TERRA beyond cancer: the biology of telomeric repeat‐containing RNAs in somatic and germ cells

The telomeric noncoding RNA TERRA is a key component of telomeres and it is widely expressed in normal as well as cancer cells. In the last 15 years, several publications have shed light on the role of TERRA in telomere homeostasis and cell survival in cancer cells. However, only few studies have investigated the regulation or the functions of TERRA in normal tissues. A better understanding of the biology of TERRA in non-cancer cells may provide unexpected insights into how these lncRNAs are transcribed and operate in cells, and their potential role in physiological processes, such as aging, age-related pathologies, inflammatory processes and human genetic diseases. In this review we aim to discuss the findings that have advanced our understanding of the biology of TERRA using non-cancer mammalian cells as a model system

Expression of Cellular and Extracellular TERRA, TERC and TERT in Hepatocellular Carcinoma

Non-coding RNAs are transcribed from telomeres and the telomeric repeat-containing RNAs (TERRA) are implicated in telomere homeostasis and in cancer. In this study, we aimed to assess in hepatocellular carcinoma (HCC) the cellular and extracellular expression of TERRA, the telomerase RNA subunit (TERC) and the telomerase catalytic subunit (TERT). We determined by qPCR the expression level of TERRA 1_2_10_13q, TERRA 15q, TERRA XpYp, TERC and of TERT mRNA in HCC tissues and in the plasma of HCC patients. Further, we profiled the same transcripts in the HCC cell lines, HA22T/VGH and SKHep1C3, and in the extracellular vesicles (EVs) derived from their secretomes. We found that the expression of TERRA and TERT mRNA was significantly deregulated in HCC, being TERRA downregulated and TERT mRNA upregulated in HCC tissues vs. the peritumoral (PT) ones, and the receiver operating characteristic (ROC) curve analyses revealed a significant ability in discriminating HCC from PT tissue. Further, the determinations of circulating TERRA and TERC showed higher amounts of these transcripts in the plasma of HCC patients vs. controls and ROC analyses gave significant results. The expression characterization of the cultured HCC cells showed their ability to produce and secrete TERRA and TERC into the EVs; the ability to produce TERT mRNA that was not detectable in the EVs; and the ability to respond to sorafenib treatment increasing TERRA expression. Our results highlight that: (i) both cellular and extracellular expressions of TERRA and TERC are dysregulated in HCC as well as the cellular expression of TERT mRNA and (ii) the combined detection of TERRA and TERC in plasma may represent a promising approach for non-invasive diagnostic molecular indicators of HCC

MicroRNA-199b-5p Impairs Cancer Stem Cells through Negative Regulation of HES1 in Medulloblastoma

BACKGROUND: Through negative regulation of gene expression, microRNAs (miRNAs) can function in cancers as oncosuppressors, and they can show altered expression in various tumor types. Here we have investigated medulloblastoma tumors (MBs), which arise from an early impairment of developmental processes in the cerebellum, where Notch signaling is involved in many cell-fate-determining stages. MBs occur bimodally, with the peak incidence seen between 3-4 years and 8-9 years of age, although it can also occur in adults. Notch regulates a subset of the MB cells that have stem-cell-like properties and can promote tumor growth. On the basis of this evidence, we hypothesized that miRNAs targeting the Notch pathway can regulated these phenomena, and can be used in anti-cancer therapies. METHODOLOGY/PRINCIPAL FINDINGS: In a screening of MB cell lines, the miRNA miR-199b-5p was seen to be a regulator of the Notch pathway through its targeting of the transcription factor HES1. Down-regulation of HES1 expression by miR-199b-5p negatively regulates the proliferation rate and anchorage-independent growth of MB cells. MiR-199b-5p over-expression blocks expression of several cancer stem-cell genes, impairs the engrafting potential of MB cells in the cerebellum of athymic/nude mice, and of particular interest, decreases the MB stem-cell-like (CD133+) subpopulation of cells. In our analysis of 61 patients with MB, the expression of miR-199b-5p in the non-metastatic cases was significantly higher than in the metastatic cases (P = 0.001). Correlation with survival for these patients with high levels of miR-199b expression showed a positive trend to better overall survival than for the low-expressing patients. These data showing the down-regulation of miR-199b-5p in metastatic MBs suggest a potential silencing mechanism through epigenetic or genetic alterations. Upon induction of de-methylation using 5-aza-deoxycytidine, lower miR-199b-5p expression was seen in a panel of MB cell lines, supported an epigenetic mechanism of regulation. Furthermore, two cell lines (Med8a and UW228) showed significant up-regulation of miR-199b-5p upon treatment. Infection with MB cells in an induced xenograft model in the mouse cerebellum and the use of an adenovirus carrying miR-199b-5p indicate a clinical benefit through this negative influence of miR-199b-5p on tumor growth and on the subset of MB stem-cell-like cells, providing further proof of concept. CONCLUSIONS/SIGNIFICANCE: Despite advances in our understanding of the pathogenesis of MB, one-third of these patients remain incurable and current treatments can significantly damage long-term survivors. Here we show that miR-199b-5p expression correlates with metastasis spread, identifying a new molecular marker for a poor-risk class in patients with MB. We further show that in a xenograft model, MB tumor burden can be reduced, indicating the use of miR199b-5p as an adjuvant therapy after surgery, in combination with radiation and chemotherapy, for the improvement of anti-cancer MB therapies and patient quality of life. To date, this is the first report that expression of a miRNA can deplete the tumor stem cells, indicating an interesting therapeutic approach for the targeting of these cells in brain tumors

miR34a targets the Notch pathway in medulloblastoma

Medulloblastoma (MB) is a highly aggressive cancer that mostly affects children, through developmental impairment of the cerebellum. Several miR34a targets belong to the Notch pathway, a signaling pathway that is involved in cerebellum development and that is aberrantly activated during MB tumorigenesis. We show here that miR34a over-expression transiently down-regulates the Notch ligand Delta-like 1 (DLL1) protein levels and also results in Notch1 activation and Notch2 signaling inhibition, an effect that we see in the MB Daoy and D283-MED cells. Moreover, ectopic expression of miR34a in MB cells results in impairment of proliferation rate and soft agar colony formation, while inducing apoptosis and differentiation processes. Noteworthy, the induction of apoptosis was inhibited by ectopic expression of DLL1, suggesting the involvement of DLL1 down-regulation in this process. We also provide evidences that the endogenous expression of miR34a can down-regulate DLL1 protein levels and contribute to the p53-induced apoptosis in Daoy cells, upon doxorubicin stimulation. Induction of miR34a correlates with DLL1 protein down-regulation also in neuroblastoma and breast cancer cells and expression of miR34b and miR34c can contribute to the DLL1 down-regulation. Finally, we report that miR34a, miR34b and miR34c, are down-regulated in human MB tumors. These results suggest that miR34a has an onco-suppressor function in MB and that it can thus be targeted for future therapeutic applications

Telomeric repeat-containing RNA TERRA: a noncoding RNA connecting telomere biology to genome integrity

Telomeres are dynamic nucleoprotein structures that protect the ends of chromosomes from degradation and activation of DNA damage response. For this reason, telomeres are essential to genome integrity. Chromosome ends are enriched in heterochromatic marks and proper organization of telomeric chromatin is important to telomere stability. Despite their heterochromatic state, telomeres are transcribed giving rise to long noncoding RNAs (lncRNA) called TERRA (telomeric repeat-containing RNA). TERRA molecules play critical roles in telomere biology, including regulation of telomerase activity and heterochromatin formation at chromosome ends. Emerging evidence indicate that TERRA transcripts form DNA-RNA hybrids at chromosome ends which can promote homologous recombination among telomeres, delaying cellular senescence and sustaining genome instability. Intriguingly, TERRA RNA-telomeric DNA hybrids are involved in telomere length homeostasis of telomerase-negative cancer cells. Furthermore, TERRA transcripts play a role in the DNA damage response (DDR) triggered by dysfunctional telomeres. We discuss here recent developments on TERRA's role in telomere biology and genome integrity, and its implication in cancer

Smc5/6 Is a Telomere-Associated Complex that Regulates Sir4 Binding and TPE

<div><p>SMC proteins constitute the core members of the Smc5/6, cohesin and condensin complexes. We demonstrate that Smc5/6 is present at telomeres throughout the cell cycle and its association with chromosome ends is dependent on Nse3, a subcomponent of the complex. Cells harboring a temperature sensitive mutant, <i>nse3</i>-1, are defective in Smc5/6 localization to telomeres and have slightly shorter telomeres. Nse3 interacts physically and genetically with two Rap1-binding factors, Rif2 and Sir4. Reduction in telomere-associated Smc5/6 leads to defects in telomere clustering, dispersion of the silencing factor, Sir4, and a loss in transcriptional repression for sub-telomeric genes and non-coding telomeric repeat-containing RNA (TERRA). <i>SIR4</i> recovery at telomeres is reduced in cells lacking Smc5/6 functionality and vice versa. However, <i>nse3</i>-1/ <i>sir4</i> Δ double mutants show additive defects for telomere shortening and TPE indicating the contribution of Smc5/6 to telomere homeostasis is only in partial overlap with SIR factor silencing. These findings support a role for Smc5/6 in telomere maintenance that is separate from its canonical role(s) in HR-mediated events during replication and telomere elongation.</p></div