Search

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.

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.

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.

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.

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.

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.