Search

Supplementary Figure 1 from Chromobox Protein Homologue 7 Protein, with Decreased Expression in Human Carcinomas, Positively Regulates E-Cadherin Expression by Interacting with the Histone Deacetylase 2 Protein

Supplementary Figure 1 from Loss of the CBX7 Gene Expression Correlates with a Highly Malignant Phenotype in Thyroid Cancer

Supplementary Figure 2 from Chromobox Protein Homologue 7 Protein, with Decreased Expression in Human Carcinomas, Positively Regulates E-Cadherin Expression by Interacting with the Histone Deacetylase 2 Protein

Chromobox protein homologue 7 (CBX7) is a chromobox family protein encoding a novel polycomb protein, the expression of which shows a progressive reduction, well related with the malignant grade of the thyroid neoplasias. Indeed, CBX7 protein levels decreased in an increasing percentage of cases going from benign adenomas to papillary, follicular, and anaplastic thyroid carcinomas. To elucidate the function of CBX7 in carcinogenesis, we searched for CBX7 interacting proteins by a proteomic analysis. By this approach, we identified several proteins. Among these proteins, we selected histone deacetylase 2 (HDAC2), which is well known to play a key role in neoplastic cell transformation and down-regulation of E-cadherin expression, the loss of which is a critical event in the epithelial-to-mesenchymal transition. We confirmed by coimmunoprecipitation that CBX7 physically interacts with the HDAC2 protein and is able to inhibit its activity. Then, we showed that both these proteins bind the E-cadherin promoter and that CBX7 up-regulates E-cadherin expression. Consistent with these data, we found a positive statistical correlation between CBX7 and E-cadherin expression in human thyroid carcinomas. Finally, we showed that the expression of CBX7 increases the acetylation status of the histones H3 and H4 on the E-cadherin promoter. Therefore, the ability of CBX7 to positively regulate E-cadherin expression by interacting with HDAC2 and inhibiting its activity on the E-cadherin promoter would account for the correlation between the loss of CBX7 expression and a highly malignant phenotype. [Cancer Res 2009;69(17):7079–87]

Supplementary Figure 2 from Loss of the CBX7 Gene Expression Correlates with a Highly Malignant Phenotype in Thyroid Cancer

Supplementary Figure Legends 1-2 from Chromobox Protein Homologue 7 Protein, with Decreased Expression in Human Carcinomas, Positively Regulates E-Cadherin Expression by Interacting with the Histone Deacetylase 2 Protein

Supplementary Methods and Figure Legends 1-2 from Loss of the CBX7 Gene Expression Correlates with a Highly Malignant Phenotype in Thyroid Cancer

Supplementary Methods from Chromobox Protein Homologue 7 Protein, with Decreased Expression in Human Carcinomas, Positively Regulates E-Cadherin Expression by Interacting with the Histone Deacetylase 2 Protein

Supplementary Table 1 from Chromobox Protein Homologue 7 Protein, with Decreased Expression in Human Carcinomas, Positively Regulates E-Cadherin Expression by Interacting with the Histone Deacetylase 2 Protein

DNA damage response has been clearly described as an anti-cancer barrier in early human tumorigenesis. Moreover, interestingly, testicular germ cell tumors (TGCTs) have been reported to lack the DNA Damage Response (DDR) pathway activation. CCDC6 is a pro-apoptotic phosphoprotein substrate of the kinase ataxia telangectasia mutated (ATM) able to sustain DNA damage checkpoint in response to genotoxic stress and is commonly rearranged in malignancies upon fusion with different partners. In our study we sought to determine whether CCDC6 could have a role in the patho-genesis of testicular germ cell tumors. To achieve this aim, analysis for CCDC6 expression has been evaluated on serial sections of the mouse testis by immunohistochemistry and on separate populations of murine testicular cells by western blot. Next, the resistance to DNA damage-induced apoptosis and the production of reactive oxygen species has been investigated in GC1 cells, derived from immortalized type B murine germ cells, following CCDC6 silencing. Finally, the CCDC6 expression in normal human testicular cells, in Intratubular Germ Cell Neoplasia Unclassified (IGCNU), in a large series of male germ cell tumours and in the unique human seminoma TCam2 cell line has been evaluated by immunohistochemistry and by Western Blot analyses. The analysis of the CCDC6 expression revealed its presence in Sertoli cells and in spermatogonial cells. CCDC6 loss was the most consistent feature among the primary tumours and TCam2 cells. Interestingly, following treatment with low doses of H2O2, the silencing of CCDC6 in GC1 cells caused a decrease in the oxidized form of cytochrome c and low detection of Bad, PARP-1 and Caspase 3 proteins. Moreover, in the silenced cells, upon oxidative damage, the cell viability was protected, the γH2AX activation was impaired and the Reactive Oxygen Species (ROS) release was decreased. Therefore, our results suggest that the loss of CCDC6 could aid the spermatogonial cells to be part of a pro-survival pathway that helps to evade the toxic effects of endogenous oxidants and contributes to testicular neoplastic growth.

We have previously studied the function of microRNAs (miRNAs) in thyroid cells using the differentiated rat thyroid PC Cl 3 cells that need thyrotropin (TSH) for their growth. The miRNA expression profile examination allowed the detection of a set of miRNAs downregulated and upregulated by TSH. Here, we first demonstrated that upregulation of miR-130b-3p occurs through a protein kinase A-cAMP-responsive element binding protein (CREB)-dependent mechanism. Then, we analyzed its expression in human thyroid follicular adenomas, where a constitutive CREB activation is frequently present. miR-130b-3p results in upregulation with a high fold-change in most thyroid follicular adenomas. Then, we identified CCDC6, coding for a protein that interacts with CREB1 leading to the transcriptional repression of CREB1 target genes, as a target of this miRNA. The targeting of CCDC6 by miR-130b-3p likely accounts for the mechanism by which its upregulation contributes to the development of thyroid adenomas increasing CREB1 activity.

Context: Micro-RNA have emerged as an important class of short endogenous RNA that act as posttranscriptional regulators of gene expression and are constantly deregulated in human cancer. MiR-1 has been found down-regulated in lung, colon, and prostate cancer. Objectives: In this study, we investigated the possible role of miR-1 in thyroid carcinogenesis. Design: We have analyzed miR-1 expression in a panel of thyroid neoplasias including benign and malignant lesions and searched for miR-1 targets. Results: Our results show that miR-1 expression is drastically down-regulated in thyroid adenomas and carcinomas in comparison with normal thyroid tissue. Interestingly, miR-1 down-regulation was also found in thyroid hyperproliferative nonneoplastic lesions such as goiters. We identified the CCND2, coding for the cyclin D2 (CCND2) protein that favors the G1/S transition, CXCR4, and SDF-1± genes, coding for the receptor for the stromal cell derived factor-1 (SDF-1)/CXCL12 chemokine and its ligand SDF-1/CXCL12, respectively, as miR-1 targets. An inverse correlation was found between miR-1 expression and CXC chemokine receptor 4 (CXCR4) and SDF-1± protein levels in papillary and anaplastic thyroid carcinomas. Consistent with a role of the CCND2 protein in cell proliferation and CXCR4 and SDF-1± proteins in cell invasion and metastasis, functional studies demonstrate that miR-1 is able to inhibit thyroid carcinoma cell proliferation and migration. Conclusions: These results indicate the involvement of miR-1 in thyroid cell proliferation and migration, validating a role of miR-1 down-regulation in thyroid carcinogenesis.

Chromobox protein homologue 7 (CBX7) is a chromobox family protein encoding a novel polycomb protein, the expression of which shows a progressive reduction, well related with the malignant grade of the thyroid neoplasias. Indeed, CBX7 protein levels decreased in an increasing percentage of cases going from benign adenomas to papillary, follicular, and anaplastic thyroid carcinomas. To elucidate the function of CBX7 in carcinogenesis, we searched for CBX7 interacting proteins by a proteomic analysis. By this approach, we identified several proteins. Among these proteins, we selected histone deacetylase 2 (HDAC2), which is well known to play a key role in neoplastic cell transformation and down-regulation of E-cadherin expression, the loss of which is a critical event in the epithelial-to-mesenchymal transition. We confirmed by coimmunoprecipitation that CBX7 physically interacts with the HDAC2 protein and is able to inhibit its activity. Then, we showed that both these proteins bind the E-cadherin promoter and that CBX7 up-regulates E-cadherin expression. Consistent with these data, we found a positive statistical correlation between CBX7 and E-cadherin expression in human thyroid carcinomas. Finally, we showed that the expression of CBX7 increases the acetylation status of the histones H3 and H4 on the E-cadherin promoter. Therefore, the ability of CBX7 to positively regulate E-cadherin expression by interacting with HDAC2 and inhibiting its activity on the E-cadherin promoter would account for the correlation between the loss of CBX7 expression and a highly malignant phenotype. [Cancer Res 2009;69(17):7079–87]

PGE2plays a critical role in colorectal carcinogenesis. We have previously shown that COX-2 expression and PGE2synthesis are mediated by IGF-II/IGF-I receptor signaling in the Caco-2 cell line and that the pathway of phosphatidylinositol 3-kinase (PI3K)/Akt protects the cell from apoptosis. In the present study, we demonstrate that PGE2has the ability to increase Ras and PI3K association and decrease the level of apoptosis in the same experimental system. The effect of PGE2on PI3K/Ras association is dependent on the activation of EP4 receptor, the increase of cAMP levels, and the activation of PKA. In fact, treatment of cells with the PKA inhibitor H89 decreases the association of Ras and PI3K and Ras-associated PI3K activity. PKA inhibitor H89 is able to decrease threonine phosphorylation of Akt and to increase serine phosphorylation of Akt by p38 MAPK and counteracts the cytoprotective effect induced by PGE2. In addition, PGE2is able to activate p38 MAPK and the inhibition of p38 MAPK, with SB203580 specific inhibitor or with dominant negative MKK6 kinase, is able to revert the apoptotic effect of H89 and serine phosphorylation of Akt. The effect of PGE2on Caco-2 cell survival through PKA activation is mediated and regulated by the balance of threonine/serine phosphorylation of Akt by p38 kinase and PI3K. In conclusion, our data elucidate a novel mechanism for regulation of colon cancer cell survival and provide evidences for new combinatory treatments of colon cancer.

We have previously studied the function of microRNAs (miRNAs) in thyroid cells using the differentiated rat thyroid PC Cl 3 cells that need thyrotropin (TSH) for their growth. The miRNA expression profile examination allowed the detection of a set of miRNAs downregulated and upregulated by TSH. Here, we first demonstrated that upregulation of miR-130b-3p occurs through a protein kinase A-cAMP-responsive element binding protein (CREB)-dependent mechanism. Then, we analyzed its expression in human thyroid follicular adenomas, where a constitutive CREB activation is frequently present. miR-130b-3p results in upregulation with a high fold-change in most thyroid follicular adenomas. Then, we identified CCDC6, coding for a protein that interacts with CREB1 leading to the transcriptional repression of CREB1 target genes, as a target of this miRNA. The targeting of CCDC6 by miR-130b-3p likely accounts for the mechanism by which its upregulation contributes to the development of thyroid adenomas increasing CREB1 activity.

// Vincenza Leone 1 , Concetta Langella 1 , Francesco Esposito 1 , Claudio Arra 2 , Giuseppe Palma 2 , Domenica Rea 2 , Orlando Paciello 3 , Francesco Merolla 1 , Davide De Biase 3 , Serenella Papparella 3 , Angela Celetti 1 , Alfredo Fusco 1, 4 1 Istituto per l’Endocrinologia ed Oncologia Sperimentale del CNR e/o Dipartimento di Medicina Molecolare e Biotecnologie Mediche, Universita degli Studi di Napoli “Federico II”, Naples, Italy 2 Istituto Nazionale per lo Studio e la Cura dei Tumori “Fondazione Giovanni Pascale”, IRCCS, Naples, Italy 3 Department of Pathology and Animal Health, University of Naples “Federico II”, Naples, Italy 4 Instituto Nacional de Câncer - INCA, Rua Andre Cavalcanti, Rio de Janeiro, CEP RJ, Brazil Correspondence to: Alfredo Fusco, e-mail: alfusco@unina.it Keywords: Ccdc6, thyroid, CREB1, knock-in mice Received: February 3, 2015 Accepted: April 14, 2015 Published: April 27, 2015 ABSTRACT CCDC6 was originally identified upon rearrangement with RET in human thyroid papillary carcinomas generating the RET/PTC1 oncogene. We have previously reported that CCDC6 interacts with CREB1 and represses its transcriptional activity. Since the function of at least one allele of CCDC6 is lost following RET/PTC1 rearrangements, we aimed at the generation of mice, carrying a CCDC6 mutant gene. Previous studies suggested that the coiled-coil domain of CCDC6, mainly encoded by human exon 2, is required for the protein function. Therefore, we engineered a murine Ccdc6 construct, carrying a deletion of the exon 2, that was able to exert only a mild repression on CREB1 transcriptional activity, with respect to the wild type Ccdc6. Subsequently, we generated Ccdc6 -ex2 knock-in mice. These mice developed thyroid hyperplasia associated with an enhanced CREB1 activity and an increased expression of the CREB-1 regulated genes. These results strongly support a CCDC6 promoting role, ascribed to its functional impairment, in the development of thyroid papillary carcinomas harboring the RET/PTC1 oncogene.

We have previously reported that the expression of the CL2/CCDC80 gene is downregulated in human papillary thyroid carcinomas, particularly in follicular variants. We have also reported that the restoration of CL2/CCDC80 expression reverted the malignant phenotype of thyroid carcinoma cell lines and that CL2/CCDC80 positively regulated E-cadherin expression, an ability that likely accounts for the role of the CL2/CCDC80 gene in thyroid cancer progression. In order to validate the tumour suppressor role of the CL2/CCDC80 gene in thyroid carcinogenesis we generated cl2/ccdc80 knock-out mice. We found that embryonic fibroblasts from cl2/ccdc80 −/− mice showed higher proliferation rate and lower susceptibility to apoptosis. Furthermore, cl2/ccdc80 −/− mice developed thyroid adenomas and ovarian carcinomas. Finally, ret/PTC1 transgenic mice crossed with the cl2/ccdc80 knock-out mice developed more aggressive thyroid carcinomas compared with those observed in the single ret/PTC1 transgenic mice. Together, these results indicate CL2/CCDC80 as a putative tumour suppressor gene in human thyroid carcinogenesis.

Aspirin displays, at millimolar concentrations, several mechanisms independent from its ability to inhibit cyclooxygenases. Occasionally, the mechanisms displayed in vitro have been clearly related to an effect of clinical relevance in vivo. An expanding literature has been focusing on the cytoprotective effect of aspirin in neurodegenerative disorders and the activation of AKT pathway in neuroprotection and induction of resistance to anticancer drugs. In this work, we tested the ability of aspirin to activate the AKT survival pathway in methylcholanthrene-induced fibrosarcoma cells (Meth A) transplanted into BALB/c nude mice and the clinical effect of aspirin cotreatment during etoposide (VP-16)-based anticancer therapy. We found that cotreatment with aspirin reduced VP-16-induced apoptosis and activated AKT in vitro and in vivo. In Meth A-bearing mice, aspirin administration also activated glycogen synthase kinase-3 and reduced the activity and the efficacy of anticancer therapy in VP-16 cotreated animals. Our data suggest that the antiapoptotic effect of aspirin operates in vivo through the activation of AKT-glycogen synthase kinase pathway causing a decrease in the outcome of VP-16-based therapy. These findings could have clinical relevance in treatment of human malignancies.

Paula Mussnich, Daniela D’Angelo, Vincenza Leone, Carlo Maria Croce, Alfredo Fusco* Istituto di Endocrinologia ed Oncologia Sperimentale del CNR, c/o Dipartimento di Biologia e Patologia Cellulare e Molecolare, Facolt a di Medicina e Chirurgia di Napoli, Universit a degli Studi di Napoli “Federico II”, via Pansini 5, 80131 Naples, Italy CAPES Foundation, Ministry of Education of Brazil, Bras ilia DF 70040-020, Brazil Department of Molecular Virology, Immunology, and Medical Genetics, Comprehensive Cancer Center, Ohio State University, 460 West 12th Avenue, Columbus, OH 43210, USA

MicroRNA (miRNA) deregulation plays a critical role in tumorigenesis. miR-23b and miR-130b are induced by thyrotropin in thyroid cells in a cAMP-dependent manner. The aim of our work has been to investigate the possible role of miR-23b and miR-130b in pituitary tumorigenesis. We have analyzed their expression in a panel of pituitary adenomas (PAs) including GH and NFPA adenomas. We report that miR-23b and miR-130b are drastically reduced in GH, gonadotroph and NFPA adenomas in comparison with normal pituitary gland. Interestingly, the overexpression of miR-23b and miR-130b inhibits cell proliferation arresting the cells in the G1 and G2 phase of the cell cycle, respectively. Moreover, we demonstrate that miR-23b and miR-130b target HMGA2 and cyclin A2 (CCNA2) genes, respectively. Finally, downregulation of miR-23b and miR-130b expression is associated with increased levels of their respective targets in human PAs. These findings suggest that miR-23b and miR-130b downregulation may contribute to pituitary tumorigenesis.

Background DNA damage response has been clearly described as an anti-cancer barrier in early human tumorigenesis. Moreover, interestingly, testicular germ cell tumors (TGCTs) have been reported to lack the DNA Damage Response (DDR) pathway activation. CCDC6 is a pro-apoptotic phosphoprotein substrate of the kinase ataxia telangectasia mutated (ATM) able to sustain DNA damage checkpoint in response to genotoxic stress and is commonly rearranged in malignancies upon fusion with different partners. In our study we sought to determine whether CCDC6 could have a role in the patho-genesis of testicular germ cell tumors. Methods To achieve this aim, analysis for CCDC6 expression has been evaluated on serial sections of the mouse testis by immunohistochemistry and on separate populations of murine testicular cells by western blot. Next, the resistance to DNA damage-induced apoptosis and the production of reactive oxygen species has been investigated in GC1 cells, derived from immortalized type B murine germ cells, following CCDC6 silencing. Finally, the CCDC6 expression in normal human testicular cells, in Intratubular Germ Cell Neoplasia Unclassified (IGCNU), in a large series of male germ cell tumours and in the unique human seminoma TCam2 cell line has been evaluated by immunohistochemistry and by Western Blot analyses. Results The analysis of the CCDC6 expression revealed its presence in Sertoli cells and in spermatogonial cells. CCDC6 loss was the most consistent feature among the primary tumours and TCam2 cells. Interestingly, following treatment with low doses of H2O2, the silencing of CCDC6 in GC1 cells caused a decrease in the oxidized form of cytochrome c and low detection of Bad, PARP-1 and Caspase 3 proteins. Moreover, in the silenced cells, upon oxidative damage, the cell viability was protected, the γH2AX activation was impaired and the Reactive Oxygen Species (ROS) release was decreased. Conclusions Therefore, our results suggest that the loss of CCDC6 could aid the spermatogonial cells to be part of a pro-survival pathway that helps to evade the toxic effects of endogenous oxidants and contributes to testicular neoplastic growth.

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.

The overexpression of the HMGA1 proteins is a feature of human malignant neoplasias and has a causal role in cell transformation. The aim of our study has been to investigate the microRNAs (miRNAs or miRs) regulated by the HMGA1 proteins in the process of cell transformation analyzing the miRNA expression profile of v‐ras‐Ki oncogene‐transformed thyroid cells expressing or not HMGA1 proteins. We demonstrate that, among the miRNAs regulated by cell transformation, there are miR‐10b, miR‐21, miR‐125b, miR‐221 and miR‐222 that are positively and miR‐34a and miR‐603 that are negatively regulated by HMGA1 expression. Then, we focused our attention on the miR‐10b and miR‐603 whose expression was dependent on the presence of HMGA1 also in other cell systems. We found that miR‐10b is able to target the PTEN gene, whereas miR‐603 targets the CCND1 and CCND2 genes coding for the cyclin D1 and cyclin D2 proteins, respectively. Moreover, functional studies showed that miR‐10b and miR‐603 regulate positively and negatively, respectively, cell proliferation and migration suggesting a role of their dysregulation in thyroid cell transformation.

CONTEXT: MicroRNA (miRNA or miR) have emerged as an important class of short endogenous RNA that act as post-transcriptional regulators of gene expression and have a critical role in cell proliferation and differentiation. OBJECTIVES: The aim of this study was to elucidate the role of miRNA in the proliferation of differentiated thyroid cells that require TSH for their growth. DESIGN: To elucidate the role of miRNA in thyroid cell proliferation, we have analyzed the miRNA expression profile of PC Cl 3 cells before and after the stimulation by TSH. RESULTS: We report the identification of two specific miRNA (miR-23b and miR-29b) whose up-regulation by TSH is required for thyroid cell growth. We identified mothers against decapentaplegic homolog 3 (Smad3), a member of the TGF-? pathway that has an inhibitor role in thyroid follicular cell proliferation as a target of miR-23b and miR-29b. Functional studies demonstrated that the overexpression of miR-23b and miR-29b promotes thyroid cell growth. Interestingly, an increased expression of both these miRNA was also detected in experimental and human goiters. CONCLUSIONS: These findings support the idea that the regulation of miRNA expression synergizes with the traditional proliferation pathways in promoting cell growth.

Associazione Italiana Ricerca sul Cancro MicroRNA (miRNA or miR) are an important class of regulators that participate in such biological functions as development, cell proliferation, differentiation, and apoptosis. the aim of this study was to elucidate the role of miRNA in cell proliferation using a unique cell system, namely thyroid cells that require thyrotropin for their growth. Here, we report the identification of a set of five specific miRNA (miR-1, miR-28-A, miR-290-5p, miR-296-3p, and miR-297a), whose down-regulation by thyrotropin is required for thyroid cell growth. in fact, overexpression of these miRNA negatively affects cell growth. We show that three of these miRNA target cAMP-responsive element binding protein (CREB)1, a thyrotropin-activated transcription factor, and that CREB1 binds the regulatory regions of the down-regulated miRNA. Hence, these data indicate that a synergistic loop involving thyrotropin, CREB1, and miRNA is required for thyroid cell proliferation. (Molecular Endocrinology 25: 1819-1830, 2011) Univ Naples Federico 2, Ist Endocrinol & Oncol Sperimentale, CNR, Dipartimento Biol & Patol Cellulare & Mol,Fac Med, I-80131 Naples, Italy CEINGE Ctr Ingn Genet Biotecnol Avanzate, Naples Oncogenom Ctr, Naples, Italy European Sch Mol Med, I-80145 Naples, Italy Universidade Federal de São Paulo, Dept Biol Sci, BR-05403 São Paulo, Brazil Ohio State Univ, Ctr Comprehens Canc, Dept Mol Virol, Columbus, OH 43210 USA Universidade Federal de São Paulo, Dept Biol Sci, BR-05403 São Paulo, Brazil Web of Science

RET/papillary thyroid carcinoma 1 (PTC1) oncogene is frequently activated in human PTCs. It is characterized by the fusion of the intracellular kinase-encoding domain of RET to the first 101 amino acids of CCDC6. The aim of our work is to characterize the function of the CCDC6 protein to better understand the function of its truncation, that results in the loss of the expression of one allele, in the process of thyroid carcinogenesis. Here, we report that CCDC6 interacts with CREB1 and represses its transcriptional activity by recruiting histone deacetylase 1 and protein phosphatase 1 proteins at the CRE site of the CREB1 target genes. Finally, we show an increased CREB1 phosphorylation and activity in PTCs carrying the RET/PTC1 oncogene. Consistently, an increased expression of two known CREB1 target genes, AREG and cyclin A, was observed in this subgroup of thyroid papillary carcinomas. Therefore, the repression of CREB1 activity by CCDC6 has a critical function in the development of human thyroid papillary carcinomas carrying RET/PTC1 activation. © 2010 Macmillan Publishers Limited All rights reserved.

Using gene expression profiling, we found that the CBX7 gene was drastically down-regulated in six thyroid carcinoma cell lines versus control cells. The aims of this study were to determine whether CBX7 is related to the thyroid cancer phenotype and to try to identify new tools for the diagnosis and prognosis of thyroid cancer. We thus evaluated CBX7 expression in various snap-frozen and paraffin-embedded thyroid carcinoma tissues of different degrees of malignancy by quantitative reverse transcription-PCR and immunohistochemistry, respectively. CBX7 expression progressively decreased with malignancy grade and neoplasia stage. Indeed, it decreased in an increasing percentage of cases going from benign adenomas to papillary (PTC), follicular, and anaplastic (ATC) thyroid carcinomas. This finding coincides with results obtained in rat and mouse models of thyroid carcinogenesis. CBX7 loss of heterozygosity occurred in 36.8% of PTC and in 68.7% of ATC. Restoration of CBX7 expression in thyroid cancer cells reduced growth rate, with a retention in the G1 phase of the cell cycle, suggesting that CBX7 can contribute to the proliferation of the transformed thyroid cells. In conclusion, loss of CBX7 expression correlates with a highly malignant phenotype in thyroid cancer patients. [Cancer Res 2008;68(16):6770–8]

We have recently reported that MicroRNAs (miR)-221 and miR-222 were up-regulated in human thyroid papillary carcinomas in comparison with the normal thyroid tissue. Bioinformatic analysis proposed the p27Kip1 protein, a key regulator of cell cycle, as a candidate target for the miR-221/222 cluster. Here, we report that the enforced expression of miR-221 and miR-222 was able to reduce p27Kip1 protein levels in thyroid carcinoma and HeLa cells in the absence of significant changes in specific p27Kip1 mRNA levels. This effect is direct as miR-221 and miR-222 negatively regulate the expression of the 3′-untranslated region-based reporter construct from the p27Kip1 gene, and is dependent on two target sites in this region. Consistent with these results, an enforced expression of the miR-221 and miR-222 induced the thyroid papillary carcinoma cell line (TPC-1) to progress to the S phase of the cell cycle. It is likely that the negative regulation of p27Kip1 by miR-221 and miR-222 might also have a role in vivo since we report an inverse correlation between miR-221 and miR-222 up-regulation and down-regulation of the p27Kip1 protein levels in human thyroid papillary carcinomas. Therefore, the data reported here demonstrate that miR-221 and miR-222 are endogenous regulators of p27Kip1 protein expression, and thereby, the cell cycle.

Thyroid carcinomas comprise a broad spectrum of tumors with different clinical behaviors. On the one side, there are occult papillary carcinomas (PTC), slow growing and clinically silent, and on the other side, rapidly growing anaplastic carcinomas (ATC), which are among the most lethal human neoplasms. We have analysed the microRNA (miR) profile of ATC in comparison to the normal thyroid using a microarray (miRNACHIP microarray). By this approach, we found an aberrant miR expression profile that clearly differentiates ATC from normal thyroid tissues and from PTC analysed in previous studies. In particular, a significant decrease in miR-30d, miR-125b, miR-26a and miR-30a-5p was detected in ATC in comparison to normal thyroid tissue. These results were further confirmed by northern blots, quantitative reverse transcription-PCR analyses and in situ hybridization. The overexpression of these four miRs in two human ATC-derived cell lines suggests a critical role of miR-125b and miR-26a downregulation in thyroid carcinogenesis, since a cell growth inhibition was achieved. Conversely, no effect on cell growth was observed after the overexpression of miR-30d and miR-30a-5p in the same cells. In conclusion, these data indicate a miR signature associated with ATC and suggest the miR deregulation as an important event in thyroid cell transformation. © 2007 Nature Publishing Group All rights reserved.

Aspirin displays, at millimolar concentrations, several mechanisms independent from its ability to inhibit cyclooxygenases. Occasionally, the mechanisms displayed in vitro have been clearly related to an effect of clinical relevance in vivo. An expanding literature has been focusing on the cytoprotective effect of aspirin in neurodegenerative disorders and the activation of AKT pathway in neuroprotection and induction of resistance to anticancer drugs. In this work, we tested the ability of aspirin to activate the AKT survival pathway in methylcholanthrene-induced fibrosarcoma cells (Meth A) transplanted into BALB/c nude mice and the clinical effect of aspirin cotreatment during etoposide (VP-16)–based anticancer therapy. We found that cotreatment with aspirin reduced VP-16-induced apoptosis and activated AKT in vitro and in vivo. In Meth A–bearing mice, aspirin administration also activated glycogen synthase kinase-3 and reduced the activity and the efficacy of anticancer therapy in VP-16 cotreated animals. Our data suggest that the antiapoptotic effect of aspirin operates in vivo through the activation of AKT-glycogen synthase kinase pathway causing a decrease in the outcome of VP-16-based therapy. These findings could have clinical relevance in treatment of human malignancies. [Mol Cancer Ther 2006;5(5):1318–24]

MiR-1 downregulation plays a critical role in thyroid cell proliferation V. Leone, D. D'Angelo, PL. Pallante, A. Ferraro. Istituto di Endocrinologia ed Oncologia Sperimentale del CNR c/o Dipartimento di Biologia e Patologia Cellulare e Molecolare, Facoltà di Medicina e Chirurgia di Napoli, Università degli Studi di Napoli “Federico II”, Naples, Italy. MicroRNAs have emerged as an important class of short endogenous RNAs that act as post-transcriptional regulators of gene expression, and their expression is constantly deregulated in human cancer. MiR-1 has been found downregulated in lung, colon and prostate cancer. The aim of our work has been to investigate the possible role of miR-1 in thyroid carcinogenesis. Therefore, we have analysed miR-1 expression in a panel of thyroid neoplasias including benign and malignant lesions. Our results show that miR-1 expression is drastically downregulated in thyroid adenomas and carcinomas in comparison with normal thyroid tissue. Interestingly, miR-1 downregulation was also found in thyroid hyperproliferative non neoplastic lesions such as goiters. These results suggested a general role of miR-1 downregulation in the control of thyroid cell proliferation. This suggestion was then confirmed by the drastic decrease in miR-1 expression following exposure of rat thyroid cells to thyrotropin (TSH), the physiological thyroid stimulator. Searching for miR-1 targets, we identified CREB1 transcriptional factor, that is the main effector of the TSH-induced thyroid cell growth, as miR-1 target, suggesting the presence of a regulatory synergistic loop including TSH, CREB1 and the miR-1. Moreover, we have demonstrated that also the CXCR4 protein and its ligand SDF1α, that are involved in cell migration and are constantly overexpressed in human thyroid carcinoma, are targets of miR-1. Functional studies demonstrate that miR-1 is able to inhibit thyroid carcinoma cell proliferation and migration validating a critical role of miR-1 downregulation in thyroid carcinogenesis. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 102nd Annual Meeting of the American Association for Cancer Research; 2011 Apr 2-6; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2011;71(8 Suppl):Abstract nr 119. doi:10.1158/1538-7445.AM2011-119

CBX7, chromobox homolog 7, is a chromobox family protein encoding a novel Polycomb protein, component of the Polycomb repressive complex 1 (PRC1). The Polycomb group (PcG) proteins are epigenetic transcriptional repressors involved in the control of cellular proliferation and oncogenesis. CBX7 protein levels show a progressive reduction, well related with the malignant grade of the thyroid neoplasias. Indeed, its expression decreased in an increasing percentage of cases going from benign adenomas to papillary, follicular and anaplastic thyroid carcinomas. To elucidate the function of CBX7 in carcinogenesis, we searched for CBX7 interacting proteins by a proteomic analysis. By this approach, we identified several proteins among which we selected Histone Deacetylase 2 (HDAC2) that is well known to play a key role in neoplastic cell transformation and to downregulate E-cadherin expression, whose loss is a critical event in the Epithelial-Mesenchymal Transition, and therefore emerging as one of the caretakers of the epithelial phenotype. We confirmed by co-immunoprecipitation that CBX7 physically interacts with the HDAC2 and demostrated that is able to inhibit its activity. Then, we showed that both these proteins bind the E-cadherin promoter, and that CBX7 is able to upregulate E-cadherin expression. Consistent with these data we found a positive statistical correlation between CBX7 and E-cadherin expression in human thyroid carcinomas. Finally, we demonstrated that the expression of CBX7 increases the acetylation status of the histones H3 and H4 on the E-cadherin promoter. Therefore, the ability of CBX7 to positively regulate E-cadherin expression, by interacting with HDAC2 and inhibiting its activity on the E-cadherin promoter, would account for the correlation between the loss of CBX7 expression and a highly malignant phenotype in thyroid cancer patients. Thus, several important interacting proteins of CBX7 and the pathways in which they are involved strongly suggest that CBX7 can be considerated a very important regulator of thyroid malignant transformation.