Targeting hTERT Research Articles

Clinical research progress of telomerase targeted cancer immunotherapy: a literature review.

Telomerase is activated or overexpressed in 85-90% of tumors, which maintains the length of telomere and has become an important anti-cancer target. Increasing clinical and preclinical data suggest that telomerase-targeted cancer immunotherapy could achieve effective killing of tumor cells in vivo. This article reviews the research progress of telomerase targeted cancer immunotherapy in clinical and pre-clinical trials, aiming to provide a reference for further clinical research and treatment of cancers. We investigated the research progress of telomerase immunotherapy in the last 20 years from four electronic databases. Telomerase-targeted immunotherapies have been developed with the arising of a new era in immuno-oncology, including peptide vaccines, DNA vaccines, dendritic cells (DCs), adoptive cell transfer (ACT) therapies, antibodies, etc. Some of them have been approved for undergoing clinical trials by the Food and Drug Administration (FDA) for the treatment of various cancers, such as pancreatic cancer, non-small cell lung cancer, melanoma, leukaemia. Of all the treatment modalities, vaccines are the primary treatment methods, some of which have been even entered into phase III clinical trials. The main clinical application direction of telomerase vaccine is the combination with other drugs and treatment modalities, including combination with other vaccines targeting human telomerase reverse transcriptase (hTERT), traditional chemotherapy drugs and immunosuppressors. We also summarized the recent findings of immunotherapy targeting hTERT, focusing on various vaccines and the current status of associated clinical trials. We further discussed the advantages, disadvantages and potential developmental directions of various telomerase-targeted immunotherapies. Telomerase-targeted cancer immunotherapy has promising prospects in improving patient survival expectancy. This review may provide data support and design ideas for all researchers and pharmaceutical enterprises in this field.

1249 Research of hiperfect-mediated RNA interference targeting hTERT on the biological behavior of the human malignant melanoma cell line A375

1249 Research of hiperfect-mediated RNA interference targeting hTERT on the biological behavior of the human malignant melanoma cell line A375

Design and Development of Nanostructured Co Delivery of Artemisinin and Chrysin for Targeting hTERT Gene Expression in Breast Cancer Cell Line: Possible Clinical Application in Cancer Treatment.

Background:Breast cancer is one of the most significant causes of female cancer death worldwide. To explore the possibility of a novel chemo-preventive strategy for improving breast cancer treatment, the anticancer effects of two natural compounds, Artemisinin (Art) and Chrysin (Chr), against T47D breast cancer cells were investigated. Methods:For this purpose, Art and Chr were co-encapsulated in PEGylated PLGA nanoparticles (NPs) and the synthesized NPs were characterized by FE-SEM, FTIR, and DLS and then, MTT assay was used to assess and compare the cytotoxicity of various concentrations of the chemotheruptic molecules in pure and nanoformulated forms as well as in alone and combination state after 48 h exposure time. Drug release study was performed using the dialysis method. Also, the mRNA levels of hTERT genes expression were studied by quantitative real-time PCR. Results:The results showed that pure and formulations drugs exhibited dose-dependent cytotoxicity against T47D cells and especially, Art/Chr–PLGA/PEG NPs had a more synergistic anti-proliferative effect and significantly arrested the growth of cancer cells than the other groups. Moreover, Real-time PCR results revealed that Art, Chr and combination of Art–Chr in pure and encapsulated forms inhibited hTERT gene expression. Conclusions: It was found that Art/Chr–PLGA/PEG NPs relative to pure combination could further decline hTERT expression in all concentrations. Our study demonstrated that Art/Chr-PLGA/PEG NPs based combinational therapy holds promising potential for the treatment of breast cancer.

Abstract 3108: Human telomerase reverse transcriptase regulates stemness of pancreatic cancer

Abstract Backgrounds: Human telomerase reverse transcriptase (hTERT) plays a key role on immortalization of cancer cells. In the present study, we clarified the effect of small interfering RNA (siRNA) targeting hTERT in pancreatic cancer cell lines. Methods: Two sequences of siRNA targeting hTERT and negative control siRNA were transfected into PANC-1 and MIA PaCa-2 human pancreatic cancer cell lines. Cell proliferation (WST-8 cell counting assay), stemness (sphere formation) and migration (scratch assay) were analyzed using hTERT-siRNA transfected cells and control-siRNA transfected cells. The expression levels of CDK1, SOX9 and nestin were analyzed by real time PCR. Results: As compared to control-siRNA, hTERT-siRNA suppressed cell proliferation and stemness of pancreatic cancer cell lines. Cell migration did not show any difference between hTERT-siRNA and control-siRNA transfected cells. The expression levels of CDK1, SOX9 and nestin in hTERT-siRNA transfected cells were lower than those in control-siRNA transfected cells. Conclusion: The present study indicates that inhibition of hTERT in pancreatic cancer cells suppressed stemness via alteration of the expression levels of CDK1, SOX9 and nestin. Citation Format: Juanjuan Ye, Keiko Yamakawa, Yuko Narusawa, Yoko Matsuda. Human telomerase reverse transcriptase regulates stemness of pancreatic cancer [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2021; 2021 Apr 10-15 and May 17-21. Philadelphia (PA): AACR; Cancer Res 2021;81(13_Suppl):Abstract nr 3108.

Telomerase inhibition, telomere attrition and proliferation arrest of cancer cells induced by phosphorothioate ASO-NLS conjugates targeting hTERC and siRNAs targeting hTERT

Telomerase activity has been regarded as a critical step in cellular immortalization and carcinogenesis and because of this, regulation of telomerase represents an attractive target for anti-tumor specific therapeutics. Recently, one avenue of cancer research focuses on antisense strategy to target the oncogenes or cancer driver genes, in a sequence specific fashion to down-regulate the expression of the target gene. The protein catalytic subunit, human telomerase reverse transcriptase (hTERT) and the template RNA component (hTERC) are essential for telomerase function, thus theoretically, inhibition of telomerase activity can be achieved by interfering with either the gene expression of hTERT or the hTERC of the telomerase enzymatic complex. The present study showed that phosphorothioate antisense oligonucleotide (sASO)-nuclear localization signal (NLS) peptide conjugates targeting hTERC could inhibit telomerase activity very efficiently at 5 μM concentration but less efficiently at 1 μM concentration. On the other hand, siRNA targeting hTERT mRNA could strongly suppress hTERT expression at 200 nM concentration. It was also revealed that siRNA targeting hTERT could induce telomere attrition and then irreversible arrest of proliferation of cancer cells.

Telomerase-Targeted Cancer Immunotherapy.

Telomerase, an enzyme responsible for the synthesis of telomeres, is activated in many cancer cells and is involved in the maintenance of telomeres. The activity of telomerase allows cancer cells to replicate and proliferate in an uncontrolled manner, to infiltrate tissue, and to metastasize to distant organs. Studies to date have examined the mechanisms involved in the survival of cancer cells as targets for cancer therapeutics. These efforts led to the development of telomerase inhibitors as anticancer drugs, drugs targeting telomere DNA, viral vectors carrying a promoter for human telomerase reverse transcriptase (hTERT) genome, and immunotherapy targeting hTERT. Among these novel therapeutics, this review focuses on immunotherapy targeting hTERT and discusses the current evidence and future perspectives.

Knockdown of human telomerase reverse transcriptase induces apoptosis in cervical cancer cell line.

Background & objectives:Human telomerase reverse transcriptase (hTERT) is the catalytic subunit of telomerase enzyme that maintains telomere ends by the addition of telomeric repeats to the ends of chromosomal DNA, and that may generate immortal cancer cells. Hence, the activity of telomerase is raised in cancer cells including cervical cancer. The present study aimed to validate the unique siRNA loaded chitosan coated poly-lactic-co-glycolic acid (PLGA) nanoparticle targeting hTERT mRNA to knock down the expression of hTERT in HeLa cells.Methods:The siRNA loaded chitosan coated polylactic-co-glycolic acid (PLGA) nanoparticles were synthesized by double emulsion solvent diffusion method. The characterization of nano-formulation was done to determine efficient siRNA delivery. MTT [3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide] assay, reverse transcriptase-polymerase chain reaction (RT-PCR) and Western blot were performed to evaluate silencing efficiency of nano-formulation.Results:Size, zeta potential and encapsulation efficiency of nanoparticles were 249.2 nm, 12.4 mV and 80.5 per cent, respectively. Sustained release of siRNA from prepared nanoparticle was studied for 72 h by ultraviolet method. Staining assays were performed to confirm senescence and apoptosis. Silencing of hTERT mRNA and protein expression were analyzed in HeLa cells by RT-PCR and Western blot.Interpretation & conclusions:The findings showed that biodegradable chitosan coated PLGA nanoparticles possessed an ability for efficient and successful siRNA delivery. The siRNA-loaded PLGA nanoparticles induced apoptosis in HeLa cells. Further studies need to be done with animal model.

The antitumor effects of oncolytic adenovirus ZD55-double-hTERT expressing double small hairpin RNA targeting hTERT gene on prostate cancer DUl45 cells.

Objective To investigate the antitumor effects of oncolytic adenovirus ZD55-double-hTERT expressing double small hairpin RNA targeting hTERT gene on prostate cancer DUl45 cells. Methods The inhibitory effect of ZD55-double-hTERT on hTERT expression was evaluated by quantitative real-time PCR and Western blot analysis.The expression of E1A protein in DUl45 cell infected with viruses respectively was detected by Western blot, The cytotoxicity of ZD55-double-hTERT was detected by MTT assay.The apoptosis effect was determined by Hoechst-33342. Results Real-time PCR and Western blot assay showed that ZD55-double-hTERT could significantly decrease the expression of hTERT in DUl45 cells. Western blot assay of the E1A expression indicated DU145 cells infected with adenoviruses could express E1A, but blank group could not. MTT assay showed that cell viability of ZD55-double-hTERT group was significantly lower than that in other groups.ZD55-double-hTERT treatment of DU145 cells increased apoptotic cell death as compared to ZD55-hTERT1, ZD55-hTERT2 and blank group. Conclusions Oncolytic adenovirus ZD55-double-hTERT can more effectively knock down the expression hTERT in DUl45 cells, and exert more dramatically antitumor effects than ZD55-hTERT1 and ZD55-hTERT2. Key words: Prostatic Neoplasms; Oncolytic Viruses; MicroRNAs; Genes

Abstract 4798: Targeting hTERT for treatment of glioblastoma (GBM)

Abstract Approximately 86% of GBM tumors exhibit a mutation at -124 or -146 bp upstream from the ATG start site in the transcription activating promoter region of Human telomerase reverse transcriptase (hTERT). Mutations in the hTERT promoter are known to impair repression, leading to overexpression of hTERT and tumor maintenance. Yet, complete understanding of mechanistic implications is still necessary. While overexpressed hTERT is associated with oncogenesis and resistance to apoptosis, we also observe phenotypes unrelated to the reverse transcription function. We characterized long-term glioma cell lines and glioma PDX models by hTERT promoter mutation status, hTERT mRNA expression, and hTERT protein expression in subcellular fractions. The -124 and -146 mutations are located in the major 5-12 G-quadruplex and result in misfolding of the silencer element, thus causing over-expression of hTERT. Using a diverse small molecule library, we identified a small drug-like pharmacological chaperone (pharmacoperone) molecule, TG-4260, which binds to the 26 mer base-pair heteroduplex loop, which is the nucleation site for cooperative folding of the major 5-12 G-quadruplex. The chaperone effect of TG-4260 corrects DNA hTERT G-quadruplex misfolding resulting from the promoter mutations and restores the silencer function of the G-quadruplex. TG-4260 directly decreases the transcription activity of the −124, −124/125, −138/139, and −146 mutants to a similar extent and suppresses the downstream gene BCL2, which activates caspase-3 and produces cell-cycle arrest, leading to cell death. This is the first example of the use of a pharmacoperone molecule to correct the misfolding of a DNA G-quadruplex element resulting from mutations in an early folding intermediate. We screened GBM cell models against this novel small molecule inhibitor that interferes with mutated hTERT promoter and demonstrated that TG-4260 selectively suppresses glioma cell viability without affecting non-transformed normal human astrocytes. Finally, telomere phenotypes from treated cells indicate non-canonical functions of telomerase may also contribute to glioma pathogenesis. Citation Format: Saumya Reddy Bollam, Hyun-Jin Kang, Sen Peng, Vijay Gokhale, Laurence Hurley, Michael Berens, Harshil Dhruv. Targeting hTERT for treatment of glioblastoma (GBM) [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2018; 2018 Apr 14-18; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2018;78(13 Suppl):Abstract nr 4798.

Telomerase reverse transcriptase coordinates with the epithelial-to-mesenchymal transition through a feedback loop to define properties of breast cancer stem cells.

ABSTRACTTelomerase and its core component, telomerase reverse transcriptase (hTERT), are critical for stem cell compartment integrity. Normal adult stem cells have the longest telomeres in a given tissue, a property mediated by high hTERT expression and high telomerase enzymatic activity. In contrast, cancer stem cells (CSCs) have short telomeres despite high expression of hTERT, indicating that the role of hTERT in CSCs is not limited to telomere elongation and/or maintenance. The function of hTERT in CSCs remains poorly understood. Here, we knocked down hTERT expression in CSCs and observed a morphological shift to a more epithelial phenotype, suggesting a role for hTERT in the epithelial-to-mesenchymal transition (EMT) of CSCs. Therefore, in this study, we systematically explored the relationship between hTERT and EMT and identified a reciprocal, bi-directional feedback loop between hTERT and EMT in CSCs. We found that hTERT expression is mutually exclusive to the mesenchymal phenotype and that, reciprocally, loss of the mesenchymal phenotype represses hTERT expression. We also showed that hTERT plays a critical role in the expression of key CSC markers and nuclear β-catenin localization, increases the percentage of cells with side-population properties, and upregulates the CD133 expression. hTERT also promotes chemoresistance properties, tumorsphere formation and other important functional CSC properties. Subsequently, hTERT knockdown leads to the loss of the above advantages, indicating a loss of CSC properties. Our findings suggest that targeting hTERT might improve CSCs elimination by transitioning them from the aggressive mesenchymal state to a more steady epithelial state, thereby preventing cancer progression.

Telomerase reverse transcriptase mediates EMT through NF-κB signaling in tongue squamous cell carcinoma.

Locoregional lymph nodes metastasis in oral tongue squamous cell carcinoma represents one of important and common prognostic factors for poor clinical outcome. The human Telomerase Reverse Transcriptase (hTERT) is one of key players in cancer metastasis and stemness, but its exact function in tongue squamous cell carcinoma remains unknown. Here, we aim to understand the role of hTERT by utilizing the CRISPR/Cas9 gene editing system to deplete hTERT in the SCC-15 cell line. Functional comparison of SCC-15 control and knockout cells (hTERT−/−) showed that loss of hTERT suppressed cell proliferation and migration/invasion. Furthermore, hTERT depletion significantly decreased tumorigenesis, including alterations in cellular morphology that areindicative for epithelial-mesenchymal transition (EMT). Mechanistically we demonstrated that the hTERT knockout attenuates NF-κB signaling via a negative feedback regulation in tumorprogression. From these results we propose a novel molecular mechanism of hTERT to promote SCC-15 invasion and metastasis via NF-κB activation. We conclude that targeting hTERT may represent a new therapeutic strategy to improve therapy and survival of tongue squamous cell carcinoma patients.

Abstract 1522: Targeted gene therapy · c-MYC and hTERT in the scope

Abstract In the past decade immunotherapy and kinase inhibitors have revolutionized the approach for cancer therapy, particularly for melanoma which has benefit from this new therapy in the last 5 years. Although, new treatment increase patient survival, there is no cure and treatments are often limited to a subtype of patients. Therefore there is still an urgent need for efficient therapy for melanoma. Among potential targets for cancer therapy, c-MYC and hTERT which are overexpressed in up-to 85% of all cancers are the most promising. The overexpression of hTERT provides indefinite division to cancer cells. Deregulation of c-MYC affect a large array of genes involved in cell cycle, proliferation/differentiation and apoptosis. Notably, both c-MYC and hTERT genes have in their promoter area a G-rich sequence that form secondary structure called G-quadruplex. These G-quadruplex structures are part of the silencers and are required for the repression of c-MYC or hTERT transcription. Mutations in the G-rich sequences of hTERT promoter destabilize the G-quadruplex and are associated with incidence of melanoma. We have shown that oligonucleotides encoding for the G-quadruples sequences of these genes promoter downregulate their respective gene expression and inhibit cell growth in different cell lines. We investigated the effect of targeting c-MYC or hTERT gene expression in melanoma cell lines using oligonucleotides targeted to the silencer G-quadruplex sequence. Four cell lines where investigated (SK-Mel-2, SK-Mel-3, SK-Mel-28 and A375) for the effect of Pu27 (targeting c-MYC) and Tert-FL (targeting hTERT) on cell proliferation using MTT assay. The treatment for 6 days resulted in 60% growth inhibition in the presence of Pu27 and 50% in the presence of Tert-FL for SK-Mel-2, SK-Mel-3, SK-Mel-28 while A375 was less sensitive. Gene expression evaluated by QRT-PCR showed down-regulation of c-MYC and hTERT in cell exposed to Pu27 and hTERT in cells exposed to Tert-FL. hTERT expression is regulated by c-MYC and in our experiment the down-regulation of c-MYC correlate with down-regulation of hTERT suggesting a cascade effect. In addition, SOX2 gene expression was also down-regulated by Pu27 suggesting an effect on the cancer stem cells (CSC). Our data demonstrate that oligonucleotides targeted to c-MYC and hTERT down-regulate gene expression associated with melanoma cell growth inhibition and possibly on metastasis. Citation Format: Francine Rezzoug, Shelia D. Thomas, Segen Tella, Donald M. Miller. Targeted gene therapy · c-MYC and hTERT in the scope [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 1522. doi:10.1158/1538-7445.AM2017-1522

Inhibitory effect of RNA interference targeting human telomerase reverse transcriptase against hepatoblastoma cells

Objective To construct a recombinant retrovirus vector expressing small interfering RNA (siRNA) targeting human telomerase reverse transcriptase (hTERT), and assess its effect on proliferation and apoptosis of human hepatoblastoma cells. Methods The sequences of the siRNA targeting hTERT, U6 promoter and enhanced green fluorescent protein (EGFP) gene were amplified by polymerase chain reaction (PCR) and inserted into the mammalian retroviral expression vector pLXSN. The PCR method was used to amplify hTERT-siRNA, EGFP, DNA fragment of U6+ 27. The retroviral expression vector pLXSN-EGFP-U6-siTERT was constructed and subjected to enzyme digestion identification. The recombinant retroviral vector pLXSN-EGFP-U6-siTERT was constructed. The vector was then used to infect human hepatoblastoma cell HepG2. The telomerase activity of the infected cells was detected by telomerase repeat amplification protocol-silver staining, and the cell apoptosis was examined using flow cytometry. The inhibition rate of HepG2 cell proliferation was analyzed by methyl thiazol tetrazolium (MTT) assay. Results hTERT-siRNA retroviral expression vectors were successfully prepared. In the control group, the telomerase activity was 2 143.06±198.69. At 24, 48 and 72 h after colony forming units (CFU) recombinant virus infection the telomerase activity was 1 632.02±116.28, 899.38±126.11 and 321.25±25.25 respectively. As compared with before infection, the telomerase activity was respectively decreased by 23.84%, 58.03% and 85.01%. There was significant difference among different time groups (P=0.046, 0.024 and 0.008). The results of flow cytometry showed that there was significant difference in the cell apoptosis rate between the experimental group and the control group with different titer of recombinant virus infection, and there was a concentration-dependent relationship. At 24 h after 1×105 CFU recombinant virus infection, the apoptosis rate was 29.05%. MTT results showed that apoptosis rate was 29.05% at 24 h after recombinant virus infection, the tumor cell death was significant, and with the increase in virus titer and prolongation of time, cell death also gradually increased. At 24, 48 and 72 h after virus infection of 6.0×105 CFU, the results of MTT assay were 0.29±0.14, 0.20±0.13 and 0.18±0.11 respectively. At 24, 48 and 72 h after virus infection of 3.0×105 CFU, MTT results were 0.32±0.11, 0.26±0.12 and 0.25±0.10. At 24, 48 and 72 h after virus infection of 1.0×105 CFU, results of MTT assay were 0.33±0.12, 0.26±0.13 and 0.26±0.12. At 24, 48 and 72 h after virus infection of 1.0×104 CFU, the results of MTT assay were 0.43±0.14, 0.35±0.10 and 0.33±0.15. At 24, 48 and 72 h after virus infection of 1.0×103 CFU, the results of MTT assay were 0.52±0.11, 0.44±0.13 and 0.44±0.10. At 24, 48 and 72 h after virus infection of 1.0×102 CFU, the results of MTT assay were 0.65±0.13, 0.61±0.15 and 0.60±0.16. In the negative control group, the results of MTT assay were 0.69±0.11, 1.01±0.14 and 2.98±0.16 respectively at 24, 48 and 72 h. There existed the concentration-effect and time-effect relationship (P=0.037, 0.034 and 0.028). Conclusion hTERT-siRNA can effectively silence hTERT gene and suppress the telomerase activity and proliferation of HepG2 cells. Key words: RNA interference; Human telomerase reverse transcriptase; Hepatoblastoma; Apoptosis

MicroRNA-138 inhibits proliferation, migration and invasion through targeting hTERT in cervical cancer.

A growing body of evidence suggests that microRNA-138 (miR-138) functions as a tumor suppressor, and is involved in tumor initiation, development and metastasis in certain types of human cancers. However, the function and underlying molecular mechanism of miR-138 in cervical cancer remains unclear. Therefore, the purpose of the present study was to investigate the clinical significance of miR-138 expression in cervical cancer, and to evaluate its role and underlying mechanisms in cervical cancer. The present study indicated that miR-138 expression was significantly downregulated in cervical cancer tissues and cell lines, and that the low miR-138 expression was negatively associated with advanced FIGO stage and lymph node metastasis (P<0.01). Functional analyses indicated that the overexpression of miR-138 in cervical cancer cells inhibited cell proliferation, migration and invasion, induced cell apoptosis in vitro, and suppressed tumor growth in a nude mice model. Luciferase reporter assays confirmed that human telomerase reverse transcriptase was a novel target gene of miR-138. The findings of the present study suggested that miR-138 could be a potential candidate for cervical cancer therapeutics.

Human telomerase reverse transcriptase (hTERT) promotes gastric cancer invasion through cooperating with c-Myc to upregulate heparanase expression.

Human telomerase reverse transcriptase (hTERT) is a central regulator of multiple hallmarks of tumors. However, the potential roles of hTERT in tumor invasion and metastasis and the underlying molecular mechanisms remain poorly understood. Here, we found that the expression of hTERT in gastric cancer (GC) was significantly associated with an advanced TNM stage, lymphatic metastasis. Survival analysis identified hTERT as an independent prognostic factor for survival of GC patients. hTERT promoted the invasion and metastasis of GC cells by binding to c-Myc and recruiting the complex to heparanase promoter to upregulate heparanase expression. In addition, our data demonstrated that hTERT activated Wnt/β-catenin signaling to promote c-Myc expression which could in turn activate hTERT transcription and expression, suggesting a positive feedback regulation in GC progression. Consistently, c-Myc and heparanase expression was positively correlated with hTERT levels, and was also an independent predictor of metastasis and survival. Collectively, our data provide a novel molecular mechanism for hTERT in promotion of GC invasion and metastasis, and highlight the molecular etiology and clinical significance of hTERT in GC progression. Targeting hTERT may represent a new therapeutic strategy to improve therapy and survival of GC patients.

MiR-512-5p suppresses tumor growth by targeting hTERT in telomerase positive head and neck squamous cell carcinoma in vitro and in vivo.

Telomerase activation has very important implications for head and neck squamous cell carcinoma (HNSCC), but the regulatory mechanisms of telomerase in HNSCC remain unclear. In our present study, we found that miR-512-5P was markedly downregulated in telomerase-positive HNSCC cell lines. Both in vitro and in vivo assays revealed that miR-512-5P mimic attenuated HNSCC cell proliferation, and tumor growth in nude mice, which exerts its tumor suppressor function through elevated apoptosis, inhibition of the telomerase activity, decrease of telomere-binding proteins and shortening of telomere length by human telomerase reverse transcriptase (hTERT) downregulation. Furthermore, the dual-luciferase reporter gene assay results demonstrated that hTERT was a direct target of miR-512-5P. We conclude that the frequently miR-512-5P overexpression can regulate hTERT and function as a tumor suppressor in HNSCC. Therefore, miR-512-5P may serve as a potential therapeutic agent for miR-based HNSCC therapy.

Abstract 3547: Systemically delivered human telomerase reverse transcriptase (hTERT)-targeting p53-laden adenovirus shows strong antitumor effects in intrahepatic Hep3B xenograft mouse model

Abstract Human telomerase reverse transcriptase (hTERT) RNA and p53 tumour suppressor were previously suggested as useful targets for cancer gene therapy. In this study, we investigated the effects of systemically delivered adenoviruses harboring trans-splicing ribozyme that could target hTERT and induce p53 transcript in targeted cancer cells in vivo using mice with Hep3B human hepatocellular carcinoma cells that metastasized to the liver. We observed that intravenous injection of this virus (0.5 X1011 Virus) produced efficient regression of Hep 3B tumor metastasized nodules. Next we performed in vitro study to know whether it has a synergistic effects with CDDP, we observed that CDDP (2.5 ug/ml) increased cytotoxic effects in vitro given with the virus (10 MOI), but in vivo study revealed that addition of CDDP or arsenic hexoxide (As4O6) did not improve the therapeutic efficacy of the virus treatment. Next we performed toxicity test with 4 doses (0.5 X1011, 1 X1011, 2 X1011, and 5 X1011, Virus), which revealed that there were no toxic death, but 2-4 fold increased in AST and ALT with no changes in bilirubin, BUN, Creatinine levels. In conclusion, this study demonstrates that the adenoviruses harboring trans-splicing ribozyme targeting hTERT and inducing p53 transcript represents a safe and promising cancer treatment for hepatocellular carcinoma. [This study was supported by a grant from the National R&amp;D Program for Cancer Control, Ministry for Health, Welfare &amp; Family Affairs, Republic of Korea. (0820050), and National Research Foundation of Korea (NRF) grant funded by the Korea government(MEST) (20120002631).] Keywords: Adenovirus, trans-splicing ribozyme, p53 tumour suppressor, Human Telomerase Reverse Transcriptase, cancer Citation Format: Won Sup Lee, Hye Lim Kang, Arulkumar Nagappan, Jeong Won Yun, GonSup Kim, Soon Chan Hong, Sang-Jin Lee, In-Hoo Kim. Systemically delivered human telomerase reverse transcriptase (hTERT)-targeting p53-laden adenovirus shows strong antitumor effects in intrahepatic Hep3B xenograft mouse model. [abstract]. In: Proceedings of the 106th Annual Meeting of the American Association for Cancer Research; 2015 Apr 18-22; Philadelphia, PA. Philadelphia (PA): AACR; Cancer Res 2015;75(15 Suppl):Abstract nr 3547. doi:10.1158/1538-7445.AM2015-3547

Expression of human telomerase reverse transcriptase mediates the senescence of mesenchymal stem cells through the PI3K/AKT signaling pathway.

Multipotent mesenchymal stem cells(MSCs) are widely used as seed cells in studies of tissue engineering and regenerative medicine; however, their clinical application is limited due to replicative senescence. It has been demonstrated that telomerase expression extends the lifespan and maintains the bone-forming ability of MSCs; however, the detailed role and the underlying molecular mechanisms in MSCs remain largely unknown. In the present study, we found that senescence was associated with human telomerase reverse transcriptase(hTERT) expression, and telomere length and telomerase activity. We established a short interfering RNA(siRNA) targeting hTERT and a gene expression vector carrying hTERT and transfected these into the MSCs to investigate the detailed role and the underlying molecular mechanisms of action of hTERT in MSCs. We found that the downregulation of hTERT by siRNA markedly decreased telomere length and telomerase activity in the MSCs, whereas the overexpression of hTERT increased telomere length and telomerase activity in the MSCs. The downregulation of hTERT inhibited cell proliferation and promoted the senescence and apoptosis of MSCs, whereas the upregulation of hTERT increased cell proliferation and decreased the senescence and apoptosis of MSCs. Of note, we also found that the activation of the PI3K/AKT signaling pathway was mediated by hTERT and that blocking this pathway using LY294002 inhibited hTERT expression, induced senescence and decreased the proliferation of MSCs. These findings reveal a previously unknown regulatory mechanism of hTERT, indicating that hTERT mediates the senescence of MSCs through the PI3K/AKT signaling pathway.

The effect of silencing human telomerase reverse transcriptase by RNA interference on U87 glioma cell proliferation and apoptosis

Objective To observe the effect of human telomerase reverse transcriptase (hTERT) on U87 glioma cell proliferation and apoptosis.Methods The small interfering RNA (siRNA) expression plasmid targeting hTERT was constructed and transfected into U87 glioma cells.The expression level of hTERT protein and mRNA was detected by Western blotting and reverse transcription-polymerase chain reaction (RT-PCR) respectively.The U87 glioma cell proliferation and apoptosis were analyzed by methyl thiazol tetrazolium (MTT) assay and flow cytometry,respectively.Results Compared with the blank control group and the negative control group,the expression level of hTERT protein in each siRNA group was significantly reduced (P ＜ 0.05).The relative expression level of hTERT mRNA in each siRNA group was 0.79 ±0.11,0.58 ±0.15 and 0.39 ±0.16 respectively,which was significantly lower than in the negative control group (P ＜ 0.05).As compared with the blank control group and the negative control group,the proliferation of U87 glioma cells after transfection was significantly inhibited and the strongest inhibitory effect was observed on the third day.The apoptosis rate of U87 glioma cells of in each siRNA group which was transfected after 48 h was (14.31 ± 0.93) ％,(17.57 ± 0.61) ％ and (22.68 ± 1.04) ％ respectively,which was significantly higher than in the blank control group and the negative control group (P ＜0.05).Conclusion The silence of hTERT gene after siRNA targeting hTERT being transfected into U87 glioma cells can significantly inhibit cell proliferation and induce cell apoptosis. Key words: RNA interference; Human telomerase reverse transcriptase; Glioma; Cell proliferation

Knockdown of hTERT by siRNA inhibits cervical cancer cell growth in vitro and in vivo.

Human telomerase reverse transcriptase (hTERT) is the catalytic component of telomerase that facilitates tumor cell invasion and proliferation. It has been reported that telomerase and hTERT are significantly upregulated in majority of cancers including cervical cancer, thus, downregulation of hTERT is a promising target in malignant tumor treatment. We established a short interfering RNA (siRNA) targeting hTERT, and transfected it into HeLa cells (a cervical cancer cell line) to investi-gate the effect of cell proliferation, apoptosis, migration and invasion in cervical cancer cells. The results showed that siRNA targeting hTERT could effectively knock down hTERT expression, remarkably suppress telomerase activity, cell proliferation, migration and invasion, and induced cell apoptosis of cervical cancers cells in vitro. In addition, we evaluated whether siRNA targeting hTERT affects tumor growth in nude mice, and found that it dramatically inhibited tumorigenesis and growth of mice injected with siRNA targeting hTERT. Furthermore, we also found that knockdown of hTERT was able to significantly suppress constitutive phosphorylation of Akt, PI3K, which might imply that reduction of hTERT inhibited tumor growth via the PI3K/Akt signaling pathway to some extent. These results suggest that the suppression of hTERT expression by siRNA inhibits cervical cancer cell growth in vitro and in vivo, and may provide a novel target for anticancer gene therapy.