Catalytic Subunit Of Telomerase Research Articles

Apoptotic Endonuclease EndoG Induces Alternative Splicing of Telomerase TERT Catalytic Subunit, Caspase-2, DNase I, and BCL-x in Human, Murine, and Rat CD4+ T Lymphocytes

Apoptotic endonuclease EndoG plays a key role in the alternative splicing of mRNA of human TERT telomerase catalytic subunit. The aim of this work was to test the ability of EndoG to induce alternative splicing of mRNA of other genes and in other organisms. To determine new mRNA splice-variants, EndoG overexpression was induced in human, mouse and rat CD4+-T-lymphocytes followed by sequencing of total RNA of these cells. Sequencing results showed that besides TERT, EndoG induced alternative splicing of deoxyribonuclease I (DNase I), caspase-2 (Casp-2) and BCL-x. The expression level of EndoG strongly correlated with mRNA splicing-variants of TERT, DNase I, Casp-2, and BCL-x in intact CD4+-T cells of healthy donors as well as different lines of mice and rats. EndoG overexpression induced down-regulation of fulllength mRNAs of TERT, DNase I, Casp-2, and BCL-x and up-regulation of their short-length mRNAs. Alternative splicing of studied mRNAs resulted in down-regulation of enzymatic activity of proteins in vitro and in vivo. The results of this work confirm the ability of endonuclease EndoG to induce alternative splicing of several mRNAs in human, mice and rats.

Pterostilbene down-regulates hTERT at physiological concentrations in breast cancer cells: Potentially through the inhibition of cMyc.

Human telomerase reverse transcriptase (hTERT) encodes the catalytic subunit of telomerase, which has been shown to be upregulated in many cancers. Pterostilbene is a naturally occurring stilbenoid and phytoalexin found primarily in blueberries that exhibits antioxidant activity and inhibits the growth of various cancer cell types. Therefore, the aim of this study was to determine whether treatment with pterostilbene, at physiologically achievable concentrations, can inhibit the proliferation of breast cancer cells and down-regulate the expression of hTERT. We found that pterostilbene inhibits the cellular proliferation of MCF-7 and MDA-MB-231 breast cancer cells in both a time- and dose-dependent manner, without significant toxicity to the MCF10A control cells. Pterostilbene was also shown to increase apoptosis in both breast cancer cell lines. Dose-dependent cell cycle arrest in G1 and G2/M phase was observed after treatment with pterostilbene in MCF-7 and MDA-231 cells, respectively. hTERT expression was down-regulated after treatment in both a time- and dose-dependent manner. Pterostilbene also reduced telomerase levels in both cell lines in a dose-dependent manner. Moreover, cMyc, a proposed target of the pterostilbene-mediated inhibition of hTERT, was down-regulated both transcriptionally and posttranscriptionally after treatment. Collectively, these findings highlight a promising use of pterostilbene as a natural, preventive, and therapeutic agent against the development and progression of breast cancer.

APIGENIN INCREASES CISPLATIN INHIBITORY EFFECTS ON THE TELOMERASE ACTIVITY OF TRIPLE NEGATIVE BREAST CANCER CELLS

Inhibition of telomerase activity has emerged as a promising strategy to combat cancer cells, especially ones with no specific molecular targets such as triple negative breast cancer (TNBC). Cisplatin, a chemotherapeutic drug, is causing DNA damage while apigenin, a plant-derived antioxidant, induces apoptosis in various cancer cell types. Little is known about their combined ability to inhibit telomerase activity in TNBC cells. In the current study, the effect of cisplatin in combination with apigenin was investigated with regards to telomerase activity and expression of the telomerase catalytic subunit hTERT as well as Heat Shock Protein 90 (Hsp90) and p23 in two types of TNBC (MDA-MB-231; HCC1806) and one non-tumorigenic (MCF10A) epithelial cell line. The results showed that the combined treatment of cisplatin and apigenin significantly down-regulated telomerase activity. The inhibition of telomerase activity was accompanied by a down-regulation of hTERT, Hsp90 and p23 at transcriptional and translational level in both TNBC cells, as compared to control cells. The results of the current study suggest that apigenin and cisplatin synergistically inhibit telomerase activity by downregulating the enzyme’s catalytic subunit. However, the exact roles of Hsp90 and p23 in the regulation of telomerase activity requires further investigation as they seem to be TNBC subtype-specific.

Mechanism of Action of G-Quadruplex–Forming Oligonucleotide Homologous to the Telomere Overhang in Melanoma

T-oligo, a guanine-rich oligonucleotide homologous to the 3'-telomeric overhang of telomeres, elicits potent DNA-damage responses in melanoma cells; however, its mechanism of action is largely unknown. Guanine-rich oligonucleotides can form G-quadruplexes (G4), which are stabilized by the hydrogen bonding of guanine residues. In this study, we confirmed the G4-forming capabilities of T-oligo using nondenaturing PAGE, nuclear magnetic resonance, and immunofluorescence. Using an anti-G-quadruplex antibody, we showed that T-oligo can form G4 in the nuclei of melanoma cells. Furthermore, using DNase I in a nuclease degradation assay, G4-T-oligo was found to be more stable than single-stranded T-oligo. G4-T-oligo had decreased antiproliferative effects compared with single-stranded T-oligo. However, G4-T-oligo has similar cellular uptake as single-stranded T-oligo, as shown by FACS analysis. Inhibition of JNK, which causes DNA damage-induced apoptosis, partially reversed the antiproliferative activity of T-oligo. T-oligo also inhibited mRNA expression of human telomerase reverse transcriptase, a catalytic subunit of telomerase that was reversed by JNK inhibition. Furthermore, two shelterin complex proteins TRF2/POT1 were found to be up-regulated and bound by T-oligo, suggesting that T-oligo may mediate dissociation of these proteins from the telomere overhang. These studies show that T-oligo can form a G-quadruplex and that the antitumor effects of T-oligo may be mediated through POT1/TRF2 and via human telomerase reverse transcriptase inhibition through JNK activation.

Expression of the human telomerase reverse transcriptase gene is modulated by quadruplex formation in its first exon due to DNA methylation

DNA secondary structures and methylation are two well-known mechanisms that regulate gene expression. The catalytic subunit of telomerase, human telomerase reverse transcriptase (hTERT), is overexpressed in ∼90% of human cancers to maintain telomere length for cell immortalization. Binding of CCCTC-binding factor (CTCF) to the first exon of the hTERT gene can down-regulate its expression. However, DNA methylation in the first exon can prevent CTCF binding in most cancers, but the molecular mechanism is unknown. The NMR analysis showed that a stretch of guanine-rich sequence in the first exon of hTERT and located within the CTCF-binding region can form two secondary structures, a hairpin and a quadruplex. A key finding was that the methylation of cytosine at the specific CpG dinucleotides will participate in quartet formation, causing the shift of the equilibrium from the hairpin structure to the quadruplex structure. Of further importance was the finding that the quadruplex formation disrupts CTCF protein binding, which results in an increase in hTERT gene expression. Our results not only identify quadruplex formation in the first exon promoted by CpG dinucleotide methylation as a regulator of hTERT expression but also provide a possible mechanistic insight into the regulation of gene expression via secondary DNA structures.

Correlation of hTERT Expression with Cervical Cytological Abnormalities and Human Papillomavirus Infection.

Telomerase Reverse Transcriptase (TERT) is the main catalytic sub-unit of telomerase, a reverse transcriptase enzyme. Telomerase expression is regulated at many levels, with numerous studies suggesting that up-regulation of human TERT gene (hTERT) at transcriptional level results in immortal cell phenotype associated with cancer. The aim of this study is to determine the correlation between hTERT expression and different cervical precursor lesions, as well as with cervical cancer in patients with confirmed Human papillomavirus (HPV) infection. The study included molecular analyzes on cervical samples from 214 women and matched Papanicolaou (Pap) test results. HPV detection and genotyping was performed by polymerase chain reaction (PCR) and genotyping. Quantitative real-time PCR (qRT-PCR) was performed using TaqMan probes and were calculated relative to the reference gene. Results showed significantly increased hTERT mRNA expression levels in high-grade and low-grade lesions compared to normal control samples (p<0.01) associated with 6.31 fold higher risk for developing ASC-US and 9.20 for LSIL. Strong correlation between HPV infection and hTERT expression in the high-grade lesions and cervical cancer was also observed. hTERT relative expression values showed 98% specificity and 100 % sensitivity as indicator of cervical lesions particularly for the ACS-H, HSIL and cervical cancer. In conclusion, hTERT expression correlate with the cytological grade of the cervical lesions and HPV infection and has a potential to be used as a diagnostic and prognostic marker.

Inhibition of telomerase activity and induction of apoptosis by Rhodospirillum rubrum L-asparaginase in cancer Jurkat cell line and normal human CD4+T lymphocytes.

Rhodospirillum rubrum L‐asparaginase mutant E149R, V150P, F151T (RrA) down‐regulates telomerase activity due to its ability to inhibit the expression of telomerase catalytic subunit hTERT. The aim of this study was to define the effect of short‐term and long‐term RrA exposure on proliferation of cancer Jurkat cell line and normal human CD4+ T lymphocytes. RrA could inhibit telomerase activity in dose‐ and time‐dependent manner in both Jurkat and normal CD4+ T cells. Continuous RrA exposure of these cells resulted in shortening of telomeres followed by cell cycle inhibition, replicative senescence, and development of apoptosis. Complete death of Jurkat cells was observed at the day 25 of RrA exposure while normal CD4+ T cells died at the day 50 due to the initial longer length of telomeres. Removal of RrA from senescent cells led to a reactivation of hTERT expression, restoration telomerase activity, re‐elongation of telomeres after 48 h of cultivation, and survival of cells. These findings demonstrate that proliferation of cancer and normal telomerase‐positive cells can be limited by continuous telomerase inhibition with RrA. Longer telomeres of normal CD4+ T lymphocytes make such cells more sustainable to RrA exposure that could give them an advantage during anti‐telomerase therapy. These results should facilitate further investigations of RrA as a potent anti‐telomerase therapeutic protein.

Telomerase Reverse Transcriptase Gene from Greasyback Shrimp Metapenaeus ensis: Isolation, Genomic Organization and Expression Pattern Analysis.

Telomerase reverse transcriptase (TERT), a catalytic subunit of telomerase, plays a key role in the activity and biological functions of telomerase. In the present study we isolated and characterized the full-length cDNA and DNA sequences of the TERT gene (MeTERT) from Metapenaeus ensis. MeTERT cDNA was 4239 bp in length, which consisted of a 369 bp 5'UTR, a 3231 bp open reading frame encoding 1076 amino acids, and a 639 bp 3'UTR. The genomic DNA of MeTERT had only two introns, similar to beetle (two introns) and silkworm (intronless). The MeTERT protein showed only 5.2-7.9% identity with other known TERTs but contained all the four primary TERT domains of the N-terminal TEN, RNA binding domain (TRBD), reverse transcriptase (RT) and C-terminus CTE. Expression pattern analysis by RT-qPCR showed that, the MeTERT mRNA transcripts could be detected in all the tested samples, with relatively higher expression level in the gill, mysis, Oka organ and egg, but lower level in muscle, ovary, in vitro cultured 3-d Oka organ cells and heart. The significant decrease of MeTERT expression in the in vitro cultured 3-d Oka organ primary cells compared with their source tissue of Oka organ may have contributed to the cellular mitosisarrest. Thus trans-activation of TERT gene may be a candidate in attempts to immortalize in vitro cultured shrimp cells. This work will lay a solid foundation for future studies of the biological functions of telomerase in crustaceans.

Alternative splicing of telomerase catalytic subunit hTERT generated by apoptotic endonuclease EndoG induces human CD4+ T cell death

Telomerase activity is regulated by alternative splicing of its catalytic subunit human Telomerase Reverse Transcriptase (hTERT) mRNA. Induction of a non-active spliced hTERT leads to inhibition of telomerase activity. However, very little is known about the mechanism of hTERT mRNA alternative splicing. The aim of this study was to determine the role of the apoptotic endonuclease EndoG in alternative splicing of hTERT and telomerase activity. A strong correlation was identified between EndoG expression levels and hTERT splice variants in human CD4+ and CD8+ T lymphocytes. Overexpression of EndoG in CD4+ T cells down-regulated the expression of the active full-length hTERT variant and up-regulated expression of the non-active spliced variant. A reduction in full-length hTERT transcripts down-regulated telomerase activity. Long-term in vitro cultivation of EndoG-overexpressing CD4+ T cells led to dramatically shortened telomeres, conversion of cells into a replicative senescence state, and activation of the BCL2/BAX-associated apoptotic pathway finally leading to cell death. These data indicated the participation of EndoG in alternative mRNA splicing of the telomerase catalytic subunit hTERT, regulation of telomerase activity and determination of cell fate.

Rhodospirillum rubruml-asparaginase targets tumor growth by a dual mechanism involving telomerase inhibition

Rhodospirillum rubruml-asparaginase mutant RrA E149R, V150P, F151T (RrA) was previously identified to down-regulate telomerase activity along with catalyzing the hydrolysis of l-asparagine. The aim of this study was to define the effect of prolonged RrA exposure on telomerase activity, maintenance of telomeres and proliferation of cancer cells in vitro and in vivo. RrA could inhibit telomerase activity in SCOV-3, SkBr-3 and A549 human cancer cell lines due to its ability to down-regulate the expression of telomerase catalytic subunit hTERT. Telomerase activity in treated cells did not exceeded 29.63 ± 12.3% of control cells. Continuous RrA exposure of these cells resulted in shortening of telomeres followed by cell death in vitro. Using real time PCR we showed that length of telomeres in SCOV-3 cells has been gradually decreasing from 10105 ± 2530 b.p. to 1233 ± 636 b.p. after 35 days of cultivation. RrA treatment of xenograft models in vivo showed slight inhibition of tumor growth accompanied with 49.5–53.3% of decrease in hTERT expression in the all tumors. However down-regulation of hTERT expression, inhibition of telomerase activity and the loss of telomeres was significant in response to RrA administration in xenograft models. These results should facilitate further investigations of RrA as a potent therapeutic protein.

Cisplatin-induced apoptotic endonuclease EndoG inhibits telomerase activity and causes malignant transformation of human CD4+ T lymphocytes

Alternative splicing of telomerase catalytic subunit hTERT pre-mRNA (human Telomerase Reverse Transcriptase) regulates telomerase activity. Increased expression of non-active splice variant hTERT results in inhibition of telomerase. Apoptotic endonuclease EndoG is known to participate in hTERT alternative splicing. Expression of EndoG can be induced in response to DNA damages. The aim of this study was to determine the ability of a DNA-damaging compound, cisplatin, to induce EndoG and its influence on alternative splicing of hTERT and telomerase activity in human CD4+ Т lymphocytes. Overexpression of EndoG in CD4+ T cells downregulated the expression of active full-length hTERT variant and upregulated its non-active spliced variant. Reduction of full-length hTERT caused downregulation of telomerase activity, shortening of telomeres length during cell divisions, converting cells to the replicative senescence state, activation of apoptosis and finally cell death. Few cells survived and underwent malignant transformation. Transformed cells have increased telomerase activity and proliferative potential compare to initial CD4+ T cells. These cells have phenotype of T lymphoblastic leukemic cells and are able to form tumors and cause death in experimental mice.

Intracellular Localization of Apoptotic Endonuclease EndoG and Splice-Variants of Telomerase Catalytic Subunit hTERT.

The activity of telomerase catalytic subunit hTERT (human telomerase reverse transcriptase) can be regulated by alternative splicing of its mRNA. The mechanism of hTERT splicing is not understood in detail. Apoptotic endonuclease EndoG is known to participate in this process. In the present work, the intracellular colocalization and mRNA levels of EndoG and hTERT splice-variants in normal and apoptotic cancer cells were studied. We found that the development of apoptosis increased the expression of EndoG and changed the ratio of hTERT splice-variants, which decreased the telomerase activity in the cells. The development of apoptosis was accompanied by changes in the amount of mRNA and in the localization of EndoG and hTERT splice-variants in the cytoplasm, nuclei, and mitochondria of the cells. The suppression of EndoG expression using RNA interference prevented induction of the α+β- splice-variant of hTERT and inhibition of the telomerase activity. A high degree of the intracellular colocalization of EndoG and hTERT was shown. The changes in the expression and localization of EndoG corresponded with changes in the amount and localization of hTERT splice-variants. These data confirm the participation of EndoG in the alternative splicing of mRNA of the telomerase catalytic subunit and in regulation of the telomerase activity.

Hippocampal TERT Regulates Spatial Memory Formation through Modulation of Neural Development.

SummaryThe molecular mechanism of memory formation remains a mystery. Here, we show that TERT, the catalytic subunit of telomerase, gene knockout (Tert−/−) causes extremely poor ability in spatial memory formation. Knockdown of TERT in the dentate gyrus of adult hippocampus impairs spatial memory processes, while overexpression facilitates it. We find that TERT plays a critical role in neural development including dendritic development and neuritogenesis of hippocampal newborn neurons. A monosynaptic pseudotyped rabies virus retrograde tracing method shows that TERT is required for neural circuit integration of hippocampal newborn neurons. Interestingly, TERT regulated neural development and spatial memory formation in a reverse transcription activity-independent manner. Using X-ray irradiation, we find that hippocampal newborn neurons mediate the modulation of spatial memory processes by TERT. These observations reveal an important function of TERT through a non-canonical pathway and encourage the development of a TERT-based strategy to treat neurological disease-associated memory impairment.

Telomerase Induction in HPV Infection and Oncogenesis.

Telomerase extends the repetitive DNA at the ends of linear chromosomes, and it is normally active in stem cells. When expressed in somatic diploid cells, it can lead to cellular immortalization. Human papillomaviruses (HPVs) are associated with and high-risk for cancer activate telomerase through the catalytic subunit of telomerase, human telomerase reverse transcriptase (hTERT). The expression of hTERT is affected by both high-risk HPVs, E6 and E7. Seminal studies over the last two decades have identified the transcriptional, epigenetic, and post-transcriptional roles high-risk E6 and E7 have in telomerase induction. This review will summarize these findings during infection and highlight the importance of telomerase activation as an oncogenic pathway in HPV-associated cancer development and progression.

Abstract 1101: Telomerase inhibitor imetelstat in combination with the BCL-2 inhibitor venetoclax enhances apoptosis in vitro and increases survival in vivo in acute myeloid leukemia

Abstract Background/Aims: Acute myeloid leukemia (AML) is an aggressive cancer with limited treatment options outside of chemotherapy. Improved therapies with novel mechanisms of action are desperately needed to fill this need. Both hTERT, the catalytic subunit of telomerase, and BCL-2, an apoptotic regulator, are overexpressed in AML, correlating with disease severity and poor prognosis respectively. Imetelstat is a novel, first-in-class competitive inhibitor of telomerase with clinical activity in hematologic malignancies. Venetoclax, an approved BCL-2 inhibitor for CLL, has shown a promising clinical benefit in AML patients. Preclinical evidence shows that downregulation of hTERT induces apoptosis via disruptions of hTERT and BCL-2 interaction; we hypothesize that inhibiting both targets would yield greater anti-tumor activity in AML compared to treatment with either agent alone. Methods: AML cell lines and AML patient’s PBMC samples were treated with imetelstat or venetoclax alone, or in combination, and viable and apoptotic populations of cells were evaluated by flow cytometry. Telomerase activity, hTERT expression and mitochondrial dysfunction were investigated for mechanism of action. Furthermore, an in vivo study in the MOLM-13 AML disseminated model was conducted to assess efficacy and survival. Results: A dose-dependent synergistic activity in inducing apoptosis was observed in multiple AML cell lines when combining imetelstat with venetoclax. In the MOLM-13 cell line, single-agent imetelstat and venetoclax had modest apoptotic activity after 48 hours (22% and 30% respectively), but the combination achieved 88% at 48 hours and nearly 100% at 96 hours. Similarly enhanced apoptotic activity was also observed in PBMCs purified from 4 AML patient whole blood samples. Molecular analyses showed combining imetelstat with venetoclax reduced hTERT expression and telomerase activity much more strongly than either agent alone. Furthermore, in vivo studies showed all mice tolerated the combination of imetelstat with ABT-199 well, with increased life span as compared to the vehicle control (68.1%, p=0.0001), to imetelstat (39.6%, p=0.0011) alone, or to venetoclax (23.3%, p=0.0001) alone. In the combination group, 40% of treated mice were alive 77-days after treatment discontinued whereas all mice of the other single agent arms died within two weeks, demonstrating a significant survival benefit. Conclusions: To our knowledge, this is the first investigation combining imetelstat with venetoclax in AML, and the results demonstrated a synergistic effect on induction of apoptosis in cell lines and patient samples in vitro, which translated into prolonged survival in xenograft models, thus providing a strong rationale for clinical exploration of this combination. Citation Format: Joshua J. Rusbuldt, Leopoldo Luistro, Diana Chin, Melissa Smith, Amy Wong, Margarita Romero, Aleksandra Rizo, Jacqueline Bussolari, Fei Huang, Amy (Kate) Sasser. Telomerase inhibitor imetelstat in combination with the BCL-2 inhibitor venetoclax enhances apoptosis in vitro and increases survival in vivo in acute myeloid leukemia [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 1101. doi:10.1158/1538-7445.AM2017-1101

Abstract 4128: Mechanism of action of G-quadruplex forming oligonucleotide homologous to the telomere overhang in melanoma

Abstract T-oligo, a guanine-rich oligonucleotide (GRO) homologous to the 3'-telomeric overhang of telomeres, elicits potent DNA-damage responses (DDRs) in cancer cells. However, the detailed molecular mechanism of action of T-oligo in cancer cells is largely unknown. Recent studies suggest that GROs can form G-quadruplexes (G4) which are stabilized by the hydrogen-bonding of guanine residues. This study aims to examine the G4-forming capabilities of T-oligo in vitro and investigates the molecular mechanism of single-stranded (SS) and G4-T-oligo induced DDRs in melanoma cells (MM-AN). G4-formation by T-oligo was confirmed using the SS-T-oligo and G4-T-oligo on a polyacrylamide gel under non-denaturing conditions. NMR studies for T-oligo in KCl confirmed that T-oligo forms G4 structures. Immunofluorescence studies conducted with an anti-G-quadruplex antibody (BG4), a G4 detecting antibody, showed 88.4% co-localization of T-oligo and BG4 in the nuclei of melanoma cells confirming the ability of T-oligo to form G-quadruplexes inside melanoma cells. While G4-T-oligo was found more stable in nuclease degradation assay by DNase I, it had a decreased anti-proliferative effects compared to SS-T-oligo. However, G4-T-oligo had similar cellular uptake as SS-T-oligo. Further, two shelterin complex proteins TRF2 and POT1 which are mainly found at the telomere ends were found to be upregulated (2.0 fold) by T-oligo suggesting TRF2 and POT1 mediated telomere overhang dissociation. We also found that T-oligo can co-localize with telomere binding proteins TRF2 and POT1 by 88.4±4.5% (n=12) and 84.5±8% (n=10) respectively. Western blot analysis results also showed upregulation of both p-JNK and total JNK by 4.0- and 2.0-fold respectively. To further confirm the involvement of p-JNK in T-oligo mediated apoptosis we used a specific JNK inhibitor SP600125. Western blot analysis showed that T-oligo mediated upregulation of p-JNK was reversed in presence of SP600125. Results from an MTT assay showed a 73.8% decrease in cell viability after T-oligo treatment alone; however, cell viability was decreased to 45.8%, and 25.3% when SP600125 was present at concentrations of 10 μM, and 12 μM respectively, in comparison to diluent. T-oligo also inhibited mRNA expression of hTERT; a catalytic subunit of telomerase by 50% .We further investigated the effect of the JNK inhibitor SP600125 on hTERT expression and found that treatment with SP600125 in presence of T-oligo partially reversed the downregulation of hTERT. We found a 16% decrease in hTERT expression in comparison to 50% reduction by T-oligo treatment alone. In conclusion, these studies demonstrate that T-oligo can form G-quadruplexes and the anti-proliferative mechanism of T-oligo may be mediated through POT1 and TRF2 as well as via JNK-activation inducing hTERT-inhibition in melanoma cells. Citation Format: Gagan Chhabra, Luke Wojdyla, Ankita Sanjali, Mark Frakes, Marko Ivancich, Pooja Vinay, Zachary Schrank, Benjamin E. Ramirez, Neelu Puri. Mechanism of action of G-quadruplex forming oligonucleotide homologous to the telomere overhang in melanoma [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 4128. doi:10.1158/1538-7445.AM2017-4128

Abstract 3469: Identification of novel human telomerase associated protein by mass spectrometry

Abstract Telomeres are nucleoprotein structures at the ends of eukaryotic chromosomes that prevent them from degradation, recombination, and end-to-end fusion. Telomere consists of DNA repeats, which in human is of the sequence TTAGGG, and it is bound by a group of proteins called shelterin complex. Telomere is synthesized by telomerase. Telomerase is absent in most human somatic cells while it is up-regulated in 90% of cancer cells, in which telomerase plays an essential role in cancer cells’ replicative immortality. However, telomerase is only present at approximately 250 molecules per cancer cell. To exert its function of elongating telomere, telomerase has to find the end of telomeres which needed to be elongated. However, how telomerase is precisely recruited to telomere ends is not clearly understood. To better understand the recruitment process of telomerase, we initially screened and subsequently identified novel binding proteins of hTERT, the catalytic subunit of telomerase, through a pull-down assay using cancer cell lines with endogenously Flag-tagged hTERT. Using mass spectrometry (MS), we were able to identify a promising interesting protein. Further, direct interaction between TERT and this protein was validated. Then, we found that this protein can also interact with the shelterin component, TRF1 (Telomeric repeat-binding factor 1), which binds to double stranded telomeric DNA. Furthermore, from previous published reports, this protein was shown to interact with single-stranded telomeric DNA binding protein POT1 (Protection of Telomeres 1) in the shelterin complex. Functionally, overexpression of this protein in cancer cell lines can result in the elongation of telomeres, while knock-down or knock-out of the protein results in the shortening of telomere. In conclusion, we identified an hTERT-binding protein that affects telomere maintenance which may be involved in telomerase recruitment process. In the long run, it could serve as a potential therapeutic target for cancers. Citation Format: Jing Wang, Xuezhi Bi, Shang Li. Identification of novel human telomerase associated protein by mass spectrometry [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 3469. doi:10.1158/1538-7445.AM2017-3469

Abstract 1521: hTERT G-quadruplex-targeted oligonucleotides inhibit glioblastoma cell growth

Abstract Glioblastoma is one of the most common and deadly forms of brain cancer, representing roughly 75% of all brain malignancies. These tumors generally have poor prognoses and are resistant to conventional therapy. It has recently been shown that as many as 80% of all glioblastomas contain mutations in a G-rich 68 base pair region of the hTERT promoter. hTERT is the catalytic subunit of telomerase, a holoenzyme responsible for lengthening the ends of chromosomes, thereby conferring immortality to cells. Normally, hTERT is not expressed in somatic cells and its expression is tightly controlled in stem cells. However, hTERT is upregulated in up-to 95% of human tumors and is considered a key activator of cancer progression and a sign of poor clinical outcome. Therefore, hTERT has been under investigation for the past decade as a potential therapeutic target. We have shown that the mutations in hTERT promoter occur in a G-rich region that is part of a silencer element which forms a secondary G-quadruplex structure required for function, and that these mutations destabilize the G-quadruplex structure, allowing hTERT expression. We have also demonstrated that oligonucleotides encoding the G-quadruplex forming sequence in the c-MYC promoter can stabilize the G-quadruplex structure and downregulate c-MYC expression. Therefore, we hypothesized that oligonucleotides targeted to the G-quadruplex of the hTERT promoter could downregulate this gene expression and inhibit glioblastoma cell proliferation in a similar manner. We designed several G-quadruplex-forming oligonucleotides covering the mutated sites in the hTERT promoter either in totality (68 nucleotides) or separately (25 nucleotides) to stabilize the G-quadruplex region. Two glioblastoma cell lines (A172 and U87) and one neuroblastoma cell line (CHP134) were exposed to these oligonucleotides and evaluated for growth inhibition using the MTT assay and for gene expression by QRT-PCR. All oligonucleotides tested were found to induce between 40 to 90% growth inhibition in the 3 cell lines. The cell growth inhibition was both time and dose dependent and showed effectiveness as early as 3 days suggesting that this effect is not solely due to telomere shortening. Four oligonucleotides with the most consistent efficacy in growth inhibition were evaluated for their effect on hTERT gene expression in the 3 cell lines at 4 day exposure and revealed that two of the G-quadruplex forming oligonucleotides significantly decreased hTERT expression compared to untreated cells. In conclusion, we have defined G-quadruplex oligonucleotides targeted to the hTERT promoter that downregulate hTERT gene expression and are effective growth inhibitors in glioblastoma cells. Our findings indicate that downregulation of hTERT with targeted oligonucleotides affects non-canonical functions of hTERT conferring an advantage to this therapeutic approach. Note: This abstract was not presented at the meeting. Citation Format: Alex West, Francine Rezzoug, Shelia D. Thomas, Donald M. Miller. hTERT G-quadruplex-targeted oligonucleotides inhibit glioblastoma cell growth [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 1521. doi:10.1158/1538-7445.AM2017-1521

Gastrointestinal cancer cells treatment with bevacizumab activates a VEGF autoregulatory mechanism involving telomerase catalytic subunit hTERT via PI3K-AKT, HIF-1α and VEGF receptors.

BackgroundTargeting angiogenesis has been considered a promising treatment of choice for a large number of malignancies, including gastrointestinal cancers. Bevacizumab is an anti-vascular endothelial growth factor (anti-VEGF) being used for this purpose. However, treatment efficacy is largely questioned. Telomerase activity, responsible for cancer cell immortality, is detected in 85–95% of human cancers and is considered a potential regulator of VEGF. The aim of our study was to investigate the interrelationship between VEGF and hTERT in gastrointestinal cancers and to explore cell response to a combined inhibition of telomerase and VEGF.MethodsAGS (gastric cancer), Caco-2 (colorectal cancer) and HepG2/C3A (hepatocellular carcinoma), were treated with telomerase inhibitors BIBR-1232 (10μM) and costunolide (10μM), with bevacizumab (Avastin® at 5 ng/ml or 100μg/ml) or with a combination of both types of inhibitors. VEGF and hTERT mRNA levels, and telomerase activity were detected by RT-PCR. VEGF levels were quantified by ELISA. Telomerase was knocked down using hTERT siRNA and hTERT was overexpressed in the telomerase negative cell line, Saos-2 (osteosarcoma), using constructs expressing either wild type hTERT (hTERT-WT) or dominant negative hTERT (hTERT-DN). Tube formation by HUVECs was assessed using ECMatrix™ (EMD Millipore).ResultsOur results showed that telomerase regulates VEGF expression and secretion through its catalytic subunit hTERT in AGS, Caco2, and HepG2/C3A, independent of its catalytic activity. Interestingly, VEGF inhibition with bevacizumab (100μg/ml) increased hTERT expression 42.3% in AGS, 94.1% in Caco2, and 52.5% in HepG2/C3A, and increased telomerase activity 30-fold in AGS, 10.3-fold in Caco2 and 8-fold in HepG2/C3A. A further investigation showed that VEGF upregulates hTERT expression in a mechanism that implicates the PI3K/AKT/mTOR pathway and HIF-1α. Moreover, bevacizumab treatment increased VEGFR1 and VEGFR2 expression in cancer cells and human umbilical vein endothelial cells (HUVECs) through hTERT. Thus, the combination of bevacizumab with telomerase inhibitors decreased VEGF expression and secretion by cancer cells, inhibited VEGFR1 and VEGFR2 upregulation, and reduced tube formation by HUVECs.ConclusionsTaken together, our results suggest that bevacizumab treatment activates a VEGF autoregulatory mechanism involving hTERT and VEGF receptors and that an inhibition of this pathway could improve tumor cell response to anti-VEGF treatment.

Functional dissection of breast cancer risk-associated TERT promoter variants.

The multi-cancer susceptibility locus at 5p15.33 includes TERT, encoding the telomerase catalytic subunit. Genome-wide association studies (GWAS) have identified six single nucleotide polymorphisms (SNPs) in the TERT promoter associated with decreased breast cancer risk, although the precise causal variants and their mechanisms of action have remained elusive. Luciferase reporter assays indicated that the protective haplotype reduced TERT promoter activity in human mammary epithelial and cancer cells in an estrogen-independent manner. Using single variant constructs, we identified rs3215401 and rs2853669 as likely functional variants. Silencing of MYC decreased TERT promoter activity but neither MYC nor ETS2 silencing conferred allele-specificity. In chromatin immunoprecipitation experiments, the ETS protein GABPA, but not ETS2 or ELF1, bound rs2853669 in an allele-specific manner in mammary epithelial cells. Investigation of open chromatin in mammoplasty samples suggested involvement of three additional variants, though not rs3215401 or rs2853669. Chromosome conformation capture revealed no interaction of the TERT promoter with regulatory elements in the locus, indicating limited local impact of candidate variants on the TERT promoter. Collectively, our functional studies of the TERT-CLPTM1L breast cancer susceptibility locus describe rs2853669 as a functional variant of this association signal among three other potentially causal variants and demonstrate the versatile mechanisms by which TERT promoter variants may affect breast cancer risk.