogenesis, IL-8 occupies a relevant place. On the other hand, it is equally well documented that IL-8 is upregulated by prostaglandin E2 (PGE2) in different pathological conditions, particularly in neoplastic disease. Here we investigated whether genistein could affect cell growth in a panel of oral, uveal and cutaneous melanoma cell lines by interfering with basal or PGE2-induced IL-8 production. To this end, experiments were performed to evaluate the effect of PGE2 treatment on IL-8 levels, the expression and the role of PGE2 receptors and whether genistein could be able to interfere with these events. Finally, it was evaluated whether the inhibition of oral, uveal and cutaneous melanoma cell proliferation in the presence of genistein could be related to a reduction of IL-8 levels. We show that PGE2 enhances IL-8 synthesis via the EP3 receptor and that genistein is able to down-regulate the latter, as well as to decrease IL-8 mRNA and protein expression, thereby inhibiting oral, uveal and cutaneous melanoma cell proliferation. Taken together, our data provide new insights into the anti-cancer properties of genistein by showing that this flavonoid may affect the development and growth of melanoma at oral, uveal and cutaneous sites. Moreover, these results provide evidence that genistein may exert its therapeutic activity through its ability to prevent PGE2-mediated IL-8 induction.

Possible protective role of the ABCA4 gene c.1268A>G missense variant in Stargardt disease and syndromic retinitis pigmentosa in a Sicilian family: Preliminary data.

In the wide horizon of ophthalmologically rare diseases among retinitis pigmentosa forms, Stargardt disease has gradually assumed a significant role due to its heterogeneity. In the present study, we aimed to support one of two opposite hypotheses concerning the causative or protective role of heterozygous c.1268A>G missense variant of the ABCA4 gene in Stargardt disease and in syndromic retinitis pigmentosa. This study was based on a family consisting of three members: proband, age 54, with high myopia, myopic chorioretinitis and retinal dystrophy; wife, age 65, with mild symptoms; daughter, age 29, asymptomatic. After genetic counseling, ABCA4 and RP1 gene analysis was performed. The results highlighted an important genetic picture. The proband was found to carry two variant RP1 SNPs, rs2293869 (c.2953A>T) and rs61739567 (c.6098G>A), and, a wild-type condition for four RP1 polymorphisms, rs444772 (c.2623G>A) and three SNPs in the 'hot-spot' region, exon 4. The proband's wife, instead, showed an opposite condition compared to her husband: a homozygous mutated condition for the first four SNPs analyzed, while the last two were wild-type. Regarding the ABCA4 gene, the proband evidenced a wild-type condition. Furthermore, the wife showed a heterozygous condition of ABCA4 rs3112831 (c.1268A>G). As expected, the daughter presented heterozygosity for all variants of both genes. In conclusion, even though the c.1268A>G missense variant of the ABCA4 gene has often been reported as causative of disease, and in other cases protective of disease, in our family case, the variant appears to reduce or delay the risk of onset of Stargardt disease.

Epidrugs in the Immunotherapy of Cutaneous and Uveal Melanoma.

Epigenetic modifications can affect numerous mechanisms used by neoplastic cells to evade immune control. In melanoma epigenetic defects, caused by dysregulations in the expression of genome writers, erasers, or readers, play a significant role in the reduced expression of molecules required for efficient immune recognition as well as antigen presentation and processing. Alterations in gene expression were identified in tumor-associated antigens (TAAs), human leukocyte antigen (HLA) complex, co-stimulatory/accessory molecules, antigen processing machinery (APM), and NKG2D ligands that have shown to be silenced or down-regulated in melanoma. In agreement with the inherent reversibility of epigenetic silencing, epigenetic drugs such as inhibitors of DNA methyltransferases (DNMTs), histone deacetylases (HDACs), histone methyltransferase enhancer of Zeste homolog 2 (EZH2), and modifiers of microRNA (miRNA) dysregulation or antagomirs can restore the expression of these molecules, favouring the recognition of cancer cells by immune responses, reducing the resistance to Natural Killer (NK) and cytotoxic T cells (CTL), and enhancing the functions of antigen presenting cells. Moreover, inhibitors of reader proteins seem to preferentially affect the NF-kB-induced activation of pro-inflammatory cytokine genes. At present an increasing interest is shown toward new combined therapeutic approaches employing epidrugs or new molecular inhibitors and in vivo immunotherapies, such as vaccines and adoptive T-cell transfer (ACT). This review summarizes the current understanding of the role of epidrugs in the modulation of molecules involved in the melanoma immune response and focuses on their future clinical use in new therapeutic combinations for melanoma treatment.

DSS1 promoter hypomethylation and overexpression predict poor prognosis in melanoma and squamous cell carcinoma patients.

Previous studies have found a link between high expression levels of the Deleted in Split hand/Split foot 1 (DSS1) gene and cancer progression. The aim of this study was to examine whether overexpression of DSS1 is a feature of melanoma and squamous cell carcinoma (SCC) and if any epigenetic modifications are involved. Evaluation of DSS1 expression profile indicated that the gene is overexpressed in 112 of 130 cutaneous melanomas (86.1%), 41 of 64 uveal melanomas (64.1%), 67 of 82 mucosal melanomas (81.7%), and 61 of 75 SCC samples (81.3%), relative to normal skin. An inverse correlation between DSS1 expression and methylation status of the promoter was found. In vitro studies showed that treatment of DSS1-methylated melanoma and SCC cells with the DNA methyltransferase inhibitor 5-aza-2'-deoxycytidine significantly increased DSS1 expression at mRNA and protein levels. Interestingly, a significant association between high DSS1 expression levels and some clinicopathological variables, such as metastasis, ulceration, and reduced overall/disease-free survival was observed. In summary, these data suggest that the extent of promoter methylation plays a role in modulating DSS1 gene expression and highlight that promoter hypomethylation is a frequent event in melanoma and SCC closely linked to poor prognosis.

miR-92a-3p and MYCBP2 are involved in MS-275-induced and c-myc-mediated TRAIL-sensitivity in melanoma cells.

Increasing evidence has demonstrated that in several tumors c-myc acts either as an oncogène or as a proapoptotic agent, depending on binding partner interactions. Recently, we showed that up-regulation of this gene by the histone deacetylase inhibitor MS-275 was responsible for sensitization to TRAIL-induced apoptosis through c-FLIP repression in melanoma. The present study aimed at investigating whether, in addition to inducing H3 hyperacetylation at the c-myc promoter, MS-275 could enhance cell death through the regulation of miRNAs involved in apoptosis, such as the miR-17-92 cluster. Following MS-275 treatment, a decrease in miR-92a-3p was observed either in TRAIL-resistant or TRAIL-sensitive cutaneous and uveal melanoma cells. Prediction tools revealed that miR-92a-3p targeted MYCBP2. Gain- and loss-of-function experiments showed that the 3'-UTR of MYCBP2 mRNA was the target of miR-92a-3p, as ectopic expression of miR-92a-3p resulted in MYCBP2 downregulation whereas miR-92a-3p knockdown markedly increased the expression of MYCBP2. Silencing of MYCBP2 counteracted the pro-apoptotic effects exerted by the down-regulation of miR-92a-3p and prevented c-myc-induced repression of c-FLIP, indicating a pivotal role of MYCBP2 as a mediator of miR-92a-3p and c-myc function. Together, our findings indicate that the MS-275-triggered downregulation of the oncogenic miR-92a-3p- which leads to the overexpression of its target gene MYCBP2 - is an event required for the enhanced susceptibility of melanoma cells to TRAIL-mediated apoptosis. Our data illustrate another epigenetic mechanism activated by MS-275 at the post-transcriptional level in melanoma, in addition to its best-known effects at the transcriptional level.

Functional characterization of a monoclonal antibody epitope using a lambda phage display-deep sequencing platform.

We have recently described a method, named PROFILER, for the identification of antigenic regions preferentially targeted by polyclonal antibody responses after vaccination. To test the ability of the technique to provide insights into the functional properties of monoclonal antibody (mAb) epitopes, we used here a well-characterized epitope of meningococcal factor H binding protein (fHbp), which is recognized by mAb 12C1. An fHbp library, engineered on a lambda phage vector enabling surface expression of polypeptides of widely different length, was subjected to massive parallel sequencing of the phage inserts after affinity selection with the 12C1 mAb. We detected dozens of unique antibody-selected sequences, the most enriched of which (designated as FrC) could largely recapitulate the ability of fHbp to bind mAb 12C1. Computational analysis of the cumulative enrichment of single amino acids in the antibody-selected fragments identified two overrepresented stretches of residues (H248-K254 and S140-G154), whose presence was subsequently found to be required for binding of FrC to mAb 12C1. Collectively, these results suggest that the PROFILER technology can rapidly and reliably identify, in the context of complex conformational epitopes, discrete "hot spots" with a crucial role in antigen-antibody interactions, thereby providing useful clues for the functional characterization of the epitope.

Epitope Mapping of a Monoclonal Antibody Directed against Neisserial Heparin Binding Antigen Using Next Generation Sequencing of Antigen-Specific Libraries.

We explore here the potential of a newly described technology, which is named PROFILER and is based on next generation sequencing of gene-specific lambda phage-displayed libraries, to rapidly and accurately map monoclonal antibody (mAb) epitopes. For this purpose, we used a novel mAb (designated 31E10/E7) directed against Neisserial Heparin-Binding Antigen (NHBA), a component of the anti-group B meningococcus Bexsero® vaccine. An NHBA phage-displayed library was affinity-selected with mAb 31E10/E7, followed by massive sequencing of the inserts present in antibody-selected phage pools. Insert analysis identified an amino acid stretch (D91-A128) in the N-terminal domain, which was shared by all of the mAb-enriched fragments. Moreover, a recombinant fragment encompassing this sequence could recapitulate the immunoreactivity of the entire NHBA molecule against mAb 31E10/E7. These results were confirmed using a panel of overlapping recombinant fragments derived from the NHBA vaccine variant and a set of chemically synthetized peptides covering the 10 most frequent antigenic variants. Furthermore, hydrogen-deuterium exchange mass-spectrometry analysis of the NHBA-mAb 31E10/E7 complex was also compatible with mapping of the epitope to the D91-A128 region. Collectively, these results indicate that the PROFILER technology can reliably identify epitope-containing antigenic fragments and requires considerably less work, time and reagents than other epitope mapping methods.

Neutrophils Directly Recognize Group B Streptococci and Contribute to Interleukin-1ß Production during Infection.

Previous studies have shown that the pro-inflammatory cytokine IL-1ß has a crucial role in host defenses against group B streptococcus (GBS), a frequent human pathogen, by recruiting neutrophils to infection sites. We examined here the cell types and mechanisms involved in IL-1ß production during infection. Using a GBS-induced peritonitis model in mice, we first found that a large proportion of exudate cells contain intracellular IL-1ß by immunofluorescence. Of the IL-1ß positive cells, 82 and 7% were neutrophils and macrophages, respectively, suggesting that the former cell type might significantly contribute to IL-1ß production. Accordingly, depletion of neutrophils with anti-Ly6G antibodies resulted in a significant reduction in the levels of IL-1ß, but not of TNF-α or IL-6. We next found that neutrophils are capable of releasing mature IL-1ß and TNF-α directly in response to in vitro stimulation with GBS. The production of pro-IL-1ß and TNF-α in these cells required the Toll-like receptor (TLR) adaptor MyD88 and the chaperone protein UNC93B1, which is involved in mobilization of a subfamily of TLRs to the endosomes. Moreover, pro-IL-1ß processing and IL-1ß release was triggered by GBS hemolysin and required components of the canonical inflammasome, including caspase-1, ASC and NLRP3. Collectively our findings indicate that neutrophils make a significant contribution to IL-1ß production during GBS infection, thereby amplifying their own recruitment. These cells directly recognize GBS by means of endosomal TLRs and cytosolic sensors, leading to activation of the caspase-1 inflammasome.

Phage display revisited: Epitope mapping of a monoclonal antibody directed against Neisseria meningitidis adhesin A using the PROFILER technology.

There is a strong need for rapid and reliable epitope mapping methods that can keep pace with the isolation of increasingly larger numbers of mAbs. We describe here the identification of a conformational epitope using Phage-based Representation OF ImmunoLigand Epitope Repertoire (PROFILER), a recently developed high-throughput method based on deep sequencing of antigen-specific lambda phage-displayed libraries. A novel bactericidal monoclonal antibody (mAb 9F11) raised against Neisseria meningitidis adhesin A (NadA), an important component of the Bexsero(®) anti-meningococcal vaccine, was used to evaluate the technique in comparison with other epitope mapping methods. The PROFILER technology readily identified NadA fragments that were capable of fully recapitulating the reactivity of the entire antigen against mAb 9F11. Further analysis of these fragments using mutagenesis and hydrogen-deuterium exchange mass-spectrometry allowed us to identify the binding site of mAb 9F11 (A250-D274) and an adjoining sequence (V275-H312) that was also required for the full functional reconstitution of the epitope. These data suggest that, by virtue of its ability to detect a great variety of immunoreactive antigen fragments in phage-displayed libraries, the PROFILER technology can rapidly and reliably identify epitope-containing regions and provide, in addition, useful clues for the functional characterization of conformational mAb epitopes.

PbsP, a cell wall-anchored protein that binds plasminogen to promote hematogenous dissemination of group B Streptococcus.

Streptococcus agalactiae (Group B Streptococcus or GBS) is a leading cause of invasive infections in neonates whose virulence is dependent on its ability to interact with cells and host components. We here characterized a surface protein with a critical function in GBS pathophysiology. This adhesin, designated PbsP, possesses two Streptococcal Surface Repeat domains, a methionine and lysine-rich region, and a LPXTG cell wall-anchoring motif. PbsP mediates plasminogen (Plg) binding both in vitro and in vivo and we showed that cell surface-bound Plg can be activated into plasmin by tissue plasminogen activator to increase the bacterial extracellular proteolytic activity. Absence of PbsP results in a decreased bacterial transmigration across brain endothelial cells and impaired virulence in a murine model of infection. PbsP is conserved among the main GBS lineages and is a major plasminogen adhesin in non-CC17 GBS strains. Importantly, immunization of mice with recombinant PbsP confers protective immunity. Our results indicate that GBS have evolved different strategies to recruit Plg which indicates that the ability to acquire cell surface proteolytic activity is essential for the invasiveness of this bacterium.

DNA methylation-induced E-cadherin silencing is correlated with the clinicopathological features of melanoma.

E-cadherin, a calcium-dependent cell-cell adhesion molecule, has an important role in epithelial cell function, maintenance of tissue architecture and cancer suppression. Loss of E-cadherin promotes tumor metastatic dissemination and predicts poor prognosis. The present study investigated the clinicopathological significance of E-cadherin expression in cutaneous, mucosal and uveal melanoma related to epigenetic mechanisms that may contribute to E-cadherin silencing. E-cadherin expression was reduced in 55/130 cutaneous (42.3%), 49/82 mucosal (59.7%) and 36/64 uveal (56.2%) melanoma samples as compared to normal skin controls and was inversely associated with promoter methylation. Of the 10 different CpG sites studied (nt 863, 865, 873, 879, 887, 892, 901, 918, 920 and 940), two sites (nt 892 and 940) were 90-100% methylated in all the melanoma specimens examined and the other ones were partially methylated (range, 53-86%). In contrast, the methylation rate of the E-cadherin gene was low in normal tissues (range, 5-24%). In all the three types of melanoma studied, a significant correlation was found between reduced levels of E-cadherin and reduced survival, high mitotic index and metastasis, accounting for the predilection of lymph nodal localization. In cutaneous and mucosal melanoma, low E-cadherin expression was positively correlated also with head/neck localization and ulceration. A high frequency of reduced E-cadherin levels occurred in choroid melanomas. In vitro experiments showed that E-cadherin transcription was restored following 5-aza-2'-deoxycytidine (5-aza-dC) treatment or DNMT1 silencing and was negatively correlated with the invasive potential of melanoma cells. The significant relationship between E-cadherin silencing and several poor prognostic factors indicates that this adhesion molecule may play an important role in melanomagenesis. Therefore, the inverse association of E-cadherin expression with promoter methylation raises the intriguing possibility that reactivation of E-cadherin expression through promoter demethylation may represent a potential therapeutic strategy for the treatment of melanoma.

Role of Genetics and Epigenetics in Mucosal, Uveal, and Cutaneous Melanomagenesis.

Melanoma prevalently occurs on parts of the body that have been overexposed to the sun. However, it can also originate in the nervous system, eye and mucous membranes. Melanoma has been thought for a long time to arise through a series of genetic mechanisms involving numerous irreversible changes within the human genome. However, recently, "epimutations" have attracted considerable attention owing to their high prevalence rate and reversible nature. These observations opened up new perspectives in the use of epidrugs with the potential for restoring the "correct" control of neoplastic genomes. Here, we focused on the common consensus on genetics and epigenetics in melanoma. We also discussed the clinical applications of regulators of epigenetic enzymes able to revert the epigenetic and metabolic hallmarks of melanoma cells. Such anti-neoplastic agents affect the expression profile of antioncogenes, proto-oncogenes, and microRNAs resulting in enhanced differentiation, apoptosis, and growth inhibition.

IL-10Rα expression is post-transcriptionally regulated by miR-15a, miR-185, and miR-211 in melanoma.

BACKGROUND: IL-10 is an immunoregulatory cytokine that increases during malignant diseases. The purpose of this study was to: i) determine the mRNA amounts of IL-10, IL-10Rα, and IL-10Rß in cutaneous and uveal melanoma cells and specimens; ii) evaluate their post-transcriptional regulation by miRNAs; iii) ascertain whether miRNA dysregulation may affect IL-10-induced proliferation. METHODS: Genome-wide miRNA expression profiling was performed using a human microarray platform. The reference gene mRNA was measured through qPCR. miRNAs/mRNAs interactions were predicted by TargetScan, microRNA, and PITA. Transfections of specific miRNA mimics/inhibitors were carried out. Cell proliferation was assessed by MTT assay in the presence of IL-10 after transfection with miRNA mimics/inhibitors. RESULTS: There were no differences in IL-10 mRNA levels between any of the 3 melanoma cell lines tested and normal melanocytes. However, lower IL-10Rα expression was found in G361 and OCM-1 cells, and higher levels of IL-10Rß were observed in G361 cells compared with normal melanocytes. GR-M cells did not exhibit any modifications in IL-10Rα and IL-10Rß expression. miR-15a, miR-185, miR-211, and miR-30d were upregulated in G361 and OCM-1 cells, remaining at similar levels in GR-M cells. miR-409-3p and miR-605were down-regulated exclusively in G361 cells. Prediction tools revealed that miR-15a, miR-185, and miR-211 targeted IL-10Rα whereas none of the miRNAs exclusively downregulated in G361 cells targeted IL-10Rß. Luciferase reporter and western blot assays showed that IL-10Rα expression is directly regulated by miR-15a, miR-185, and miR-211, either alone or in combination. An inverse expression pattern between IL-10Rα, on one side, and miR-15a, miR-185, and miR-211 on the other one was also shown in melanoma samples. Ectopic expression of individual miR-15a, miR-185, and miR-211, and even more their co-expression, caused a marked decrease in the proliferation rate of all the cell lines. Likewise, inhibition of any specific miRNA promoted cell growth, an effect that further increased when inhibition concerned all three miRNA. Moreover, specific knockdown of IL-10Rα prevented the proliferative effect of miRNA inhibitors. CONCLUSIONS: Our results support a key role of IL-10Rα in the development and progression of melanoma and suggest that the IL-10/IL-10 receptor system may become a new therapeutic target for melanoma treatment.

The overriding of TRAIL resistance by the histone deacetylase inhibitor MS-275 involves c-myc up-regulation in cutaneous, uveal, and mucosal melanoma.

Malignant melanoma is a highly aggressive tumor which may occur in the skin, eye, and mucous membranes. The prognosis of melanoma remains poor in spite of therapeutic advances, emphasizing the importance of innovative treatment modalities. Currently, tumor necrosis factor-related apoptosis-inducing ligand (TRAIL) is showing promising clinical responses, however its use is hampered by intrinsic or acquired melanoma resistance to apoptosis. Recently, we showed that the combination of TRAIL with the class I-specific histone deacetylase inhibitor (HDACi) MS-275 was a privileged way to override TRAIL resistance through down-regulation of cellular Fas-associated death domain (FADD)-like interleukin-1beta-converting enzyme-inhibitory protein (c-FLIP). Here, we elucidated the underlying mechanism and provided evidence that a crucial step in the c-FLIP downregulation triggered by MS-275 implies the up-regulation of c-myc, a transcriptional repressor of c-FLIP. Notably, MS-275 caused H3 histone acetylation at the promoter of c-myc and increased its binding to the c-FLIP promoter, that in turn led to reduced c-FLIP gene transcription. Knockdown of c-myc prevented the MS-275-mediated downregulation of c-FLIP and hindered TRAIL-plus MS-275-induced apoptosis. Findings reported here provide additional knowledge tools for a more aware and effective molecular therapy of melanoma.

Cellular Mechanisms of Oxidative Stress and Action in Melanoma.

Most melanomas occur on the skin, but a small percentage of these life-threatening cancers affect other parts of the body, such as the eye and mucous membranes, including the mouth. Given that most melanomas are caused by ultraviolet radiation (UV) exposure, close attention has been paid to the impact of oxidative stress on these tumors. The possibility that key epigenetic enzymes cannot act on a DNA altered by oxidative stress has opened new perspectives. Therefore, much attention has been paid to the alteration of DNA methylation by oxidative stress. We review the current evidence about (i) the role of oxidative stress in melanoma initiation and progression; (ii) the mechanisms by which ROS influence the DNA methylation pattern of transformed melanocytes; (iii) the transformative potential of oxidative stress-induced changes in global and/or local gene methylation and expression; (iv) the employment of this epimutation as a biomarker for melanoma diagnosis, prognosis, and drug resistance evaluation; (v) the impact of this new knowledge in clinical practice for melanoma treatment.

Involvement of epimutations in meningioma.

Epimutations are heritable and reversible cell markers, which can influence cell function going beyond the effects of DNA mutations. They result from multiple and coordinated mechanisms able to modulate gene expression. Regarding the significance of epigenetics in meningioma, few and somehow contradictory results are available, although promising information has been obtained. Here we highlight the most recent advances about the impact of DNA methylation, histone modifications, and microRNA regulation on meningioma development as well as the interplay between genetic and epigenetic alterations. Data indicate that epigenetics can help to identify novel candidate genes for the management and treatment of meningioma.

Epigenetic marks responsible for cadmium-induced melanoma cell overgrowth.

Cadmium (Cd) is a human carcinogen that likely acts via epigenetic mechanisms. However, the precise role of Cd in melanoma remains to be defined. The goals of this study are to: (i) examine the effect of Cd on the proliferation rate of cutaneous and uveal melanoma cells; (ii) identify the genes affected by Cd exposure; (iii) understand whether epigenetic changes are involved in the response to Cd. The cell growth capacity increased at 48 h after Cd treatment at doses ranging from 0.5 to 10 µM. The research on the key genes regulating proliferation has shown that aberrant methylation is responsible for silencing of p16(INK4A) and caspase 8 in uveal and cutaneous melanoma cells, respectively. The methylation and expression patterns of p14(ARF), death receptors 4/5, and E-cadherin remained unmodified after Cd treatment in all the cell lines analyzed. Ectopic expression of p16(INK4A) abolished the overgrowth of uveal melanoma cells in response to Cd and the overexpression of caspase 8 drastically increased the apoptotic rate of Cd-treated cutaneous melanoma cells. In conclusion, our data suggest that hypermethylation of p16(INK4A) and caspase 8 represents the most common event linked to Cd-induced stimulation of cell growth and inhibition of cell death pathway in melanoma.

Epigenetic regulation of p14ARF and p16INK4A expression in cutaneous and uveal melanoma.

Inactivation of p14ARF and p16INK4A by epigenetic changes in cutaneous and uveal melanoma has been here investigated. Compared with melanocytes, p14ARF mRNA reduction and p16INK4A inactivation were frequently noticed. No association between p14ARF promoter methylation and mRNA levels was found, whereas aberrant p16INK4A methylation was associated with gene silencing (p<0.001). Comparative analysis within melanomas of different Breslow's thicknesses showed that drastic reductions in p14ARF and p16INK4A expression appeared at the level of thin/intermediate and intermediate/thick transitions. The effects of 5-aza-2'-deoxycytidine (5-aza-dC) and suberanilohydroxamic acid (SAHA) on in vivo binding of DNA methyltransferases (DNMTs) and acetyl histone H3/H4 to p14ARF and p16INK4A promoters were tested together with the impact of ectopic expression of p14ARF and p16INK4A on cell proliferation, migration, and invasion. SAHA treatment induced H3 and H4 hyperacetylation at the p14ARF promoter followed by increased p14ARF expression, whereas exposure to 5-aza-dC decreased the recruitment of DNMT1 and DNMT3b at the p16INK4A promoter and reactivated p16INK4A. Studies on promoter-associated di-methyl histone H3 (Lys4) levels ruled out an involvement of this epigenetic trait on p14ARF and p16INK4A expression. The enforced expression of p14ARF or p16INK4A and, even more so, their co-expression, significantly reduced cell proliferation, migration and invasion. Our data pinpoint: i) a frequent impairment of p14ARF and p16INK4A gene expression by epigenetic modifications in melanoma; ii) histone hypoacetylation as the dominant mechanism of p14ARF silencing; and iii) 5' CpG promoter methylation as the major mechanism of p16INK4A gene inactivation. Collectively, our data suggest that selected epi-drugs may be useful in melanoma treatment.