Thyroid Cancer Persistence in Patients with Unreliable Thyroglobulin Measurement: Circulating microRNA as Candidate Alternative Biomarkers.

BACKGROUND: We aimed to evaluate the role of circulating miRNAs as a biomarker of the persistence of papillary thyroid cancer (PTC) in patients with an "uninformative" thyroglobulin (Tg) measurement. METHODS: We prospectively enrolled 49 consecutive PTC patients with Tg-positive antibodies (TgAb) who had undergone a (near)-total thyroidectomy and 131I therapy (RIT). The serum thyroid stimulating hormone (TSH), Tg, and TgAb levels were measured before and at 6 and 12 months after RIT, respectively. The serum miRNA (221, 222, 375, 155, and 146b) levels were measured simultaneously. RESULTS: The response to the initial therapy was assessed according to the 2015 ATA criteria. A decrease in 50% or more of serum miRNA over time was observed in 41/49 PTC patients, who showed an excellent response (ER), but six and two patients were classified to have an indeterminate/incomplete biochemical or incomplete structural response to initial therapy. CONCLUSION: Serum miRNA kinetics emerge as a promising biomarker for the early detection of a persistent disease in PTC patients with uninformative Tg results.

The First Identification in Italy of SARS-CoV-2 Omicron BA.4 Harboring KSF141_del: A Genomic Comparison with Omicron Sub-Variants.

The rapid emergence and worldwide detection of the SARS-CoV-2 Omicron variant underscore the importance of robust genomic surveillance systems and prompt information sharing among global public health partners. The Omicron variant has rapidly replaced the Delta variant as a dominating SARS-CoV-2 variant because of natural selection, favoring the variant with higher infectivity and stronger vaccine breakthrough capability. The Omicron variant is also known as B.1.1.529. It has four sub-variants, indicated as BA.1, BA.2, BA.3 and BA.4. Among them, BA.1 is the currently prevailing sub-variant, and BA.2 has been found to be able to alarmingly re-infect patients initially infected by Omicron BA.1. The BA.3 sub-variant is a combination of mutations of BA.1 and BA.2, especially in the spike protein. Today, the BA.4 variant is emerging, which is herein described, and it was the first detected in Italy. Via bioinformatic analysis, we are reporting that the BA.4 that was identified harbors a new mutation, specifically a deletion in the ORF1ab gene, corresponding to KSF141_del in non-structural protein 1 (nsp1), a critical virulence factor able to suppress host translation. The bioinformatics comparison analysis with the other three sub-variants reveals that the deletion was not present before and was never reported until now. Therefore, we can speculate that Omicron BA.4 will become a new dominating "variant of concern" and may also break vaccine protection. Moreover, we show that other proteins are mutated in the BA.4. In particular, seven mutations are recognized in the nucleocapsid (N) protein, and the capability of five different types of rapid antigenic tests are used to identify it.

A new phenotype of muscle glycogen synthase deficiency (GSD0B) characterized by an adult onset myopathy without cardiomyopathy.

Muscle Glycogenosis type 0 (GSD0B) is an extremely rare disorder first recognized in 2007 in three siblings with childhood onset and severe cardiomyopathy. Since then, a few cases with severe cardiac involvement and premature death have been reported. We describe two unrelated cases presenting with an adult-onset myopathy with no heart involvement. Clinical features were quite similar in both patients, mainly characterized by early fatigability, myalgia and muscle weakness. Muscle biopsy revealed marked glycogen depletion in nearly all myofibers. Biochemical assay demonstrated a marked reduction of Glycogen Synthase (GS) activity. Sequence analysis of GYS1 revealed two new variants: a homozygous G to C substitution in the splice donor consensus site (c.678+1G>C) in patient1 and a homozygous missense variant c.630G>C in exon 3 (p. Asp145His) in patient 2. This study describes a new phenotype of muscle GSD0B presenting with adult onset, proximal myopathy, no cardiac abnormalities and a quite benign disease course. This report highlights the importance of a systematic diagnostic approach that includes muscle morphology and enzymatic assay to facilitate the identification of adult patients with GSD0B.

Methylome Analysis in Nonfunctioning and GH-Secreting Pituitary Adenomas.

Pituitary adenomas (PAs), usually benign lesions, can sometimes present with "aggressive" features (rapid growth, local invasiveness, scarce response to conventional treatments). Despite the fact that a few genetic alterations have been associated to this clinical behavior, the role of epigenetic modifications, mainly methylation and miRNAs activity, is now opening new frontiers in this field. We evaluated the methylation profile of 21 PA (11 GH-omas, 10 nonfunctioning tumors-NFPAs) samples from TNS surgery and 5 normal pituitaries, collected at our neurosurgery between 2015 and 2017. DNA was extracted and sequenced, selecting 184,841 target regions. Moreover, methylation profiles were correlated with demographic, radiological, and clinicopathological features. NFPAs showed higher methylation levels vs. GH-omas, with 178 differentially methylated regions (DMRs) mainly consisting of noncoding and intronic sequences, and mostly localized in the open sea regions. We also found three hypermethylated genes (C7orf50, GNG7, and BAHCC1) involved in tumorigenesis processes and potentially influencing pituitary tumor pathophysiology. Among the clinicopathological features, only the maximum diameter resulted significantly higher in NFPAs. Our data provide further evidence of the complex epigenetic background of pituitary tumors. In line with the current literature, we confirmed a significant prevalence of hypermethylation in NFPAs vs. GH-omas, whose pathophysiological consequence is yet to be defined.

Diagnostic Challenges in Late Onset Multiple Acyl-CoA Dehydrogenase Deficiency: Clinical, Morphological, and Genetic Aspects.

Background: Multiple acyl-CoA dehydrogenase deficiency (MADD) is an autosomal recessive disorder of fatty acid oxidation due to deficiency of the mitochondrial electron transfer chain. The late-onset form is characterized by exercise intolerance, muscle weakness, and lipid storage in myofibers. Most MADD patients greatly benefit from riboflavin supplementation. Patients and methods: A retrospective study was conducted on patients with a diagnosis of vacuolar myopathy with lipid storage followed in our neuromuscular unit in the last 20 years. We selected 10 unrelated patients with the diagnosis of MADD according to clinical, morphological, and biochemical aspects. Clinical features, blood tests including serum acylcarnitines, EMG, and ENG were revised. Muscle biopsy was performed in all, and one individual underwent also a sural nerve biopsy. Gene sequencing of ETFA, ETFB, and ETFDH was performed as a first-tier genetic analysis followed by next-generation sequencing of an hyperCKemia gene panel in patients with undefined genotypes. Results: Clinical evaluation at onset in all our patients showed fatigue and muscle weakness; four patients showed difficulties in chewing, three patients complained of dysphagia, two patients had a dropped head, and a patient had an unexpected ataxia with numbness and dysesthesia. Laboratory blood tests revealed a variable increase in serum CK (266-6,500) and LDH levels (500-2,000). Plasma acylcarnitine profile evidenced increased levels of different chains intermediates. EMG was either normal or showed myogenic or neurogenic patterns. NCS demonstrated sensory neuropathy in two patients. Muscle biopsies showed a vacuolar myopathy with a variable increase in lipid content. Nerve biopsy evidenced an axonal degeneration with the loss of myelinated fibers. ETFDH genetic analysis identifies 14 pathogenic variants. Patients were treated with high doses of riboflavin (400 mg/die). All of them showed a rapid muscle strength improvement and normalization of abnormal values in laboratory tests. Neuropathic symptoms did not improve. Conclusion: Our data confirmed that clinical features in MADD patients are extremely variable in terms of disease onset and symptoms making diagnosis difficult. Laboratory investigations, such as serum acylcarnitine profile and muscle biopsy evaluation, may strongly address to a correct diagnosis. The favorable response to riboflavin supplementation strengthens the importance of an early diagnosis of these disorders among the spectrum of metabolic myopathies.

CXCL12/CXCR4 axis supports mitochondrial trafficking in tumor myeloma microenvironment.

Mesenchymal stromal cells (MSCs) within the protective microenvironment of multiple myeloma (MM) promote tumor growth, confer chemoresistance and support metabolic needs of plasma cells (PCs) even transferring mitochondria. In this scenario, heterocellular communication and dysregulation of critical signaling axes are among the major contributors to progression and treatment failure. Here, we report that myeloma MSCs have decreased reliance on mitochondrial metabolism as compared to healthy MSCs and increased tendency to deliver mitochondria to MM cells, suggesting that this intercellular exchange between PCs and stromal cells can be consider part of MSC pro-tumorigenic phenotype. Interestingly, we also showed that PCs promoted expression of connexin 43 (CX43) in MSCs leading to CXCL12 activation and stimulation of its receptor CXCR4 on MM cells favoring protumor mitochondrial transfer. Consistently, we observed that selective inhibition of CXCR4 by plerixafor resulted in a significant reduction of mitochondria trafficking. Moreover, intracellular expression of CXCR4 in myeloma PCs from BM biopsy specimens demonstrated higher CXCR4 colocalization with CD138+ cells of non-responder patients to bortezomib compared with responder patients, suggesting that CXCR4 mediated chemoresistance in MM. Taken together, our data demonstrated that CXCL12/CXCR4 axis mediates intercellular coupling thus suggesting that the myeloma niche may be exploited as a target to improve and develop therapeutic approaches.

Nusinersen Modulates Proteomics Profiles of Cerebrospinal Fluid in Spinal Muscular Atrophy Type 1 Patients.

Spinal muscular atrophy (SMA) type 1 is a severe infantile autosomal-recessive neuromuscular disorder caused by a survival motor neuron 1 gene (SMN1) mutation and characterized by progressive muscle weakness. Without supportive care, SMA type 1 is rapidly fatal. The antisense oligonucleotide nusinersen has recently improved the natural course of this disease. Here, we investigated, with a functional proteomic approach, cerebrospinal fluid (CSF) protein profiles from SMA type 1 patients who underwent nusinersen administration to clarify the biochemical response to the treatment and to monitor disease progression based on therapy. Six months after starting treatment (12 mg/5 mL × four doses of loading regimen administered at days 0, 14, 28, and 63), we observed a generalized reversion trend of the CSF protein pattern from our patient cohort to that of control donors. Notably, a marked up-regulation of apolipoprotein A1 and apolipoprotein E and a consistent variation in transthyretin proteoform occurrence were detected. Since these multifunctional proteins are critically active in biomolecular processes aberrant in SMA, i.e., synaptogenesis and neurite growth, neuronal survival and plasticity, inflammation, and oxidative stress control, their nusinersen induced modulation may support SMN improved-expression effects. Hence, these lipoproteins and transthyretin could represent valuable biomarkers to assess patient responsiveness and disease progression.

SIRT2 and SIRT3 expression correlates with redox imbalance and advanced clinical stage in patients with multiple myeloma.

OBJECTIVES: Sirtuins comprise seven family elements (SIRT1-7) involved in various cell signalling pathways comprising cancer inhibition and tumorigenesis. The present study aims to evaluate SIRT2 and SIRT3 gene expression and potential redox reactions in patients with multiple myeloma (MM) at onset and its correlation with disease status, extent and presence of organ damage secondary to myeloma. DESIGN & METHODS: Total RNA was extracted from 17 MM patients and 10 controls to assess gene expression using real-time PCR. The NAD+/NADH ratio as well as the levels of glutathione peroxidase (GPx) and hydrogen peroxide (HP) in peripheral blood mononuclear cells (PBMCs) were determined using established biochemical assays. RESULTS: SIRT2 and SIRT3 expression is reduced in MM patients compared to healthy controls. Correlational analysis demonstrated that SIRT2 reduction is associated with advanced clinical stage and with more advanced bone lesions than in the remaining patients. SIRT3 expression is correlated with lytic bone lesions. Biochemical analysis indicated an imbalance of oxidative stress biomarkers with low concentrations of the antioxidant enzyme GPx, low amounts of NAD + and higher concentrations of pro-oxidant enzyme HP in PBMCs of MM patients compared to controls. Moreover, MM patients with bone lesions had lower concentrations of NAD + and GPx in PBMCs than patients without signs of bone disease. In addition, MM patients had higher quantities of intracellular HP than controls. CONCLUSIONS: Our results demonstrate that SIRT2 and SIRT3 are downregulated in MM and that lower concentrations correlate with an advanced stage of disease and redox imbalance. We conclude that SIRT2 and SIRT3 together with oxidative stress biomarkers, may be useful for improved risk stratification of MM patients.

Serum levels of miRNA-21-5p in vitiligo patients and effects of miRNA-21-5p on SOX5, beta-catenin, CDK2 and MITF protein expression in normal human melanocytes.

BACKGROUND: Epigenetics of vitiligo was evaluated in few studies. In particular, the role of miR-21, a microRNA involved in various processes, including melanogenesis, was never investigated. OBJECTIVE: Evaluation of serum levels of miR-21-5p in vitiligo patients and miR-21-5p effects on melanogenesis. METHODS: We measured serum levels of miR-21-5p in 40 patients affected by nonsegmental vitiligo and 40 sex- and age-matched healthy controls. Next, normal human melanocytes were transfected with miR-21-5p to study the effects of this microRNA, which targeted some proteins involved in melanogenesis pathway like SOX5, beta-catenin, cyclin-dependent kinase 2 (CDK2), and MITF. RESULTS: The expression of miR-21-5p in vitiligo patients was 3.6-4454.4 fold (mean 990.4 ± 1397.9) higher than in controls. The relative expression of miR-21-5p was directly and significantly correlated with disease severity, defined by VASI (Vitiligo Area and Severity Index) score (Rho = 0.89, p = 10-7), but not other individual or clinical characteristics. In the second part of the study, a significant reduction of SOX5, beta-catenin and CDK2 protein expression and increase of MITF protein expression was observed in cultured melanocytes after 24 h trasfection with miR-21-5p. CONCLUSION: According to literature, miR-21-5p upregulation and consequent SOX5 downregulation should upregulate melanogenesis, while vitiligo is characterized by skin depigmentation. Our results suggest that current knowledge of the pathogenesis of vitiligo is probably incomplete. Clinical manifestations could result from an altered balance between metabolic pathways with contrasting effects. In this view, miR-21-5p upregulation might be a tentative compensation mechanism. Further studies appear necessary to confirm and better understand our results and their importance.

microRNA-10 and -221 modulate differential expression of Hippo signaling pathway in human astroglial tumors.

Gliomas represent over 70% of all brain tumors, they are highly invasive and structurally vascular neoplasms. Despite the latest technological advance in neuro-surgery the survival of patients with high-grade glioma remains poor. The lack of robust treatment options has propelled the search for new markers that may able allow the identification of patients who can benefit from molecularly targeted therapies. The Hippo signaling pathway is considered as a key regulator of tissue homeostasis, cell proliferation and apoptosis, and alterations of this pathway seem to contribute to tumorigenesis. Yes-associated protein (YAP1) is a downstream target of the Hippo pathway which acts as a transcription co-activator. In cancer, YAP1 has been reported to function either as an oncogene or tumor suppressor, depending on the cell context. The aim of this study was to examine the expression of YAP1, Survivin and LATS1 kinase activity in human astroglial tumors with different grades of malignancy. Moreover, we also investigated the expression of miR-221 and miR-10b and their relationship with core molecules of the Hippo pathway. Our results showed the overexpression of YAP1 and Survivin as well as a decreased activity of large tumor suppressor 1 (LATS1) in high-grade glioblastoma versus anaplastic astrocytoma and low-grade glioma. Furthermore, we also demonstrated that miR-221 and miR-10b are specifically involved in Hippo signaling via LATS1 regulation and that their knockdown significantly decreased glioma cell proliferation. This preliminary data confirmed the crucial role of the Hippo pathway in cancer and suggested that miR-221 and miR-10b could be potential therapeutic targets for glioma treatment.

Circulating microRNAs Profile in Patients With Transthyretin Variant Amyloidosis.

Transthyretin variant amyloidosis (ATTRv) is a rare autosomal dominant disease characterized by the accumulation of amyloid in many organs, mostly causing a sensory-motor neuropathy, cardiomyopathy, and dysautonomia. The aim of the study was to report microRNAs (miRNAs) expression profile identified in the blood of ATTRv patients. Ten ATTRv patients, 10 asymptomatic carriers of transthyretin variant (TTRv), 10 patients with Charcot-Marie-Tooth (CMT) disease, and 10 healthy controls were studied. Human Schwann cells cultures were used to study the regulatory effects of miR-150-5p on the expression of cAMP response element-binding protein (CREB), brain-derived neurotrophic factor (BDNF), and nerve growth factor (NGF). ATTRv patients had 33 miRNAs up-regulated and 48 down-regulated versus healthy controls; 9 miRNAs were up-regulated and 30 down-regulated versus CMT patients; 19 miRNAs were up-regulated and 38 down-regulated versus asymptomatic TTRv carriers. Twelve out of the 19 upregulated miRNAs had a fold increase higher than 100. The validation experiment indicated miR-150-5p as a valuable biomarker to differentiate ATTRv patients from asymptomatic TTRv carriers (AUC: 0.9728; p < 0.0001). Schwann cells culture model demonstrated that miR-150-5p is a powerful negative regulator of CREB, BDNF, and NGF genes. Identification of deregulated miRNAs can help in understanding the complex pathomechamism underlying the development of ATTRv and related multisystemic pathology. Further investigations are needed on the role of circulating miR-150-5p to predict the shift of TTRv carriers from an asymptomatic status to symptoms appearance.

Effect of exercise on telomere length and telomere proteins expression in mdx mice.

In Duchenne muscular dystrophy (DMD), telomere shortening has been postulated to contribute to the failure of regenerative activity promoting the premature senescence of satellite cells. The aim of the present study was to investigate the telomere length and the expression of telomeric repeat-binding factor-1 (TRF1), poly (ADP-ribose) polymerase-1 (PARP1) and mouse telomerase reverse transcriptase (MTERT) in gastrocnemius, tibialis anterior and diaphragm muscles of the murine model of DMD, the mdx mouse and whether a chronic protocol of forced exercise impacts on them. Our results confirmed a telomere shortening in mdx muscles, more evident in the diaphragm, in which exercise induced a greater shortening than in wild-type mice. Moreover, we showed for the first time in mdx an increased TRF1 and PARP1 expression and an augmented activity of MTERT, further enhanced by exercise. These results reinforce the hypothesis that a deregulation of mechanisms involved in telomere length occurs and may pave the way for the test of compounds targeting proteins modulating telomere maintenance as a novel strategy to treat dystrophinopathies.

Circulating miRNAs expression as potential biomarkers of mild traumatic brain injury.

TBI is the main cause of death and disability in individuals aged 1-45 in Western countries. One of the main challenges of TBI at present is the lack of specific diagnostic biomarkers, especially for mild TBI (mTBI), which remains currently difficult to value in clinical practice. In this context MiRNAs may be important mediators of the profound molecular and cellular changes that occur after TBI in both the short and the long term. Recently, plasma miRNAs profiling in human TBI, have revealed dynamic temporal regulation of miRNA expression within the cortex. Aim of this study was to select a specific miRNAs panel for mTBI, by focusing the research on the prognostic meaning of miRNAs in the hours following the trauma, in order to be able to use this MIRNAs as potential biomarkers useful for monitoring the follow up of mild TBI. Serum levels of 17 miRNAs were measured by RT-quantitative polymerase chain reaction (qPCR) in 20 patients with mTBI at three different time-points (0 h, 24 h, 48 h) and in 10 controls. For 15 miRNAs we found a significant differences in the comparison among the three time points: for each of these miRNAs the values were greater at baseline and progressively reduced at 24 h and 48 h. These data allow us to consider the miRNAs included in panel as sensitive and specific biomarkers for mTBI, useful in monitoring the post-trauma period.

Vacuolated PAS-Positive Lymphocytes on Blood Smear: An Easy Screening Tool and a Possible Biomarker for Monitoring Therapeutic Responses in Late Onset Pompe Disease (LOPD).

Background: Primary aim was to investigate the diagnostic value of PAS-positive vacuolated lymphocytes on blood smear in Late Onset Pompe Disease (LOPD) patients and, secondly, to evaluate its potential utility in monitoring treatment effects. Methods: We examined blood smear of 26 LOPD patients. We evaluated 10 treated and 16 untreated LOPD patients. Among the latter group, 7 patients later initiated ERT and were tested again 6 months after start. Blood smear was also sampled from 82 controls and 19 patients with other muscle glycogenoses (MGSDs). PAS staining was used to evaluate: (1) presence of lymphocytes with glycogen-filled vacuoles, (2) quantification of vacuolated lymphocytes. Results: We found that PAS-positive lymphocytes were significantly higher in LOPD patients than in controls or other MGSDs (p < 0.05 and p < 0.001, respectively). ROC curve for discriminating between untreated LOPD patients and controls yielded an AUC of 1.00 (95%CI 1.00-1.00; p < 0.0001). PAS-positive lymphocyte cutoff level of >10 yielded sensitivity of 100% (95%CI 78-100%), specificity of 100% (95%CI 96-100%), and positive predictive value of 100%. Patients studied before and after ERT showed a dramatic decrease of PAS-positive vacuolated lymphocytes number (p = 0.016). In other MGSDs, PAS-positive lymphocytes were significantly lower that untreated LOPD patients but higher than controls. Conclusions: Our data suggest that the Blood Smear Examination (BSE) for PAS-positive lymphocytes quantification could be used as a simple and sensitive test for a quick screening of suspected Pompe disease. The quantification of vacuolated lymphocytes appears to be also a valuable tool for monitoring the efficacy of treatment in LOPD patients.

Hippo signaling pathway is altered in Duchenne muscular dystrophy.

Hippo signaling pathway is considered a key regulator of tissue homeostasis, cell proliferation, apoptosis and it is involved in cancer development. In skeletal muscle, YAP, a downstream target of the Hippo pathway, is an important player in myoblast proliferation, atrophy/hypertrophy regulation, and in mechano-trasduction, transferring mechanical signals into transcriptional responses. We studied components of Hippo pathway in muscle specimens from patients with Duchenne muscular dystrophy (DMD), Becker muscular dystrophy, limb-girdle muscular dystrophy type 2A and type 2B and healthy subjects. Only DMD muscles had decreased YAP1 protein expression, increased LATS1/2 kinase activity, low Survivin mRNA expression and high miR-21 expression. In light of our novel results, a schematic model is postulated: low levels of YOD1 caused by increased inhibition by miR-21 lead to an increase of LATS1/2 activity which in turn augments phosphorylation of YAP. Reduced amount of active YAP, which is also a target of increased miR-21, causes decreased nuclear expression of YAP-mediated target genes. Since it is known that YAP has beneficial roles in promoting tissue repair and regeneration after injury so that its activation may be therapeutically useful, our results suggest that some components of Hippo pathway could become novel therapeutic targets for DMD treatment.

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.

miRNA expression profiling regulates necroptotic cell death in hepatocellular carcinoma.

Hepatocellular carcinoma (HCC) is one of the most aggressive types of cancer and is among the leading causes of cancer-related mortality worldwide. Although the dysregulation of microRNAs (miRNAs or miRs) has often been reported in HCC, the precise molecular mechanisms by which miRNAs modulate the process of tumorigenesis and the behavior of cancer cells are not yet clearly understood. In this study, we identified a novel three­miRNA signature, including miR­371-5p, miR­373 and miR­543, that appears to orchestrate programmed cell necrosis in HCC by directly targeting the caspase­8 gene (Casp­8). Our results demonstrated that miR­371-5p, miR­373 and miR­543 were overexpressed in HCC tissues compared with paired adjacent normal tissues. The upregulation of these miRNAs specifically and markedly downregulated the expression of Casp­8, as well as significantly enhanced the Z-VAD/TNF­α-induced necroptosis of HCC cells. By contrast, the selective knockdown of miRNA expression led to a significant increase in Casp­8 levels and a marked reduction in programmed cell necrosis. Intriguingly, the sustained overexpression of Casp­8 reversed the pro­necroptotic effects exerted by miRNA mimics. Finally, a strong inverse association between the level of miR­223 and the expression levels of nucleotide-binding oligomerization domain-like receptor family, pyrin domain-containing-3 inflammasome was observed in our HCC specimens. On the whole, the present study revealed a molecular link between the three­miRNA signature, comprising miR­371-5p, miR­373 and miR­543, and the negative necroptotic regulator Casp­8, and presents evidence for its employment as a novel potential diagnostic, prognostic and therapeutic target in HCC.

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.

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.

Class I-specific histone deacetylase inhibitor MS-275 overrides TRAIL-resistance in melanoma cells by downregulating c-FLIP.

Tumor-necrosis factor-related apoptosis-inducing ligand (TRAIL) has selective killing effect toward malignant cells; however some human melanomas are intrinsically resistant. In this study, we have shown that class I-specific histone deacetylase inhibitor (HDACi) MS-275 can synergize with TRAIL to induce apoptosis in TRAIL-resistant cell lines and to enhance susceptibility of sensitive cells. Conversely, class II-selective HDACi MC1575 has shown no effect on the resistance of melanoma cells and was able exclusively to increase TRAIL-induced cell death in responsive cells. Both the HDACis variably increased DR4, DR5, and procaspase 8 expression, regardless whether cells were TRAIL-sensitive or TRAIL-resistant. However, only MS-275 markedly decreased the expression levels of both the long and short c-FLIP isoforms. RNAi-mediated c-FLIP silencing resulted in caspase 8-dependent apoptosis in survivor cells which was comparable to that observed following MS-275 treatment. Accordingly, enforced expression of ectopic c-FLIP has abolished the cooperative induction of apoptosis by the combination of MS-275 and TRAIL. These data indicate that c-FLIP is a critical regulator of death ligand sensitivity in melanoma. Inhibition of class I HDAC isoenzymes 1, 2 and 3 has resulted to be functionally important for c-FLIP downregulation by MS-275. In contrast, knockdown of class II HDACs has had no effect on c-FLIP expression, thus explaining the dual incapacity of MC1575 to inhibit c-FLIP expression and sensitize cells resistant to TRAIL. The data reported here suggest that MS-275 represents a promising therapeutic approach in combination with TRAIL for treatment of cutaneous and uveal melanoma due to its ability to reduce c-FLIP expression.