Antonella Federico

MiR-1 Is a Tumor Suppressor in Thyroid Carcinogenesis Targeting CCND2, CXCR4, and SDF-1α

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-1α 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-1α protein levels in papillary and anaplastic thyroid carcinomas. Consistent with a role of the CCND2 protein in cell proliferation and CXCR4 and SDF-1α 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.

Loss of the CBX7 Gene Expression Correlates with a Highly Malignant Phenotype in Thyroid Cancer

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 G(1) 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.

CBX7 is a tumor suppressor in mice and humans

The CBX7 gene encodes a polycomb group protein that is known to be downregulated in many types of human cancers, although the role of this protein in carcinogenesis remains unclear. To shed light on this issue, we generated mice null for Cbx7. Mouse embryonic fibroblasts derived from these mice had a higher growth rate and reduced susceptibility to senescence compared with their WT counterparts. This was associated with upregulated expression of multiple cell cycle components, including cyclin E, which is known to play a key role in lung carcinogenesis in humans. Adult Cbx7-KO mice developed liver and lung adenomas and carcinomas. In in vivo and in vitro experiments, we demonstrated that CBX7 bound to the CCNE1 promoter in a complex that included HDAC2 and negatively regulated CCNE1 expression. Finally, we found that the lack of CBX7 protein expression in human lung carcinomas correlated with CCNE1 overexpression. These data suggest that CBX7 is a tumor suppressor and that its loss plays a key role in the pathogenesis of cancer.

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.

Loss of the CBX7 protein expression correlates with a more aggressive phenotype in pancreatic cancer.

Polycomb group (PcG) proteins function as multiprotein complexes and are part of a gene regulatory mechanism that determines cell fate during normal and pathogenic development. Several studies have implicated the deregulation of different PcG proteins in neoplastic progression. Pancreatic ductal adenocarcinoma is an aggressive neoplasm that follows a multistep model of progression through precursor lesions called pancreatic intraepithelial neoplasia (PanIN). Aim of this study was to investigate the role of PcG protein CBX7 in pancreatic carcinogenesis and to evaluate its possible diagnostic and prognostic significance. We analysed by immunohistochemistry the expression of CBX7 in 210 ductal pancreatic adenocarcinomas from resection specimens, combined on a tissue microarray (TMA) including additional 40 PanIN cases and 40 normal controls. The results were evaluated by using receiver operating characteristic (ROC) curve analysis for the selection of cut-off scores and correlated to the clinicopathological parameters of the tumours and the outcome of the patients. Expression of E-cadherin, a protein positively regulated by CBX7, was also assessed. A significantly differential, and progressively decreasing CBX7 protein expression was found between normal pancreatic tissue, PanINs and invasive ductal adenocarcinoma. Loss of CBX7 expression was associated with increasing malignancy grade in pancreatic adenocarcinoma, whereas the maintenance of CBX7 expression showed a trend toward a longer survival. Moreover, loss of E-cadherin expression was associated with loss of CBX7 and with a trend towards worse patient survival. These results suggest that CBX7 plays a role in pancreatic carcinogenesis and that its loss of expression correlates to a more aggressive phenotype.

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.

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.

Hmga1/Hmga2 double knock-out mice display a “superpygmy” phenotype

ABSTRACT The HMGA1 and HMGA2 genes code for proteins belonging to the High Mobility Group A family. Several genes are negatively or positively regulated by both these proteins, but a number of genes are specifically regulated by only one of them. Indeed, knock-out of the Hmga1 and Hmga2 genes leads to different phenotypes: cardiac hypertrophy and type 2 diabetes in the former case, and a large reduction in body size and amount of fat tissue in the latter case. Therefore, to better elucidate the functions of the Hmga genes, we crossed Hmga1-null mice with mice null for Hmga2. The Hmga1−/−/Hmga2−/− mice showed reduced vitality and a very small size (75% smaller than the wild-type mice); they were even smaller than pygmy Hmga2-null mice. The drastic reduction in E2F1 activity, and consequently in the expression of the E2F-dependent genes involved in cell cycle regulation, likely accounts for some phenotypic features of the Hmga1−/−/Hmga2−/− mice.

miR-142–3p Down-Regulation Contributes to Thyroid Follicular Tumorigenesis by Targeting ASH1L and MLL1

CONTEXT A previous micro-RNA expression profile of thyroid follicular adenomas identified miR-142 precursor among the miRNAs downregulated in the neoplastic tissues compared to normal thyroid gland. OBJECTIVE The aim of this work has been to assess the expression of miR-142-3p in a large panel of follicular thyroid adenomas and carcinomas and evaluate its effect on thyroid cell proliferation and target expression. DESIGN The expression of miR-142-3p was analyzed by qRT-PCR in thyroid follicular adenomas and carcinomas, compared to normal thyroids. MiR-142-3p expression was restored in WRO cells and the effects on cell proliferation and target expression were evaluated. RESULTS Here we show that miR-142-3p is downregulated in FTAs, FTCs, and FVPTCs. MiR-142-3p was demonstrated to reduce the proliferation rate of WRO and FTC133 cells, supporting its tumor suppressor role in thyroid cancerogenesis. Moreover, this microRNA was able to downregulate the expression of ASH1L and MLL1, by direct and indirect mechanisms, respectively. Consistently, an inverse correlation between miR-142-3p expression and ASH1L and MLL1 proteins was found in thyroid follicular adenomas and carcinomas. ASH1L and MLL1, which belong to the Trithorax group (TrxG) proteins and are major regulators of Homeobox gene expression, maintain active target gene transcription by histone 3 lysine 4 methylation. Interestingly, we found that FTCs and FTC cell lines express tumor specific, shorter forms of the two proteins. The capability of miR-142-3p to modulate the levels of these tumor-associated forms and to reactivate thyroid-specific Hox gene expression, likely contributes to its tumor suppressive function. CONCLUSIONS These data demonstrate that miR-142-3p downregulation has a role in thyroid tumorigenesis, by regulating ASH1L and MLL1.

Ibuprofen delivered by poly(lactic-co-glycolic acid) (PLGA) nanoparticles to human gastric cancer cells exerts antiproliferative activity at very low concentrations

Purpose Epidemiological, clinical, and laboratory studies have suggested that ibuprofen, a commonly used nonsteroidal anti-inflammatory drug, inhibits the promotion and proliferation of certain tumors. Recently, we demonstrated the antiproliferative effects of ibuprofen on the human gastric cancer cell line MKN-45. However, high doses of ibuprofen were required to elicit these antiproliferative effects in vitro. The present research compared the antiproliferative effects of ibuprofen delivered freely and released by poly(lactic-co-glycolic acid) (PLGA) nanoparticles (NPs) in MKN-45 cells. Methods MKN-45 human gastric adenocarcinoma cells were treated with ibuprofen-loaded PLGA NPs. The proliferation of MKN-45 cells was then assessed by cell counting. The uptake of NPs was imaged by fluorescence microscopy and flow cytometry. The release of ibuprofen from ibuprofen-loaded PLGA NPs in the cells was evaluated by gas chromatography–mass spectrometry. Results Dramatic inhibition of cellular proliferation was observed in cells treated with ibuprofen-loaded PLGA NPs versus those treated with free ibuprofen at the same concentration. The localization of NPs was cytoplasmic. The initiation of ibuprofen release was rapid, commencing within 2 hours, and then increased slowly over time, reaching a maximum concentration at 24 hours. The inhibition of proliferation was confirmed to be due to the intracellular release of ibuprofen from the NPs. Using PLGA NPs as carriers, ibuprofen exerted an antiproliferative activity at concentrations > 100 times less than free ibuprofen, suggesting greater efficiency and less cellular toxicity. In addition, when carried by PLGA NPs, ibuprofen more quickly induced the expression of transcripts involved in proliferation and invasiveness processes. Conclusion Ibuprofen exerted an antiproliferative effect on MKN-45 cells at low concentrations. This effect was achieved using PLGA NPs as carriers of low doses of ibuprofen.