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Low-density lipoproteins (LDLs) are the most abundant circulating lipoproteins and the most critical factor in the development of atherosclerosis. This protocol allows the staining of LDLs with oil red O to monitor particle uptake in bright-field microscopy. Here, we describe how to stain isolated LDLs using oil red O and how to use them to monitor LDL uptake in time-lapse experiments or fixed cells., Competing Interests: Declaration of interests M.A. received research grant support from Amryt Pharmaceutical, Amgen, Ionis, Akcea Therapeutics, Daiichi-Sankyo, Novartis, Pfizer, and Regeneron; served as a consultant for Amgen, Akcea Therapeutics, Daiichi-Sankyo, and Ultragenyx; and received fees for lecturing, congress participation, and advisory board participation from Amgen, Amryt Pharmaceutical, Daiichi-Sankyo, Regeneron, Sanofi, Amarin, and Ultragenyx. S.B. received fees for lectures from Sobi; detains stock options of Eli Lilly, UnitedHealth, Novo Nordisk, Merck, and Thermo Fisher Scientific; and received grants for meeting participation from Novartis and Chiesi. L.D. received personal fees for public speaking, consultancy, or grant support from Amryt Pharmaceutical, Akcea Therapeutics, Sobi, Aurora Biopharma, Novartis, Amarin, Daiichi-Sankyo, Bayer, and Sandoz. D.T. received fees for lectures from Sobi and grants for meeting participation from Ionis., (Copyright © 2024 The Author(s). Published by Elsevier Inc. All rights reserved.)

Purpose: Papillary thyroid carcinoma (PTC) is the most common malignant thyroid neoplasm, accounting for approximately 85% of all follicular cell-derived thyroid nodules. This study aimed to assess the diagnostic potential of circulating microRNA-146a-5p and microRNA-221-3p as biomarkers for PTC and their usefulness in monitoring disease progression during patient follow-up., Methods: An observational study was conducted on two cohorts of PTC patients and healthy controls (HCs) using digital PCR. We collected patients' clinical, biochemical, and imaging data during the post-surgery surveillance. We analyzed the levels of circulating miRNAs in serum samples of patients before surgery and during the follow-up, including those with indeterminate/biochemical incomplete response (IndR/BIR) and residual thyroid tissues (Thy Residue)., Results: Both miR-146a-5p and miR-221-3p were confirmed as effective biomarkers for PTC diagnosis. They enabled differentiation between pre-surgery PTC patients and HCs with an area under the curve (AUC) of 92% and 87.3%, respectively, using a threshold level of 768,545 copies/uL for miR-146a-5p and 389,331 copies/uL for miR-221-3p. It was found that miRNA fold change levels, rather than absolute levels, can be useful during patient follow-up. In particular, we found that a fold change of 2 for miR-146a-5p and 2.2 for miR-221-3p can identify a progressive disease, regardless of the presence of TgAbs or remnant thyroid., Conclusion: MiRNA-146a-5p and miRNA-221-3p, particularly the former, could be valuable diagnostic biomarkers for PTCs. They also seem to be effective in monitoring disease progression during patient follow-up by evaluating their fold change, even when thyroglobulin is uninformative., (© 2024. The Author(s).)

Medullary thyroid carcinoma (MTC) is a malignant tumor with challenging management. Multi-targeted kinase inhibitors (MKI) and tyrosine-kinase inhibitors (TKI) with high specificity for RET protein are approved for advanced MTC treatment. However, their efficacy is hindered by evasion mechanisms of tumor cells. Thus, the aim of this study was the identification of an escape mechanism in MTC cells exposed to a highly selective RET TKI. TT cells were treated with TKI, MKI, and/or the HH-Gli inhibitors, GANT61 and Arsenic Trioxide (ATO), in the presence or absence of hypoxia. RET modifications, oncogenic signaling activation, proliferation and apoptosis were assessed. Additionally, cell modifications and HH-Gli activation were also evaluated in pralsetinib-resistant TT cells. Pralsetinib inhibited RET autophosphorylation and RET downstream pathways activation in normoxic and hypoxic conditions. Additionally, pralsetinib impaired proliferation, induced the activation of apoptosis and, in hypoxic cells, downregulated HIF-1α. Focusing on escape molecular mechanisms associated with therapy, we observed increased Gli1 levels in a subset of cells. Indeed, pralsetinib stimulated the re-localization of Gli1 into the cell nuclei. Treatment of TT cells with both pralsetinib and ATO resulted in Gli1 down-regulation and impaired cell viability. Moreover, pralsetinib-resistant cells confirmed Gli1 activation and up-regulation of its transcriptionally regulated target genes. Altogether, we showed that pralsetinib impairs MTC cell growth and induces cell death, also in hypoxic conditions. The HH-Gli pathway is a new molecular mechanism of escape to pralsetinib therapy that can be overcome through combined therapy., Competing Interests: Declaration of Competing Interest None., (Copyright © 2023 The Authors. Published by Elsevier Masson SAS.. All rights reserved.)

Purpose: In this study, we evaluated the biological role of miRNA-31-5p in papillary thyroid cancer (PTC)., Methods: By using the real-time PCR, we measured miRNA-31-5p expression levels in 25 PTC tissues and in two human PTC cell lines (K1 and TPC-1). Then, K1 cells were transiently transfected with mirVana inhibitor or mirVana mimic to miRNA-31-5-p. Cell proliferation was determined by MTT and colony formation assays. The in vitro metastatic ability of thyroid cancer cells was evaluated by adhesion, migration and invasion assays. Epithelial mesenchymal transition (EMT) and Hippo pathway related gene and protein levels were evaluated by using the TaqMan™ Gene Expression Assays and western blot analysis, respectively., Results: We found a significant increase of miR-31-5-p expression in tumor tissue and in K1 cells harboring the BRAF p.V600E mutation. Knockdown of miR-31-5p determined a reduction of cell proliferation, associated with a significant decrease in cell adhesion, migration and invasion properties. A downregulation of EMT markers and YAP/β-catenin axis was also observed., Conclusions: Our findings suggest that miRNA-31-5p acts as oncogenic miRNA in human thyrocytes and its overexpression may be involved in the BRAF-related tumorigenesis in PTCs, providing new understanding into its pathological role in PTC progression and invasiveness., (© 2022. The Author(s), under exclusive licence to Springer Science+Business Media, LLC, part of Springer Nature.)

Background and Aim: Angiopoietin-like 3 (ANGPTL3) and 4 (ANGPTL4) are regulators of triglyceride storage and utilization. Bariatric surgery (BS) leads to profound changes in adipose tissue composition and energy metabolism. We evaluated the impact of BS on plasma levels of ANGPTL3 and ANGPTL4., Methods and Results: Twenty-seven subjects affected by morbid obesity with or without type 2 diabetes (T2D) underwent Roux-en-Y gastric bypass (RYGB) and 18 patients with advanced T2D received Biliopancreatic Diversion (BPD). Fasting ANGPTL proteins levels, insulin sensitivity (evaluated by euglycemic hyperinsulinemic clamp), total bile acids (TBA) and free fatty acids (FFA) were measured at baseline and 1 year after surgery. Both surgical procedures resulted in the loss of fat mass, improved glucose control, and a ∼2-fold increase of insulin sensitivity. ANGPTL4 levels decreased significantly with both RYGB (26.6 ± 0.6 to 24.4 ± 0.3 ng/mL, p = 0.001) and BPD (27.9 ± 1.5 to 24.0 ± 0.5 ng/mL, p = 0.003). In contrast, ANGPTL3 concentrations did not change after RYGB but rose following BPD (225 ± 20 to 300 ± 15 ng/mL, p = 0.003). By multiple regression analysis, changes after BS in ANGPTL4 were independently associated with changes in blood glucose, (p = 0.0169) whereas changes in ANGPTL3 were associated with variations in FFA (p = 0.008) and insulin sensitivity (p = 0.043)., Conclusion: Circulating ANGPTL4 is reduced by BS, probably due to the loss of fat mass and improved insulin sensitivity. Conversely, ANGPTL3 levels increased after BPD, but not after RYGB, presumably because of the metabolic changes induced by the malabsorptive effect of BPD., Competing Interests: Declaration of competing interest LD has received personal fees for public speaking, consultancy or grant support from Amryt Pharmaceuticals, Akcea Therapeutics, Pfizer, Amgen and Sanofi; MA has received research grant support from Amryt Pharmaceutical, Amgen, IONIS, Akcea Therapeutics, Pfizer and Sanofi; has served as a consultant for Amgen, Aegerion, Akcea Therapeutics, Regeneron, Sanofi and Alfasigma and received lecturing fees from Amgen, Amryth Pharmaceutical, Pfizer, Sanofi and AlfaSigma. EF reports receiving consultancy/speaker fees, outside the present work, from Boehringer Ingelheim, Lilly&Co., AstraZeneca, and Sanofi. Other authors have declared no conflict of interest., (Copyright © 2022 The Italian Diabetes Society, the Italian Society for the Study of Atherosclerosis, the Italian Society of Human Nutrition and the Department of Clinical Medicine and Surgery, Federico II University. All rights reserved.)

Aberrant activation of the RET proto-oncogene is implicated in a plethora of cancers. RET gain-of-function point mutations are driver events in multiple endocrine neoplasia 2 (MEN2) syndrome and in sporadic medullary thyroid cancer, while RET rearrangements are driver events in several non-medullary thyroid cancers. Drugs able to inhibit RET have been used to treat RET -mutated cancers. Multikinase inhibitors were initially used, though they showed modest efficacy and significant toxicity. However, new RET selective inhibitors, such as selpercatinib and pralsetinib, have recently been tested and have shown good efficacy and tolerability, even if no direct comparison is yet available between multikinase and selective inhibitors. The advent of high-throughput technology has identified cancers with rare RET alterations beyond point mutations and fusions, including RET deletions, raising questions about whether these alterations have a functional effect and can be targeted by RET inhibitors. In this mini review, we focus on tumors with RET deletions, including deletions/insertions (indels), and their response to RET inhibitors., Competing Interests: The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest., (Copyright © 2022 Verrienti, Grani, Sponziello, Pecce, Damante, Durante, Russo and Filetti.)

Here, we describe a protocol to generate organoids from human thyroid cancer cells. Starting from the same patient-derived cells, we establish both organoids and primary lines. The organoid medium is supplemented with conditioned medium obtained from the primary cell line. This modification enables culture of the organoid lines for up to 10 months. Even after long-term culture, the organoids retain the genetic and phenotypic characteristics of their tissue of origin., Competing Interests: The authors declare no competing interests., (© 2022 The Author(s).)

The release of molecules in exosomal cargoes is involved in tumor development and progression. We compared the profiles of exosomal microRNAs released by two thyroid cancer cell lines (TPC-1 and K1) with that of non-tumorigenic thyroid cells (Nthy-ori-3-1), and we explored the network of miRNA-target interaction. After extraction and characterization of exosomes, expression levels of microRNAs were investigated using custom TaqMan Advanced array cards, and compared with those expressed in the total cell extracts. The functional enrichment and network-based analysis of the miRNAs' targets was also performed. Five microRNAs (miR-21-5p, miR-31-5p, miR-221-3p, miR-222-3p, and let-7i-3p) were significantly deregulated in the exosomes of tumor cells vs. non-tumorigenic cells, and three of them (miR-31-5p, miR-222-3p, and let-7i-3p) in the more aggressive K1 compared to TPC-1 cells. The network analysis of the five miRNAs identified some genes as targets of more than one miRNAs. These findings permitted the identification of exosomal microRNAs secreted by aggressive PTC cells, and indicated that their main targets are regulators of the tumor microenvironment. A deeper analysis of the functional role of the targets of exosomal miRNAs will provide further information on novel targets of molecular treatments for these neoplasms.

Background: ANGPTL3 stimulates lipolysis in adipocytes, but the underlying molecular mechanism is yet unknown. The C-terminal fibrinogen-like domain of ANGPTL3 (ANGPTL3-Fld) activates the AKT pathway in endothelial cells. Hence, we evaluated whether ANGPTL3-Fld stimulates lipolysis in adipocytes through the MAPK kinase pathway., Materials and Methods: 3T3-L1 adipocytes were treated with isoproterenol (ISO), ANGPTL3-Fld, or both. Lipolysis was evaluated through the release of free fatty acids (FFAs) in the culture medium. The activation status of intracellular kinases was evaluated with and without the inhibition of the BRAF-ERK arm of the MAPK pathway., Results: ANGPTL3-Fld alone was not able to activate lipolysis, while the combination of ANGPTL3-Fld and ISO determined a 10-fold enrichment of the FFA concentration in the culture medium with an incremental effect (twofold) when compared with ISO alone. ANGPTL3-Fld alone inhibited hormone-sensitive lipase (HSL), whereas the treatment with ISO induced the activation of HSL. The net balance of ANGPTL3-Fld and ISO cotreatment resulted in HSL activation. The results indicate that ANGPTL3-Fld generated an intracellular activation signal involving the MAPK-ERK pathway, possibly through the PDGFRβ-PLCγ-AMPK axis., Conclusion: ANGPTL3-Fld appears to act as a facilitator of lipolysis in adipocytes, and this effect was driven by a signal mediated by a pathway that is different from the canonical β-adrenergic stimulus.

Purpose: In this study, we investigated the profile of microRNAs (miRNAs) contained in exosomes secreted in the serum of patients with papillary thyroid cancer (PTC)., Methods: Exosome were isolated by adding ExoQuick Exosome Precipitation Solution. Dynamic light scattering (DLS) and western blotting analysis were used to ensure the quality of exosomes. The expression levels of miRNAs were investigated using custom-designed TaqMan Advanced miRNA Array Cards in the screening cohort and using specific TaqMan Advanced MicroRNA Assays in the validation cohort., Results: We identified miR24-3p, miR146a-5p, miR181a-5p and miR382-5p with different expression levels in two different series of 56 and 58 PTC patients as compared with healthy controls. Significant differences in the expression of three PTC exosomal miRNAs, depending on the presence of lymph node metastasis, were detected in only one PTC series. When comparing the expression levels of some PTC-specific exosomal miRNAs with those of the same miRNAs circulating free of any encapsulation, we found a significant correlation for only miR24-3p, suggesting that only select miRNAs are secreted in exosomes., Conclusions: Our findings demonstrate that four miRNAs are differently secreted in the exosomes of PTC patients, whereas no conclusive results were found to characterize PTCs with lymph node metastasis, suggesting caution in the use of circulating exosomal miRNA expression levels as lymph node metastasis biomarkers. Further investigation into the mechanisms governing miRNA secretion in tumor cells are required., (© 2021. The Author(s), under exclusive licence to Springer Science+Business Media, LLC, part of Springer Nature.)

The SARS-CoV-2 pandemic is a worldwide public health emergency. Despite the beginning of a vaccination campaign, the search for new drugs to appropriately treat COVID-19 patients remains a priority. Drug repurposing represents a faster and cheaper method than de novo drug discovery. In this study, we examined three different network-based approaches to identify potentially repurposable drugs to treat COVID-19. We analyzed transcriptomic data from whole blood cells of patients with COVID-19 and 21 other related conditions, as compared with those of healthy subjects. In addition to conventionally used drugs (e.g., anticoagulants, antihistaminics, anti-TNFα antibodies, corticosteroids), unconventional candidate compounds, such as SCN5A inhibitors and drugs active in the central nervous system, were identified. Clinical judgment and validation through clinical trials are always mandatory before use of the identified drugs in a clinical setting., (Copyright © 2021 The Authors. Published by Elsevier Masson SAS.. All rights reserved.)

Background: Understanding the molecular mechanisms underlying papillary thyroid cancer (PTC) proved to be vital not only for diagnostic purposes but also for tailored treatments. Despite the strong evidence of heritability, only a small subset of alterations has been implicated in PTC pathogenesis. To this reason, we used targeted next-generation sequencing (NGS) to identify candidate variants implicated in PTC pathogenesis, progression, and invasiveness., Methods: A total of 42 primary PTC tissues were investigated using a targeted next-generation sequencing (NGS) panel enlisting 47 genes involved in DNA repair and tumor progression., Results: We identified 57 point mutations in 78.5% of samples (n = 32). Thirty-two somatic mutations were identified exclusively in known thyroid cancer genes (BRAF, KRAS, NRAS, and TERT). Unpredictably, 45% of the all identified mutations (n = 25) resulted to be germline, most affecting DNA repair genes. Interestingly, none of the latter variants was in the main population databases. Following ACMG classification, 20% of pathogenic/likely pathogenic and 68% of variant of unknown significance were identified., Conclusions: Overall, our results support the hypothesis that rare germline variants in DNA repair genes are accountable for PTC susceptibility. More data, including the segregation analysis in affected families, should be collected before definitely annotate these alterations and to establish their potential prognostic and treatment implications., (© 2021. The Author(s).)

Cancer stem-like cells (CSCs) have self-renewal abilities responsible for cancer progression, therapy resistance, and metastatic growth. The glioblastoma stem-like cells are the most studied among CSC populations. A recent study identified four transcription factors (SOX2, SALL2, OLIG2, and POU3F2) as the minimal core sufficient to reprogram differentiated glioblastoma (GBM) cells into stem-like cells. Transcriptomic data of GBM tissues and cell lines from two different datasets were then analyzed by the SWItch Miner (SWIM), a network-based software, and FOSL1 was identified as a putative regulator of the previously identified minimal core. Herein, we selected NTERA-2 and HEK293T cells to perform an in vitro study to investigate the role of FOSL1 in the reprogramming mechanisms. We transfected the two cell lines with a constitutive FOSL1 cDNA plasmid. We demonstrated that FOSL1 directly regulates the four transcription factors binding their promoter regions, is involved in the deregulation of several stemness markers, and reduces the cells' ability to generate aggregates increasing the extracellular matrix component FN1. Although further experiments are necessary, our data suggest that FOSL1 reprograms the stemness by regulating the core of the four transcription factors., (© 2021. The Author(s).)

Purpose: In the search for novel effective compounds to use in thyroid cancer (TC) unresponsive to current treatment, attention has recently focused on plant-derived compounds with anticancer activity. In this review, we discuss the preclinical studies demonstrating phytochemical activity against thyroid cancer cells., Results/conclusions: In particular, we describe their antiproliferative properties or ability to re-induce iodine retention, thus supporting their potential use as single agents or adjuvants in radioiodine-resistant thyroid cancer treatment.

Angiopoietin-like proteins, namely ANGPTL3-4-8, are known as regulators of lipid metabolism. However, recent evidence points towards their involvement in the regulation of adipose tissue function. Alteration of adipose tissue functions (also called adiposopathy) is considered the main inducer of metabolic syndrome (MS) and its related complications. In this review, we intended to analyze available evidence derived from experimental and human investigations highlighting the contribution of ANGPTLs in the regulation of adipocyte metabolism, as well as their potential role in common cardiometabolic alterations associated with adiposopathy. We finally propose a model of ANGPTLs-based adipose tissue dysfunction, possibly linking abnormalities in the angiopoietins to the induction of adiposopathy and its related disorders.

Context: Approximately 60% of adults harbor 1 or more thyroid nodules. The possibility of cancer is the overriding concern, but only about 5% prove to be malignant. The widespread use of diagnostic imaging and improved access to health care favor the discovery of small, subclinical nodules and small papillary cancers. Overdiagnosis and overtreatment is associated with potentially excessive costs and nonnegligible morbidity for patients., Evidence Acquisition: We conducted a PubMed search for the recent English-language articles dealing with thyroid nodule management., Evidence Synthesis: The initial assessment includes an evaluation of clinical risk factors and sonographic examination of the neck. Sonographic risk-stratification systems (e.g., Thyroid Imaging Reporting and Data Systems) can be used to estimate the risk of malignancy and the need for biopsy based on nodule features and size. When cytology findings are indeterminate, molecular analysis of the aspirate may obviate the need for diagnostic surgery. Many nodules will not require biopsy. These nodules and those that are cytologically benign can be managed with long-term follow-up alone. If malignancy is suspected, options include surgery (increasingly less extensive), active surveillance or, in selected cases, minimally invasive techniques., Conclusion: Thyroid nodule evaluation is no longer a 1-size-fits-all proposition. For most nodules, the likelihood of malignancy can be confidently estimated without resorting to cytology or molecular testing, and low-frequency surveillance is sufficient for most patients. When there are multiple options for diagnosis and/or treatment, they should be discussed with patients as frankly as possible to identify an approach that best meets their needs., (© Endocrine Society 2020.)

Purpose: The identification of somatic mutations in cancer specimens enables detection of molecular markers for personalized treatment. We recently developed a novel molecular assay and evaluated its clinical performance as an ancillary molecular method for indeterminate thyroid nodule cytology. Herein we describe the analytical validation of the novel targeted next-generation sequencing (NGS) assay in thyroid samples from different sources., Methods: We present validation data of a novel NGS-based panel on 463 thyroid samples, including 310 fine-needle aspiration (FNA) specimens from different sources (FNA collected in preservative solution, liquid-based, and stained smears), 10 fresh frozen, and 143 formalin-fixed paraffin-embedded (FFPE) thyroid tissue specimens. Sequencing performance in the different samples was evaluated along with reproducibility, repeatability, minimum nucleic acid input to detect variants, and analytical sensitivity of the assay., Results: All thyroid samples achieved high sequencing performance, with a mean base coverage depth ranging from 2228 × (in liquid-based FNA) to 3661 × (in FNA stained smears), and coverage uniformity ranging from 86% (in FFPE) to 95% (in FNA collected in preservative solution), with all target regions covered above the minimum depth required to call a variant (500×). The minimum nucleic acid input was 1 ng. Analytic sensitivity for mutation detection was 2-5% mutant allele frequency., Conclusions: This validation study of a novel NGS-based assay for thyroid nodules demonstrated that the assay can be reliably used on multiple thyroid sample types, including FNA from different sources and FF and FFPE thyroid samples, thus providing a robust and reliable assay to genotype thyroid nodules, which may improve thyroid cancer diagnosis and care.

Hürthle cell carcinomas (HCC) are rare differentiated thyroid cancers that display low avidity for radioactive iodine and respond poorly to kinase inhibitors. Here, using next-generation sequencing, we analyzed the mutational status of primary tissue and poorly differentiated metastatic tissue from two HCC patients. In both cases, metastatic tissues harbored a mutation of SETD2, each resulting in loss of the SRI and WW domains of SETD2, a methyltransferase that trimethylates H3K36 (H3K36me3) and also interacts with p53 to promote its stability. Functional studies of the novel p.D1890fs6* mutation (case 1) revealed significantly reduced H3K36me3 levels in SETD2-mutated tissue and primary cell cultures and decreased levels of the active form of p53. Restoration of SETD2-wildtype expression in the SETD2-mutant cells significantly reduced the expression of four well-known stemness markers (OCT-4, SOX2, IPF1, Goosecoid). These findings suggest potential roles for SETD2 loss-of-function mutations in HCC progression, possibly involving p53 destabilization and promotion of stemness. Their prevalence and potential treatment implications in thyroid cancer, especially HCC, require further study.

Purpose: Deciding whether patients with a cytologically indeterminate thyroid nodule should be referred for surgery or for active surveillance is an important challenge for clinicians. The aim of this study was to evaluate the performance of a novel dual-component molecular assay as an ancillary molecular method for resolving indeterminate thyroid nodule cytology., Methods: We selected 156 thyroid nodules from those that had undergone fine-needle aspiration processed by liquid-based cytology and surgical resection between June 2016 and December 2017. The sample set included 63 nodules cytologically classified as indeterminate, and 93 other nodules randomly selected from those with non-diagnostic, benign, suspicious, or malignant cytology. Nucleic acids from each nodule were subjected to next-generation sequencing analysis for mutation detection in 23 genes and to digital polymerase chain reaction (PCR) evaluation for miR-146b-5p expression levels., Results: Used alone, mutation analysis in the indeterminate subset (cancer prevalence: 22.5%) displayed high sensitivity (89%) and NPV (96%). In contrast, the miR-146b-5p assay offered high specificity (93%) and PPV (93%). Combined use of both analyses improved panel performance by eliminating false-negative results., Conclusions: These preliminary data suggest that a dual-component molecular test can increase the diagnostic accuracy of thyroid cytology alone by reducing the number of nodules that will be classified as indeterminate and increasing those that can be reliably classified as benign. If these findings are confirmed, this test can be considered for use in clinical practice and is expected to reduce diagnostic surgery and health care costs, and to improve patient quality of life.

Purpose: Several studies have shown that different tumour types sharing a driver gene mutation do not respond uniformly to the same targeted agent. Our aim was to use an unbiased network-based approach to investigate this fundamental issue using BRAF V600E mutant tumours and the BRAF inhibitor vemurafenib., Methods: We applied SWIM, a software able to identify putative regulatory (switch) genes involved in drastic changes to the cell phenotype, to gene expression profiles of different BRAF V600E mutant cancers and their normal counterparts in order to identify the switch genes that could potentially explain the heterogeneity of these tumours' responses to vemurafenib., Results: We identified lung adenocarcinoma as the tumour with the highest number of switch genes (298) compared to its normal counterpart. By looking for switch genes encoding for kinases with homology sequences similar to known vemurafenib targets, we found that thyroid cancer and lung adenocarcinoma have a similar number of putative targetable switch gene kinases (5 and 6, respectively) whereas colorectal cancer has just one., Conclusions: We are persuaded that our network analysis may aid in the comprehension of molecular mechanisms underlying the different responses to vemurafenib in BRAF V600E mutant tumours.

Purpose: 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)., Methods: 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., Results: 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., Conclusions: These findings demonstrate that hTERT may represent an excellent therapeutic target in subgroups of aggressive PTCs.

Background: Obesity has been hypothesized to contribute to the aggressiveness of thyroid cancer through the production of abnormal levels of serum adipokines. Leptin receptor (OB-R) expression has also been documented in papillary thyroid cancer (PTC)., Aim: In this translational study, we analyzed in vitro the effects of leptin on the growth and migration of thyroid cancer cells (TPC-1 and K1), the molecular mechanisms underlying leptin's action, and the influence of prolonged leptin exposure on cell response to a protein kinase inhibitor lenvatinib. The expression levels of OB-R mRNA and protein were also investigated in vivo in a series of aggressive PTCs divided into two groups based on the presence of the BRAF mutation., Results: In TPC-1 and K1 cells, prolonged treatment with leptin (500 ng/ml for 96 h) resulted in a mild increase in the proliferation (about 20% over control only in K1 cells, p < 0.05) and in the migration of both cancer cell lines. Immunoblot analysis revealed a slight increase in the phosphorylation of AKT, but no effect on β -catenin and phospho-ERK expressions. The inhibitory effects of lenvatinib on the viability of both cell lines were not influenced by the leptin treatment . OB-R transcript (in fresh tissues) and proteins (in formalin-fixed and paraffin-embedded specimens) were expressed in all PTC tissues examined, with no significant differences between BRAF- mutated and BRAF -wild-type tumors., Conclusions: These results demonstrate leptin's role in mildly increasing the aggressive phenotype of PTC cells but without influencing the action of lenvatinib. Further studies will clarify whether it is possible to target OB-R, expressed in all aggressive PTCs, as an adjuvant treatment approach for these malignancies.

Synonymous mutations continue to be filtered out from most large-scale cancer genome studies, but several lines of evidence suggest they can play driver roles in neoplastic disease. We investigated a case of an aggressive, apparently sporadic medullary thyroid carcinoma (MTC) harboring a somatic RET p.Cys634Arg mutation (a known MTC driver). A germ-line RET substitution (p.Cys630=) had also been found but was considered clinically irrelevant because of its synonymous nature. Next generation sequencing (NGS) of the tumor tissues revealed that the RET mutations were in cis. There was no evidence of gene amplification. Expression analysis found an increase of RET transcript in p.Cys630=;p.Cys634Arg patient compared with that found in 7 MTCs harboring p.Cys634 mutations. Minigene expression assays demonstrated that the presence of the synonymous RET mutation was sufficient to explain the increased RET mRNA level. In silico analyses and RNA immunoprecipitation experiments showed that the p.Cys630 = variant created new exonic splicing enhancer motifs that enhanced SRp55 recruitment to the mutant allele, leading to more efficient maturation of its pre-mRNA and an increased abundance of mature mRNA encoding a constitutively active RET receptor. These findings document a novel mechanism by which synonymous mutations can contribute to cancer progression., Competing Interests: The authors have declared that no competing interests exist.

Whole exome sequencing (WES) was used to investigate two Italian siblings with wild-type RET genotype, who developed medullary thyroid cancers (MTCs) and, later, primary prostate and breast cancers, respectively. The proband's MTC harbored a p.Met918Thr RET mutation; his sister's MTC was RET/RAS wild-type. Both siblings had a germline mutation (p.Arg417Gln) in the extracellular Sema domain of the proto-oncogene MET. Experiments involving ectopic expression of MET p.Arg417Gln in MET-negative T47D breast cancer cells documented the mutant receptor's functionality and its ability to enhance cell migration and invasion. Our findings highlight a possible link between MET germline mutations and MTCs and suggest that MET p. Arg417Gln may promote an invasive malignant phenotype. The possibility that MTC can be driven/co-driven by a MET mutation has potential management implications, since the tyrosine-kinase inhibitor cabozantinib-approved for treating advanced MTCs-is a specific MET inhibitor., (© 2017 Wiley Periodicals, Inc.)