Mami Yasukawa

An RNA-dependent RNA polymerase formed by TERT and the RMRP RNA

Constitutive expression of telomerase in human cells prevents the onset of senescence and crisis by maintaining telomere homeostasis. However, accumulating evidence suggests that the human telomerase reverse transcriptase catalytic subunit (TERT) contributes to cell physiology independently of its ability to elongate telomeres. Here we show that TERT interacts with the RNA component of mitochondrial RNA processing endoribonuclease (RMRP), a gene that is mutated in the inherited pleiotropic syndrome cartilage–hair hypoplasia. Human TERT and RMRP form a distinct ribonucleoprotein complex that has RNA-dependent RNA polymerase (RdRP) activity and produces double-stranded RNAs that can be processed into small interfering RNA in a Dicer (also known as DICER1)-dependent manner. These observations identify a mammalian RdRP composed of TERT in complex with RMRP.

Maintenance of tumor initiating cells of defined genetic composition by nucleostemin

Recent work has identified a subset of cells resident in tumors that exhibit properties similar to those found in normal stem cells. Such cells are highly tumorigenic and may be involved in resistance to treatment. However, the genes that regulate the tumor initiating cell (TIC) state are unknown. Here, we show that overexpression of either of the nucleolar GTP-binding proteins nucleostemin (NS) or GNL3L drives the fraction of genetically defined tumor cells that exhibit markers and tumorigenic properties of TICs. Specifically, cells that constitutively express elevated levels of NS or GNL3L exhibit increased TWIST expression, phosphorylation of STAT3, expression of genes that induce pluripotent stem cells, and enhanced radioresistance; in addition, they form tumors even when small numbers of cells are implanted and exhibit an increased propensity to metastasize. GNL3L/NS forms a complex with the telomerase catalytic subunit [human telomerase reverse transcriptase (hTERT)] and the SWItch-Sucrose NonFermentable (SWI-SNF) complex protein brahma-related gene 1 (BRG1), and the expression of each of these components is necessary to facilitate the cancer stem cell state. Together, these observations define a complex composed of TERT, BRG1, and NS/GNL3L that maintains the function of TICs.

Identification of serum anti-human telomerase reverse transcriptase (hTERT) auto-antibodies during progression to hepatocellular carcinoma.

Human telomerase reverse transcriptase, hTERT, is the catalytic component of human telomerase. Expression of hTERT confers telomerase activity, indicating that hTERT is the rate-limiting component of human telomerase. Here we report the detection of anti-hTERT auto-antibodies in the sera derived patients with hepatocellular carcinoma using recombinant, purified hTERT as an antigen in an enzyme linked immunosorbent assay (ELISA). The levels of anti-hTERT antibodies in serum correlated with progression to hepatocellular carcinoma. In contrast, we detected only low levels of anti-hTERT auto-antibodies in the sera derived from 18 normal volunteers. The observation of hTERT auto-antibodies in the sera derived from cancer patients suggests that such auto-antibodies constitute novel and specific tumor marker.

Involvement of Telomerase Reverse Transcriptase in Heterochromatin Maintenance

ABSTRACT In the fission yeast Schizosaccharomyces pombe, centromeric heterochromatin is maintained by an RNA-directed RNA polymerase complex (RDRC) and the RNA-induced transcriptional silencing (RITS) complex in a manner that depends on the generation of short interfering RNA. In association with the telomerase RNA component (TERC), the telomerase reverse transcriptase (TERT) forms telomerase and counteracts telomere attrition, and without TERC, TERT has been implicated in the regulation of heterochromatin at locations distinct from telomeres. Here, we describe a complex composed of human TERT (hTERT), Brahma-related gene 1 (BRG1), and nucleostemin (NS) that contributes to heterochromatin maintenance at centromeres and transposons. This complex produced double-stranded RNAs homologous to centromeric alpha-satellite (alphoid) repeat elements and transposons that were processed into small interfering RNAs targeted to these heterochromatic regions. These small interfering RNAs promoted heterochromatin assembly and mitotic progression in a manner dependent on the RNA interference machinery. These observations implicate the hTERT/BRG1/NS (TBN) complex in heterochromatin assembly at particular sites in the mammalian genome.

Telomerase Reverse Transcriptase Regulates microRNAs

MicroRNAs are small non-coding RNAs that inhibit the translation of target mRNAs. In humans, most microRNAs are transcribed by RNA polymerase II as long primary transcripts and processed by sequential cleavage of the two RNase III enzymes, DROSHA and DICER, into precursor and mature microRNAs, respectively. Although the fundamental functions of microRNAs in RNA silencing have been gradually uncovered, less is known about the regulatory mechanisms of microRNA expression. Here, we report that telomerase reverse transcriptase (TERT) extensively affects the expression levels of mature microRNAs. Deep sequencing-based screens of short RNA populations revealed that the suppression of TERT resulted in the downregulation of microRNAs expressed in THP-1 cells and HeLa cells. Primary and precursor microRNA levels were also reduced under the suppression of TERT. Similar results were obtained with the suppression of either BRG1 (also called SMARCA4) or nucleostemin, which are proteins interacting with TERT and functioning beyond telomeres. These results suggest that TERT regulates microRNAs at the very early phases in their biogenesis, presumably through non-telomerase mechanism(s).

Eribulin mesylate targets human telomerase reverse transcriptase in ovarian cancer cells.

Treatment of advanced ovarian cancer involves platinum-based chemotherapy. However, chemoresistance is a major obstacle. Cancer stem cells (CSCs) are thought to be one of the causes of chemoresistance, but the underlying mechanism remains elusive. Recently, human telomerase reverse transcriptase (hTERT) has been reported to promote CSC-like traits. In this study, we found that a mitotic inhibitor, eribulin mesylate (eribulin), effectively inhibited growth of platinum-resistant ovarian cancer cell lines. Eribulin-sensitive cells showed a higher efficiency for sphere formation, suggesting that these cells possess an enhanced CSC-like phenotype. Moreover, these cells expressed a higher level of hTERT, and suppression of hTERT expression by siRNA resulted in decreased sensitivity to eribulin, suggesting that hTERT may be a target for eribulin. Indeed, we found that eribulin directly inhibited RNA-dependent RNA polymerase (RdRP) activity, but not telomerase activity of hTERT in vitro. We propose that eribulin targets the RdRP activity of hTERT and may be an effective therapeutic option for CSCs. Furthermore, hTERT may be a useful biomarker to predict clinical responses to eribulin.

De Novo RNA Synthesis by RNA-Dependent RNA Polymerase Activity of Telomerase Reverse Transcriptase

ABSTRACT RNA-dependent RNA polymerase (RdRP) plays key roles in RNA silencing to generate double-stranded RNAs. In model organisms, such as Caenorhabditis elegans and Neurospora crassa, two types of small interfering RNAs (siRNAs), primary siRNAs and secondary siRNAs, are expressed; RdRP produces secondary siRNAs de novo, without using either Dicer or primers, while primary siRNAs are processed by Dicer. We reported that human telomerase reverse transcriptase (TERT) has RdRP activity and produces endogenous siRNAs in a Dicer-dependent manner. However, de novo synthesis of siRNAs by human TERT has not been elucidated. Here we show that the TERT RdRP generates short RNAs that are complementary to template RNAs and have 5′-triphosphorylated ends, which indicates de novo synthesis of the RNAs. In addition, we confirmed short RNA synthesis by TERT in several human carcinoma cell lines and found that TERT protein levels are positively correlated with RdRP activity.

ATF7 mediates TNF-α–induced telomere shortening

Abstract Telomeres maintain the integrity of chromosome ends and telomere length is an important marker of aging. The epidemiological studies suggested that many types of stress including psychosocial stress decrease telomere length. However, it remains unknown how various stresses induce telomere shortening. Here, we report that the stress-responsive transcription factor ATF7 mediates TNF-α–induced telomere shortening. ATF7 and telomerase, an enzyme that elongates telomeres, are localized on telomeres via interactions with the Ku complex. In response to TNF-α, which is induced by various stresses including psychological stress, ATF7 was phosphorylated by p38, leading to the release of ATF7 and telomerase from telomeres. Thus, a decrease of ATF7 and telomerase on telomeres in response to stress causes telomere shortening, as observed in ATF7-deficient mice. These findings give credence to the idea that various types of stress might shorten telomere.

Semi-quantitative Detection of RNA-dependent RNA Polymerase Activity of Human Telomerase Reverse Transcriptase Protein

Human telomerase reverse transcriptase (TERT) is the catalytic subunit of telomerase, and it elongates telomere through RNA-dependent DNA polymerase activity. Although TERT is named as a reverse transcriptase, structural and phylogenetic analyses of TERT demonstrate that TERT is a member of right-handed polymerases, and relates to viral RNA-dependent RNA polymerases (RdRPs) as well as viral reverse transcriptase. We firstly identified RdRP activity of human TERT that generates complementary RNA stand to a template non-coding RNA and contributes to RNA silencing in cancer cells. To analyze this non-canonical enzymatic activity, we developed RdRP assay with recombinant TERT in 2009, thereafter established in vitro RdRP assay for endogenous TERT. In this manuscript, we describe the latter method. Briefly, TERT immune complexes are isolated from cells, and incubated with template RNA and rNTPs including radioactive rNTP for RdRP reaction. To eliminate single-stranded RNA, reaction products are treated with RNase I, and the final products are analyzed with polyacrylamide gel electrophoresis. Radiolabeled RdRP products can be detected by autoradiography after overnight exposure.

Abstract 3117: Eribulin is a novel TERT-targeting inhibitor that penetrates into intracerebrally grown glioblastomas and suppresses their growth in mice

[Introduction] No effective molecular targeting therapy has been clinically developed for patients with glioblastoma (GBM) despite of intense genome analysis. Approximately 60-80% of GBM harbor mutations in the promoter region of TERT that leads to its upregulation, making it the most common single genetic abnormalities in GBM. TERT, a reverse-transcriptase subunit of telomerase, has been an attractive candidate for therapy target because of their observed upregulation in a wide variety of cancers, including GBMs, a phenomenon that presumably helps maintain telomeres for their indefinite proliferation. It has been reported recently that TERT has an RNA-dependent RNA polymerase (RdRP) activity, with which TERT is involved in maintenance of stem cell phenotype and mitotic progression. We have previously identified eribulin as a specific inhibitor of RdRP activity of TERT through drug screening and shown that eribulin suppressed growth of ovarian cancers with high TERT expression. In order to investigate an efficacy of eribulin as a novel TERT-targeted therapy against GBM, we investigated the antitumor effect of eribulin in cultured cell and brain tumor model using GBM cell lines. [Methods] The TERT promoter status was examined in 20 glioma cell lines. Seven cell lines were subjected to in vitro cytotoxicity assay. U87MG was either subcutaneously or intracranially transplanted in athymic mice. Tissue or plasma concentration of eribulin was measured by LC-MS/MS. [Results] All GBM cell lines tested that harbored TERT promoter mutations including U87MG, U251MG, U118MG as well as several patient-derived glioblastoma sphere culture cells were highly sensitive to eribulin, IC50 below 1nM. Intraperitoneal administration of eribulin completely suppressed the growth of U87MG subcutaneous tumor in nude mice, and the RdRP activities in treated tumors were significantly decreased in a dose-dependent manner. In the brain tumor model, a high concentration of eribulin was detected in the brain tumor tissues as early as 15 minutes after intravenous injection of eribulin, which remained stable even 24 hours later when the plasma concentration of eribulin became undetectable. Finally, intraperitoneal administration of eribulin significantly prolonged the survival of mice with intracerebrally transplanted U87MG xenograft (p [Conclusion] Our results showed that eribulin efficiently transfers into brain tumor tissues and has a strong antitumor effect against GBM cells through inhibition of RdRP activity. Eribulin thus appears to be a promising novel TERT-targeting therapeutic agent against GBM. A clinical trial is being scheduled. Citation Format: Masamichi Takahashi, Shunichiro Miki, Yuko Matsushita, Kohei Fukuoka, Yoshiko Maida, Mami Yasukawa, Mitsuhiro Hayashi, Akinobu Hamada, Akitake Mukasa, Ryo Nishikawa, Kenji Tamura, Yoshitaka Narita, Kenkichi Masutomi, Koichi Ichimura. Eribulin is a novel TERT-targeting inhibitor that penetrates into intracerebrally grown glioblastomas and suppresses their growth in mice [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2017; 2017 Apr 1-5; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2017;77(13 Suppl):Abstract nr 3117. doi:10.1158/1538-7445.AM2017-3117