KLHL14 is a tumor suppressor downregulated in undifferentiated thyroid cancer.

KLHL14 is a substrate-binding subunit of Cullin-RING ligase 3 ubiquitin ligase complex, highly enriched in thyroid since early embryonic development, together with its antisense RNA KLHL14-AS. We have previously demonstrated that Klhl14-AS is a competing endogenous RNA regulating several differentiation and survival factors in thyroid cancer, acting as tumor suppressor. Recently, also KLHL14 has been shown to function as tumor suppressor in diffuse large B-cell lymphoma and in malignant mesothelioma. Here we show that KLHL14 expression is strongly reduced in anaplastic thyroid cancer, the less differentiated and most aggressive type of thyroid neoplasia. Such reduction is reproduced in different in vivo and in vitro models of thyroid cancer, being invariably associated with loss of differentiation. When Klhl14 expression is rescued in thyroid transformed cells, it reduces the cell proliferation rate and increase the number of apoptotic cells. On the other side, Klhl14 loss of function in normal thyroid cells affects the expression of several regulatory as well as functional thyroid markers. All these findings suggest that KLHL14 could be considered as a novel tumor suppressor in thyroid cancer, by also revealing its physiological role in the maintenance of a fully differentiated and functional thyroid phenotype.

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.

Genetics and Epigenetics of Sex Bias: Insights from Human Cancer and Autoimmunity.

High-throughput sequencing and genome-wide association studies have revealed a sex bias in human diseases. The underlying molecular mechanisms remain, however, unknown. Here, we cover recent advances in cancer and autoimmunity focusing on intrinsic genetic and epigenetic differences underlying sex biases in human disease. These studies reveal a central role of genome regulatory mechanisms including genome repair, chromosome folding, and epigenetic regulation in dictating the sex bias. These highlight the importance of considering sex as a variable in both basic science and clinical investigations. Understanding the molecular mechanisms underlying sex bias in human diseases will be instrumental in making a first step forwards into the era of personalized medicine.

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.

Double knock-out of Hmga1 and Hipk2 genes causes perinatal death associated to respiratory distress and thyroid abnormalities in mice.

The serine-threonine kinase homeodomain-interacting protein kinase 2 (HIPK2) modulates important cellular functions during development, acting as a signal integrator of a wide variety of stress signals, and as a regulator of transcription factors and cofactors. We have previously demonstrated that HIPK2 binds and phosphorylates High-Mobility Group A1 (HMGA1), an architectural chromatinic protein ubiquitously expressed in embryonic tissues, decreasing its binding affinity to DNA. To better define the functional role of HIPK2 and HMGA1 interaction in vivo, we generated mice in which both genes are disrupted. About 50% of these Hmga1/Hipk2 double knock-out (DKO) mice die within 12 h of life (P1) for respiratory failure. The DKO mice present an altered lung morphology, likely owing to a drastic reduction in the expression of surfactant proteins, that are required for lung development. Consistently, we report that both HMGA1 and HIPK2 proteins positively regulate the transcriptional activity of the genes encoding the surfactant proteins. Moreover, these mice display an altered expression of thyroid differentiation markers, reasonably because of a drastic reduction in the expression of the thyroid-specific transcription factors PAX8 and FOXE1, which we demonstrate here to be positively regulated by HMGA1 and HIPK2. Therefore, these data indicate a critical role of HIPK2/HMGA1 cooperation in lung and thyroid development and function, suggesting the potential involvement of their impairment in the pathogenesis of human lung and thyroid diseases.

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.

Tissue- and Cell Type-Specific Expression of the Long Noncoding RNA Klhl14-AS in Mouse.

lncRNAs are acquiring increasing relevance as regulators in a wide spectrum of biological processes. The extreme heterogeneity in the mechanisms of action of these molecules, however, makes them very difficult to study, especially regarding their molecular function. A novel lncRNA has been recently identified as the most enriched transcript in mouse developing thyroid. Due to its genomic localization antisense to the protein-encoding Klhl14 gene, we named it Klhl14-AS. In this paper, we highlight that mouse Klhl14-AS produces at least five splicing variants, some of which have not been previously described. Klhl14-AS is expressed with a peculiar pattern, characterized by diverse relative abundance of its isoforms in different mouse tissues. We examine the whole expression level of Klhl14-AS in a panel of adult mouse tissues, showing that it is expressed in the thyroid, lung, kidney, testis, ovary, brain, and spleen, although at different levels. In situ hybridization analysis reveals that, in the context of each organ, Klhl14-AS shows a cell type-specific expression. Interestingly, databases report a similar expression profile for human Klhl14-AS. Our observations suggest that this lncRNA could play cell type-specific roles in several organs and pave the way for functional characterization of this gene in appropriate biological contexts.