Eleonora Borbone

Down-Regulation of the miR-25 and miR-30d Contributes to the Development of Anaplastic Thyroid Carcinoma Targeting the Polycomb Protein EZH2

CONTEXT We have previously demonstrated that a set of micro-RNA (miRNA) is significantly down-regulated in anaplastic thyroid carcinomas with respect to normal thyroid tissues and to differentiated thyroid carcinomas. OBJECTIVE The objective was to evaluate the role of two of these down-regulated miRNA, miR-25 and miR-30d, in thyroid carcinogenesis. DESIGN miR-25 and miR-30d expression was restored in the ACT-1, 8505c, and FRO anaplastic thyroid cell lines, and their effects on cell proliferation, migration, and target expression were evaluated. RESULTS We report that miR-25 and miR-30d target the polycomb protein enhancer of zeste 2 (EZH2) that has oncogenic activity and is drastically up-regulated in anaplastic thyroid carcinomas but not in the differentiated ones. Ectopic expression of miR-25 and miR-30d inhibited proliferation and colony formation of anaplastic thyroid carcinoma cells by inducing G2/M-phase cell-cycle arrest. Finally, we found an inverse correlation between the expression of these miRNA and the EZH2 protein levels in anaplastic thyroid carcinomas, suggesting a critical role of these miRNA in regulating EZH2 expression also in vivo. CONCLUSION The down-regulation of miR-25 and miR-30d could contribute to the process of thyroid cancer progression, leading to the development of anaplastic carcinomas targeting EZH2 mRNA.

Oncogenic alterations in papillary thyroid cancers of young patients.

BACKGROUND Papillary thyroid carcinoma (PTC) in young people usually has an aggressive initial presentation, though a good general prognosis despite recurrences in 10%-20% of patients. A number of genetic alterations that activate the mitogen-activated protein kinase (MAPK) pathway have been found in PTC. Some of these alterations have been identified as prognostic factors of PTC in adults. The objective of the current study was to comprehensively characterize all known oncogenic alterations of the MAPK pathway in young people. METHODS One hundred three PTCs removed from 9 children, 19 adolescents, and 75 young adults were submitted to molecular analyses. RESULTS Altogether, 57 alterations were found in 56 PTCs (55%) corresponding to V600E BRAF in 20.3%, RAS mutations in 12.6%, RET/PTC 1 in 11.6%, RET/PTC 3 in 8.7%, and rearrangement of NTRK in 1.9%. The prevalence of all alterations increased with age (22.2% in children; 52.6% in adolescents, 51.4% in adults 20-25 years, and 55.1% in adults 25-35 years). Prevalence increased from 39.2% earlier to 61.3% after 20 years mainly due to BRAF mutations. Classic-type PTC was associated with a larger prevalence of alterations, predominantly BRAF and RET/PTC, whereas the follicular variant was chiefly associated with RAS. RET/PTC (1 and 3) was significantly associated with extrathyroid extension (ET) and lymph node metastasis (es) (LNM). This association was found in the adult group. There were no associations of BRAF or RAS mutations with ET or LNM. A 3-year median follow up was available for 90 patients. RET/PTC 1 and 3 was associated with short-term disease dissemination (cervical lymph node recurrences and distant metastases) in young adults (p=0.001). Persistent illness was more prevalent in patients with (15%) than in patients without (7%) genetic alterations. CONCLUSION PTCs in young patients display a low prevalence of the already identified oncogenic alterations. The increasing prevalence with age is mainly due to V600E BRAF mutation. There is no relation between tumor aggressiveness and BRAF mutation. There is a relation between the presence of RET/PTC (1 and 3) and the histological and clinical short-term aggressiveness of PTC in the population of young adults. Such a relation is not found in children and adolescents.

miR-191 Down-Regulation Plays a Role in Thyroid Follicular Tumors through CDK6 Targeting

CONTEXT Well-differentiated thyroid carcinomas include papillary (PTC) and follicular (FTC) carcinomas. FTC is usually a more aggressive form of cancer than the more common papillary type. miR-191 expression is frequently altered in several neoplasias, being up-regulated in some cases, such as pancreatic carcinomas, and down-regulated in other carcinomas, such as melanomas. OBJECTIVE The objective was to evaluate the expression and the role of miR-191 in thyroid carcinogenesis. DESIGN The expression of miR-191 was analyzed in tissues from patients with follicular adenoma (n = 24), FTC (n = 24), PTC (n = 15), anaplastic thyroid carcinoma (n = 8), and the follicular variant of PTC (n = 6) compared with normal thyroid tissues by quantitative RT-PCR. miR-191 expression was restored in the follicular thyroid cell line WRO, and the effects on cell proliferation, migration, and target expression were evaluated. RESULTS miR-191 is down-regulated in follicular adenoma, FTC, and follicular variant of PTC. We identified CDK6, a serine-threonine kinase involved in the control of cell cycle progression, as a novel target of miR-191. Restoration of miR-191 expression in WRO cells reduces cell growth and migration rate on vitronectin. CDK6 overexpression, correlated with miR-191 down-regulation, was found in follicular adenoma and FTC, suggesting a role of miR-191 down-regulation in the generation of these neoplasias. CONCLUSIONS Our results suggest that miR-191 down-regulation plays a role in thyroid neoplasias of the follicular histotype, likely by targeting CDK6.

Enhancer of zeste homolog 2 overexpression has a role in the development of anaplastic thyroid carcinomas.

CONTEXT Enhancer of zeste homolog 2 (EZH2) is a histone lysine methyltransferase belonging to the polycomb group protein family. Overexpression of EZH2 has been found in several human malignancies including hematological and solid tumors. OBJECTIVES In this study we investigated the expression levels of EZH2 and its polycomb group protein partners in thyroid carcinoma tissues with different degrees of malignancy to identify potential new therapeutic targets for anaplastic thyroid carcinoma (ATC). RESULTS We show that high EZH2 expression levels are characteristic of undifferentiated ATC, whereas no significant changes were observed in well-differentiated papillary and follicular thyroid carcinomas as compared with normal thyroid. Knockdown of EZH2 in ATC cell lines results in cell growth inhibition, loss of anchorage-independent growth, migration, and invasion properties. Moreover, we demonstrate that EZH2 directly controls differentiation of ATC cells by silencing the thyroid specific transcription factor paired-box gene 8 (PAX8). CONCLUSIONS EZH2 is specifically overexpressed in ATC, and it directly contributes to transcriptional silencing of PAX8 gene and ATC differentiation.

Let-7a down-regulation plays a role in thyroid neoplasias of follicular histotype affecting cell adhesion and migration through its ability to target the FXYD5 (Dysadherin) gene.

CONTEXT Thyroid neoplasias of the follicular histotype include the benign follicular adenomas and the malignant follicular carcinomas. Although several genetic lesions have already been described in human thyroid follicular neoplasias, the mechanisms underlying their development are still far from being completely elucidated. MicroRNAs (miRs or miRNAs) have recently emerged as important regulators of gene expression, also playing a key role in the process of carcinogenesis. OBJECTIVE The aim of our work has been to identify the miRNAs differentially expressed in human thyroid follicular neoplasias and define their role in thyroid carcinogenesis. DESIGN The miRNA expression profile of 10 human thyroid follicular adenomas was compared to that of 10 normal thyroid tissues. RESULTS The miRNA expression profiles revealed the down-regulation of let-7a in thyroid follicular adenomas compared to normal thyroid. Then, quantitative RT-PCR analyses validated the microarray data and showed a significantly higher decrease in let-7a expression in follicular carcinomas. Enforced let-7a expression in the follicular thyroid carcinoma cell line WRO induces an epithelial-like phenotype, increases cell adhesion, and decreases cell migration. Conversely, silencing of let-7a in the normal rat thyroid cell line PC Cl 3 has opposite effects. We identified dysadherin (FXYD5), a cell membrane glycoprotein, correlated with tumor progression and invasiveness, as a target of let-7a. Consistently, an inverse correlation between dysadherin and let-7a expression levels was found in human thyroid follicular adenomas and carcinomas. CONCLUSIONS These results suggest a role of let-7a down-regulation in the development of thyroid neoplasias of the follicular histotype, likely regulating dysadherin protein expression levels.

Role of PTPRJ genotype in papillary thyroid carcinoma risk

The strong genetic predisposition to papillary thyroid carcinoma (PTC) might be due to a combination of low-penetrance susceptibility variants. Thus, the research into gene variants involved in the increase of susceptibility to PTC is a relevant field of investigation. The gene coding for the receptor-type tyrosine phosphatase PTPRJ has been proposed as a cancer susceptibility gene, and its role as a tumor suppressor gene is well established in thyroid carcinogenesis. In this study, we want to ascertain the role of PTPRJ genotype in the risk for PTC. We performed a case-control study in which we determined the PTPRJ genotype for the non-synonymous Gln276Pro and Asp872Glu polymorphisms by PCR amplification and sequencing. We calculated allele and genotype frequencies for the considered polymorphisms of PTPRJ in a total sample of 299 cases (PTC patients) and 339 controls (healthy subjects) selected from Caucasian populations. We observed a significantly higher frequency of homozygotes for the Asp872 allele in the group of PTC patients than in the control group (odds ratio=1.61, 95% confidence interval 1.15-2.25, P=0.0053). We observed a non-significant increased frequency of homozygotes for Gln276Pro polymorphism in PTC cases in two distinct Caucasian populations. Therefore, the results reported here show that the homozygous genotype for Asp872 of PTPRJ is associated with an increased risk to develop PTC.

The impairment of the High Mobility Group A (HMGA) protein function contributes to the anticancer activity of trabectedin.

Trabectedin (Ecteinascidin-743 or ET-743) is a novel antitumour agent of marine origin with potent antitumour activity both in vitro and in vivo. It interacts with the minor groove of DNA, interfering with transcriptional activity and DNA repair pathways. Here, we report a novel mechanism by which trabectedin exerts its cytotoxic effects on carcinoma cells. It is based on its ability to impair the function of the High-Mobility Group A (HMGA) proteins. These proteins have a key role in cell transformation, and their overexpression is a common feature of human malignant neoplasias, representing a poor prognostic index often correlated to anti-cancer drug resistance. They bind the minor groove of DNA, alter chromatin structure and, thus, regulate the transcription of several genes by enhancing or suppressing the activity of transcription factors. We first report that trabectedin has a higher cytotoxic effect on thyroid and colon carcinoma cells expressing abundant levels of HMGAs in comparison with cells not expressing them. Then, we have shown that trabectedin treatment displaces HMGA proteins from the HMGA-responsive promoters, including ATM promoter, impairing their transcriptional activity. Finally, we report a synergism between Ionising Radiations and trabectedin treatment restricted to the HMGA-overexpressing cancer cells. This result might have important clinical implications since it would suggest the use of trabectedin for the treatment of neoplasias expressing abundant HMGA levels that are frequently associated to chemoresistance and poor prognosis.

Tumor suppressor role of the CL2/DRO1/CCDC80 gene in thyroid carcinogenesis.

CONTEXT Thyroid carcinoma is one of the most common malignancies of the endocrine system, and, despite the high frequency of oncogene activation in thyroid neoplastic lesions, the tumor suppressor genes involved in thyroid carcinogenesis remain unidentified. Our previous data implicated a link between the CL2/CCDC80 gene and thyroid cancer. OBJECTIVE The objective of the study was to examine the expression of the CL2/CCDC80 gene in human thyroid carcinomas in the attempt to determine whether it plays a role in thyroid carcinogenesis. DESIGN We evaluated the expression of CL2/CCDC80 in a large number of thyroid neoplastic tissue samples differing in degree of malignancy. We also investigated the effects of its restoration in 2 human thyroid carcinoma cell lines characterized by very low levels of CL2/CCDC80 expression. RESULTS CL2/CCDC80 expression was much lower in almost all the thyroid carcinomas analyzed than in normal thyroid tissues and was lowest in follicular variants of papillary carcinomas. Loss of heterozygosity partially accounted for CL2/CCDC80 down-regulation in thyroid carcinoma samples. Restoration of CL2/CCDC80 expression in the 2 human thyroid anaplastic carcinoma cell lines resulted in a higher susceptibility to apoptosis and suppression of the malignant phenotype. CL2/CCDC80 expression positively regulated the expression of E-cadherin, thereby halting cancer progression. CONCLUSIONS These results indicate that CL2/CCDC80 is a putative tumor suppressor gene in thyroid carcinogenesis.

HAND1 gene expression is negatively regulated by the High Mobility Group A1 proteins and is drastically reduced in human thyroid carcinomas

HMGA1 proteins exert their major physiological function during embryonic development and play a critical role in neoplastic transformation. Here, we show that Hand1 gene, which codes for a transcription factor crucial for differentiation of trophoblast giant cells and heart development, is upregulated in hmga1 minus embryonic stem cells. We demonstrate that HMGA1 proteins bind directly to Hand1 promoter both in vitro and in vivo and inhibit Hand1 promoter activity. We have also investigated HAND1 expression in human thyroid carcinoma cell lines and tissues, in which HMGA proteins are overexpressed, with respect to normal thyroid; an inverse correlation between HMGA1 and HAND1 expression was found in all thyroid tumor histotypes. A correlation between HAND1 gene repression and promoter hypermethylation was found in anaplastic carcinomas but not in other thyroid tumor histotypes. Therefore, we can hypothesize that HMGA1 overexpression plays a key role on HAND1 silencing in differentiated thyroid carcinomas and that promoter hypermethylation occurs in later stages of thyroid tumor progression. Finally, the restoration of the HAND1 gene expression reduces the clonogenic ability of two human thyroid carcinoma-derived cell lines, suggesting that HAND1 downregulation may have a role in the process of thyroid carcinogenesis.

Mycalol: A Natural Lipid with Promising Cytotoxic Properties against Human Anaplastic Thyroid Carcinoma Cells

The high-mobility group A (HMGA, types 1 and 2) proteins are low-molecular-weight nuclear factors that orchestrate the assembly of nucleoprotein complexes involved in gene transcription, replication, and chromatin structure. HMGAs possess oncogenic activity 2] and proteins of type 1 (HMGA1) have been correlated to cellular invasiveness and drug-resistance in human malignancies. In particular, blockage of expression of these proteins significantly enhances the responsiveness of tumor cell lines that are otherwise resistant to cytotoxic agents. Thus, phenotypic assays based on cells with reduced levels of HMGA are a possible tool for a rational search of novel compounds against tumors whose aggressiveness and resistance reduce the success of normal screening methods. Herein, we report the elucidation of the structure of mycalol (1), a novel polyoxygenated ether lipid that showed a promising in vitro specific activity against different cell lines derived from human anaplastic thyroid carcinoma (ATC), the most aggressive human thyroid gland malignancy. Mycalol was identified by a novel screening method based on the parallel use of FRO cells, which are human ATC-derived cells with high constitutive levels of HMAG1, but not HMGA2, and FRO-asHMGA1 cells, a genetically modified population of FRO cells that stably express an anti-HMGA1 antisense construct that blocks HMGA1 synthesis. The effects of extracts and fractions were measured on the paired cell lines by MTS proliferation assay. Mycalol was isolated from a chloroform extract of the sponge Mycale (Oxymycale) acerata Kirkpatrick 1907 collected along the coasts of Terra Nova Bay (Antarctica) during the Austral summer of 2005. The sponge, frozen soon after collection, was extracted with MeOH and fractionated according to a modified Kupchan method. The chloroform extract showed no activity against FRO cells up to 50 mgmL , but gave a good response (IC50 = 7.5 mgmL ) against HMGA1-silenced FRO cells (FRO-asHMGA), thus supporting the potential of a novel screening method based on HMGA-interference. Sequential steps of silica gel radial chromatography and reverse-phase HPLC (see the Supporting information) gave alkyl glyceryl ether 1 together with a number of minor compounds that are still under study.