The lncRNA RMST is drastically downregulated in anaplastic thyroid carcinomas where exerts a tumor suppressor activity impairing epithelial-mesenchymal transition and stemness.

Thyroid cancer is the most prevalent endocrine malignancy and comprises a wide range of lesions subdivided into differentiated (DTC) and undifferentiated thyroid cancer (UTC), mainly represented by the anaplastic thyroid carcinoma (ATC). This is one of the most lethal malignancies in humankind leading invariably to patient death in few months. Then, a better comprehension of the mechanisms underlying the development of ATC is required to set up new therapeutic approaches. Long non-coding RNAs (lncRNAs) are transcripts over 200 nucleotides in length that do not code for proteins. They show a strong regulatory function at both transcriptional and post-transcriptional level and are emerging as key players in regulating developmental processes. Their aberrant expression has been linked to several biological processes, including cancer, making them potential diagnostic and prognostic markers. We have recently analyzed the lncRNA expression profile in ATC through a microarray technique and have identified rhabdomyosarcoma 2-associated transcript (RMST) as one of the most downregulated lncRNA in ATC. RMST has been reported to be deregulated in a series of human cancers, to play an anti-oncogenic role in triple-negative breast cancer, and to modulate neurogenesis by interacting with SOX2. Therefore, these findings prompted us to investigate the role of RMST in ATC development. In this study we show that RMST levels are strongly decreased in ATC, but only slightly in DTC, indicating that the loss of this lncRNA could be related to the loss of the differentiation and high aggressiveness. We also report a concomitant increase of SOX2 levels in the same subset of ATC, that inversely correlated with RMST levels, further supporting the RMST/SOX2 relationship. Finally, functional studies demonstrate that the restoration of RMST in ATC cells reduces cell growth, migration and the stemness properties of ATC stem cells. In conclusion, these findings support a critical role of RMST downregulation in ATC development.

Differential Expression of LncRNA in Bladder Cancer Development.

Bladder cancer (BC) is the tenth most common cancer, with urothelial carcinoma representing about 90% of all BC, including neoplasms and carcinomas of different grades of malignancy. Urinary cytology has a significant role in BC screening and surveillance, although it has a low detection rate and high dependence on the pathologist's experience. The currently available biomarkers are not implemented into routine clinical practice due to high costs or low sensitivity. In recent years, the role of lncRNAs in BC has emerged, even though it is still poorly explored. We have previously shown that the lncRNAs Metallophosphoesterase Domain-Containing 2 Antisense RNA 1 (MPPED2-AS1), Rhabdomyosarcoma-2 Associated Transcript (RMST), Kelch-like protein 14 antisense (Klhl14AS) and Prader Willi/Angelman region RNA 5 (PAR5) are involved in the progression of different types of cancers. Here, we investigated the expression of these molecules in BC, first by interrogating the GEPIA database and observing a different distribution of expression levels between normal and cancer specimens. We then measured them in a cohort of neoplastic bladder lesions, either benign or malignant, from patients with suspicion of BC undergoing transurethral resection of bladder tumor (TURBT). The total RNA from biopsies was analyzed using qRT-PCR for the expression of the four lncRNA genes, showing differential expression of the investigated lncRNAs between normal tissue, benign lesions and cancers. In conclusion, the data reported here highlight the involvement of novel lncRNAs in BC development, whose altered expression could potentially affect the regulatory circuits in which these molecules are involved. Our study paves the way for testing lncRNA genes as markers for BC diagnosis and/or follow-up.

FOXE1-Dependent Regulation of Macrophage Chemotaxis by Thyroid Cells In Vitro and In Vivo.

Forkhead box E1 (FOXE1) is a lineage-restricted transcription factor involved in thyroid cancer susceptibility. Cancer-associated polymorphisms map in regulatory regions, thus affecting the extent of gene expression. We have recently shown that genetic reduction of FOXE1 dosage modifies multiple thyroid cancer phenotypes. To identify relevant effectors playing roles in thyroid cancer development, here we analyse FOXE1-induced transcriptional alterations in thyroid cells that do not express endogenous FOXE1. Expression of FOXE1 elicits cell migration, while transcriptome analysis reveals that several immune cells-related categories are highly enriched in differentially expressed genes, including several upregulated chemokines involved in macrophage recruitment. Accordingly, FOXE1-expressing cells induce chemotaxis of co-cultured monocytes. We then asked if FOXE1 was able to regulate macrophage infiltration in thyroid cancers in vivo by using a mouse model of cancer, either wild type or with only one functional FOXE1 allele. Expression of the same set of chemokines directly correlates with FOXE1 dosage, and pro-tumourigenic M2 macrophage infiltration is decreased in tumours with reduced FOXE1. These data establish a novel link between FOXE1 and macrophages recruitment in the thyroid cancer microenvironment, highlighting an unsuspected function of this gene in the crosstalk between neoplastic and immune cells that shape tumour development and progression.

FOXE1 Gene Dosage Affects Thyroid Cancer Histology and Differentiation In Vivo.

The transcription factor Forkhead box E1 (FOXE1) is a key player in thyroid development and function and has been identified by genome-wide association studies as a susceptibility gene for papillary thyroid cancer. Several cancer-associated polymorphisms fall into gene regulatory regions and are likely to affect FOXE1 expression levels. However, the possibility that changes in FOXE1 expression modulate thyroid cancer development has not been investigated. Here, we describe the effects of FOXE1 gene dosage reduction on cancer phenotype in vivo. Mice heterozygous for FOXE1 null allele (FOXE1+/-) were crossed with a BRAFV600E-inducible cancer model to develop thyroid cancer in either a FOXE1+/+ or FOXE1+/- genetic background. In FOXE1+/+ mice, cancer histological features are quite similar to that of human high-grade papillary thyroid carcinomas, while cancers developed with reduced FOXE1 gene dosage maintain morphological features resembling less malignant thyroid cancers, showing reduced proliferation index and increased apoptosis as well. Such cancers, however, appear severely undifferentiated, indicating that FOXE1 levels affect thyroid differentiation during neoplastic transformation. These results show that FOXE1 dosage exerts pleiotropic effects on thyroid cancer phenotype by affecting histology and regulating key markers of tumor differentiation and progression, thus suggesting the possibility that FOXE1 could behave as lineage-specific oncogene in follicular cell-derived thyroid cancer.

HMGA1-pseudogene7 transgenic mice develop B cell lymphomas.

We have recently identified and characterized two pseudogenes (HMGA1P6 and HMGA1P7) of the HMGA1 gene, which has a critical role in malignant cell transformation and cancer progression. HMGA1P6 and HMGAP17 act as microRNA decoy for HMGA1 and other cancer-related genes upregulating their protein levels. We have previously shown that they are upregulated in several human carcinomas, and their expression positively correlates with a poor prognosis and an advanced cancer stage. To evaluate in vivo oncogenic activity of HMGA1 pseudogenes, we have generated a HMGA1P7 transgenic mouse line overexpressing this pseudogene. By a mean age of 12 months, about 50% of the transgenic mice developed splenomegaly and accumulation of lymphoid cells in several body compartments. For these mice FACS and immunohistochemical analyses suggested the diagnosis of B-cell lymphoma that was further supported by clonality analyses and RNA expression profile of the pathological tissues of the HMGA1P7 transgenic tissues. Therefore, these results clearly demonstrate the oncogenic activity of HMGA1 pseudogenes in vivo.

A ceRNA Circuitry Involving the Long Noncoding RNA Klhl14-AS, Pax8, and Bcl2 Drives Thyroid Carcinogenesis.

Klhl14-AS is a long noncoding RNA expressed since early specification of thyroid bud and is the most enriched gene in the mouse thyroid primordium at E10.5. Here, we studied its involvement in thyroid carcinogenesis by analyzing its expression in cancer tissues and different models of neoplastic transformation. Compared with normal thyroid tissue and cells, Klhl14-AS was significantly downregulated in human thyroid carcinoma tissue specimens, particularly the anaplastic histotype, thyroid cancer cell lines, and rodent models of thyroid cancer. Downregulating the expression of Klhl14-AS in normal thyroid cells decreased the expression of thyroid differentiation markers and cell death and increased cell viability. These effects were mediated by the binding of Klhl14-AS to two miRNAs, Mir182-5p and Mir20a-5p, which silenced Pax8 and Bcl2, both essential players of thyroid differentiation. MIR182-5p and MIR20a-5p were upregulated in human thyroid cancer and thyroid cancer experimental models and their effects on Pax8 and Bcl2 were rescued by Klhl14-AS overexpression, confirming Klhl14-AS as a ceRNA for both Pax8 and Bcl2. This work connects deregulation of differentiation with increased proliferation and survival in thyroid neoplastic cells and highlights a novel ceRNA circuitry involving key regulators of thyroid physiology. SIGNIFICANCE: This study describes a new ceRNA with potential tumor suppression activity and helps us better understand the regulatory mechanisms during thyroid differentiation and carcinogenesis.

The Metallophosphoesterase-Domain-Containing Protein 2 (MPPED2) Gene Acts as Tumor Suppressor in Breast Cancer.

Background: We have recently reported the downregulation of the Metallophosphoesterase-domain-containing protein 2 (MPPED2) gene and its cognate long non-coding RNA, MPPED2-AS1, in papillary thyroid carcinomas. Functional studies supported a tumor suppressor role of both these genes in thyroid carcinogenesis. We then decided to investigate their role in breast carcinogenesis. Methods: In order to verify MPPED2 expression, 45 human breast carcinoma samples have been investigated by quantitative real-time polymerase chain reaction (qRT-PCR). Then, MPPED2 has been transfected in several human breast carcinoma cell lines, analyzing its role in cell proliferation, migration and invasion. To study the regulation of MPPED2 expression the methylation of its promoter was investigated by targeted bisulfite sequencing. Results: MPPED2 expression was decreased in breast cancer samples, and this was confirmed by the analysis of data available in The Cancer Genome Atlas (TCGA). Interestingly, the hypermethylation of MPPED2 promoter likely accounted for its downregulation in breast cancer. Additionally, MPPED2-AS1 was also found downregulated in breast cancer tissues and, intriguingly, its expression decreased the hypermethylation of the MPPED2 promoter by inhibiting DNA methyltransferase 1 (DNMT1). Furthermore, the restoration of MPPED2 expression reduced cell proliferation, migration and invasion capability of breast carcinoma cell lines. Conclusion: Taken together, these results propose MPPED2 downregulation as a critical event in breast carcinogenesis.

A Toxicogenomic Approach Reveals a Novel Gene Regulatory Network Active in In Vitro and In Vivo Models of Thyroid Carcinogenesis.

Epidemiological and experimental studies emphasize the link between environmental chemicals exposure and thyroid cancer. However, this association is strongly debated and the mechanisms of action of environmental thyroid carcinogens still need to be identified. The analysis of in vitro transcriptomic data developed to investigate the effects of chlorpyrifos on immortalized thyrocytes highlighted the impaired expression of genes involved in endodermal carcinogenesis. This endodermal carcinogenic gene-network (ECGN, including Zfp36l2, Dmbt1, Ddit4), was validated in cellular and mouse models of thyroid carcinogenesis, characterized by the constitutive activation of the mitogen-activated protein kinase (MAPK) pathway and in immortalized thyrocytes exposed to tetrachlorodibenzo-p-dioxin (TCDD) and chlorpyrifos (CPF). The mRNA levels of Zfp36l2, Dmbt1 and Ddit4 were increased in models characterized by MAPK activation or following TCDD exposure, whereas they were inhibited by CPF exposure. Overall, the ECGN transcripts identify a novel gene-regulatory network associated with thyroid carcinogenesis promoted by genetic mutation or by environmental carcinogens. The latter have opposite effects on the modulation of the ECGN transcripts according to their mechanisms of action in promoting carcinogenesis. Therefore, the analyses of ECGN might be helpful in discriminating compounds that promote cellular survival associated or not to proliferation of thyrocytes.

Role of Dicer1 in thyroid cell proliferation and differentiation.

DICER1 plays a central role in the biogenesis of microRNAs and it is important for normal development. Altered microRNA expression and DICER1 dysregulation have been described in several types of tumors, including thyroid carcinomas. Recently, our group identified a new somatic mutation (c.5438A>G; E1813G) within DICER1 gene of an unknown function. Herein, we show that DICER1 is overexpressed, at mRNA level, in a significant-relative number of papillary (70%) and anaplastic (42%) thyroid carcinoma samples, whereas is drastically downregulated in all the analyzed human thyroid carcinoma cell lines (TPC-1, BCPAP, FRO and 8505c) in comparison with normal thyroid tissue samples. Conversely, DICER1 is downregulated, at protein level, in PTC in comparison with normal thyroid tissues. Our data also reveals that DICER1 overexpression positively regulates thyroid cell proliferation, whereas its silencing impairs thyroid cell differentiation. The expression of DICER1 gene mutation (c.5438A>G; E1813G) negatively affects the microRNA machinery and cell proliferation as well as upregulates DICER1 protein levels of thyroid cells but has no impact on thyroid differentiation. In conclusion, DICER1 protein is downregulated in papillary thyroid carcinomas and affects thyroid proliferation and differentiation, while DICER1 gene mutation (c.5438A>G; E1813G) compromises the DICER1 wild-type-mediated microRNA processing and cell proliferation.

Mice lacking microRNAs in Pax8-expressing cells develop hypothyroidism and end-stage renal failure.

BACKGROUND: Non-coding RNAs have gained increasing attention during the last decade. The first large group of non-coding RNAs to be characterized systematically starting at the beginning of the 21st century were small oligonucleotides--the so-called microRNAs (miRNAs). By now we have learnt that microRNAs are indispensable for most biological processes including organogenesis and maintenance of organ structure and function. The role of microRNAs has been studied extensively in the development of a number of organs, so far most studies focussed on e.g. the heart or the brain whilst the role of microRNAs in the development and maintenance of complex epithelial organs is less well understood. Furthermore most analyses regarding microRNA function in epithelial organs employed conditional knockout mouse models of the RNAse III Dicer to abrogate microRNA biogenesis. However, there is increasing evidence for Dicer to have multiple functions independent from microRNA maturation. Therefore Dicer independent models are needed to gain further insight into the complex biology of miRNA dependent processes. RESULTS: Here we analyze the contribution of microRNA-dependent transcriptional control in Pax8-expressing epithelial cells. Pax8 is a transcription factor that is crucial to the development of epithelial organs. The miRNA machinery was disrupted by crossing conditional DiGeorge syndrome critical region 8 (Dgcr8) fl/fl mice to Pax8Cre mice. The Dgcr8/Drosha complex processes pri-miRNAs in the nucleus before they are exported as pre-miRNAs for further maturation by Dicer in the cytoplasm. Dgcr8 fl/fl; Pax8Cre+ knockout mice died prematurely, developed massive hypothyroidism and end stage renal disease due to a loss of miRNAs in Pax8 expressing tissue. CONCLUSION: Pax8Cre-mediated conditional loss of DiGeorge syndrome critical region 8 (Dgcr8), an essential component of the nuclear machinery that is required for microRNA biogenesis, resulted in severe hypothyroidism, massively reduced body weight and ultimately led to renal failure and death of the animals. These data provide further insight into the importance of miRNAs in organ homeostasis using a Dicer independent model.

PATZ1 is a target of miR-29b that is induced by Ha-Ras oncogene in rat thyroid cells.

The regulatory transcriptional factor PATZ1 is constantly downregulated in human thyroid cancer where it acts as a tumour suppressor by targeting p53-dependent genes involved in Epithelial-Mesenchymal Transition and cell migration. The aim of the present work was to elucidate the upstream signalling mechanisms regulating PATZ1 expression in thyroid cancer cells. The bioinformatics search for microRNAs able to potentially target PATZ1 led to the identification of several miRNAs. Among them we focused on the miR-29b since it was found upregulated in rat thyroid differentiated cells transformed by the Ha-Ras oncogene towards a high proliferating and high migratory phenotype resembling that of anaplastic carcinomas. Functional assays confirmed PATZ1 as a target of miR-29b, and, consistently, an inverse correlation between miR-29b and PATZ1 protein levels was found upon induction of Ha-Ras oncogene expression in these cells. Interestingly, restoration of PATZ1 expression in rat thyroid cells stably expressing the Ha-Ras oncogene decreased cell proliferation and migration, indicating a key role of PATZ1 in Ras-driven thyroid transformation. Together, these results suggest a novel mechanism regulating PATZ1 expression based on the upregulation of miR-29b expression induced by Ras oncogene.

Clinical relevance of thyroid cell models in redox research.

BACKGROUND: Thyroid-derived cell models are commonly used to investigate the characteristics of thyroid cancers. It is noteworthy that each in vitro single cell model system imitates only a few characteristics of thyroid cancer depending on e.g. source of cells or oncogene used to transform the cells. METHODS: In the current work we utilized rat thyroid cancer cell models to determine their clinical relevance in redox gene studies by comparing in vitro expression data to thyroid Oncomine microarray database. To survey the cell lines we analyzed mRNA expression of genes that produce superoxide anion (nox family), genes that catalyze destruction of superoxide anion to hydrogen peroxide (sod family), and genes that remove hydrogen peroxide from cellular environment (catalase, gpx family and prdx family). RESULTS: Based on the current results, rat thyroid PC Cl3, PC PTC1, PC E1A, or FRLT5 cell models can be used to study NOX2, NOX4, SOD2, SOD3, CATALASE, GPX1, GPX2, GPX5, PRDX2, and PRDX3 gene expression and function. CONCLUSIONS: Redox gene expression in rat originated single cell model systems used to study human thyroid carcinogenesis corresponds only partly with human redox gene expression, which may be caused by differences in redox gene activation stimulus. The data suggest careful estimation of the data observed in rat thyroid in vitro models.

Ras oncogene-mediated progressive silencing of extracellular superoxide dismutase in tumorigenesis.

Extracellular superoxide dismutase (SOD3) is a secreted enzyme that uses superoxide anion as a substrate in a dismutase reaction that results in the formation of hydrogen peroxide. Both of these reactive oxygen species affect growth signaling in cells. Although SOD3 has growth-supporting characteristics, the expression of SOD3 is downregulated in epithelial cancer cells. In the current work, we studied the mechanisms regulating SOD3 expression in vitro using thyroid cell models representing different stages of thyroid cancer. We demonstrate that a low level of RAS activation increases SOD3 mRNA synthesis that then gradually decreases with increasing levels of RAS activation and the decreasing degree of differentiation of the cancer cells. Our data indicate that SOD3 regulation can be divided into two classes. The first class involves RAS-driven reversible regulation of SOD3 expression that can be mediated by the following mechanisms: RAS GTPase regulatory genes that are responsible for SOD3 self-regulation; RAS-stimulated p38 MAPK activation; and RAS-activated increased expression of the mir21 microRNA, which inversely correlates with sod3 mRNA expression. The second class involves permanent silencing of SOD3 mediated by epigenetic DNA methylation in cells that represent more advanced cancers. Therefore, the work suggests that SOD3 belongs to the group of ras oncogene-silenced genes.

Selective dicer suppression in the kidney alters GSK3ß/ß-catenin pathways promoting a glomerulocystic disease.

Dicer is a crucial enzyme for the maturation of miRNAs. Mutations in the Dicer gene are highly associated with Pleuro Pulmonary Blastoma-Family Dysplasia Syndrome (PPB-FDS, OMIM 601200), recently proposed to be renamed Dicer syndrome. Aside from the pulmonary phenotype (blastoma), renal nephroma and thyroid goiter are frequently part of Dicer syndrome. To investigate the renal phenotype, conditional knockout (cKO) mice for Dicer in Pax8 expressing cells were generated. Dicer cKO mice progressively develop a glomerulocystic phenotype coupled with urinary concentration impairment, proteinuria and severe renal failure. Higher cellular turnover of the parietal cells of Bowman's capsule precedes the development of the cysts and the primary cilium progressively disappears with cyst-enlargement. Upregulation of GSK3ß precedes the development of the glomerulocystic phenotype. Downregulation of ß-catenin in the renal cortex and its cytosolic removal in the cells lining the cysts may be associated with observed accumulation of GSK3ß. Alterations of ß-catenin regulating pathways could promote cystic degeneration as in other models. Thus, miRNAs are fundamental in preserving renal morphology and function. Alteration of the GSK3ß/ß-catenin pathway could be a crucial mechanism linking miRNA dysregulation and the development of a glomerulocystic disease.

A genetic screen implicates miRNA-372 and miRNA-373 as oncogenes in testicular germ cell tumors.

Endogenous small RNAs (miRNAs) regulate gene expression by mechanisms conserved across metazoans. While the number of verified human miRNAs is still expanding, only few have been functionally annotated. To perform genetic screens for novel functions of miRNAs, we developed a library of vectors expressing the majority of cloned human miRNAs and created corresponding DNA barcode arrays. In a screen for miRNAs that cooperate with oncogenes in cellular transformation, we identified miR-372 and miR-373, each permitting proliferation and tumorigenesis of primary human cells that harbor both oncogenic RAS and active wild-type p53. These miRNAs neutralize p53-mediated CDK inhibition, possibly through direct inhibition of the expression of the tumorsuppressor LATS2. We provide evidence that these miRNAs are potential novel oncogenes participating in the development of human testicular germ cell tumors by numbing the p53 pathway, thus allowing tumorigenic growth in the presence of wild-type p53.

A mammalian microRNA expression atlas based on small RNA library sequencing.

MicroRNAs (miRNAs) are small noncoding regulatory RNAs that reduce stability and/or translation of fully or partially sequence-complementary target mRNAs. In order to identify miRNAs and to assess their expression patterns, we sequenced over 250 small RNA libraries from 26 different organ systems and cell types of human and rodents that were enriched in neuronal as well as normal and malignant hematopoietic cells and tissues. We present expression profiles derived from clone count data and provide computational tools for their analysis. Unexpectedly, a relatively small set of miRNAs, many of which are ubiquitously expressed, account for most of the differences in miRNA profiles between cell lineages and tissues. This broad survey also provides detailed and accurate information about mature sequences, precursors, genome locations, maturation processes, inferred transcriptional units, and conservation patterns. We also propose a subclassification scheme for miRNAs for assisting future experimental and computational functional analyses.

p38alpha MAP kinase as a sensor of reactive oxygen species in tumorigenesis.

p38alpha is a stress-activated protein kinase that negatively regulates malignant transformation induced by oncogenic H-Ras, although the mechanisms involved are not fully understood. Here, we show that p38alpha is not a general inhibitor of oncogenic signaling, but that it specifically modulates transformation induced by oncogenes that produce reactive oxygen species (ROS). This inhibitory effect is due to the ROS-induced activation of p38alpha early in the process of transformation, which induces apoptosis and prevents the accumulation of ROS and their carcinogenic effects. Accordingly, highly tumorigenic cancer cell lines have developed a mechanism to uncouple p38alpha activation from ROS production. Our results indicate that oxidative stress sensing plays a key role in the inhibition of tumor initiation by p38alpha.

RET/papillary thyroid carcinoma oncogenic signaling through the Rap1 small GTPase.

RET/papillary thyroid carcinoma (PTC) oncoproteins result from the in-frame fusion of the RET receptor tyrosine kinase with protein dimerization motifs encoded by heterologous genes. Here, we show that RET/PTC1 activates the Rap1 small GTPase. The activation of Rap1 was dependent on the phosphorylation of RET Tyr(1062). RET/PTC1 recruited a complex containing growth factor receptor binding protein 2-associated binding protein 1 (Gab1), CrkII (v-crk sarcoma virus CT10 oncogene homologue II), and C3G (Rap guanine nucleotide exchange factor 1). By using dominant-negative and small interfering duplex (small interfering RNA) oligonucleotides, we show that RET/PTC1-mediated Rap1 activation was dependent on CrkII, C3G, and Gab1. Activation of Rap1 was involved in the RET/PTC1-mediated stimulation of the BRAF kinase and the p42/p44 mitogen-activated protein kinases. Proliferation and stress fiber formation of RET/PTC1-expressing PC Cl 3 thyroid follicular cells were inhibited by the dominant-negative Rap1(N17) and by Rap1-specific GTPase-activating protein. Thus, Rap1 is a downstream effector of RET/PTC and may contribute to the transformed phenotype of RET/PTC-expressing thyrocytes.