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According to the American College of Medical Genetics (ACMG) classification, variants of uncertain significance (VUS) are gene variations whose impact on the disease risk is not yet known. VUS, therefore, represent an unmet need for genetic counselling. Aim of the study is the use the AlphaFold artificial intelligence algorithm to predict the impact of novel mutations of the IGFALS gene, detected in a subject with short stature and initially classified as VUS according to the ACMG classification.A short-stature girl and her parents have been investigated. IGFALS mutations have been detected through clinical exome and confirmed by Sanger sequencing. The potential presence of co-occurring gene alterations was investigated in the proband by whole exome and CGH array. Structure of the ALS protein (encoded by the IGFALS gene) was evaluated through the AlphaFold artificial intelligence algorithm.Two IGFALS variants were found in the proband: c.1349T C (p.Leu450Pro) and c.1363_1365delCTC (p.Leu455del), both classified as VUS, according to ACMG. Parents' analysis highlighted the in trans position of the two variants. AlphaFold showed that the mutated positions were found the concave side a horseshoe structure of the ALS protein, likely interfering with protein-protein interactions. According to a loss of function (LoF) effect of the two variants, reduced levels of the IGF1 and IGFBP-3 proteins, as well as a growth hormone (GH) excess were detected in the proband's serum.By using the AlphaFold structure we were able to predict two IGFALS gene mutations initially classified as VUS, as potentially pathogenetic. Our proof-of-concept showed a potential application of AlphaFold as tool to a better inform VUS interpretation of genetic tests.

Severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) infection has rapidly spread worldwide from the beginning of 2020. The presence of viral RNA in samples by nucleic acid (NA) molecular analysis is the only method available to diagnose COVID-19 disease and to assess patients’ viral load. Since the demand for laboratory reagents has increased, there has been a worldwide shortage of RNA extraction kits. We, therefore, developed a fast and cost-effective viral genome isolation method that, combined with quantitative RT-PCR assay, detects SARS-CoV-2 RNA in patient samples. The method relies on the addition of Proteinase K followed by a controlled heat-shock incubation and, then, E gene evaluation by RT-qPCR. It was validated for sensitivity, specificity, linearity, reproducibility, and precision. It detects as low as 10 viral copies/sample, is rapid, and has been characterized in 60 COVID-19-infected patients. Compared to automated extraction methods, our pretreatment guarantees the same positivity rate with the advantage of shortening the time of the analysis and reducing its cost. This is a rapid workflow meant to aid the healthcare system in the rapid identification of infected patients, such as during a pathogen-related outbreak. For its intrinsic characteristics, this workflow is suitable for large-scale screenings.

Exosomes released from tumor cells are instrumental in shaping the local tumor microenvironment to allow cancer progression. Recently, it has been shown that tumor exosomes carry large fragments of dsDNA, which may reflect the mutational status of parental cells. Although it has been described that a stressful microenvironment can influence exosomal cargo, the effects on DNA packing and its transfer into recipient cells have yet to be investigated. Here, we report that exosomes derived from SW480 (human colorectal adenocarcinoma cell line) cells can carry dsDNA fragments containing the entire coding sequence of both TP53 and KRAS genes, harboring the SW480-related TP53 c.818G &gt, A and KRAS c.35G &gt, T typical mutations. We also report the following: that cell stimulation with lipopolysaccharides (LPS) promotes the selective packaging of the TP53 gene, but not the KRAS gene, that exosomes secreted by SW480 cells efficiently transfer the mutated sequences into normal CCD841-CoN colon epithelial and THLE-2 hepatic cells, that this mechanism is more efficient when the cells had been previously incubated with pro-inflammatory cytokines, that the TP53 gene appears actively transcribed in both recipient cells, and that mutated mRNA levels are not influenced by cytokine treatment. Our data strongly suggest that pro-inflammatory stimulation promotes the horizontal transfer of an oncogene by exosomes, although this remains a rare event. Further studies are needed to assess the impact of the oncogenic transfer by exosomes in malignant transformation and its role in tumor progression.

Congenital hypothyroidism (CH) is a neonatal endocrine disorder that might occur as itself or be associated to congenital extra-thyroidal defects. About 85% of affected subjects experience thyroid dysgenesis (TD), characterized by defect in thyroid gland development. In vivo experiments on null mice paved the way for the identification of genes involved thyroid morphogenesis and development, whose mutation has been strongly associated to TD. Most of them are thyroid-specific transcription factors expressed during early thyroid development. Despite the arduous effort in unraveling the genetics of TD in animal models, up to now these data have been discontinuously confirmed in humans and only 5% of TD have associated with known null mice-related mutations (mainly PAX8 and TSHR). Notwithstanding, the advance in genetic testing represented by the next-generation sequencing (NGS) approach is steadily increasing the list of genes whose highly penetrant mutation predisposes to TD. In this review we intend to outline the molecular bases of TD, summarizing the current knowledge on thyroid development in both mice and humans and delineating the genetic features of its monogenetic forms. We will also highlight current strategies to enhance the insight into the non-Mendelian mechanisms of abnormal thyroid development.

Toward the discovery of novel reliable biomarkers, epigenetic alterations have been repeatedly proposed for the diagnosis and the development of therapeutic strategies against cancer. Indeed, for promoter methylation to actively become a tumor marker for clinical use, it must be combined with a highly informative technology evaluated in an appropriate biospecimen. Methodological standardization related to epigenetic research is, in fact, one of the most challenging tasks. Moreover, tissue-based biopsy is being complemented and, in some cases, replaced by liquid biopsy. This review will highlight the advancements made for both pre-analytical and analytical implementation for the prospective use of methylation biomarkers in clinical settings, with particular emphasis on liquid biopsy.

Bromodomain and Extra-Terminal (BET) proteins are historically involved in regulating gene expression and BRD4 was recently found to be involved in DNA damage regulation. Aims of our study were to assess BRD4 regulation in homologous recombination-mediated DNA repair and to explore novel clinical strategies through the combinations of the pharmacological induction of epigenetic BRCAness in BRCA1 wild-type triple negative breast cancer (TNBC) cells by means of BET inhibitors and compounds already available in clinic. Performing a dual approach (chromatin immunoprecipitation and RNA interference), the direct relationship between BRD4 and BRCA1/RAD51 expression was confirmed in TNBC cells. Moreover, BRD4 pharmacological inhibition using two BET inhibitors (JQ1 and GSK525762A) induced a dose-dependent reduction in BRCA1 and RAD51 levels and is able to hinder homologous recombination-mediated DNA damage repair, generating a BRCAness phenotype in TNBC cells. Furthermore, BET inhibition impaired the ability of TNBC cells to overcome the increase in DNA damage after platinum salts (i.e., CDDP) exposure, leading to massive cell death, and triggered synthetic lethality when combined with PARP inhibitors (i.e., AZD2281). Altogether, the present study confirms that BET proteins directly regulate the homologous recombination pathway and their inhibition induced a BRCAness phenotype in BRCA1 wild-type TNBC cells. Noteworthy, being this strategy based on drugs already available for human use, it is rapidly transferable and could potentially enable clinicians to exploit platinum salts and PARP inhibitors-based treatments in a wider population of TNBC patients and not just in a specific subgroup, after validating clinical trials.

SPG6 accounts for 1% of autosomal dominant Hereditary Spastic Paraplegia (HSP) and is caused by pathogenic variants in NIPA1, which encodes a magnesium transporter located in plasma membrane and early endosomes, implicated in neuronal development and maintenance. Here we report a 39-year-old woman affected by progressive gait disturbance associated to absence seizures episodes within childhood. Clinical exome sequencing identified a likely pathogenic de novo heterozygous variant in NIPA1 (NM_144599.5 c.249 C > G; p.Asn83Lys). Molecular modelling was performed to evaluate putative functional consequence of the NIPA1 protein. Indeed, the Asn83Lys modification is predicted to induce a significant perturbation of the protein structure, altering signal transduction or small-molecule transport by modulating the length of the second transmembrane domain. This is the first study reporting a SPG6-affected patient harbouring the NIPA1 p.Asn83Lys mutation.

Background. Severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) infection has rapidly spread worldwide from the beginning of 2020. Quantitative reverse transcription-PCR (RT-qPCR) is, to this day, the preferred methodology for viral RNA detection, even if not without problems. To overcome some of the limitations still existing for the detection and quantification of nucleic acids in various applications, the use of one-step reverse transcription-droplet digital PCR (RT-ddPCR) has been established. The purpose of this study was, then, to evaluate the efficacy of ddPCR for the detection of SARS-CoV-2 RNA in nasopharyngeal swabs, optimizing the detection of low-viral load-burdened samples. Methods. The RT-ddPCR workflow was validated for sensitivity, specificity, linearity, reproducibility, and precision using samples from 90 COVID-19-infected patients referred to the Department of Laboratory Medicine of the University Hospital of Udine (Italy). Results. The present study shows that RT-ddPCR allows the detection of as low as 10.3 copies of a SARS-COV-2 E-gene per sample with a higher level of accuracy and precision, especially at low concentration. Conclusion. During the postpeak phase of the SARS-CoV-2 pandemic, it is essential to rely on a highly robust molecular biology method to identify infected subjects, whether they have symptoms or not, in order to prepare appropriate containment measures.

Background Colorectal cancer (CRC) represents the fourth leading cause of cancer-related deaths. The heterogeneity of CRC identity limits the usage of cell lines to study this type of tumor because of the limited representation of multiple features of the original malignancy. Patient-derived colon organoids (PDCOs) are a promising 3D-cell model to study tumor identity for personalized medicine, although this approach still lacks detailed characterization regarding molecular stability during culturing conditions. Correlation analysis that considers genomic, transcriptomic, and proteomic data, as well as thawing, timing, and culturing conditions, is missing. Methods Through integrated multi–omics strategies, we characterized PDCOs under different growing and timing conditions, to define their ability to recapitulate the original tumor. Results Whole Exome Sequencing allowed detecting temporal acquisition of somatic variants, in a patient-specific manner, having deleterious effects on driver genes CRC-associated. Moreover, the targeted NGS approach confirmed that organoids faithfully recapitulated patients’ tumor tissue. Using RNA-seq experiments, we identified 5125 differentially expressed transcripts in tumor versus normal organoids at different time points, in which the PTEN pathway resulted of particular interest, as also confirmed by further phospho-proteomics analysis. Interestingly, we identified the PTEN c.806_817dup (NM_000314) mutation, which has never been reported previously and is predicted to be deleterious according to the American College of Medical Genetics and Genomics (ACMG) classification. Conclusion The crosstalk of genomic, transcriptomic and phosphoproteomic data allowed to observe that PDCOs recapitulate, at the molecular level, the tumor of origin, accumulating mutations over time that potentially mimic the evolution of the patient’s tumor, underlining relevant potentialities of this 3D model.

Background The Bosch‐Boonstra‐Schaaf optic atrophy syndrome (BBSOAS) is an autosomal‐dominant disorder (OMIM615722) mostly characterized by optic atrophy and/or hypoplasia, mild intellectual disability, hypotonia, seizures/infantile epilepsy. This disorder is caused by loss‐of‐function alterations of NR2F1 (i.e., either whole gene deletions or single nucleotide variants) and, to date, 40 patients have been identified with deletions or mutations in this gene. Here we describe two monozygotic twins harboring a de novo missense variant in the DNA‐binding domain of NR2F1 (c.313G>A, p.Gly105Ser), with well‐characterized features associated to BBSOAS. Methods Patients’ DNA was analyzed by exome sequencing identifying the missense variant c.313G>A in NR2F1 (NM_005654.4). Furthermore, molecular modeling was performed to evaluate putative differences in DNA binding between wild‐type and mutated NR2F1. Results The missense variant is predicted to be likely pathogenetic following the ACMG (American College of Medical Genetics and Genomics)/AMP (Association for Molecular Pathology) guidelines. Indeed, dynamic simulation experiments highlighted that the Gly105Ser substitution let the formation of a hydrogen bond between the S105 side chain and R142 and a base (G5) of the DNA sequence, allowing us to hypothesize that the G105 residue might be evolutionary conserved due to the absence of a side chain, besides glycine conformational features. Therefore, the G105S variation seems to cause a stiffening and a possible deformation in the protein‐DNA complex due to the interaction of residues R142‐S105 and G5 on the DNA, compared to the wild‐type. Conclusion In summary, we described two monozygotic twins harboring a novel Gly105Ser mutation in NR2F1 DNA binding domain, displaying the classical phenotype of BBSOAS‐affected patients. Our computational data suggest a dominant negative effect of this newly characterized missense variant. To date, this is the first genetic report analyzing in silico structural consequences of NR2F1 Gly105Ser substitution., Here we describe two monozygotic twins harboring a de novo missense variant in the DNA binding domain of NR2F1, with well‐characterized features associated to BBSOAS. Following the American College of Medical Genetics and Genomics (ACMG) guidelines, the variant is classified as likely pathogenic, matching 4 out of 6 specific criteria. Furthermore, molecular dynamics simulations highlighted that the complexes mutation introduces bonds in the triad R142‐S105‐G5 DNA base which could lead to a distortion and stiffening in this region, compromising the receptor functions.

In the current pandemic, the presence of SARS-CoV-2 RNA in samples by nucleic acid (NA) molecular analysis is the only method available to diagnose COVID-19 disease and to assess patients’ infectiveness. Recently, the demand for laboratory reagents has greatly increased; in particular, there is a worldwide shortage of RNA extraction kits. Here, we describe a fast, simple and inexpensive method for the detection of SARS CoV-2 RNA, which includes a pretreatment with Proteinase K and a heating-cooling cycle before the amplification. This method bypasses the RNA extraction step; it leads to a higher amount of available viral RNA compared to the automated extraction methods, and generates the same profile in the subsequent amplification phase.

The Silver–Russell syndrome (SRS) is a rare disorder characterized by heterogeneous clinical features, including growth retardation, typical facial dysmorphisms, and body asymmetry. Genetic alterations causative of SRS mostly affect imprinted genes located on chromosomes 7 or 11. Hypomethylation of the Imprinting Center 1 (IC1) of the chromosome 11p15.5 is the most common cause of SRS, while the Imprinting Center 2 (IC2) has been more rarely involved. Specifically, maternally inherited 11p15.5 deletions including the IC2 have been associated with the Beckwith–Wiedemann Syndrome (BWS), while paternal deletions with a variable spectrum of phenotypes. Here, we describe the case of a girl with a mild SRS phenotype associated with a paternally inherited 1.4 kb deletion of IC2. The father of the proband inherited the deletion from his mother and showed normal growth, while the paternal grandmother had the deletion on her paternal chromosome and exhibited short stature. Together with previous findings obtained in mouse and humans, our data support the notion that deletion of the paternal copy of IC2 can cause SRS.

We identified a de novo 44.7 Kb interstitial 12p13.33 micro-deletion that involves solely the first exon of the CACNA1C (MIM 114205), using microarray-based comparative genomic hybridization (aCGH). The associated main phenotype is characterized by expressive language impairment, tremors, fine motor-skills delay, muscular hypotonia, and joint laxity. A careful comparison between the clinical and genomic characteristics between our proband and 20 previously reported patients, led us to propose CACNA1C haploinsufficiency as the main cause of both expressive language delay and motor-skills impairment. Pathogenic variants of CACNA1C have been associated to a plethora of clinical phenotypes, such as Timothy syndrome (TS, OMIM 601005), Brugada syndrome (BRGDA3, OMIM 611875) and a variety of neuropsychiatric disorders (bipolar disorder, major depression, schizophrenia, autism spectrum disorder, psychotic manifestations). In this report we describe a 12p13.33 micro-deletion involving one coding gene only, in contrast with previous studies that mostly concluded that a multi-genes deletion in the 12p13.33 sub-telomeric region is responsible of the minimum clinical phenotype of patients with 12p13.33 monosomy. Certainly, larger deletions spanning multiple Mb in 12p13.33 are responsible for more severe phenotypes, associated to a variable degree of dysmorphic features.

The anaplastic thyroid carcinoma (ATC) is the most aggressive thyroid cancer with a high mortality rate. Since nutraceuticals may exert beneficial effects on tumor biology, here, effects of four of these compounds [resveratrol, genistein, curcumin and epigallocatechin-3-gallate (EGCG)] on ATC cell lines were investigated. Two ATC-derived cell lines were used: SW1736 and 8505C. Cell viability and in vitro aggressiveness was tested by MTT and soft agar assays. Apoptosis was investigated by Western Blot, using an anti-cleaved-PARP antibody. mRNA and miRNA levels were quantified by real-time PCR. All tested nutraceuticals caused in both cell lines decrease of cell viability and increase of apoptosis. In contrast, only curcumin reduced in vitro aggressiveness in both SW1736 and 8505C cell lines, while genistein and EGCG determined a reduction of colony formation only in 8505C cells. Effects on genes related to the thyroid-differentiated phenotype were also tested: resveratrol and genistein administration determined the increment of almost all tested mRNAs in both cell lines. Instead curcumin and EGCG treatments had opposite effects in the two cell lines, causing the increment of almost all the mRNAs in 8505C cells and their reduction in SW1736. Finally, effects of nutraceuticals on levels of several miRNAs, known as important in thyroid cancer progression (hsa-miR-221, hsa-miR-222, hsa-miR-21, hsa-miR-146b, hsa-miR-204), were tested. Curcumin induced a strong and significant reduction of all miR analyzed, except for has-miR-204, in both cell lines. Altogether, our results clearly indicate the anti-cancer proprieties of curcumin, suggesting the promising use of this nutraceutical in ATC treatment. Resveratrol, genistein and EGCG have heterogeneous effects on molecular features of ATC cells.

The epigenetic machinery deputed to control histone post-translational modifications is frequently dysregulated in cancer cells. With epigenetics being naturally reversible, it represents a good target for therapies directed to restore normal gene expression. Since the discovery of Bromodomain and Extra Terminal (BET) inhibitors, a great effort has been spent investigating the effects of chromatin readers’ inhibition, specifically the class of proteins assigned to bind acetylated and methylated residues. So far, focused studies have been produced on epigenetic regulation, dissecting a specific class of epigenetic-related proteins or investigating epigenetic therapy in a specific tumor type. In this review, recent steps toward drug discovery on the different classes of chromatin readers have been outlined, highlighting the pros and cons of current therapeutic approaches.

The pathogenesis of colorectal cancer (CRC) involves different mechanisms, such as genomic and microsatellite instabilities. Recently, a contribution of the base excision repair (BER) pathway in CRC pathology has been emerged. In this context, the involvement of APE1 in the BER pathway and in the transcriptional regulation of genes implicated in tumor progression strongly correlates with chemoresistance in CRC and in more aggressive cancers. In addition, the APE1 interactome is emerging as an important player in tumor progression, as demonstrated by its interaction with Nucleophosmin (NPM1). For these reasons, APE1 is becoming a promising target in cancer therapy and a powerful prognostic and predictive factor in several cancer types. Thus, specific APE1 inhibitors have been developed targeting: i) the endonuclease activity; ii) the redox function and iii) the APE1-NPM1 interaction. Furthermore, mutated p53 is a common feature of advanced CRC. The relationship between APE1 inhibition and p53 is still completely unknown. Here, we demonstrated that the inhibition of the endonuclease activity of APE1 triggers p53-mediated effects on cell metabolism in HCT-116 colon cancer cell line. In particular, the inhibition of the endonuclease activity, but not of the redox function or of the interaction with NPM1, promotes p53 activation in parallel to sensitization of p53-expressing HCT-116 cell line to genotoxic treatment. Moreover, the endonuclease inhibitor affects mitochondrial activity in a p53-dependent manner. Finally, we demonstrated that 3D organoids derived from CRC patients are susceptible to APE1-endonuclease inhibition in a p53-status correlated manner, recapitulating data obtained with HCT-116 isogenic cell lines. These findings suggest the importance of further studies aimed at testing the possibility to target the endonuclease activity of APE1 in CRC.

Mutations in TERT promoter have been detected in the more aggressive papillary thyroid cancers (PTCs). To elucidate the role of TERT as an eligible molecular target in these tumors, the expression of hTERT was analyzed in a series of PTCs and the effects of both pharmacological and RNA-interference-induced hTERT silencing were investigated in two human PTC cell lines (K1 and BCPAP). The expression levels of hTERT mRNA and protein were evaluated by real-time PCR and western blot assays, respectively. Effects of hTERT silencing on PTC cell lines were analyzed by MTT, migration and western blot assays. Pharmacological inhibition of hTERT was performed using two bromodomain and extra-terminal (BET) inhibitors, JQ1 and I-BET762. hTERT expression results increased in 20 out of 48 PTCs, including tumors either positive or negative for the presence of hTERT promoter and/or BRAF mutations. In K1 and BCPAP cells, hTERT silencing determined a reduction in cell viability (~50% for K1 and ~70%, for BCPAP, vs control) and migration properties that were associated with a decrease of AKT phosphorylation and β-Catenin expression. Moreover, hTERT mRNA levels were down-regulated by two BET inhibitors, JQ1 and I-BET762, which at the same dosage (0.5 and 5 µM) reduced the growth of these thyroid cancer cells. These findings demonstrate that hTERT may represent an excellent therapeutic target in subgroups of aggressive PTCs.

Williams syndrome (WS) is a rare neurodevelopmental disorder associated to a hemizygous deletion of 28 genes located on chromosome 7q11.23. WS affected subjects frequently suffer from several endocrine abnormalities including hypothyroidism due to defects in thyroid morphology. To date, several genes involved in thyroid dysgenesis have been identified, nonetheless, none of them is located in the 7q11.23 region. Thus, the hypothyroidism-linked molecular features in WS are not yet known. In this study we focused on one of the WS deleted gene, BAZ1B, demonstrating that its downregulation in thyroid cells leads to cell viability and survival decrement. Taking together, our results show that BAZ1B could be the mainly responsible for thyroid defects observed in some of WS patients and that these alterations are activated by PTEN-mediated mechanisms.

Inhibition of bromo-and extra-terminal domain (BET) proteins, epigenetic regulators of genes involved in cell viability, has been efficiently tested in preclinical models of triple negative breast cancer (TNBC). However, the use of the selective BET-inhibitor JQ1 on humans is limited by its very short half-life. Herein, we developed, characterized and tested a novel formulation of nanoparticles containing JQ1 (N-JQ1) against TNBC in vitro and in vivo. N-JQ1, prepared using the nanoprecipitation method of preformedpoly-lactid-co-glycolic acid in an aqueous solution containing JQ1 and poloxamer-188 as a stabilizer, presented a high physico-chemical stability. Treatment of MDA-MB 157 and MDA-MB 231 TNBC cells with N-JQ1 determined a significant decrease in cell viability, adhesion and migration. Intra-peritoneal administration (5 days/week for two weeks) of N-JQ1 in nude mice hosting a xenograft TNBC after flank injection of MDA-MB-231 cells determined a great reduction in the growth and vascularity of the neoplasm. Moreover, the treatment resulted in a minimal infiltration of nearby tissues. Finally, the encapsulation of JQ1 in nanoparticles improved the anticancer efficacy of this epigenetic compound against TNBC in vitro and in vivo, opening the way to test it in the treatment of TNBC.

Genomic and epigenetic alterations are now being exploited as molecular targets in cancer treatment. Abnormalities involving the post-translational modification of histones have been demonstrated in thyroid cancer, and they are regarded as promising molecular targets for novel drug treatment of tumors that are resistant to conventional therapies. After a brief overview of the histone modifications most commonly associated with human malignancies, we will review recently published preclinical and clinical findings regarding the use of histone-activity modulators in thyroid cancers. Particular attention will be focused on their use as re-differentiating or anti-proliferating agents, the differential effects observed when they are used alone and in combination with other targeted drugs, and current prospects for their use in the treatment of thyroid cancer.

Anaplastic thyroid carcinoma (ATC) represents the most lethal thyroid cancer sub-type, currently unresponsive to standard treatments. Recently, bromodomain and extra-terminal (BET) proteins have emerged as attractive therapeutic targets in several diseases, including cancer. In different cancer models, the anti-neoplastic activity of BET inhibitors such as JQ1, I-BET762 and I-BET151 have already been established, due to both direct and indirect effects. miRNAs are 20-22 nucleotide transcriptional regulators which play important roles in proliferation, differentiation and apoptosis. Hitherto, the relationship between JQ1 and miRNAs has not been explored. The goal of this study was to delineate JQ1-associated miRNA regulation in ATC cells. Two ATC-derived cell lines (SW1736 and 8505c) were treated with either 5 µM JQ1 or vehicle for 48 or 72 h. A non-tumorigenic thyroid cell line (Nthy-ori 3-1) was used as a control. miRNome analysis displayed a JQ1-related dysregulation of several miRNAs, 7 of which turned out to be commonly dysregulated in both cell lines at both time-points. Furthermore, miR-4516 turned out to be downregulated in both ATC cell lines, when compared to the non-tumorigenic ones and notably, JQ1 treatment in both ATC cell lines induced its upregulation, restoring, in some way, its basal expression levels. We, therefore, focused on miR-4516 expression and STAT3 levels, since it was previously predicted to be a putative target of this microRNA. Consistently, phospho-STAT3 and its target p21Waf1/Cip1 turned out to be downregulated and upregulated in both JQ1-treated ATC cell lines, respectively. Thus, our data revealed that modulation of miRNA expression is one of the multiple mechanisms of the effect of JQ1 in thyroid cancer cells.

Anaplastic thyroid cancer (ATC) constitutes one of the most aggressive types of human solid cancer, and is characterized by the absence of thyroid differentiation features and a marked degree of invasiveness. We have previously demonstrated that the RNA-binding protein Hu antigen R (HuR) is overexpressed in thyroid carcinoma; thus, the biological role of this RNA-binding protein was investigated in the present study using the ATC cell lines SW1736 and 8505C. In both cell lines, HuR protein levels were higher compared with in the non-tumorigenic thyroid cell line Nthy-ori-3.1. HuR silencing by RNA interference in both ATC cell lines decreased cell viability, increased apoptosis rates and reduced the capability to form colonies in soft agar. Thus, HuR plays an important role in the proliferation and aggressiveness of ATC cells. The histone deacetylase inhibitor suberoylanilide hydroxamic acid (SAHA) was able to reduce the viability of ATC cells. The results demonstrated that SAHA was able to decrease HuR expression in SW1736 and 8505C cells. Furthermore, since it is known that the transcription factor nuclear factor (NF)-κB modulates HuR expression, whether SAHA affects the nuclear (active) fraction of NF-κB in ATC cells was investigated. The data suggested that SAHA decreases ATC cell viability by reducing the active form of NF-κB, which, in turn, modulates HuR expression.

Application of distinct technologies of cancer genome analysis has provided important information for the molecular characterization of several human neoplasia, including follicular cell-derived thyroid carcinoma. Among them, comparative genomic hybridization (CGH)-based procedures have been extensively applied to evaluate genomic imbalances present in these tumours, obtaining data leading to an increase in the understanding of their complexity and diversity. In this review, after a brief overview of the most commonly used CGH-based technichs, we will describe the major results deriving from the most influential studies in the literature which used this approach to investigate the genomic aberrations of thyroid cancer cells. In most studies a small number of patients have been analyzed. Deletions and duplications at different chromosomal regions were detected in all investigated cohorts. A higher number of genomic imbalances has been detected in anaplastic or poorly differentiated thyroid carcinomas compared to well differentiated ones. Limitations in the interpretation of the results, as well the potential impact in the clinical practice are discussed. Though a quite heterogeneous picture arises from results so far available, CGH array, combined with other methodologies as well as an accurate clinical management, may offer novel opportunities for a better stratification of thyroid cancer patients.

// Federica Baldan 1 , Catia Mio 1 , Lorenzo Allegri 1 , Ketty Conzatti 1 , Barbara Toffoletto 1 , Cinzia Puppin 1 , Slobodanka Radovic 2 , Carlo Vascotto 1 , Diego Russo 3 , Carla Di Loreto 1, 4 , Giuseppe Damante 1, 5 1 Department of Medical and Biological Sciences, University of Udine, 33100 Udine, Italy 2 IGA Technology Services Srl, 33100 Udine, Italy 3 Department of Health Sciences, University of Catanzaro “Magna Graecia”, 88100 Catanzaro, Italy 4 Institute of Anatomic Pathology, University Hospital “S. Maria della Misericordia”, 33100 Udine, Italy 5 Institute of Medical Genetics, University Hospital “S. Maria della Misericordia”, 33100 Udine, Italy Correspondence to: Giuseppe Damante, email: giuseppe.damante@uniud.it Keywords: HuR, RNA-binding proteins, RNA-seq, RIP-seq Received: December 21, 2015 Accepted: July 26, 2016 Published: August 12, 2016 ABSTRACT RNA binding proteins (RBPs) play a central role in cell physiology and pathology. Among them, HuR is a nuclear RBP, which shuttles to the cytoplasm to allow its RNA targets processing. HuR over-expression and delocalization are often associated to cell transformation. Numerous cancers display increased HuR protein levels and its high cytoplasmic levels has been associated with a worse prognosis. In our study, we first evaluated HuR expression in normal and cancer thyroid tissues and then evaluated its function in thyroid cell lines. HuR is over-expressed in all thyroid tumor tissues; high cytoplasmic levels are detected in all thyroid carcinomas. HuR silencing decreased cell viability and determined apoptotic cell death, in a non-tumorigenic (Nthy-ori-3.1) and a tumorigenic (BCPAP) thyroid cell line. Global transcriptome analysis indicated that HuR silencing, though having similar biological effects, induces distinct gene expression modifications in the two cell lines. By using the RIP-seq approach, the HuR-bound RNA profiles of different thyroid cell lines were evaluated. We show that in distinct cell lines HuR-bound RNA profiles are different. A set of 114 HuR-bound RNAs distinguishing tumorigenic cell lines from the non-tumorigenic one was identified. Altogether, our data indicate that HuR plays a role in thyroid tumorigenesis. Moreover, our findings are a proof of concept that RBP targets differ between cells with the same origin but with distinct biological behavior.

Anaplastic thyroid carcinoma (ATC) is an extremely aggressive thyroid cancer subtype, refractory to the current medical treatment. Among various epigenetic anticancer drugs, bromodomain and extra-terminal inhibitors (BETis) are considered to be an appealing novel class of compounds. BETi target the bromodomain and extra-terminal of BET proteins that act as regulators of gene transcription, interacting with histone acetyl groups. The goal of this study is to delineate which pathway underlies the biological effects derived from BET inhibition, in order to find new potential therapeutic targets in ATC. We investigated the effects of BET inhibition on two human anaplastic thyroid cancer-derived cell lines (FRO and SW1736). The treatment with two BETis, JQ1 and I-BET762, decreased cell viability, reduced cell cycle S-phase, and determined cell death. In order to find BETi effectors, FRO and SW1736 were subjected to a global transcriptome analysis after JQ1 treatment. A significant portion of deregulated genes belongs to cell cycle regulators. Among them, MCM5 was decreased at both mRNA and protein levels in both tested cell lines. Chromatin immunoprecipitation (ChIP) experiments indicate thatMCM5is directly bound by the BET protein BRD4.MCM5silencing reduced cell proliferation, thus underlining its involvement in the block of proliferation induced by BETis. Furthermore, MCM5 immunohistochemical evaluation in human thyroid tumor tissues demonstrated its overexpression in several papillary thyroid carcinomas and in all ATCs. MCM5 was also overexpressed in a murine model of ATC, and JQ1 treatment reducedMcm5mRNA expression in two murine ATC cell lines. Thus, MCM5 could represent a new target in the therapeutic approach against ATC.

PAX8 is a thyroid-specific transcription factor whose expression is dysregulated in thyroid cancer. A recent study using a conditional knock-out mouse model identified 58 putative PAX8 target genes. In the present study, we evaluated the expression of 11 of these genes in normal and tumoral thyroid tissues from patients with papillary thyroid cancer (PTC). ATP1B1, GPC3, KCNIP3, and PRLR transcript levels in tumor tissues were significantly lower in PTCs than in NT, whereas LCN2, LGALS1 and SCD1 expression was upregulated in PTC compared with NT. Principal component analysis of the expression of the most markedly dysregulated PAX8 target genes was able to discriminate between PTC and NT. Immunohistochemistry was used to assess levels of proteins encoded by the two most dyregulated PAX8 target genes, LCN2 and GPC3. Interestingly, GPC3 was detectable in all of the NT samples but none of the PTC samples. Collectively, these findings point to significant PTC-associated dysregulation of several PAX8 target genes, supporting the notion that PAX8-regulated molecular cascades play important roles during thyroid tumorigenesis.

Anaplastic thyroid carcinoma (ATC) is an extremely aggressive human malignancy characterized by a marked degree of invasiveness, absense of features of thyroid differentiation and resistance to current medical treatment. It is well known that ATCs are characterized by deregulation of genes related to cell cycle regulation, i.e., cyclin-dependent kinases (CDKs) and endogenous cyclin-dependent kinase inhibitors (CDKIs). Therefore, in the present study, the effect of a novel exogenous cyclin-dependent kinase inhibitor, BP-14, was investigated in three human ATC cell lines. The ATC-derived cell lines FRO, SW1736 and 8505C were treated with BP-14 alone or in combination with the mTOR inhibitor everolimus. In all ATC cell lines, treatment with BP-14 decreased cell viability and, in two of them, BP-14 modified expression of genes involved in epithelial-mesenchymal transition. Thus, our data indicate that BP-14 is a potential new compound effective against ATC. Combined treatment with BP-14 and the mTOR inhibitor everolimus had a strong synergistic effect on cell viability in all three cell lines, suggesting that the combined used of CDK and mTOR inhibitors may be a useful strategy for ATC treatment.

Circadian clock regulation in mammals is controlled by feedback loops of a set of circadian genes. One of these circadian genes, NPAS2, encodes for a member of the bHLH-PAS class of transcription factors and is expressed in the forebrain and in some peripheral organs such as liver and skin. Other biological processes are also regulated by circadian genes. For example, NPAS2 is involved in cell proliferation, DNA damage repair and malignant transformation. Aberrant expression of clock genes has been previously observed in melanoma which led to our effort to sequence the NPAS2 promoter region in this cancer type. The NPAS2 putative promoter and 5′ untranslated region of ninety-three melanoma patients and ninety-six control subjects were sequenced and several variants were identified. Among these is a novel microsatellite comprising a GGC repeat with different alleles ranging from 7 to 13 repeats located in the 5′ untranslated exon. Homozygosity of an allele with nine repeats (9/9) was more prevalent in melanoma than in control subjects (22.6% and 13.5%, respectively, P: 0.0206) suggesting that some NPAS2 variants might contribute to melanoma susceptibility. Impact statement This report describes a variable microsatellite repeat sequence located in the 5′ untranslated exon of NSPAS2, a gene encoding a clock transcription factor. Significantly, this study is the first to show that a variant copy number GGC repeat sequence in the NPAS2 clock gene associates with melanoma risk and which may be useful in the assessment of melanoma predisposition.

Anaplastic thyroid carcinoma (ATC) is a very aggressive human malignancy, having a marked degree of invasiveness and no features of thyroid differentiation. It is known that either HDAC inhibitors or PARP inhibitors have antiproliferative effects on thyroid cancer cells. Therefore, in this study the possible synergy between the two types of compounds has been investigated. The ATC-derived cell line SW1736 has been treated with the HDAC inhibitor suberoylanilide hydroxamic acid (SAHA) and the PARP inhibitor PJ34, alone or in combination. In terms of cell viability, the combination index value was always lower than 1 at various tested dosages, indicating, therefore, synergy in a wide range of doses for both compounds. Synergy was also observed in induction of apoptosis. In terms of thyroid-specific gene expression, synergy was observed for TSHR mRNA levels but not for NIS, TTF1, TTF2, and PAX8 mRNA levels. Altogether, these data suggest that the combined use of HDAC and PARP inhibitors may be a useful strategy for treatment of ATC.

Studies on stem cell differentiation led to the identification of paused genes, characterized by the contemporary presence of both activator and repressor epigenetic markers (bivalent marking). TXNIP is an oncosuppressor gene the expression of which was reduced in breast cancer. In the present study, we evaluated whether the concept of epigenetic bivalent marking can be applied to TXNIP gene in breast cancer cells. Using chromatin immunoprecipitation (ChIP), three histone modifications were investigated: two associated with transcriptional activation, lysines 9-14 acetylation of H3 histone (H3K9K14ac) and lysine 4 trimethylation of H3 histone (H3K4me3), and one associated with transcriptional silencing, lysine 27 trimethylation of H3 histone (H3K27me3). According to the bivalent marking model, TXNIP gene appears to be paused in MDA157 cells (markers of active and repressed transcription are present), but are definitively silenced in MDA468 cells (presence of only markers of transcription repression). This was proven by evaluating TXNIP mRNA and protein levels after the treatment of cell lines with a histone deacetylase inhibitor (SAHA) and a poly-ADP-ribose polymerases inhibitor (PJ34). In MDA157 cells, SAHA and PJ34 showed a synergistic effect: a large increment was observed in TXNIP mRNA and protein levels. By contrast, in MDA468 cells, synergy between the two compounds was not observed. Therefore, the pausing epigenetic signature was permissive for synergy between SAHA and PJ34 on TXNIP gene expression. The synergy between SAHA and PJ34 on TXNIP expression was associated with variation in cell viability and apoptosis. In MDA157 cells, but not in MDA468 cells, combined treatment of SAHA and PJ34 induced a decrease in cell viability and an increase of apoptosis. Thus, our data support the hypothesis that TXNIP is an effective target for the treatment of breast cancer.