Telomerase Reverse Transcriptase Expression Research Articles

Interplay between TERT promoter mutations and methylation culminates in chromatin accessibility and TERT expression.

The telomerase reverse transcriptase (TERT) gene is responsible for telomere maintenance in germline and stem cells, and is re-expressed in 90% of human cancers. CpG methylation in the TERT promoter (TERTp) was correlated with TERT mRNA expression. Furthermore, two hotspot mutations in TERTp, dubbed C228T and C250T, have been revealed to facilitate binding of transcription factor ETS/TCF and subsequent TERT expression. This study aimed to elucidate the combined contribution of epigenetic (promoter methylation and chromatin accessibility) and genetic (promoter mutations) mechanisms in regulating TERT gene expression in healthy skin samples and in melanoma cell lines (n = 61). We unexpectedly observed that the methylation of TERTp was as high in a subset of healthy skin cells, mainly keratinocytes, as in cutaneous melanoma cell lines. In spite of the high promoter methylation fraction in wild-type (WT) samples, TERT mRNA was only expressed in the melanoma cell lines with either high methylation or intermediate methylation in combination with TERT mutations. TERTp methylation was positively correlated with chromatin accessibility and TERT mRNA expression in 8 melanoma cell lines. Cooperation between epigenetic and genetic mechanisms were best observed in heterozygous mutant cell lines as chromosome accessibility preferentially concerned the mutant allele. Combined, these results suggest a complex model in which TERT expression requires either a widely open chromatin state in TERTp-WT samples due to high methylation throughout the promoter or a combination of moderate methylation fraction/chromatin accessibility in the presence of the C228T or C250T mutations.

An institutional experience evaluating hTERT immunostaining in 100 consecutive ThinPrep urine specimens

Studies have shown that expression of human telomerase reverse transcriptase (hTERT) in mature urothelial cells indicates an increased risk of urothelial carcinoma. We evaluated the utility of immunocytochemistry with a commercially available anti-hTERT antibody (SCD-A7) in 100 consecutive urine cytology specimens using ThinPrep processing. ThinPrep slides prepared from 100 consecutive urine specimens were stained using anti-hTERT antibody (SCD-A7) after staining optimization had been successfully completed. Patient demographics, cytology diagnoses, histologic follow-up data, and anti-hTERT staining results were recorded. The cytology diagnoses included 7 cases of high-grade urothelial carcinoma (HGUC), 2 cases suspicious for HGUC (SHGUC), 24 cases of atypical urothelial cells (AUCs), and 67 cases negative for HGUC (NHGUC). Of 92 samples, 68 (74%) were positive and 24 (26%) were negative for anti-hTERT staining. Although 31 of 32 specimens (97%) with a diagnosis of AUCs and greater showed positive staining, 37 of 60 NHGUC cases (62%) were also positive for anti-hTERT. Although the HGUC and suspicious for HGUC cases were more likely to show strong staining (6 of 9; 67%), 7 AUC (32%) and 8 NHGUC (22%) cases also demonstrated strong staining. Eight samples (8%) were unsatisfactory for interpretation. Anti-hTERT staining of nonurothelial cells was seen in 77 of 92 samples (84%). Interpretation of anti-hTERT immunocytochemical staining of ThinPrep material is challenging owing to obscuring of nonurothelial cell staining and difficulty discerning individual urothelial cell cytomorphology when the cells are stained. The significance of the large number of anti-hTERT-positive but cytology-negative cases in our study is uncertain.

Telomerase Reverse Transcriptase (TERT) Expression, Telomerase Activity, and Expression of Matrix Metalloproteinases (MMP)-1/-2/-9 in Feline Oral Squamous Cell Carcinoma Cell Lines Associated With Felis catus Papillomavirus Type-2 Infection.

Telomerase activity contributes to cell immortalization by avoiding telomere shortening at each cell division; indeed, its catalytic subunit telomerase reverse transcriptase (TERT) is overexpressed in many tumors, including human oral squamous cell carcinoma (hOSCC). In these tumors, matrix metalloproteinases (MMPs), a group of zinc-dependent endopeptidases involved in cell migration, contribute to invasive potential of cancer cells. A proportion of hOSCC is associated with infection by high-risk human papillomavirus (HR-HPVs), whose E6 oncogene enhances TERT and MMPs expression, thus promoting cancer progression. Feline oral squamous cell carcinoma (FOSCC) is a malignant tumor with highly invasive phenotype; however, studies on telomerase activity, TERT, and MMPs expression are scarce. In this study, we demonstrate telomerase activity, expression of TERT, and its transcriptional activator cMyc along with expression of MMP-1, -2, and -9 in FOSCC-derived cell lines SCCF2 and SCCF3, suggesting a contribution by these pathways in cell immortalization and invasion in these tumors. Recent studies suggest that a sub-group of FOSCC as well as SCCF2 and SCCF3 are associated with Felis catus PV type-2 (FcaPV-2) infection. However, in this work, FcaPV-2 E6 gene knock-down caused no shift in either TERT, cMyc, or MMPs levels, suggesting that, unlike its human counterpart, the viral oncogene plays no role in their regulation.

The Solo Play of TERT Promoter Mutations.

The reactivation of telomerase reverse transcriptase (TERT) protein is the principal mechanism of telomere maintenance in cancer cells. Mutations in the TERT promoter (TERTp) are a common mechanism of TERT reactivation in many solid cancers, particularly those originating from slow-replicating tissues. They are associated with increased TERT levels, telomere stabilization, and cell immortalization and proliferation. Much effort has been invested in recent years in characterizing their prevalence in different cancers and their potential as biomarkers for tumor stratification, as well as assessing their molecular mechanism of action, but much remains to be understood. Notably, they appear late in cell transformation and are mutually exclusive with each other as well as with other telomere maintenance mechanisms, indicative of overlapping selective advantages and of a strict regulation of TERT expression levels. In this review, we summarized the latest literature on the role and prevalence of TERTp mutations across different cancer types, highlighting their biased distribution. We then discussed the need to maintain TERT levels at sufficient levels to immortalize cells and promote proliferation while remaining within cell sustainability levels. A better understanding of TERT regulation is crucial when considering its use as a possible target in antitumor strategies.

Epigenetic Targeting of TERT-Associated Gene Expression Signature in Human Neuroblastoma with TERT Overexpression

Neuroblastoma is a deadly pediatric solid tumor with infrequent recurrent somatic mutations. Particularly, the pathophysiology of tumors without MYCN amplification remains poorly defined. Utilizing an unbiased approach, we performed gene set enrichment analysis of RNA-sequencing data from 498 patients with neuroblastoma and revealed a differentially overexpressed gene signature in MYCN nonamplified neuroblastomas with telomerase reverse transcriptase (TERT) gene overexpression and coordinated activation of oncogenic signaling pathways, including E2Fs, Wnt, Myc, and the DNA repair pathway. Promoter rearrangement of the TERT gene juxtaposes the coding sequence to strong enhancer elements, leading to TERT overexpression and poor prognosis in neuroblastoma, but TERT-associated oncogenic signaling remains unclear. ChIP-seq analysis of the human CLB-GA neuroblastoma cells harboring TERT rearrangement uncovered genome-wide chromatin co-occupancy of Brd4 and H3K27Ac and robust enrichment of H3K36me3 in TERT and multiple TERT-associated genes. Brd4 and cyclin-dependent kinases (CDK) had critical regulatory roles in the expression and chromatin activation of TERT and multiple TERT-associated genes. Epigenetically targeting Brd4 or CDKs with their respective inhibitors suppressed the expression of TERT and multiple TERT-associated genes in neuroblastoma with TERT overexpression or MYCN amplification. ChIP-seq and ChIP-qPCR provided evidence that the CDK inhibitor directly inhibited Brd4 recruitment to activate chromatin globally. Therefore, inhibiting Brd4 and CDK concurrently with AZD5153 and dinaciclib would be most effective in tumor growth suppression, which we demonstrated in neuroblastoma cell lines, primary human cells, and xenografts. In summary, we describe a unique mechanism in neuroblastoma with TERT overexpression and an epigenetically targeted novel therapeutic strategy. SIGNIFICANCE: Epigenetically cotargeting Brd4 and Cdks suppresses human neuroblastoma with TERT overexpression by inhibiting the TERT-associated gene expression networks.

The crustacean Armadillidium vulgare (Latreille, 1804) (Isopoda: Oniscoidea), a new promising model for the study of cellular senescence

AbstractSenescence, the decline of physiological parameters with increasing age, is a quasi-ubiquitous phenomenon in the living world. The observed patterns of senescence, however, can markedly differ across species and populations, between sexes, and even among individuals. To identify the drivers of this variation in senescence, experimental approaches are essential and involve the development of tools and new study models. Current knowledge of the senescence process is mostly based on studies on vertebrates and the main information about senescence in invertebrates is mostly limited to model organisms such as Caenorhabditis elegans or Drosophila melanogaster. In this context, we tested whether biomarkers of vertebrate ageing could be used to study senescence in a new invertebrate model: the common woodlouse Armadillidium vulgare (Latreille, 1804). More specifically, we looked for the effect of age in woodlouse on three well-established physiological biomarkers of ageing in vertebrates: immune cells (cell size, density, and viability), β-galactosidase activity, and the gene expression of telomerase reverse transcriptase (TERT), an essential subunit of telomerase protein. We found that the size of immune cells was higher in older individuals, whereas their density and viability decreased, and that the β-galactosidase activity increased with age, whereas the TERT gene expression decreased. These findings demonstrate that woodlouse displays age-related changes in biomarkers of vertebrate senescence, with different patterns depending on gender. The tools used in studies of vertebrate senescence can thus be successfully used in studies of senescence of invertebrates such as the woodlouse. The application of commonly used tools to new biological models offers a promising approach to assess the diversity of senescence patterns across the tree of life.

Expression of telomerase reverse transcriptase positively correlates with duration of lithium treatment in bipolar disorder

Bipolar disorder (BD) may be associated with accelerated cellular aging. However, previous studies on telomere length (TL), an important biomarker of cellular aging, have yielded mixed results in BD. We aimed to evaluate the hypothesis that BD is associated with telomere shortening and whether this is counteracted by long-term lithium treatment. We also sought to determine whether long-term lithium treatment is associated with increased expression of telomerase reverse transcriptase (TERT), the catalytic subunit of telomerase. We determined TL and TERT expression in 100 BD I patients and 100 healthy controls. We also genotyped three single nucleotide polymorphisms associated with TL. TERT expression was significantly increased in BD I patients currently on lithium treatment. TERT expression was also significantly positively correlated with duration of lithium treatment in patients treated for 24 months or more. However, we did not find any significant effect of lithium treatment on TL. Neither did we find significant differences in TL between BD patients and controls. We suggest that long-term lithium treatment is associated with an increase in the expression of TERT. We hypothesize that an increase in TERT expression may contribute to lithium's mood stabilizing and neuroprotective properties by improving mitochondrial function and decreasing oxidative stress.

Establishment and characterization of an immortalized human chondrocyte cell line.

Following a specific number of mitotic divisions, primary chondrocytes undergo proliferative senescence, thwarting efforts to expand sufficient populations in vitro suitable to meet the needs of scientific research or medical therapies. Therefore, the human telomerase reverse transcriptase (TERT) was used to immortalize human chondrocyte and establish a cell line that escape from cellular senescence. The human chondrocytes were successfully immortalized by ectopic stable expression of TERT. The established TERT-Chondrocyte cell line showed robust proliferation capacity, even in late passages up to P20, and displayed little cellular senescence. Moreover, TERT-Chondrocyte cells at 20th passage showed similar chondrocyte properties to normal chondrocytes at early passages. Ectopic stable expression of TERT is an effective way to immortalized human chondrocyte. The immortalized chondrocytes displayed little cellular senescence, showed promise as an in vitro model to investigate osteoarthritis, and may be a promising resource for cell-based therapy for damaged cartilage.

Major depressive disorder and accelerated aging from a peripheral IGF-1 overexpression perspective

Over the past decades, data on the relationship between major depressive disorder (MDD) and accelerated aging processes were accumulated. Specific cognitive impairments, alterations in associated brain areas, a significant decrease in telomere length and expression of telomerase reverse transcriptase, oxidative and nitrosative stress, neuroinflammatory machinery, deficiency of growth factors (BDNF, IGF-1) in the brain of MDD patients resemble those that occur in aging. A decrease in the brain IGF-1 expression has been proven to disrupt mechanisms of neuroplasticity and promote cerebral inflammatory pathways, leading to morphological deterioration in the brain areas responsible for emotional and cognitive processing. From this point of view, the increase in systemic IGF-1 levels observed in most patients with MDD can be considered as a compensatory mechanism of enhancing the hypothalamic-pituitary-somatotropic axis activity in response to insufficient cerebral IGF-1 concentrations. Nevertheless, this compensatory mechanism may have a pathogenic value, improving the aging process in this patients population. This point of view has been confirmed by the evidence on the IGF-1-induced stimulation of the intracellular kinase-dependent pathways (Rho/ROCK kinase) involved in proliferation and telomere attrition in various organs, which can be considered as the underlying mechanism of aging. In line with these data, there is evidence that decreased IGF-1 signaling extends longevity in a wide range of biological species, including humans. Therefore, we hypothesized that systemic IGF-1 overexpression could be one of the pathological factors of accelerating aging in MDD patients. In this article, we analyzed data supporting this hypothesis.

Protocatechuic acid decreased telomerase reverse transcriptase (TERT) expression in DMBA-induced liver carcinogenesis mice model

Protocatechuic acid decreased telomerase reverse transcriptase (TERT) expression in DMBA-induced liver carcinogenesis mice model

Human telomerase reverse transcriptase is a novel target of Hippo-YAP pathway.

Telomerase plays a pivotal role in tumorigenesis by maintaining telomere homeostasis, a hallmark of cancer. However, the mechanisms by which telomerase is reactivated or upregulated during tumorigenesis remain incompletely understood. Here, we report that the Hippo pathway effector Yes-associated protein (YAP) regulates the expression of human telomerase reverse transcriptase (hTERT). Ectopic expression or physiological activation of YAP increases hTERT expression, whereas knockdown of YAP decreases the expression of hTERT. YAP binds to the hTERT promoter through interaction with the TEA domain family transcription factors and activates hTERT transcription. Furthermore, sustained YAP hyperactivation promotes telomerase activity and extends telomere length, with increased hTERT expression. In addition, we show that hTERT expression is positively correlated with YAP activation in human liver cancer tissues. Together, our results demonstrate that YAP promotes hTERT expression, which could contribute to tumor progression.

Poly(U) binding splicing factor 60 promotes renal cell carcinoma growth by transcriptionally upregulating telomerase reverse transcriptase.

Background: Abnormal transcriptional upregulation of telomerase reverse transcriptase (TERT) plays a dominant role in telomerase activation in various cancers. TERT promoter mutations (TPMs) have been identified as a key mechanism in TERT upregulation. However, the mechanism of TERT upregulation in cancers with low frequency of TPMs are not fully elucidated so far.Methods: The expression of PUF60 and TERT was detected by real-time PCR, western blot and immunohistochemistry. TERT promoter binding proteins were identified by streptavidin-agarose pulldown assay and mass spectrum (MS) analysis. The role of PUF60/TERT in renal cancer was evaluated on cell growth in vitro and in vivo.Results: In this study, we identify the regulation mechanism of TERT in renal cell carcinoma (RCC) cells which have rare TPMs but exert significant upregulation of TERT. We found that TERT was highly expressed in RCC tumor tissues, and elevated TERT expression was associated with poor prognosis for patients. We also detected the relatively rare TPM status in both RCC tumor tissues and RCC cell lines. Mechanistically, PUF60, a RNA binding protein, was identified as a novel TERT regulator which bound to the TERT and transcriptionally upregulated TERT expression in RCC cells. The in vitro and in vivo experiments also demonstrated that PUF60 could promote RCC cell growth through activation of TERT expression in a TPM status independent way. Furthermore, we showed that there was a strong correlation of the expression of PUF60 and TERT in RCC tumor tissues and RCC cell lines, and the patients with high expression of PUF60 and TERT had significantly shorter survival.Conclusions: Collectively, these results indicated that PUF60 transcriptionally upregulated TERT expression to promote RCC growth and progression in a TPM status independent way, suggesting that the PUF60/TERT signaling pathway may serve as potential prognostic biomarkers and therapeutic targets for RCC.

Expression of microRNA-155 and human telomerase reverse transcriptase in patients with bladder cancer

ABSTRACT Bladder cancer (BC) is the most common malignant tumor in the urinary system, with a high incidence and recurrence rate. microRNA-155 (miR-155), and human telomerase reverse transcriptase (hTERT) had a role in BC pathogenesis. In our study, we investigated the usability of miR-155, and hTERT in BC detection, muscle invasion presence, and grades of non-muscle invasive BC (NMIBC). miR-155 and hTERT were determined in urine sediment of 30 BC patients and 15 control patients with normal urologic workup by using real-time polymerase chain reaction (RT-qPCR) technique. A receiver operating characteristic (ROC) was plotted to choose the best cut-off point. Sensitivity, specificity, positive predictive value (PPV), and negative predictive value (NPV) were calculated for each marker. BC patients had lower expression of miR-155 (p= 0.005) and higher expression of hTERT (p ˂ 0.001) than control patients. Both markers had higher sensitivity than cytology for BC diagnosis (p˂ 0.001). Except for miR-155, hTERT could differentiate between NMIBC and MIBC (p˂ 0.001). miR-155 and hTERT showed no significant difference between different grades of NMIBC with p= 0.73 and p=0.07, respectively. Our results indicated that these urinary markers have the potential to be developed as non-invasive markers for BC.

Establishment of Immortalized Laryngeal Epithelial Cells Transfected with Bmi1

Primary laryngeal epithelial cells are essential to exploring the mechanisms of laryngeal and voice disorders; however, they are difficult to study and apply because of their limited life span. The purpose of this study was to develop a stable and reliable in vitro model for the comprehensive study of the pathogenesis of laryngeal and voice diseases. The pLVTHM-Bmi1 plasmid was constructed and used to immortalize primary laryngeal epithelial cells by lentiviral infection. The expressions of Bmi1, human telomerase reverse transcriptase (hTERT), p53, and pRB pathway proteins were detected by western blotting. Functional characteristics of the immortalized cell lines were verified by cell senescence β-galactosidase staining, 5-ethynyl-2′-deoxyuridine cell proliferation test, and flow cytometry. We successfully introduced Bmi into human subglottic (hSG) cells and human ventricle (hV) cells. Both the human immortalized subglottic Bmi1 (hSG-Bmi1) cell line and the human immortalized ventricle Bmi1 (hV-Bmi1) cell line maintained normal epithelial morphology and divided successfully after more than 20 culture passages. As Bmi1 was overexpressed in these cells, the expression of human telomerase reverse transcriptase (hTERT) and phosphorylated Rb increased while p16 and p21 decreased. Following Bmi1-mediated immortalization, cell senescence decreased significantly, and cell proliferation was accelerated. Tumor formation was not observed for hSG, hV, or hSG-Bmi1, and hV-Bmi1 cells in nude mice. hSG-Bmi1 cells dominated by stratified squamous epithelium and hV-Bmi1 cells dominated by columnar cells were established. The new cell lines lay a foundation for the study of the pathogenic mechanisms of laryngeal and voice diseases.

Shorter Leukocyte Telomere Length coupled with lower expression of Telomerase Genes in patients with Essential Hypertension.

Background: The essential hypertension (EH) pathophysiology remains poorly understood. Many studies indicate that reduced leukocyte telomere length (LTL) is involved in the EH pathogenesis, however, the direct analysis of arterial telomere length (ATL) from EH patients and normotensive individuals did not show a difference. To address these discrepant observations between LTL and ATL, we performed comprehensive analyses of LTL, telomerase gene expression and their genetic variants in healthy normotensive controls and EH patients.Methods: Sex-matched 206 EH patients and equal numbers of healthy controls were recruited. LTL, and the expression of two key telomerase components, telomerase reverse transcriptase (TERT) and internal RNA template (TERC) were determined using qPCR. Genetic variants of rs2736100 at the TERT and rs12696304 at the TERC loci were determined using TaqMan genotyping kits.Results: LTL was significantly shorter in EH patients than in their normotensive controls (0.96 ± 0.52 vs 1.19 ± 0.58, P = 0.001). Moreover, TERT and TERC expression in patients' leukocytes were substantially lower compare to that in healthy controls (TERT, 0.98 ± 0.98 vs 1.76 ± 1.75, P = 0.003; TERC, 1.26 ± 1.62 vs 4.69 ± 3.61, P < 0.001). However, there were no differences in the genetic variants of rs2736100 and rs12696304 between patient and control groups.Conclusions: EH patients have significantly shorter LTL, which may result from defective TERT and TERC expression in leukocytes. Collectively, lower telomerase expression contributes to shorter LTL observed in EH patients, and telomerase activators may be considered for EH therapy.

STAT5: From Pathogenesis Mechanism to Therapeutic Approach in Acute Leukemia.

Based on the results of multiple studies, multiple signaling pathways is a major cause of resistence to chemotherapy in leukemia cells. Signal transducer and activator of transcription 5 (STAT5) is among these factors; it plays an essential role in proliferation of leukemic cells. We obtained the materials used in our study via PubMed search from 1996 through 2019. The key search terms included "STAT5," "acute leukemia," "leukemogenesis," and "mutation." On activation, STAT5 not only inhibits apoptosis of leukemic cells via activating the B-cell lymphoma 2 (BCL-2) gene but also inhibits resistance to chemotherapy by enhancing human telomerase reverse transcriptase (hTERT) expression and maintaining telomere length in cells. It has also been shown that a number of mutations in the STAT5 gene and in related genes alter the expression of STAT5. The identification of STAT5 and the factors activated in its up- or downstream expression, affecting its function, contribute to better treatments such as targeted therapy rather than chemotherapy, improving the quality of life patients.

Impact of Mothers' Age on Telomere Length and Human Telomerase Reverse Transcriptase Expression in Human Fetal Membrane-Derived Mesenchymal Stem Cells.

Age-related cellular changes and limited replicative capacity of adult mesenchymal stem cells (MSCs) are few of the challenges confronting stem cell research. MSCs from human fetal membranes (hFM-MSCs), including placental, umbilical cord, and amniotic membrane, are considered an alternative to adult MSCs. However, the effect of mothers' age on hFM-MSC cellular properties is still not clearly established. This study aimed to evaluate the effect of mothers' age on hFM-MSC telomere length, telomerase activity, and proliferation ability in three different age groups: GI (20-29 years), GII (30-39 years), and GIII (≥40 years). hFM samples were collected from pregnant women ≤37 weeks after obtaining consent. hFM-MSCs were isolated and cultured to characterize them by flow cytometry and assess proliferation by MTT assay and doubling time. Telomere length and expression levels of human telomerase reverse transcriptase were assessed by quantitative real-time polymerase chain reaction (qRT-RCR). hFM-MSCs in the three age groups were spindle-shaped, plastic-adherent, and exhibited high proliferation rates and strong expression of hMSC markers. GI showed the longest telomere length in hMSCs in various FM regions, whereas GIII showed the highest level of telomerase expression. There was no difference in telomere length between GII and GIII, and both groups showed the same hMSC characteristics. In conclusion, although the hFM-MSCs derived from different fetal membranes maintained the MSC characteristics in all study groups, the hFM-MSCs of older mothers had shorter telomeres and higher telomerase activity and proliferation rate than did those derived from younger mothers. Thus, the hFM-MSCs of older mothers could be unsuitable for expansion in vitro or stem cell therapy. Determination of telomere length and telomerase expression level of hFM might help characterizing and understanding the biological differences of hFM-MSCs in different age groups.

Increasing telomerase enhanced steroidogenic genes expression and steroid hormones production in rat and human granulosa cells and in mouse ovary

Telomerase, a ribonucleoprotein responsible for telomere re-elongation, is important for male and female fertility. Several factors, including the steroid hormone estrogen, regulate the expression of Telomerase Reverse Transcriptase (TERT), which one of its non-canonical functions is gene expression regulation. The steroidogenesis process is regulated principally by transcription of genes encoding steroidogenic enzymes, but it is not clear if TERT non-canonical functions affect the expression of steroidogenic genes. Here we investigated this new notion by increasing TERT expression and activity in granulosa cells (GCs) derived from rat and from women that underwent in vitro fertilization (IVF) procedures and in vivo in mouse ovary. We show that gonadotropin enhanced the expression of TERT in rat GCs. Overexpression of human- TERT enhanced the expression of steroidogenesis genes in gonadotropin-stimulated rat GCs. Moreover, treatment with TERT increasing compounds (AGS) alone enhanced the expression of the steroidogenic genes in both rat and human GCs and in vivo in mouse ovary, while telomerase inhibitor reduced their expression. Treatment with AGS compounds, together with gonadotropin stimulation, additively increased steroidogenic gene expression. Enhancing TERT expression and activity increased the level of progesterone in mouse blood and in the medium of rat GCs and estrogen in women derived pre-ovulatory luteinized GCs. These data suggest that increasing TERT in GCs by pharmaceutical compounds enhanced steroidogenesis and the production of steroid hormones that are essential processes in human and animal reproduction. These data also suggest a novel possible strategy for the enhancement of the production of steroid hormones.

CBMT-23. NON-CANONICAL FUNCTIONS OF TERT IN GLIOBLASTOMA

Abstract Telomerase is an enzyme with a catalytic subunit, telomerase reverse transcriptase (TERT), that is in charge of telomere elongation in the nucleus. Promotor region of TERT is commonly mutated across cancers, especially in glioblastoma (GBM) with over 80% frequency. In the absence of any effective molecular targeting therapy for GBM, elucidating oncogenic signaling of TERT could open new avenues in GBM treatment. Canonically, mutations of TERT, which result in TERT upregulation, maintain telomere length in the nucleus and promote indefinite proliferation of cancer cells. However, a non-canonical function of TERT in the mitochondria has recently been suggested. We screened GBM cell models against a novel small molecule inhibitor (RG1534, Reglagene Inc.) that interferes with the functionality of a mutated hTERT promoter. RG1534 selectively suppresses glioma cell viability without affecting non-transformed normal human astrocytes. More interestingly, RG1534 treatment leads to rapid apoptosis induction in glioma cell lines that does not correlate with the time course of the telomere shortening effect. We further validated this rapid apoptosis behavior in glioma cell lines using siRNA and CRISPR/Cas-9 mediated hTERT knockdown. We also measured the protein expression of TERT in subcellular fractions of glioma cell lines and demonstrated the presence of higher TERT expression in mitochondrial extract compared to the nucleus. Finally, using MitoSOX dye we assessed ROS generation in glioma cells in response to an oxidant with or without TERT expresssion. In summary, our results demonstrate that non-canonical functions of TERT may play critical role in glioma pathobiology and require more detail investigation.

CSIG-24. GABP LINKS AMPK SIGNALING TO TERT REGULATION IN A TERT PROMOTER MUTATION DEPENDENT MANNER

Abstract Over 90% of human tumors achieve immortality by reactivating telomerase reverse transcriptase (TERT), the catalytic subunit of telomerase. Two mutually exclusive, heterozygous mutations that activate the TERT promoter (TERTp) occur in a wide range of cancers including ~80% of glioblastomas (GBM). The mutations and TERTp activation enable cells to avoid replicative senescence and continue to divide beyond the normal limits on cellular lifespan, thus becoming immortal. Our lab has demonstrated that each mutation creates a novel E26-transformation specific binding site that allows the tetrameric form of the GA-binding protein GABP to bind and aberrantly activate the mutant TERTp. We further showed that reducing a tetramer-forming subunit of GABP, the GABPB1L transactivating subunit, reverses cellular immortality in vitro and reduces tumor growth in vivo. Beyond GABP, little is known about pathways that contribute to immortality via activation of the mutant TERTp. The heterotrimeric 5’ AMP-activated protein kinase (AMPK) complex is activated in human GBM compared to normal brain, and was previously implicated in regulating GABP expression. Here, we have determined that AMPK inhibition decreases transcription of both subunits of the tetrameric GABP complex (GABPA and GABPB1L) in GBM cells, and is sufficient to reduce TERT expression and telomerase activity in a TERT promoter mutation dependent manner. Together, these data suggest a novel and targetable connection between the AMPK signaling axis, telomerase activity and potentially tumor cell immortality.