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Anaplastic thyroid carcinoma (ATC) is a rare and aggressive endocrine tumor with highly undifferentiated morphology. It has been suggested that cancer stem cells (CSCs) might play a central role in ATC. The objectives of this study were (i) to characterize CSCs from ex vivo ATC specimens by investigating the expression of several pluripotent stem cell markers, and (ii) to evaluate in vitro drug resistance modifications after specific CSC transcription factor switch-off.In ex vivo experiments, eight formalin-fixed, paraffin-embedded ATC specimens were analyzed by reverse-transcription and real-time quantitative PCR and immunohistochemistry. In in vitro experiments using ATC SW1736 cells, the expression levels of OCT-4, NANOG, and ABCG2 and the sensitivity to either cisplatin or doxorubicin were evaluated after silencing.OCT-4, KLF4, and SOX2 transcription factors and C-KIT and THY-1 stem surface antigens showed variable up-regulation in all ATC cases. The SW1736 cell line was characterized by a high percentage of stem population (10.4±2.1% of cells were aldehyde dehydrogenase positive) and high expression of several CSC markers (SOX2, OCT4, NANOG, C-MYC, and SSEA4). SOX2 silencing down-regulated OCT-4, NANOG, and ABCG2. SOX2 silencing sensitized SW1736 cells, causing a significant cell death increase (1.8-fold) in comparison to control cells with 10 μM cisplatin (93.9±3.4% vs. 52.6±9.4%, p0.01) and 2.7 fold with 0.5 μM doxorubicin (45.8±9.9% vs. 17.1±3.4% p0.01). ABCG2 silencing caused increased cell death with both cisplatin (74.9±1.4%) and doxorubicin treatment (74.1±0.1%) vs. no-target-treated cells (respectively, 45.8±1.0% and 48.6±1.0%, p0.001).The characterization of CSCs in ATC through the analysis of multiple pluripotent stem cell markers might be useful in identifying cells with a stem-like phenotype capable of resisting conventional chemotherapy. In addition, our data demonstrate that SOX2 switch-off through ABCG2 transporter down-regulation has a major role in overcoming CSC chemotherapy resistance.

Following publication of the original article in Stem Cell Research and Therapy [1], it was brought to our attention that panel 5E in Fig. 5 is a duplicate of panel 5F. Please find below the figure with the correct panel E. We apologize for the inconvenience this may have caused.

RNA mediated trans-Activation is a proposed mechanism involved in the setting of the epigenetic state of chromatin. It has been studied first in Drosophila melanogaster where it seems that at least some transcribed loci are marked as transcriptionally active by their own transcripts. This effect acts in trans and in some cases could give rise to a transgenerational paramutational-like effect. This work studies RNA mediated trans-activation in the light of one of its possible applications, specifically its use as a therapeutic strategy for the incurable and highly aggressive ATC (anaplastic thyroid cancer). We explore the possibility to reactivate the Thyroid specific NIS (Natrium-Iodine symport) via the expression of ncRNAs with sequence homology with transcripts from the NIS coding locus itself. This work suggests that RNA trans-activation is a conserved mechanism and it may be used in human to manipulate the gene expression.

We present a classic interactome bioinformatic analysis and a study on competing endogenous (ce) RNAs for hTERT. The hTERT gene codes for the catalytic subunit and limiting component of the human telomerase complex. Human telomerase reverse transcriptase (hTERT) is essential for the integrity of telomeres. Telomere dysfunctions have been widely reported to be involved in aging, cancer, and cellular senescence. The hTERT gene network has been analyzed using the BioGRID interaction database (http://thebiogrid.org/) and related analysis tools such as Osprey (http://biodata.mshri.on.ca/osprey/servlet/Index) and GeneMANIA (http://genemania.org/). The network of interaction of hTERT transcripts has been further analyzed following the competing endogenous (ce) RNA hypotheses (messenger [m] RNAs cross-talk via micro [mi] RNAs) using the miRWalk database and tools (www.ma.uni-heidelberg.de/apps/zmf/mirwalk/). These analyses suggest a role for Akt, nuclear factor-κB (NF-κB), heat shock protein 90 (HSP90), p70/p80 autoantigen, 14-3-3 proteins, and dynein in telomere functions. Roles for histone acetylation/deacetylation and proteoglycan metabolism are also proposed.

HSP90 activities decline during ageing. Noteworthy, HSP90 mediated retrotransposon silencing seems to have a key role in the maintenance of genome integrity. Furthermore, inhibition of HSP90 activities lead to cell senescence and apoptosis. Original bioinformatics analyses and literature data will be discussed in the light of the potential involvement of HSP90 in ageing progression and ageing related diseases.

Cancer stem cells (CSC) are believed to play a central role in oncogenesis, but until today their isolation and characterization is still particularly complex. Anaplastic thyroid cancer (ATC) presents several characteristics suggestive of a tumour highly enriched in CSC (high mitotic rate, poor prognosis, high aggressiveness, resistance to treatments, etc). For these reasons ATC represents a good candidate to study CSCs. SOX2 is a key stem transcriptional factor, usually only transiently expressed, that plays a fundamental role in stem cell identity. SOX2 proved to be constitutively expressed in SW1736 cell line, a well established and recognized ATC cell line. The bioinformatics ceRNA analysis permits to discover gene transcripts that may be post-transcriptionally positively co-regulated with the 3’UTR probe. We aimed to perform ceRNA analysis in SW1736 to identify candidate genes that might be involved in SOX2 functional network and in turn in stemness and CSC biogenesis. A competing endogenous RNA (ceRNA) in silico analysis was performed on 3’UTR of SOX2 mRNA. Our analysis harvested several genes involved in the RNA interference mechanism (DICER1, DROSHA, AGO2) and in cell cycle control (TP53, CCND1). To further validate the in silico analysis, in vitro analysis via RT-PCR was performed. Knocking down SOX2 in SW1736 (0.417 expression rate), the interacting ceRNA transcripts were coherently downregulated with significant differences (expression rates from 0.355 to 0.705, p < 0.05). Moreover, a statistical analysis of the ceRNAs in other cell lines and clinical specimens revealed a positive correlation with the expression of TP53, DICER1, DROSHA and AGO2, suggesting the existence of a fine regulatory network.

Thyroid cancer is the most frequent endocrine malignancy with a global increasing incidence. Many evidences show that sulphoraphane (SF), a natural isothiocyante found in cruciferous vegetables has a wide range of chemopreventive as well as apoptosis inducing properties. The ability of SF in inducing apoptosis and cell cycle arrest is associated with the regulation of many proteins including Bcl-2 family proteins, caspases, p21, and cyclin dependent kinases. In the present work, we investigated in vitro the activity of SF in three human thyroid cancer cell lines. For this purpose we studied SW1736 (ATC), BC-PAP (PTC) and TT (MTC) cell line by MTT assay, after addition of SF ranging from 0 to 20M. Cell lines were treated with SF at different concentration (1M, 5M, 10M, 20M) for 72 hours and after treatment we observed a SF- induced toxicity in all thyroid cancer cell lines, whereas SF had not significant effects on nonmalignant cells. In order to demonstrate the role of SF in the apoptotic pathway, we analyzed its effect on Bcl-2 and BAX protein expression by western blot. The increase of SF concentration induce an upregulation of BAX followed by down-regulation of Bcl-2, confirming its pro-apoptotic role in all three cell lines. So, our preliminary data, suggest its possible use in prevention trial in high-risk areas and as enhancer in anti-neoplastic therapy of thyroid cancer.

Messenger RNA (mRNA) translation efficiency is regulated by microRNAs. Each microRNA is able to regulate the translation of multiple mRNAs and each mRNA is regulated by multiple microRNAs. Thus, cellular mRNAs pool competed for microRNAs pool and viceversa. The regulatory network between mRNAs and microRNAs can be studied in the perspective of Competing Endogenous RNAs or ceRNAs. Here it is presented a bioinformatic study on ceRNAs for human telomerase (hTERT). Several genes potentially involved in the regulatory network of hTERT have been harvested by this study. hTERT is essential for the telomeres integrity. Telomere dysfunctions have been widely reported to be involved in Ageing, Cancer and Cellular Senescence. Amongst the gene collected, the oncosuppressor PTEN and the dynein heavy chain coding gene DNHD1 are top level interactors. Interestingly, many genes of unknow functions results as predicted interactors, suggesting that hTERT may be involved in unexplored network and scenarios.

In the last decade non-coding RNAs (ncRNAs) have emerged as cellular key regulators. The attention of the scientific community has focused on ncRNAs with repressive features on eukaryotic transcriptional regulation. Many experimental evidences suggest that ncRNAs could also positively regulate transcription. The RNA-Memory Model (Arancio W. Rejuvenation Res. 2010 Apr-Jun;13(2-3):365-72.) gives possible explanations to several biological phenomena via trans-acting ncRNAs (memRNAs) able to orchestrate chromatin remodelling and in turn enhance transcription. memRNAs assert their functions especially during the post-mitotic chromatin remodelling. memRNAs can mark the genes transcribed in the mother cell that must be re-activate after the cell division. During the M phase of the cell cycle the chromatin is almost totally collapsed and the transcription is turned off. RNA memory model explains easily how the epigenetic state can be re-established after mitosis. RNA memory, e. g., can easily explain why the interference machinery is needed for the establishment of the silenced state of a gene but it is not needed for its maintenance: the interference machinery drives the degradation of specific memRNAs in the mother cell; so in the next cell generation the gene is maintained silenced, and so on through generations, if no external stimuli perturb the state. RNA memory model fits perfectly with other models on the epigenetic inheritance (epigenetic histone code readers/writers, histone variants, PcG/trxG interaction, structural epigenetic memory, etc). The relationship of RNA memory with other models will be discussed specifically. An RNA-Memory model explanation of controversial biological phenomena will be suggested. If the model is correct, the impact in the comprehension of transcriptional regulation events could be enormous.

Hutchinson-Gilford progeria syndrome (HGPS) is a rare human genetic disease that leads to premature ageing and ageing-associated phenotype. HGPS is caused by mutation in the lamin A (LMNA) gene that leads, in affected young individuals, to the generation of progerin, a splicing mutant of lamin A. A bioinformatics analysis of the LMNA gene network of interactions is presented. Lamin A seems to be involved in epigenetic regulation of transcription, chromatin remodelling, DNA repair, with key roles in stemness regulation, normal ageing and telomere functions. The study suggests particular relevance of chromatin remodellers and histones covalent modifiers in the LMNA network. Specifically, HTATIP histone acetylase seems to be of particular relevance in the network.