Correction: Sepe et al. The Long Non-Coding RNA RP5-1024C24.1 and Its Associated-Gene MPPED2 Are Down-Regulated in Human Thyroid Neoplasias and Act as Tumour Suppressors. Cancers 2018, 10, 146.

In the original publication [...].

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.

Drug-induced inhibition of HMGA and EZH2 activity as a possible therapy for anaplastic thyroid carcinoma.

Anaplastic thyroid carcinoma (ATC) is one of the most aggressive and lethal neoplasms in humans, and just limited progresses have been made to extend patient survival and decrease ATC-associated mortality. Thus, the identification of novel therapeutic strategies for treating ATC is needed. Recently, our group has identified two proteins with oncogenic activity, namely HMGA1 and EZH2, with pivotal roles in ATC cancer progression. Therefore, we tested the ability of trabectedin, a HMGA1-targeting drug, and GSK126, an inhibitor of EZH2 enzymatic activity, to impair cell viability of four ATC-derived cell lines. In the present study, we first confirmed the overexpression of HMGA1 and EZH2 in all ATC-derived cell lines and tissues compared to the normal primary thyroid cells and tissues. Then, treatment of the ATC cell lines with trabectedin and GSK126 resulted in a drastic induction of apoptotic cell death, which increased when the ATC cell lines were treated with a combination of both drugs. Conversely, normal primary human thyroid cells did not show any significant reduction in their viability when exposed to the same drugs. Noteworthy, both drugs induced the deregulation of EZH2- and HMGA1-controlled genes. Altogether, these findings propose the combination of trabectedin and GSK126 as possible novel strategy for ATC therapy.

Noncoding RNAs in Thyroid-Follicular-Cell-Derived Carcinomas.

Among the thyroid neoplasias originating from follicular cells, we can include well-differentiated carcinomas, papillary (PTC) and follicular (FTC) thyroid carcinomas, and the undifferentiated anaplastic (ATC) carcinomas. Several mutations in oncogenes and tumor suppressor genes have already been observed in these malignancies; however, we are still far from the comprehension of their full regulation-altered landscape. Even if only 2% of the human genome has the ability to code for proteins, most of the noncoding genome is transcribed, constituting the heterogeneous class of noncoding RNAs (ncRNAs), whose alterations are associated with the development of several human diseases, including cancer. Hence, many scientific efforts are currently focused on the elucidation of their biological role. In this review, we analyze the scientific literature regarding the involvement of microRNAs (miRNAs), long noncoding RNAs (lncRNAs), and pseudogenes in FTC, PTC, and ATC. Recent findings emphasized the role of lncRNAs in all steps of cancer progression. In particular, lncRNAs may control progression steps by regulating the expression of genes and miRNAs involved in cell proliferation, apoptosis, epithelial-mesenchymal transition, and metastatization. In conclusion, the determination of the diagnosis, prognosis, and treatment of cancer based on the evaluation of the ncRNA network could allow the implementation of a more personalized approach to fighting thyroid tumors.

The role of HMGA1 protein in gastroenteropancreatic neuroendocrine tumors.

Neuroendocrine tumors (NETs) are neoplasms derived from neuroendocrine cells. One of their main features is to often remain asymptomatic and clinically undetectable. High Mobility Group A (HMGA) proteins belong to a family of non-histone chromatinic proteins able to modulate gene expression through the interaction with DNA and transcription factors. They are overexpressed in most of the human malignancies, playing a critical role in carcinogenesis. However, their expression levels and their role in neuroendocrine carcinogenesis has not been exhaustively evaluated until now. Therefore, in this study, we have addressed the validity of using the expression of HMGA1 as a diagnostic marker and have investigated its role in NET carcinogenesis. The expression of HMGA1 has been evaluated by qRT-PCR and immunohistochemistry, using NET tissue microarrays, in a cohort of gastroenteropancreatic (GEP)-NET samples. The expression levels of HMGA1 have been then correlated with the main clinical features of NET samples. Finally, the contribution of HMGA1 overexpression to NET development has been addressed as far as the modulation of proliferation and migration abilities of NET cells is concerned. Here, we report that HMGA1 is overexpressed in GEP-NET samples, at both mRNA and protein levels, and that the silencing of HMGA1 protein expression interferes with the ability of NET cells to proliferate and migrate through the downregulation of Cyclin E, Cyclin B1 and EZH2. These results propose the HMGA proteins as new diagnostic and prognostic markers.

Critical role of the high mobility group A proteins in hematological malignancies.

The high mobility group A (HMGA) protein family is composed of three non-histone chromatin remodeling proteins that act as architectural transcriptional factors. Indeed, although HMGA proteins lack transcriptional activity per se, they bind the minor groove of DNA at AT-rich sequences, and, interacting with the transcription machinery, are able to modify chromatin modeling, thus regulating the expression of several genes. HMGA proteins have been deeply involved in embryogenesis process, and a large volume of studies has pointed out their key role in human cancer. Here, we review the studies on the role of the HMGA proteins in human hematological malignancies: they are overexpressed in most of the cases and their expression correlates with a reduced survival. In some cases, such as in acute lymphoblastic leukemia and acute myelogenous leukemia, HMGA2 gene rearrangements have been also described. Finally, recent studies evidence a synergism between HMGA and EZH2 in diffuse B-cell lymphomas, suggesting an innovative therapy for this disease based on the inhibition of the function of both these proteins.

HMGA1 induces EZH2 overexpression in human B-cell lymphomas.

EZH2 is an enzymatic subunit of PRC2, an epigenetic regulator that triggers the methylation of the histone H3 lysine 27 silencing the transcription of several genes. EZH2 has a critical role in cancer progression, since its overexpression has been associated with increased cancer cell invasiveness, drug resistance and poor patient survival. However, the mechanisms accounting for EZH2 overexpression in cancer remain still unclear. Intriguingly, also HMGA protein overexpression is a feature of many human malignancies and correlates with the presence of metastases and a poor outcome. The HMGA proteins, including HMGA1 and HMGA2, belong to the architectural transcription factors that play a key role in the organization of chromatin structure. Here, we report a statistically significant correlation between HMGA1 and EZH2 expression in human lymphomas. We demonstrate that HMGA1 is able to bind EZH2 promoter and induce its activity. Consistently, silencing of HMGA1 expression results in the downregulation of the EZH2 levels leading to a decreased proliferation and migration rate of human lymphoma cell lines. Therefore, these data identify HMGA1 as an EZH2 activator, suggesting a novel molecular mechanism contributing to EZH2 overexpression in human malignancies and a synergism of these proteins in cancer progression.

MPPED2 is downregulated in glioblastoma, and its restoration inhibits proliferation and increases the sensitivity to temozolomide of glioblastoma cells.

Glioblastoma (GBM) is the most aggressive and lethal neoplasia of the central nervous system in adults. Based on the molecular signature genes, GBM has been classified in proneural, neural, mesenchymal and classical subtypes. The Metallophosphoesterase-domain-containing protein 2 (MPPED2) gene encodes a metallophosphodiesterase protein highly conserved throughout the evolution. MPPED2 downregulation, likely due to its promoter hypermethylation, has been found in several malignant neoplasias and correlated with a poor prognosis. In this study, we aimed to investigate the expression and the functional role of MPPED2 in GBM. TCGA and Gravendeel databases were employed to explore the MPPED2 expression levels in this type of tumor. We have found that MPPED2 expression is downregulated in GBM patients, showing a positive correlation with survival. Moreover, TCGA and Gravendeel data also revealed that MPPED2 expression negatively correlates with the most aggressive mesenchymal subtype. Additionally, the restoration of MPPED2 expression in U251 and GLI36 GBM cell lines decreases cell growth, migration and enhanced the sensitivity to the temozolomide, inducing apoptotic cell death, of GBM cells. These findings suggest that the restoration of MPPED2 function can be taken into consideration for an innovative GBM therapy.

Interplay between HMGA and TP53 in cell cycle control along tumor progression.

The high mobility group A (HMGA) proteins are found to be aberrantly expressed in several tumors. Studies (in vitro and in vivo) have shown that HMGA protein overexpression has a causative role in carcinogenesis process. HMGA proteins regulate cell cycle progression through distinct mechanisms which strongly influence its normal dynamics along malignant transformation. Tumor protein p53 (TP53) is the most frequently altered gene in cancer. The loss of its activity is recognized as the fall of a barrier that enables neoplastic transformation. Among the different functions, TP53 signaling pathway is tightly involved in control of cell cycle, with cell cycle arrest being the main biological outcome observed upon p53 activation, which prevents accumulation of damaged DNA, as well as genomic instability. Therefore, the interaction and opposing effects of HMGA and p53 proteins on regulation of cell cycle in normal and tumor cells are discussed in this review. HMGA proteins and p53 may reciprocally regulate the expression and/or activity of each other, leading to the counteraction of their regulation mechanisms at different stages of the cell cycle. The existence of a functional crosstalk between these proteins in the control of cell cycle could open the possibility of targeting HMGA and p53 in combination with other therapeutic strategies, particularly those that target cell cycle regulation, to improve the management and prognosis of cancer patients.

Identification of HMGA2 inhibitors by AlphaScreen-based ultra-high-throughput screening assays.

The mammalian high mobility group protein AT-hook 2 (HMGA2) is a multi-functional DNA-binding protein that plays important roles in tumorigenesis and adipogenesis. Previous results showed that HMGA2 is a potential therapeutic target of anticancer and anti-obesity drugs by inhibiting its DNA-binding activities. Here we report the development of a miniaturized, automated AlphaScreen ultra-high-throughput screening assay to identify inhibitors targeting HMGA2-DNA interactions. After screening the LOPAC1280 compound library, we identified several compounds that strongly inhibit HMGA2-DNA interactions including suramin, a century-old, negatively charged antiparasitic drug. Our results show that the inhibition is likely through suramin binding to the "AT-hook" DNA-binding motifs and therefore preventing HMGA2 from binding to the minor groove of AT-rich DNA sequences. Since HMGA1 proteins also carry multiple "AT-hook" DNA-binding motifs, suramin is expected to inhibit HMGA1-DNA interactions as well. Biochemical and biophysical studies show that charge-charge interactions and hydrogen bonding between the suramin sulfonated groups and Arg/Lys residues play critical roles in the binding of suramin to the "AT-hook" DNA-binding motifs. Furthermore, our results suggest that HMGA2 may be one of suramin's cellular targets.

The HMGA1-pseudogene7 shows oncogenic activity in vivo.

We have recently reported that transgenic mice overexpressing the HMGA1-pseudogene7 develop hematological neoplasia marked by monoclonal B-cell populations, and diagnosed as Diffuse Large B-cell Lymphoma. These findings prove the HMGA1-pseudogene7 oncogenic role in vivo.

Characterization of HMGA1P6 transgenic mouse embryonic fibroblasts.

Latest studies have shown that deregulated pseudogene transcripts contribute to cancer working as competing endogenous RNAs. Our research group has recently demonstrated that the overexpression of two HMGA1 pseudogenes, HMGA1P6 and HMGA1P7, has a critical role in cancer progression. These pseudogenes work sustaining the expression of HMGA1 and other cancer-related genes. We generated a mouse model overexpressing HMGA1P6 to better study the HMGA1-pseudogene function in a more physiological context. Here, we show the proliferation rate and the susceptibility to senescence of mouse embryonic fibroblasts obtained from HMGA1P6-overexpressing mice to better characterize the HMGA1-pseudogene function. Indeed, our study reports that mouse embryonic fibroblasts (MEFs) derived from HMGA1P6 mice express higher HMGA1 mRNA and protein levels. Moreover, these cells grow faster and senesce later than wild-type sustaining the oncogenic role of ceRNA crosstalk mediated by HMGA1Ps.

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.

HMGA1-Regulating microRNAs Let-7a and miR-26a are Downregulated in Human Seminomas.

BACKGROUND: Recent studies have underlined HMGA protein's key role in the onset of testicular germ cell tumors, where HMGA1 is differently expressed with respect to the state of differentiation, suggesting its fine regulation as master regulator in testicular tumorigenesis. Several studies have highlighted that the HMGA1 transcript is strictly regulated by a set of inhibitory microRNAs. Thus, the aim of this study is to test whether HMGA1 overexpression in human seminomas may be induced by the deregulation of miR-26a and Let-7a-two HMGA1-targeting microRNAs. METHODS: HMGA1 mRNA and Let-7a and miR-26a levels were measured in a seminoma dataset available in the Cancer Genome Atlas database and confirmed in a subset of seminomas by qRT-PCR and western blot. A TCam-2 seminoma cell line was then transfected with Let-7a and miR-26a and tested for proliferation and motility abilities. RESULTS: an inverse correlation was found between the expression of miR-26a and Let-7a and HMGA1 expression levels in seminomas samples, suggesting a critical role of these microRNAs in HMGA1 levels regulation. Accordingly, functional studies showed that miR-26a and Let-7a inhibited the proliferation, migration and invasion capabilities of the human seminoma derived cell line TCam-2. CONCLUSIONS: these data strongly support that the upregulation of HMGA1 levels occurring in seminoma is-at least in part-due to the downregulation of HMGA1-targeting microRNAs.

The Long Non-Coding RNA Prader Willi/Angelman Region RNA5 (PAR5) Is Downregulated in Anaplastic Thyroid Carcinomas Where It Acts as a Tumor Suppressor by Reducing EZH2 Activity.

Anaplastic thyroid carcinoma (ATC) represents one the most aggressive neoplasias in humans, and, nowadays, limited advances have been made to extend the survival and reduce the mortality of ATC. Thus, the identification of molecular mechanism underlying its progression is needed. Here, we evaluated the long non-coding RNA (lncRNA) expression profile of nine ATC in comparison with five normal thyroid tissues by a lncRNA microarray. By this analysis, we identified 19 upregulated and 28 downregulated lncRNAs with a fold change >1.1 or <-1.1 and p-value < 0.05, in ATC samples. Some of them were subsequently validated by qRT-PCR. Then, we investigated the role of the lncRNA Prader Willi/Angelman region RNA5 (PAR5), drastically and specifically downregulated in ATC. The restoration of PAR5 reduces proliferation and migration rates of ATC-derived cell lines indicating that its downregulation contributes to thyroid cancer progression. Our results suggest that PAR5 exerts its anti-oncogenic role by impairing Enhancer of Zeste Homolog 2 (EZH2) oncogenic activity since we demonstrated that PAR5 interacts with it in thyroid cancer cell lines, reducing EZH2 protein levels and its binding on the E-cadherin promoter, relieving E-cadherin from the negative regulation by EZH2. Consistently, EZH2 is overexpressed in ATC, but not in differentiated thyroid carcinomas. The results reported here define a tumor suppressor role for PAR5 in undifferentiated thyroid neoplasias, further highlighting the pivotal role of lncRNAs in thyroid carcinogenesis.