Pierlorenzo Pallante

Specific microRNAs are downregulated in human thyroid anaplastic carcinomas.

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.

Deregulation of microRNA expression in follicular cell-derived human thyroid carcinomas

Carcinoma of the thyroid gland is an uncommon cancer, but one of the most frequent malignancies of the endocrine system. Most thyroid cancers are derived from the follicular cells. Follicular carcinoma is considered more malignant than papillary thyroid carcinoma (PTC), and anaplastic thyroid cancer (ATC) is one of the most lethal human cancers. Even though several genetic lesions have been already described in human thyroid cancer, particularly in the papillary histotype, the mechanisms underlying the development of these neoplasias are still far from being completely elucidated. Some years ago, several studies were undertaken to analyze the expression of microRNAs (miRNAs or miRs) in thyroid carcinoma to evaluate a possible role of their deregulation in the process of carcinogenesis. These studies showed an aberrant microRNA expression profile that distinguishes unequivocally among PTC, ATC, and normal thyroid tissue. Here, other than summarizing the current findings on microRNA expression in human thyroid carcinomas, we discuss the mechanisms by which microRNA deregulation may play a role in thyroid carcinogenesis, and the possible use of microRNA knowledge in the diagnosis and therapy of thyroid neoplasms.

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.

The tyrosine phosphatase PTPRJ/DEP-1 genotype affects thyroid carcinogenesis.

We recently isolated the r-PTPeta gene, which encodes a receptor-type tyrosine phosphatase protein that suppresses the neoplastic phenotype of retrovirally transformed rat thyroid cells. The human homologue gene PTPRJ/DEP-1 is deleted in various tumors. Moreover, the Gln276Pro polymorphism, located in the extracellular region of the gene, seems to play a critical role in susceptibility to some human neoplasias. Here we report the loss of heterozygosity (LOH) of PTPRJ in 11/76 (14.5%) informative thyroid tumors (including adenomas and carcinomas). We also looked for the Gln276Pro, Arg326Gln and Asp872Glu polymorphisms in exons 5, 6 and 13 of PTPRJ in 88 patients with thyroid tumors and in 54 healthy individuals. We found that the PTPRJ genotypes homozygous for the Gln276Pro and Arg326Gln polymorphisms, and the Asp872 allele were more frequent in thyroid carcinoma patients than in healthy individuals (P=0.032). In addition, PTPRJ LOH was more frequent in thyroid carcinomas of heterozygotes for Gln276Pro and Arg326Gln compared with homozygotes (P=0.006). This suggests that the presence of hemizygosity for these polymorphisms in the tumor facilitates tumor progression. These results indicate that the genotypic profile of PTPRJ affects susceptibility to thyroid carcinomas, and that allelic loss of this gene is involved in thyroid carcinogenesis.

Deregulation of microRNA expression in thyroid neoplasias

MicroRNAs (miRNAs) have emerged as a class of powerful gene expression regulators. Acting at the post-transcriptional level, miRNAs modulate the expression of at least one-third of the mRNAs that are encoded by the human genome. The expression of a single gene can be regulated by several miRNAs, and every miRNA has more than one target gene. Thus, the miRNA regulatory circuit, which affects essential cellular functions, is of enormous complexity. Moreover, a fundamental role for miRNAs has been determined in the onset and progression of human cancers. Here, we summarize the main alterations in miRNA expression that have been identified in thyroid neoplasias and examine the mechanisms through which miRNA deregulation might promote thyroid cell transformation. We also discuss how the emerging knowledge on miRNA deregulation could be harnessed for the diagnosis and treatment of thyroid neoplasias.

UbcH10 expression may be a useful tool in the prognosis of ovarian carcinomas

The UbcH10 gene codes for a protein that belongs to the ubiquitin-conjugating enzyme family. Previous studies of our group suggest UbcH10 expression as a valid indicator of the proliferative and aggressive status of thyroid carcinomas. Therefore, to better understand the process of ovarian carcinogenesis, and to look for possible tools to be used as prognostic markers in these neoplasias, we decided to extend the analysis of the UbcH10 expression to the ovarian neoplastic disease. We found that the UbcH10 gene was upregulated in some ovarian carcinoma cell lines analysed. Then, immunohistochemical studies demonstrate that UbcH10 expression significantly correlates with the tumor grade and the undifferentiated histotype of the ovarian carcinomas. Furthermore, a significant relationship between UbcH10 expression and overall survival was observed. Finally, the block of UbcH10 protein synthesis by RNA interference inhibited the growth of ovarian carcinoma cell lines, suggesting a role of UbcH10 overexpression in ovarian carcinogenesis. Therefore, all these data taken together suggest the possibility to use UbcH10 detection as a marker for the diagnosis and prognosis of these neoplastic diseases and open the perspective of a therapy of some ovarian carcinomas based on the suppression of the UbcH10 synthesis and/or function.

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.