Daniela D'Angelo

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.

A TSH-CREB1-microRNA Loop Is Required for Thyroid Cell Growth

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.

The High Mobility Group A proteins contribute to thyroid cell transformation by regulating miR-603 and miR-10b expression

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.

Thyrotropin Regulates Thyroid Cell Proliferation by Up-Regulating miR-23b and miR-29b that Target SMAD3

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-β 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.

HMGA1-pseudogene expression is induced in human pituitary tumors.

Numerous studies have established that High Mobility Group A (HMGA) proteins play a pivotal role on the onset of human pituitary tumors. They are overexpressed in pituitary tumors, and, consistently, transgenic mice overexpressing either the Hmga1 or the Hmga2 gene develop pituitary tumors. In contrast with HMGA2, HMGA1 overexpression is not related to any rearrangement or amplification of the HMGA1 locus in these tumors. We have recently identified 2 HMGA1 pseudogenes, HMGA1P6 and HMGA1P7, acting as competitive endogenous RNA decoys for HMGA1 and other cancer related genes. Here, we show that HMGA1 pseudogene expression significantly correlates with HMGA1 mRNA levels in growth hormone and nonfunctioning pituitary adenomas likely inhibiting the repression of HMGA1 through microRNAs action. According to our functional studies, these HMGA1 pseudogenes enhance the proliferation and migration of the mouse pituitary tumor cell line, at least in part, through their upregulation. Our results point out that the overexpression of HMGA1P6 and HMGA1P7 could contribute to increase HMGA1 levels in human pituitary tumors, and then to pituitary tumorigenesis.

Downregulation of miR-410 targeting the cyclin B1 gene plays a role in pituitary gonadotroph tumors.

MicroRNAs (miRNAs) are small noncoding RNAs that act as posttranscriptional regulators of gene expression, and are frequently altered in human neoplasias. Here, we have analyzed the miRNA expression profile of human gonadotroph adenomas versus normal pituitary tissue using a miRNACHIP microarray. We demonstrate that miRNA-410 is downregulated in gonadotroph adenomas when compared with normal pituitary gland. We validate CCNB1 as target of miRNA-410 since its overexpression reduces CCNB1 at protein and mRNA levels, decreasing cell proliferation. In conclusion, our study suggess that the downregulation of miRNA-410 plays a role in the behavior of gonadotroph tumors.

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.

HMGA2 overexpression plays a critical role in the progression of esophageal squamous carcinoma

Esophageal Squamous Cell Carcinoma (ESCC) is the most common esophageal tumor worldwide. However, there is still a lack of deeper knowledge about biological alterations involved in ESCC development. High Mobility Group A (HMGA) protein family has been related with poor outcome and malignant cell transformation in several tumor types. In this way, the aim of this study was to analyze the expression of HMGA1 and HMGA2 expression in ESCC and their role in crucial cellular features. We evaluated HMGA1 and HMGA2 mRNA expression in 52 paired ESCC and normal surrounding tissue samples by qRT-PCR. Here, we show that HMGA2, but not HMGA1, is overexpressed in ESCC samples. This result was further confirmed by the immunohistochemical analysis. Indeed, accordingly to mRNA expression data, HMGA2, but not HMGA1, was overexpressed in approximately 90% of ESCC samples, while it was barely expressed in the respective control. Conversely, HMGA1, but not HMGA2, was overexpressed in esophageal adenocarcinoma samples. Interestingly, HMGA2 abrogation attenuated the malignant phenotype of two ESCC cell lines, suggesting that HMGA2 overexpression is involved in ESCC progression.

Analysis of miRNA profiles identified miR-196a as a crucial mediator of aberrant PI3K/AKT signaling in lung cancer cells

Hyperactivation of the PI3K/AKT pathway is observed in most human cancer including lung carcinomas. Here we have investigated the role of miRNAs as downstream targets of activated PI3K/AKT signaling in Non Small Cell Lung Cancer (NSCLC). To this aim, miRNA profiling was performed in human lung epithelial cells (BEAS-2B) expressing active AKT1 (BEAS-AKT1-E17K), active PI3KCA (BEAS-PIK3CA-E545K) or with silenced PTEN (BEAS-shPTEN). Twenty-four differentially expressed miRNAs common to BEAS-AKT1-E17K, BEAS-PIK3CA-E545K and BEAS-shPTEN cells were identified through this analysis, with miR-196a being the most consistently up-regulated miRNA. Interestingly, miR-196a was significantly overexpressed also in human NSCLC-derived cell lines (n=11) and primary lung cancer samples (n=28). By manipulating the expression of miR-196a in BEAS-2B and NCI-H460 cells, we obtained compelling evidence that this miRNA acts downstream the PI3K/AKT pathway, mediating some of the proliferative, pro-migratory and tumorigenic activity that this pathway exerts in lung epithelial cells, possibly through the regulation of FoxO1, CDKN1B (hereafter p27) and HOXA9.