Positive Linear Relationship between Nucleophosmin Protein Expression and the Viral Load in HPV-Associated Oropharyngeal Squamous Cell Carcinoma: A Possible Tool for Stratification of Patients.

Most oropharyngeal squamous cell carcinomas (OPSCCs) are human papillomavirus (HPV)-associated, high-risk (HR) cancers that show a better response to chemoradiotherapy and are associated with improved survival. Nucleophosmin (NPM, also called NPM1/B23) is a nucleolar phosphoprotein that plays different roles within the cell, such as ribosomal synthesis, cell cycle regulation, DNA damage repair and centrosome duplication. NPM is also known as an activator of inflammatory pathways. An increase in NPM expression has been observed in vitro in E6/E7 overexpressing cells and is involved in HPV assembly. In this retrospective study, we investigated the relationship between the immunohistochemical (IHC) expression of NPM and HR-HPV viral load, assayed by RNAScope in situ hybridization (ISH), in ten patients with histologically confirmed p16-positive OPSCC. Our findings show that there is a positive correlation between NPM expression and HR-HPV mRNA (Rs = 0.70, p = 0.03), and a linear regression (r2 = 0.55; p = 0.01). These data support the hypothesis that NPM IHC, together with HPV RNAScope, could be used as a predictor of transcriptionally active HPV presence and tumor progression, which is useful for therapy decisions. This study includes a small cohort of patients and, cannot report conclusive findings. Further studies with large series of patients are needed to support our hypothesis.

Cytoplasmic HDAC4 regulates the membrane repair mechanism in Duchenne muscular dystrophy.

BACKGROUND: Histone deacetylase 4 (HDAC4) is a stress-responsive factor that mediates multiple cellular responses. As a member of class IIa HDACs, HDAC4 shuttles between the nucleus and the cytoplasm; however, HDAC4 cytoplasmic functions have never been fully investigated. Duchenne muscular dystrophy (DMD) is a genetic, progressive, incurable disorder, characterized by muscle wasting, which can be treated with the unspecific inhibition of HDACs, despite this approach being only partially effective. More efficient strategies may be proposed for DMD only after the different HDAC members will be characterized. METHODS: To fully understand HDAC4 functions, we generated dystrophic mice carrying a skeletal muscle-specific deletion of HDAC4 (mdx;KO mice). The progression of muscular dystrophy was characterized in mdx and age-matched mdx;KO mice by means of histological, molecular, and functional analyses. Satellite cells (SCs) from these mice were differentiated in vitro, to identify HDAC4 intrinsic functions influencing the myogenic potential of dystrophic SCs. Gain-of-function experiments revealed the cytoplasmic functions of HDAC4 in mdx;KO muscles. RESULTS: Histone deacetylase 4 increased in the skeletal muscles of mdx mice (~3-fold; P < 0.05) and of DMD patients (n = 3, males, mean age 13.3 ± 1.5 years), suggesting that HDAC4 has a role in DMD. Its deletion in skeletal muscles importantly worsens the pathological features of DMD, leading to greater muscle fragility and degeneration over time. Additionally, it impairs SC survival, myogenic potential, and muscle regeneration, ultimately compromising muscle function (P < 0.05-0.001). The impaired membrane repair mechanism in muscles and SCs accounts for the mdx;KO phenotype. Indeed, the ectopic expression of Trim72, a major player in the membrane repair mechanism, prevents SC death (~20%; P < 0.01) and increases myogenic fusion (~40%; P < 0.01) in vitro; in vivo it significantly reduces myofibre damage (~10%; P < 0.005) and improves mdx;KO muscle function (P < 0.05). The mdx;KO phenotype is also fully rescued by restoring cytoplasmic levels of HDAC4, both in vitro and in vivo. The protective role of HDAC4 in the cytoplasm of mdx;KO muscles is, in part, independent of its deacetylase activity. HDAC4 expression correlates with Trim72 mRNA levels; furthermore, Trim72 mRNA decays more rapidly (P < 0.01) in mdx;KO muscle cells, compared with mdx ones. CONCLUSIONS: Histone deacetylase 4 performs crucial functions in the cytoplasm of dystrophic muscles, by mediating the muscle repair response to damage, an important role in ensuring muscle homeostasis, probably by stabilizing Trim72 mRNA. Consequently, the cytoplasmic functions of HDAC4 should be stimulated rather than inhibited in muscular dystrophy treatments, a fact to be considered in future therapeutic approaches.

microRNAs involved in psoriasis and cardiovascular diseases.

Psoriasis is a chronic inflammatory disease involving the skin. Both genetic and environmental factors play a pathogenic role in psoriasis and contribute to the severity of the disease. Psoriasis, in fact, has been associated with different comorbidities such as diabetes, metabolic syndrome, gastrointestinal or kidney diseases, cardiovascular disease (CVD), and cerebrovascular diseases (CeVD). Indeed, life expectancy in severe psoriasis is reduced by up to 5 years due to CVD and CeVD. Moreover, patients with severe psoriasis have a higher prevalence of traditional cardiovascular (CV) risk factors, including dyslipidemia, diabetes, smoking, and hypertension. Further, systemic inflammation is associated with oxidative stress increase and induces endothelial damage and atherosclerosis progression. Different miRNA have been already described in psoriasis, both in the skin tissues and in the blood flow, to play a role in the progression of disease. In this review, we will summarize and discuss the most important miRNAs that play a role in psoriasis and are also linked to CVD.

miR-200c-3p Regulates Epitelial-to-Mesenchymal Transition in Epicardial Mesothelial Cells by Targeting Epicardial Follistatin-Related Protein 1.

Recent findings suggest that epithelial to mesenchymal transition (EMT), a key step during heart development, is involved in cardiac tissue repair following myocardial infarction (MI). MicroRNAs (miRNAs) act as key regulators in EMT processes; however, the mechanisms by which miRNAs target epicardial EMT remain largely unknown. Here, by using an in vitro model of epicardial EMT, we investigated the role of miRNAs as regulators of this process and their potential targets. EMT was induced in murine epicardial-mesothelial cells (EMCs) through TGF ß1 treatment for 48, 72, and 96 h as indicated by the expression of EMT-related genes by qRT-PCR, WB, and immunofluorescence. Further, enhanced expression of stemness genes was also detected. Among several EMT-related miRNAs, miR-200c-3p expression resulted as the most strongly suppressed. Interestingly, we also found a significant upregulation of Follistatin-related protein 1 (FSTL1), a miR-200c predicted target already identified as a potent cardiogenic factor produced by epicardial cells that promotes regeneration following MI. Dual-luciferase reporter assay demonstrated that miR-200c-3p directly targeted the 3'-untranslated region of FSTL1 in EMCs. Consistently, WB analysis showed that knockdown of miR-200c-3p significantly increased FSTL1 expression, whereas overexpression of miR-200c-3p counteracted TGF ß1-mediated FSTL1 upregulation. Importantly, FSTL1 silencing maintained epithelial features in EMCs, despite EMT induction by TGF ß1, and attenuated EMT-associated traits, including migration and stemness. In conclusion, epicardial FSTL1, an important cardiogenic factor in its secreted form, induces EMT, stemness, and migration of EMCs in a miR-200c-3p dependent pathway.

Doxorubicin induces an alarmin-like TLR4-dependent autocrine/paracrine action of Nucleophosmin in human cardiac mesenchymal progenitor cells.

BACKGROUND: Doxorubicin (Dox) is an anti-cancer anthracycline drug that causes double-stranded DNA breaks. It is highly effective against several types of tumours; however, it also has adverse effects on regenerative populations of normal cells, such as human cardiac mesenchymal progenitor cells (hCmPCs), and its clinical use is limited by cardiotoxicity. Another known effect of Dox is nucleolar disruption, which triggers the ubiquitously expressed nucleolar phosphoprotein Nucleophosmin (NPM) to be released from the nucleolus into the cell, where it participates in the orchestration of cellular stress responses. NPM has also been observed in the extracellular space in response to different stress stimuli; however, the mechanism behind this and its functional implications are as yet largely unexplored. The aim of this study was to establish whether Dox could elicit NPM secretion in the extracellular space and to elucidate the mechanism of secretion and the effect of extracellular NPM on hCmPCs. RESULTS: We found that following the double-strand break formation in hCmPCs caused by Dox, NPM was rapidly secreted in the extracellular space by an active mechanism, in the absence of either apoptosis or necrosis. Extracellular release of NPM was similarly seen in response to ultraviolet radiation (UV). Furthermore, we observed an increase of NPM levels in the plasma of Dox-treated mice; thus, NPM release also occurred in vivo. The treatment of hCmPCs with extracellular recombinant NPM induced a decrease of cell proliferation and a response mediated through the Toll-like receptor (TLR)4. We demonstrated that NPM binds to TLR4, and via TLR4, and nuclear factor kappa B (NFkB) activation/nuclear translocation, exerts proinflammatory functions by inducing IL-6 and COX-2 gene expression. Finally, we found that in hCmPCs, NPM secretion could be driven by an autophagy-dependent unconventional mechanism that requires TLR4, since TLR4 inhibition dramatically reduced Dox-induced secretion. CONCLUSIONS: We hypothesise that the extracellular release of NPM could be a general response to DNA damage since it can be elicited by either a chemical agent such as Dox or a physical genotoxic stressor such as UV radiation. Following genotoxic stress, NPM acts similarly to an alarmin in hCmPCs, being rapidly secreted and promoting cell cycle arrest and a TLR4/NFκB-dependent inflammatory response.

MicroRNAs in Cancer Treatment-Induced Cardiotoxicity.

Cancer treatment has made significant progress in the cure of different types of tumors. Nevertheless, its clinical use is limited by unwanted cardiotoxicity. Aside from the conventional chemotherapy approaches, even the most newly developed, i.e., molecularly targeted therapy and immunotherapy, exhibit a similar frequency and severity of toxicities that range from subclinical ventricular dysfunction to severe cardiomyopathy and, ultimately, congestive heart failure. Specific mechanisms leading to cardiotoxicity still remain to be elucidated. For instance, oxidative stress and DNA damage are considered key players in mediating cardiotoxicity in different treatments. microRNAs (miRNAs) act as key regulators in cell proliferation, cell death, apoptosis, and cell differentiation. Their dysregulation has been associated with adverse cardiac remodeling and toxicity. This review provides an overview of the cardiotoxicity induced by different oncologic treatments and potential miRNAs involved in this effect that could be used as possible therapeutic targets.

Extracellular Vesicles-Encapsulated MicroRNA-125b Produced in Genetically Modified Mesenchymal Stromal Cells Inhibits Hepatocellular Carcinoma Cell Proliferation.

Hepatocellular carcinoma (HCC) is the most frequent type of primary liver cancer and one of the prominent causes of cancer mortality, leading to approximately 780,000 deaths per year worldwide. Down-regulation of microRNA-125b (miR-125b) is a prognostic indicator in HCC patients. Conversely, over-expression of miR-125b in HCC cells induces cell cycle arrest, inhibits proliferation, migration and invasion. Extracellular vesicles (EVs) function as intercellular messengers transferring proteins, RNAs, DNAs, carbohydrates, and lipids. Since EVs protect their cargo from degradation, delivery of therapeutic bioactive molecules, in particular miRNAs, through EVs represents an innovative avenue for cancer therapy. In this study, we evaluated a replacement strategy for the treatment of HCC via delivery of EVs secreted from human adipose tissue-derived mesenchymal stromal/medicinal signaling cells (ASCs) genetically modified with a lentiviral vector expressing miR-125b with a specific ExoMotif sequence tag to enhance the loading into extracellular vesicles. In particular, we determined that the delivery of miR-125b-loaded EVs produced in engineered ASCs specifically reduces HCC cell proliferation in vitro modulating a series of miR-125b targets, which belong to the p53 signaling pathway. This proof-of-concept study supports the development of innovative therapeutic strategies for HCC via EV-mediated miRNA delivery.

Transcriptional activation of the miR-17-92 cluster is involved in the growth-promoting effects of MYB in human Ph-positive leukemia cells.

MicroRNAs, non-coding regulators of gene expression, are likely to function as important downstream effectors of many transcription factors including MYB. Optimal levels of MYB are required for transformation/maintenance of BCR-ABL-expressing cells. We investigated whether MYB silencing modulates microRNA expression in Philadelphia-positive (Ph+) leukemia cells and if MYB-regulated microRNAs are important for the "MYB addiction" of these cells. Thirty-five microRNAs were modulated by MYB silencing in lymphoid and erythromyeloid chronic myeloid leukemia-blast crisis BV173 and K562 cells; 15 of these were concordantly modulated in both lines. We focused on the miR-17-92 cluster because of its oncogenic role in tumors and found that: i) it is a direct MYB target; ii) it partially rescued the impaired proliferation and enhanced apoptosis of MYB-silenced BV173 cells. Moreover, we identified FRZB, a Wnt/ß-catenin pathway inhibitor, as a novel target of the miR-17-92 cluster. High expression of MYB in blast cells from 2 Ph+leukemia patients correlated positively with the miR-17-92 cluster and inversely with FRZB. This expression pattern was also observed in a microarray dataset of 122 Ph+acute lymphoblastic leukemias. In vivo experiments in NOD scid gamma mice injected with BV173 cells confirmed that FRZB functions as a Wnt/ß-catenin inhibitor even as they failed to demonstrate that this pathway is important for BV173-dependent leukemogenesis. These studies illustrate the global effects of MYB expression on the microRNAs profile of Ph+cells and supports the concept that the "MYB addiction" of these cells is, in part, caused by modulation of microRNA-regulated pathways affecting cell proliferation and survival.

Peripheral Blood Mononuclear Cells Therapy for Treatment of Lower Limb Ischemia in Diabetic Patients: A Single-Center Experience.

BACKGROUND: The aim of this study is to analyze the effects of peripheral blood mononuclear cells (PBMNCs) therapy in diabetic patients with critical limb ischemia (CLI), with particular regard to its application, as adjuvant therapy in patients underwent endovascular revascularization. METHODS: Fifty diabetic patients affected by CLI were enrolled. All patients underwent PBMNCs therapy. Thirty-two patients underwent PBMNCs therapy associated with endovascular revascularization (adjuvant therapy group). In 18 patients, who were considered nonrevascularizable or underwent unsuccessful revascularization, regenerative therapy with PBMNCs was performed as the therapeutic choice (PBMNCs therapy group). RESULTS: The median follow-up period was 10 months. The baseline and end point results in adjuvant group were as follows. The mean transcutaneous partial pressure of oxygen (TcPO2) improved from 25 ± 9.2 mmHg to 45.6 ± 19.1 mmHg (P < 0.001), and visual analogue scale (VAS) score means decreased from 8.6 ± 2.1 to 3.8 ± 3.5 (P = 0.001). In PBMNCs therapy group, the mean TcPO2 improved from 16.2 ± 7.2 mmHg to 23.5 ± 8.4 mmHg (P < 0.001), and VAS score means decreased from 9 ± 1.1 to 4.1 ± 3.3 (P = 0.001). Major amputation was observed in 3 cases (9.4%), both in adjuvant therapy group and in PBMNCs therapy one (16.7%) (P = 0.6). CONCLUSIONS: The role of cellular therapy with PBMNCs is decisive in the patients that are not susceptible to revascularization. In diabetic patients with CLI and healing resistant ulcers, the adjuvant PBMNCs therapy could represent a valid therapeutic option.

Central role of the p53 pathway in the noncoding-RNA response to oxidative stress.

Oxidative stress plays a fundamental role in many conditions. Specifically, redox imbalance inhibits endothelial cell (EC) growth, inducing cell death and senescence. We used global transcriptome profiling to investigate the involvement of noncoding-RNAs in these phenotypes. By RNA-sequencing, transcriptome changes were analyzed in human ECs exposed to H2O2, highlighting a pivotal role of p53-signaling. Bioinformatic analysis and validation in p53-silenced ECs, identified several p53-targets among both mRNAs and long noncoding-RNAs (lncRNAs), including MALAT1 and NEAT1. Among microRNAs (miRNAs), miR-192-5p was the most induced by H2O2 treatment, in a p53-dependent manner. Down-modulated mRNA-targets of miR-192-5p were involved in cell cycle, DNA repair and stress response. Accordingly, miR-192-5p overexpression significantly decreased EC proliferation, inducing cell death. A central role of the p53-pathway was also confirmed by the analysis of differential exon usage: Upon H2O2 treatment, the expression of p53-dependent 5'-isoforms of MDM2 and PVT1 increased selectively. The transcriptomic alterations identified in H2O2-treated ECs were also observed in other physiological and pathological conditions where redox control plays a fundamental role, such as ECs undergoing replicative senescence, skeletal muscles of critical limb-ischemia patients and the peripheral-blood mononuclear cells of long-living individuals. Collectively, these findings indicate a prominent role of noncoding-RNAs in oxidative stress response.

Doxorubicin upregulates CXCR4 via miR-200c/ZEB1-dependent mechanism in human cardiac mesenchymal progenitor cells.

Doxorubicin (DOXO) treatment is limited by its cardiotoxicity, since it causes cardiac-progenitor-cell depletion. Although the cardioprotective role of the stromal cell-derived factor-1/C-X-C chemokine receptor type 4 (SDF1/CXCR4) axis is well established, its involvement during DOXO-induced cardiotoxicity has never been investigated. We showed that in a mouse model of DOXO-induced cardiomyopathy, CXCR4+ cells were increased in response to DOXO, mainly in human cardiac mesenchymal progenitor cells (CmPC), a subpopulation with regenerative potential. Our in vitro results showed a CXCR4 induction after 24 h of DOXO exposure in CmPC. SDF1 administration protected from DOXO-induced cell death and promoted CmPC migration. CXCR4 promoter analysis revealed zinc finger E-box binding homeobox 1 (ZEB1) binding sites. Upon DOXO treatment, ZEB1 binding decreased and RNA-polymerase-II increased, suggesting a DOXO-mediated transcriptional increase in CXCR4. Indeed, DOXO induced the upregulation of miR-200c, that directly targets ZEB1. SDF1 administration in DOXO-treated mice partially reverted the adverse remodeling, decreasing left ventricular (LV) end diastolic volume, LV ejection fraction and LV anterior wall thickness in diastole, recovering LV end systolic pressure and reducing±dP/dt. Moreover, in vivo administration of SDF1 partially reverted DOXO-induced miR-200c and p53 protein upregulation in mouse hearts. In addition, downmodulation of ZEB1 mRNA and protein by DOXO was significantly increased by SDF1. In keeping, p21 mRNA, that is induced by p53 and inhibited by ZEB1, is induced by DOXO treatment and is decreased by SDF1 administration. This study showed new players of the DOXO-induced cardiotoxicity, that can be exploited to ameliorate DOXO-associated cardiomyopathy.

Circulating miR-200c is up-regulated in paediatric patients with familial hypercholesterolaemia and correlates with miR-33a/b levels: implication of a ZEB1-dependent mechanism.

Hypercholesterolaemia provokes reactive oxygen species (ROS) increase and is a major risk factor for cardiovascular disease (CVD) development. We previously showed that circulating miR-33a/b expression levels were up-regulated in children with familial hypercholesterolaemia (FH). miR-33a/b control cholesterol homoeostasis and recently miR-33b has been demonstrated to directly target the transcription factor zinc finger E-box-binding homeobox 1 (ZEB1). The latter acts in a negative feedback loop with the miR-200 family. Our previous studies showed that the ROS-dependent miR-200c up-regulation induces endothelial dysfunction and provokes a ZEB1-dependent apoptosis and senescence. In the present study, we aimed to verify whether circulating miR-200c was induced in FH children, and whether a correlation existed with miR-33a/b Total RNA was extracted from plasma of 28 FH children and 25 age-matched healthy subjects (HS) and miR-200c levels were measured. We found that miR-200c was up-regulated in FH compared with HS (4.00 ± 0.48-fold increase, P<0.05) and exhibited a positive correlation with miR-33a/b. miR-200c did not correlate with plasma lipids, but correlated with C-reactive protein (CRP) plasma levels and glycaemia (GLI). Ordinary least squares (OLS) regression analysis revealed that miR-200c was significantly affected by GLI and by miR-33a (P<0.01; P<0.001 respectively). Moreover, we found that miR-33 overexpression, in different cell lines, decreased ZEB1 expression and up-regulated both the intracellular and the extracellular miR-200c expression levels. In conclusion, circulating miR-200c is up-regulated in FH, probably due to oxidative stress and inflammation and via a miR-33a/b-ZEB1-dependent mechanism. The present study could provide the first evidence to point to the use of miR-33a/b and miR-200c, as early biomarkers of CVD, in paediatric FH.

The laminA/NF-Y protein complex reveals an unknown transcriptional mechanism on cell proliferation.

Lamin A is a component of the nuclear matrix that also controls proliferation by largely unknown mechanisms. NF-Y is a ubiquitous protein involved in cell proliferation composed of three subunits (-YA -YB -YC) all required for the DNA binding and transactivation activity. To get clues on new NF-Y partner(s) we performed a mass spectrometry screening of proteins that co-precipitate with the regulatory subunit of the complex, NF-YA. By this screening we identified lamin A as a novel putative NF-Y interactor. Co-immunoprecipitation experiments and confocal analysis confirmed the interaction between the two endogenous proteins. Interestingly, this association occurs on euchromatin regions, too. ChIP experiments demonstrate lamin A enrichment in several promoter regions of cell cycle related genes in a NF-Y dependent manner. Gain and loss of function experiments reveal that lamin A counteracts NF-Y transcriptional activity. Taking advantage of a recently generated transgenic reporter mouse, called MITO-Luc, in which an NF-Y-dependent promoter controls luciferase expression, we demonstrate that lamin A counteracts NF-Y transcriptional activity not only in culture cells but also in living animals. Altogether, our data demonstrate the occurrence of lamin A/NF-Y interaction and suggest a possible role of this protein complex in regulation of NF-Y function in cell proliferation.

Oxidative Stress-Induced miR-200c Disrupts the Regulatory Loop Among SIRT1, FOXO1, and eNOS.

AIMS: Reactive oxygen species (ROS) play a pivotal role in different pathologic conditions, including ischemia, diabetes, and aging. We previously showed that ROS enhance miR-200c expression, causing endothelial cell (EC) apoptosis and senescence. Herein, we dissect the interaction among miR-200c and three strictly related proteins that modulate EC function and ROS production: sirtuin 1 (SIRT1), endothelial nitric oxide synthase (eNOS), and forkhead box O1 (FOXO1). Moreover, the role of miR-200c on ROS modulation was also investigated. RESULTS: We demonstrated that miR-200c directly targets SIRT1, eNOS, and FOXO1; via this mechanism, miR-200c decreased NO and increased the acetylation of SIRT1 targets, that is, FOXO1 and p53. FOXO1 acetylation inhibited its transcriptional activity on target genes, that is, SIRT1 and the ROS scavengers, catalase and manganese superoxide dismutase. In keeping, miR-200c increased ROS production and induced p66Shc protein phosphorylation in Ser-36; this mechanism upregulated ROS and inhibited FOXO1 transcription, reinforcing this molecular circuitry. These in vitro results were validated in three in vivo models of oxidative stress, that is, human skin fibroblasts from old donors, femoral arteries from old mice, and a murine model of hindlimb ischemia. In all cases, miR-200c was higher versus control and its targets, that is, SIRT1, eNOS, and FOXO1, were downmodulated. In the mouse hindlimb ischemia model, anti-miR-200c treatment rescued these targets and improved limb perfusion. Innovation and Conclusion: miR-200c disrupts SIRT1/FOXO1/eNOS regulatory loop. This event promotes ROS production and decreases NO, contributing to endothelial dysfunction under conditions of increased oxidative stress such as aging and ischemia. Antioxid. Redox Signal. 27, 328-344.

Epigenetics and cardiovascular risk in childhood.

Cardiovascular disease (CVD) can arise at the early stages of development and growth. Genetic and environmental factors may interact resulting in epigenetic modifications with abnormal phenotypic expression of genetic information without any change in the nucleotide sequence of DNA. Maternal dietary imbalance, inadequate to meet the nutritional needs of the fetus can lead to intrauterine growth retardation, decreased gestational age, low birth weight, excessive post-natal growth and metabolic alterations, with subsequent appearance of CVD risk factors. Fetal exposure to high cholesterol, diabetes and maternal obesity is associated with increased risk and progression of atherosclerosis. Maternal smoking during pregnancy and exposure to various environmental pollutants induce epigenetic alterations of gene expression relevant to the onset or progression of CVD. In children with hypercholesterolemia and/or obesity, oxidative stress activates platelets and monocytes, which release proinflammatory and proatherogenic substances, inducing endothelial dysfunction, decreased Doppler flow-mediated dilation and increased carotid intima-media thickness. Primary prevention of atherosclerosis should be implemented early. It is necessary to identify, through screening, high-risk apparently healthy children and take care of them enforcing healthy lifestyle (mainly consisting of Mediterranean diet and physical activity), prescribing nutraceuticals and eventual medications, if required by a high-risk profile. The key issue is the restoration of endothelial function in the reversible stage of atherosclerosis. Epigenetics may provide new markers for an early identification of children at risk and thereby develop innovative therapies and specific nutritional interventions in critical times.

Expression of the FGFR2 mesenchymal splicing variant in epithelial cells drives epithelial-mesenchymal transition.

The FGFRs are receptor tyrosine kinases expressed by tissue-specific alternative splicing in epithelial IIIb or mesenchymal IIIc isoforms. Deregulation of FGF/FGFR signaling unbalances the epithelial-stromal homeostasis and may lead to cancer development. In the epithelial-context, while FGFR2b/KGFR acts as tumor suppressor, FGFR2c appears to play an oncogenic role. Based on our recent observation that the switching of FGFR2b versus FGFR2c induces EMT, here we investigated the biological outcome of the ectopic expression of FGFR2c in normal human keratinocytes. Morphological analysis showed that, differently from FGFR2b overexpression, the forced expression and activation of FGFR2c drive the epithelial cells to acquire a mesenchymal-like shape and actin reorganization. Moreover, the appearance of invasiveness and anchorage-independent growth ability in FGFR2c transfected keratinocytes was consistent with the potential tumorigenic role proposed for this receptor variant. Biochemical and molecular approaches revealed that the observed phenotypic changes were accompanied by modulation of EMT biomarkers and indicated the involvement of EMT transcription factors and miRs. Finally, the analysis of the expression pattern of discriminating markers strongly suggested that activation of FGFR2c triggers a process corresponding to the initiation of the pathological type III EMT, but not to the more physiological type II EMT occurring during FGFR2b-mediated wound healing.

Circulating miR-33a and miR-33b are up-regulated in familial hypercholesterolaemia in paediatric age.

Hypercholesterolaemia is one of the major causes of CVD (cardiovascular disease). It is associated with enhanced oxidative stress, leading to increased lipid peroxidation which in turn determines endothelial dysfunction and susceptibility to coronary vasoconstriction and atherosclerosis. Different miRNAs are involved in the pathogenesis of CVD and play an important role in inflammatory process control, therefore, together with atherogenic factors, they can stimulate atherosclerotic degeneration of the vessel walls of arteries. miR-33a and miR-33b play a pivotal role in a variety of biological processes including cholesterol homoeostasis, HDL (high-density lipoprotein)-cholesterol formation, fatty acid oxidation and insulin signalling. Our study aimed to determine whether circulating miR-33a and miR-33b expression was altered in familial hypercholesterolaemic children. Total RNA was extracted from plasma, and miR-33a and miR-33b were measured by quantitative real-time PCR. We found that miR-33a and miR-33b were significantly up-regulated in the plasma of 28 hypercholesterolaemic children compared with 25 healthy subjects (4.49±0.27-fold increase, P<0.001, and 3.21±0.39-fold increase, P<0.05 respectively), and for both miRNAs, a positive correlation with total cholesterol, LDL (low-density lipoprotein)-cholesterol, LDL-cholesterol/HDL-cholesterol ratio, apolipoprotein B, CRP (C-reactive protein) and glycaemia was found. OLS (ordinary least squares) regression analysis revealed that miR-33a was significantly affected by the presence of FH (familial hypercholesterolaemia), glycaemia and CRP (P<0.001, P<0.05 and P<0.05 respectively). The same analysis showed that miR-33b was significantly related to FH and CRP (P<0.05 and P<0.05 respectively). Although it is only explorative, the present study could be the first to point to the use of miR-33a and miR-33b as early biomarkers for cholesterol levels in childhood, once validated in independent larger cohorts.

HPV16 E5 expression induces switching from FGFR2b to FGFR2c and epithelial-mesenchymal transition.

The E5 oncoprotein of the human papillomavirus type 16 (HPV16 E5) deregulates epithelial homeostasis through the modulation of receptor tyrosine kinases and their signaling. Accordingly, the fibroblast growth factor receptor 2b (FGFR2b/KGFR), epithelial splicing transcript variant of the FGFR2, is down-modulated by the viral protein expression, leading to impairment of keratinocyte differentiation. Here, we report that, in cell models of transfected human keratinocytes as well as in cervical epithelial cells containing episomal HPV16, the down-regulation of FGFR2b induced by 16E5 is associated with the aberrant expression of the mesenchymal FGFR2c isoform as a consequence of splicing switch: in fact, quantitative RT-PCR analysis showed that this molecular event is transcriptionally regulated by the epithelial splicing regulatory proteins 1 and 2 (ESRP1 and ESRP2) and is able to produce effects synergistic with those caused by TGFß treatment. Immunofluorescence analysis revealed that this altered FGFR2 splicing leads to changes in the specificity for the ligands FGFs and in the cellular response, triggering epithelial-mesenchymal transition (EMT). Through 16E5 or FGFR2 silencing as well as inhibition of FGFR2 activity we demonstrated the direct role of the viral protein in the receptor isoform switching and EMT, suggesting that these early molecular events during HPV infection might represent additional mechanisms driving cervical transformation and tumor progression.

Platelet-derived growth factor-receptor alpha strongly inhibits melanoma growth in vitro and in vivo.

Cutaneous melanoma is the most aggressive skin cancer; it is highly metastatic and responds poorly to current therapies. The expression of platelet-derived growth factor receptors (PDGF-Rs) is reported to be reduced in metastatic melanoma compared with benign nevi or normal skin; we then hypothesized that PDGF-Ralpha may control growth of melanoma cells. We show here that melanoma cells overexpressing PDGF-Ralpha respond to serum with a significantly lower proliferation compared with that of controls. Apoptosis, cell cycle arrest, pRb dephosphorylation, and DNA synthesis inhibition were also observed in cells overexpressing PDGF-Ralpha. Proliferation was rescued by PDGF-Ralpha inhibitors, allowing to exclude nonspecific toxic effects and indicating that PDGF-Ralpha mediates autocrine antiproliferation signals in melanoma cells. Accordingly, PDGF-Ralpha was found to mediate staurosporine cytotoxicity. A protein array-based analysis of the mitogen-activated protein kinase pathway revealed that melanoma cells overexpressing PDGF-Ralpha show a strong reduction of c-Jun phosphorylated in serine 63 and of protein phosphatase 2A/Balpha and a marked increase of p38gamma, mitogen-activated protein kinase kinase 3, and signal regulatory protein alpha1 protein expression. In a mouse model of primary melanoma growth, infection with the Ad-vector overexpressing PDGF-Ralpha reached a significant 70% inhibition of primary melanoma growth (P < .001) and a similar inhibition of tumor angiogenesis. All together, these data demonstrate that PDGF-Ralpha strongly impairs melanoma growth likely through autocrine mechanisms and indicate a novel endogenous mechanism involved in melanoma control.