Stefania Bulotta

Different expression of TSH receptor and NIS genes in thyroid cancer: role of epigenetics

The TSH receptor (TSHR) and sodium/iodide symporter (NIS) are key players in radioiodine-based treatment of differentiated thyroid cancers. While NIS (SLC5AS) expression is diminished/lost in most thyroid tumors, TSHR is usually preserved. To examine the mechanisms that regulate the expression of NIS and TSHR genes in thyroid tumor cells, we analyzed their expression after inhibition of ras-BRAF-MAPK and PI3K-Akt-mTOR pathways and the epigenetic control occurring at the gene promoter level in four human thyroid cancer cell lines. Quantitative real-time PCR was used to measure NIS and TSHR mRNA in thyroid cancer cell lines (TPC-1, BCPAP, WRO, and FTC-133). Western blotting was used to assess the levels of total and phosphorylated ERK and Akt. Chromatin immunoprecipitation was performed for investigating histone post-translational modifications of the TSHR and NIS genes. ERK and Akt inhibitors elicited different responses of the cells in terms of TSHR and NIS mRNA levels. Akt inhibition increased NIS transcript levels and reduced those of TSHR in FTC-133 cells but had no significant effects in BCPAP. ERK inhibition increased the expression of both genes in BCPAP cells but had no effects in FTC-133. Histone post-translational modifications observed in the basal state of the four cell lines as well as in BCPAP treated with ERK inhibitor and FTC-133 treated with Akt inhibitor show cell- and gene-specific differences. In conclusion, our data indicate that in thyroid cancer cells the expression of TSHR and NIS genes is differently controlled by multiple mechanisms, including epigenetic events elicited by major signaling pathways involved in thyroid tumorigenesis.

Nucleophosmin Delocalization in Thyroid Tumour Cells

Nucleophosmin (NPM) is a multifunctional nucleolar protein that, depending on the context, can act as oncogene or tumour suppressor. Mutations of the NPM1 gene induce delocalization of NPM in acute myeloid leukaemia. Differently, in solid tumours, only NPM overexpression, but not delocalization, has been so far reported. Here, NPM localization in thyroid tumours was investigated. By using immunohistochemistry, we show increase of NPM cytoplasmic localization in follicular adenomas and papillary carcinomas compared to normal thyroid tissue (pu2009=u20090.0125 and <0.0001, respectively). NPM1 mutations commonly found in human leukaemia are not present in thyroid tumours. Immunofluorescence in cultured cell lines was utilized to discriminate between nucleolar and nuclear localization. We show that in thyroid cancer cell lines NPM localizes both in the nucleolus and in nucleus, while in non-tumorigenic thyroid cell lines localizes only in nucleolus. Either presence of the histone deacetylase inhibitor trichostatin A or absence of thyroid-stimulating hormone induces NPM nuclear localization in non-tumorigenic thyroid cell lines.

Fibronectin-1 expression is increased in aggressive thyroid cancer and favors the migration and invasion of cancer cells

In this study we analyzed the expression levels of markers of epithelial-to-mesenchymal transition (EMT) in several papillary thyroid carcinomas (PTCs) and the relation with tumor genotypes and clinicopathological characteristics. The role of fibronectin-1 (FN1) was investigated by analyzing the effects of FN1 silencing in two human thyroid cancer cell lines. Most of EMT markers were significantly over-expressed in a group of 36xa0PTCs. In particular, FN1 mRNA levels were higher in tumor vs non-tumor tissue (117.3, pxa0<xa00.001) and also in aggressive and BRAF(V600E) samples. Similar results were observed (and confirmed at the protein level) when FN1 expression was analyzed in a validation group of 50xa0PTCs and six lymph node (LN) metastases. Silencing of FN1 in TPC-1 and BCPAP thyroid cancer cells significantly reduced proliferation, adhesion, migration, and invasion in both cell lines. Collectively, our data indicate that FN1 overexpression is an important determinant of thyroid cancer aggressiveness.

Sodium iodide symporter (NIS) in extrathyroidal malignancies: focus on breast and urological cancer

BackgroundExpression and function of sodium iodide symporter (NIS) is requisite for efficient iodide transport in thyrocytes, and its presence in cancer cells allows the use of radioiodine as a diagnostic and therapeutic tool in thyroid neoplasia. Discovery of NIS expression in extrathyroidal tissues, including transformed cells, has opened a novel field of research regarding NIS-expressing extrathyroidal neoplasia. Indeed, expression of NIS may be used as a biomarker for diagnostic, prognostic, and therapeutic purposes. Moreover, stimulation of endogenous NIS expression may permit the radioiodine treatment of extrathyroidal lesions by concentrating this radioisotope.ResultsThis review describes recent findings in NIS research in extrathyroidal malignancies, focusing on breast and urological cancer, emphasizing the most relevant developments that may have clinical impact.ConclusionsGiven the recent progress in the study of NIS regulation as molecular basis for new therapeutic approaches in extrathyroidal cancers, particular attention is given to studies regarding the relationship between NIS and clinical-pathological aspects of the tumors and the regulation of NIS expression in the experimental models.

Emerging strategies for managing differentiated thyroid cancers refractory to radioiodine

AbstractnEfficient treatment of radio refractory thyroid cancer is still a major challenge. The recent identification of genetic and epigenetic alterations present in almost all differentiated tumors has revealed novel molecular targets, which can hopefully be exploited to create new treatments for these tumors. This review looks briefly at some of the innovative strategies currently being investigated for the treatment the radioiodine-resistant thyroid cancers.

Expression of adenylyl cyclase types III and VI in human hyperfunctioning thyroid nodules

Hyperfunctioning thyroid nodules are characterized by the presence of spontaneous somatic mutations responsible for constitutive activation of the cAMP pathway. However, alterations affecting other elements of the cAMP signaling system may counteract the effects of the mutations. In this study, the expression of the adenylyl cyclase (AC) types III and VI was investigated by Western blot in 18 hyperfunctioning thyroid nodules; in 12 samples, we also assessed the presence of TSH receptor (TSHR) or gsp mutations and levels of AC VI and III mRNA. We found that the expression of nodular AC VI (but not AC III) was significantly lower (85.1% of normal, P=0.014) than the expression of both adenylyl cycles types of perinodular tissue from the same patients. Slightly, but not significant differences were detected in nodules with or without mutations and AC protein levels generally showed correlation with the levels of the transcripts detected by RT-PCR. In addition, AC III and AC VI expression levels within a given nodule were characterized by a significant positive correlation. These findings indicate that a diminished expression of AC type VI may be part of the mechanisms occurring in the hyperfunctioning nodules, independently of the presence of TSHR or gsp mutations, which influence the resulting phenotype.

APE/Ref-1 is increased in nuclear fractions of human thyroid hyperfunctioning nodules.

Apurinic/apyrimidinic endonuclease APE/Ref-1 is a multifunctional protein provided with DNA repair, transcription-factor regulation and anti-apoptotic activities. We have previously reported that, in thyroid cells, TSH regulates both the synthesis and nuclear translocation of APE/Ref-1. We have also shown that nuclear levels of this protein are reduced both in thyroid carcinoma tissues and cell lines. In the present study, APE/Ref-1 expression and cellular localization were analysed by Western blot in hyperfunctioning thyroid nodules from patients with toxic adenoma and/or toxic multinodular goiter. The total content of APE/Ref-1 protein was increased in the majority of the hyperfunctioning tissues with respect to normal adjacent tissue. There was also an increase in the nuclear levels of APE/Ref-1, suggesting enhanced cytoplasm-to-nucleus translocation of the protein in addition to its increased rate of synthesis. These results demonstrate that the phenomenon of nuclear translocation of APE/Ref-1 hypothesized on the basis of cell culture experiments does actually occur in vivo. Together with previous observations in thyroid carcinomas and tumoral cell lines, our findings suggest a two-stage model of APE/Ref-1 behaviour during malignant thyrocyte transformation: an early stage characterized by simple hyperplasia and upregulation of APE/Ref-1 in the nuclear compartment of the cell and a later stage in which nuclear levels of the protein drop to below-normal levels as the cell becomes progressively undifferentiated.

Silencing of hTERT blocks growth and migration of anaplastic thyroid cancer cells

Mutations in the hTERT promoter responsible for constitutive telomerase activity are the most frequent genetic alteration detected in anaplastic thyroid cancer (ATC), and proposed as diagnostic and prognostic biomarker in these tumours. In this study we analyzed hTERT expression in a series of human ATCs and investigated the effects of small-interfering RNA-mediated silencing of hTERT on viability and migration and invasive properties of three human ATC cell lines. Expression of hTERT mRNA resulted increased in 8/10 ATCs compared to normal thyroid tissues. Silencing of hTERT in CAL-62, 8505C and SW1736xa0cells did not modify telomere length but determined a significant decrease (about 50%) of cell proliferation in all cell lines and a great reduction (about 50%) of migration and invasion capacity. These finding demonstrate that hTERT may be considered as a molecular target for ATC treatment.

Anti-hTERT siRNA-loaded nanoparticles block the growth of anaplastic thyroid cancer xenograft

The high frequency of hTERT-promoting mutations and the increased expression of hTERT mRNA in anaplastic thyroid cancer (ATC) make TERT a suitable molecular target for the treatment of this lethal neoplasm. In this study, we encapsulated an anti-hTERT oligonucleotide in biocompatible nanoparticles and analyzed the effects of this novel pharmaceutical preparation in preclinical models of ATC. Biocompatible nanoparticles were obtained in an acidified aqueous solution containing chitosan, anti-hTERT oligoRNAs, and poloxamer 188 as a stabilizer. The effects of these anti-hTERT nanoparticles (Na-siTERT) were tested in vitro on ATC cell lines (CAL-62 and 8505C) and in vivo on xenograft tumors obtained by flank injection of CAL-62 cells into SCID mice. The Na-siTERT reduced the viability and migration of CAL-62 and 8505C cells after 48-hour incubation. Intravenous administration (every 48 hours for 13 days) of this encapsulated drug in mice hosting a xenograft thyroid cancer determined a great reduction in the growth of the neoplasm (about 50% vs. untreated animals or mice receiving empty nanoparticles), and decreased levels of Ki67 associated with lower hTERT expression. Moreover, the treatment resulted in minimal invasion of nearby tissues and reduced the vascularity of the xenograft tumor. No signs of toxicity appeared following this treatment. Telomere length was not modified by the Na-siTERT, indicating that the inhibitory effects of neoplasm growth were independent from the enzymatic telomerase function. These findings demonstrate the potential suitability of this anti-TERT nanoparticle formulation as a novel tool for ATC treatment. Mol Cancer Ther; 17(6); 1187–95. ©2018 AACR.

Targeting post-translational histone modifications for the treatment of non-medullary thyroid cancer

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.