Downregulation Of hTERT Research Articles

Inhibition of hTERT/telomerase/telomere mediates therapeutic efficacy of osimertinib in EGFR mutant lung cancer.

The inevitable acquired resistance to osimertinib (AZD9291), an FDA-approved third-generation EGFR tyrosine kinase inhibitor (EGFR-TKI) for the treatment of patients with advanced non-small cell lung cancer (NSCLC) harboring EGFR activating or T790M resistant mutations, limits its long-term clinical benefit. Telomere maintenance via telomerase reactivation is linked to uncontrolled cell growth and is a cancer hallmark and an attractive cancer therapeutic target. Our effort toward understanding the action mechanisms, including resistance mechanisms, of osimertinib has led to the identification of a novel and critical role in maintaining c-Myc-dependent downregulation of hTERT, a catalytic subunit of telomerase, and subsequent inhibition of telomerase/telomere and induction of telomere dysfunction in mediating therapeutic efficacy of osimertinib. Consequently, osimertinib combined with the telomere inhibitor, 6-Thio-dG, which is currently tested in a phase II trial, effectively inhibited the growth of osimertinib-resistant tumors, regressed EGFRm NSCLC patient-derived xenografts, and delayed the emergence of acquired resistance to osimertinib, warranting clinical validation of this strategy to manage osimertinib acquired resistance.

The Effect of Dual Bioactive Compounds Artemisinin and Metformin Co-loaded in PLGA-PEG Nano-particles on Breast Cancer Cell lines: Potential Apoptotic and Anti-proliferative Action.

The most prevalent malignancy among women is breast cancer. Phytochemicals and their derivatives are rapidly being recognized as possible cancer complementary therapies because they can modify signaling pathways that lead to cell cycle control or directly alter cell cycle regulatory molecules. The phytochemicals' poor bioavailability and short half-life make them unsuitable as anticancer drugs. Applying PLGA-PEG NPs improves their solubility and tolerance while also reducing drug adverse effects. According to the findings, combining anti-tumor phytochemicals can be more effective in regulating several signaling pathways linked to tumor cell development. The point of the study was to compare the anti-proliferative impacts of combined artemisinin and metformin on cell cycle arrest and expression of cyclin D1 and apoptotic genes (bcl-2, Bax, survivin, caspase-7, and caspase-3), and also hTERT genes in breast cancer cells. T-47D breast cancer cells were treated with different concentrations of metformin (MET) and artemisinin (ART) co-loaded in PLGA-PEG NPs and free form. The MTT test was applied to assess drug cytotoxicity in T47D cells. The cell cycle distribution was investigated using flow cytometry and the expression levels of cyclin D1, hTERT, Bax, bcl-2, caspase-3, and caspase-7, and survivin genes were then determined using real-time PCR. The findings of the MTT test and flow cytometry revealed that each state was cytotoxic to T47D cells in a time and dose-dependent pattern. Compared to various state of drugs (free and nano state, pure and combination state) Met-Art-PLGA/PEG NPs demonstrated the strongest anti-proliferative impact and considerably inhibited the development of T-47D cells; also, treatment with nano-formulated forms of Met-Art combination resulted in substantial downregulation of hTERT, Bcl-2, cyclin D1, survivin, and upregulation of caspase-3, caspase-7, and Bax, in the cells, as compared to the free forms, as indicated by real-time PCR findings. The findings suggested that combining an ART/MET-loaded PLGA-PEG NP-based therapy for breast cancer could significantly improve treatment effectiveness.

Angiotensin-(1–7) promotes mitochondrial translocation of human telomerase reverse transcriptase in HUVECs through the TOM20 complex

BackgroundAngiotensin (Ang) (1–7) is a vasodilator peptide that ameliorates microcirculation dysfunction, increases telomerase activity in cells, and exerts vasodilatory, anti-inflammatory, antioxidative stress, and antiapoptotic effects. Mitochondrial human telomerase reverse transcriptase (hTERT) plays an important role in the processes of antiapoptosis, antioxidative stress, and immortalization. This study aimed to investigate the effect of Ang(1–7) on the mitochondrial translocation of hTERT. MethodsAn in vitro model of lipopolysaccharide (LPS)-induced inflammation was established in human umbilical vein endothelial cells (HUVECs). Ang(1–7) was added to cells 30 min before LPS stimulation. The Ang(1–7)/Mas receptor antagonist A779 plus Ang(1–7) were added to the cells 30 min before LPS stimulation. The translocase outer membrane (TOM)20-overexpression HUVECs (HUVEC-TOM20OE), TOM20-knockdown HUVECs (HUVEC-TOM20KD), and the corresponding negative control cell lines were constructed by lentiviral transfection of HUVECs. Cells subjected to LPS stimulation alone, LPS plus Ang(1–7), LPS plus Ang(1–7) and A779, vehicle and no treatment were termed the LPS group, LPS + A group, LPS + A + A779 group, Con group and Neg group, respectively. Immunofluorescence staining was used to detect the distribution of hTERT in the nuclei and mitochondria of HUVECs and to locate TOM20, TOM40, and translocase inner membrane (TIM)23 in the mitochondria. The protein expression levels of total hTERT, mitochondrial hTERT, TOM20, TOM40, and TIM23 were measured by Western blot. The mRNA expression levels of hTERT, TOM20, TOM40, and TIM23 were assessed by quantitative reverse transcription–polymerase chain reaction (qRT-PCR). ResultshTERT colocalized with TOM40, TOM20 and TIM23 in the mitochondria. The mitochondrial hTERT protein level of the LPS + A group was significantly greater than that of the LPS group (P = 0.001), and the LPS group showed significantly increased expression of mitochondrial hTERT compared with that of the control group (P = 0.001). No significant difference in the level of total hTERT was observed between the LPS + A and LPS groups. The mitochondrial hTERT protein level of the LPS + A + A779 group was significantly lower than that of the LPS + A group (P = 0.021). The protein level of mitochondrial hTERT in HUVEC-TOM20KD treated with or without LPS alone or LPS + A was significantly decreased compared with the corresponding groups of control HUVECs (HUVEC-TOM20KD-Con vs. HUVEC-Con, P = 0.035; HUVEC-TOM20KD-LPS vs. HUVEC-LPS, P = 0.003; HUVEC-TOM20KD-LPS + A vs. HUVEC-LPS + A, P = 0.001), and treatment with Ang(1–7) did not restore the downregulation of mitochondrial hTERT in HUVEC-TOM20KD. HUVEC-TOM20OE showed a significantly increased level of mitochondrial hTERT (HUVEC-TOM20OE-Con vs. HUVEC-Con, P = 0.010), which was further elevated by Ang(1–7) stimulation (HUVEC-TOM20OE-LPS + A vs. HUVEC-TOM20OE-Con, P = 0.011). Lastly, the protein expression levels of TOM40 (HUVEC-TOM20KD-Con vs. HUVEC-Con, P = 0.007) and TIM23 (HUVEC-TOM20KD-Con vs. HUVEC-Con, P = 0.001) were significantly increased in HUVEC-TOM20KD in comparison to HUVECs. ConclusionsAng(1–7) effectively promoted mitochondrial translocation of hTERT in HUVECs via TOM20, indicating that hTERT may be transported to the mitochondria through the TOM20 complex. In addition, A779 could block the effects of Ang(1–7) in HUVECs.

Human Telomerase Reverse Transcriptase as a Therapeutic Target of Dihydroartemisinin for Esophageal Squamous Cancer

Objective: To elucidate the oncogenic role of human telomerase reverse transcriptase (hTERT) in esophageal squamous cancer and unravel the therapeutic role and molecular mechanism of dihydroartemisinin (DHA) by targeting hTERT. Methods: The expression of hTERT in esophageal squamous cancer and the patients prognosis were analyzed by bioinformatic analysis from TCGA database, and further validated with esophageal squamous cancer tissues in our cohort. The Cell Counting Kit-8 (CCK8) and colony formation assay were used to evaluate the proliferation of esophageal squamous cancer cell lines (Eca109, KYSE150, and TE1) after hTERT overexpression or treated with indicated concentrations of DHA. Transwell migration assay and scratch assay were employed to determine the migration abilities of cancer cells. Fluorescence microscopy and flow cytometry were conducted to measure the intracellular reactive oxygen species (ROS) levels in cancer cells after treated with DHA. Moreover, RT-PCR and Western blot were performed to test the alteration of associated genes on mRNA and protein level in DHA treated esophageal squamous cancer cell lines, respectively. Furthermore, tumor-bearing nude mice were employed to evaluate the anticancer effect of DHA in vivo. Results: We found that hTERT was significantly upregulated in esophageal squamous cancer both from TCGA database and our cohort also. Overexpression of hTERT evidently promoted the proliferation and migration of esophageal squamous cancer cells in vitro. Moreover, DHA could significantly inhibit the proliferation and migration of esophageal cancer cell lines Eca109, KYSE150, and TE1 in vitro, and significantly down-regulate the expression of hTERT on both mRNA and protein level in a time- and dose-dependent manner as well. Further studies showed that DHA could induce intracellular ROS production in esophageal cancer cells and down-regulate SP1 expression, a transcription factor that bound to the promoter region of hTERT gene. Moreover, overexpression of SP1 evidently promoted the proliferation and migration of Eca109 and TE1 cells. Intriguingly, rescue experiments showed that inhibiting ROS by NAC alleviated the downregulation of SP1 and hTERT in cells treated with DHA. Furthermore, overexpression of SP1 or hTERT could attenuate the inhibition effect of DHA on the proliferation and migration of Eca109 cells. In tumor-bearing nude mice model, DHA significantly inhibited the growth of esophageal squamous cancer xenografts, and downregulated the expression of SP1 and hTERT protein, while no side effects were observed from heart, kidney, liver, and lung tissues by HE stain. Conclusion: hTERT plays an oncogenic role in esophageal squamous cancer and might be a therapeutic target of DHA through regulating ROS/SP1 pathway.

Oligonucleotides and microRNAs Targeting Telomerase Subunits in Cancer Therapy.

Telomerase provides cancer cells with replicative immortality, and its overexpression serves as a near-universal marker of cancer. Anti-cancer therapeutics targeting telomerase have garnered interest as possible alternatives to chemotherapy and radiotherapy. Oligonucleotide-based therapies that inhibit telomerase through direct or indirect modulation of its subunits, human telomerase reverse transcriptase (hTERT) and human telomerase RNA gene (hTERC), are a unique and diverse subclass of telomerase inhibitors which hold clinical promise. MicroRNAs that play a role in the upregulation or downregulation of hTERT and respective progression or attenuation of cancer development have been effectively targeted to reduce telomerase activity in various cancer types. Tumor suppressor miRNAs, such as miRNA-512-5p, miRNA-138, and miRNA-128, and oncogenic miRNAs, such as miRNA-19b, miRNA-346, and miRNA-21, have displayed preclinical promise as potential hTERT-based therapeutic targets. Antisense oligonucleotides like GRN163L and T-oligos have also been shown to uniquely target the telomerase subunits and have become popular in the design of novel cancer therapies. Finally, studies suggest that G-quadruplex stabilizers, such as Telomestatin, preserve telomeric oligonucleotide architecture, thus inhibiting hTERC binding to the telomere. This review aims to provide an adept understanding of the conceptual foundation and current state of therapeutics utilizing oligonucleotides to target the telomerase subunits, including the advantages and drawbacks of each of these approaches.

Atorvastatin-induced senescence of hepatocellular carcinoma is mediated by downregulation of hTERT through the suppression of the IL-6/STAT3 pathway

Hepatocellular carcinoma (HCC), a hepatic malignancy, has a poor prognosis and contributes to cancer-related death worldwide. Cellular senescence is an anticancer therapeutic strategy that causes irreversible cell cycle arrest and enables immune-mediated clearance of cancer cells. Atorvastatin, an HMG-CoA reductase inhibitor, has been shown to inhibit tumor growth and induce apoptosis or autophagy in malignant tumors. However, whether atorvastatin can induce HCC cell senescence and the mechanisms involved are poorly understood. The effects of atorvastatin-induced senescence were examined in both HCC cells and mouse xenograft models. The phenomenon and mechanism of senescence were examined by cell cycle analysis, senescence-associated β-galactosidase (SA-β-gal) staining and western blotting in HCC cells, and HCC tissues from mice were analyzed by immunohistochemical (IHC) staining. We demonstrated that atorvastatin induced cell growth inhibition and G0/G1 phase cell cycle arrest, leading to senescence in HCC cells. Atorvastatin-induced senescence was independent of p53, p14, and p16, and atorvastatin not only decreased the secretion of IL-6, a major senescence-associated secretory phenotype (SASP) factor, and the phosphorylation of STAT3 but also inhibited the expression of hTERT, a catalytic subunit of telomerase. Supplementation with exogenous IL-6 reversed both atorvastatin-induced suppression of STAT3 phosphorylation and hTERT expression and atorvastatin-induced senescence. Overexpression of constitutively activated STAT3 rescued HCC cells from atorvastatin-induced hTERT suppression and senescence. Moreover, atorvastatin decreased tumor growth in mouse xenograft models. Consistent with these results, atorvastatin decreased the IL-6, p-STAT3, and hTERT levels and increased β-gal expression in tumor sections. Taken together, these data indicate that atorvastatin can induce atypical cellular senescence in HCC cells to inhibit tumor growth, an effect mediated by downregulation of hTERT through suppression of the IL-6/STAT3 pathway.

Potent Suppression of Proliferation of Breast Carcinoma Cells by a Novel Anthranilic Acid Derivative

Background/objective: Chemotherapy remains the mainstay therapy for the most intrusive-type of breast carcinoma, triple-negative breast cancer (TNBC) that has a higher tendency for visceral metastases, relapses, and poor prognoses. It is well recognized that the reactivation of a ribonucleoprotein enzyme, telomerase is among the key determinants of breast carcinogenesis, cellular immortalization and metastatic progression. Therefore, our objectives were to assess (i) short- and (ii) long-term effects of a novel anthranilic acid (GV6) developed at our institute and compare it to a known analogue, BIBR1532 on TNBC (MDA-MB 231) and non-TNBC (MCF-7) cells. Methods: Seeded TNBC and non-TNBC flasks were supplemented with 25 µM of either BIBR 1532 or GV6 or solvent (DMSO) alone for 14 (Short-term) or 27 (Long-term) days. Trypan-blue dye exclusion test was utilized to determine viable cells, senescence-associated β-galactosidase activity was employed to detect senescent cells and qPCR was used to quantitate transcript abundance. Results: Cell viability assay revealed that short- and long-term growth inhibitions of TNBC and non-TNBC cells were comparable between BIBR1532 and GV6. Cytochemical detection of βGalactosidase demonstrated that GV6 was equally effective in inducing replicative senescence in treated TNBC and non-TNBC cells. Short- and long-term regimen of BIBR1532 and GV6 showed similar drug-induced downregulation of hTERT in TNBC and non-TNBC cells. Conclusion: Results indicate that GV6 is an equally potent inhibitor of hTERT that induces growth impedance and triggers senescence in TNBC, as well as non-TNBC cells and merits further studies for improved treatment options.

Downregulation of hTERT contributes to ovarian cancer apoptosis and inhibits proliferation of ovarian cancer cells.

BackgroundOur study aims to study the effects of the exogenous human telomerase reverse transcriptase (hTERT) interfering gene on the ovarian cancer cell line SKOV3 through proliferation and apoptosis.MethodsLipofectamine TM2000 was used to transfer the hTERT interfering gene into the SKOV3 cells. After a predetermined amount of time after transfection with the hTERT interfering genes, the expression of the tumor-related genes was detected using real-time quantitative polymerase chain reaction (RT-qPCR), and relative protein level was detected using western blot analysis. Cell morphology was acquired by microscopy. Cell viability was detected by middle-time-spray (MTS), and cell cycle and apoptosis were detected by flow cytometry.ResultsForty-eight hours after transfection, the expression of tumor-related proteins in the experimental group was increased compared with the control group, and the difference was statistically significant (P<0.05). The cell morphology showed a significant difference between the control group and the hTERT shRNA group. Furthermore, 48 and 72 h after transfection with the hTERT interfering gene, the cell viability inhibition rates of the hTERT shRNA group were 0.77±0.02 and 0.88±0.01 respectively. Compared with the control group, the cell viability inhibition rates were 11.97±2.37 (%) and 18.72±1.01 (%), respectively, with statistically significant differences (P=0.009, P=0.004). Flow cytometry detected the apoptosis peak of SKOV3 cells in the experimental group 48 h after transfection with the hTERT interfering gene. Propidium iodide (PI) and Annexin V-FITC revealed that the apoptotic cells accounted for 18.13% of the total number, which was significantly higher than the 3.85% demonstrated by the control group.ConclusionsThe amount and activity of SKOV3 cells in ovarian cancer was decreased after the exogenous introduction of the hTERT interfering gene.

The effect of maslinic acid on apoptotic genes in u266 multiple myeloma cell line

BackgroundMultiple myeloma is a clonal plasma cell malignancy which is characterized by renal failure, bone lesions, hypercalcemia and anemia. It has very poor prognosis and no effective drug has been discovered yet. Multiple myeloma patients eventually end up relapse at some point of their lifetime. Maslinic acid (MA) is a pentacyclic triterpene, found in the skin of some plants. The aim of current study was to investigate the apoptotic effects of Maslinic acid on U266 multiple myeloma cell line. MethodsViability and metabolic activity of U266 cells were examined following treatment with MA. The cell apoptosis was determined by flowcytometry analysis using Annexin V/7AAD apoptosis detection kit. The expression of hTERT, c-MYC, BCL-XL and genes were studied using Real-Time PCR. ResultsThe viability of U266 cells significantly decreased following treatment with MA in a dose-dependent manner. Metabolic activity of the cells also decreased in a dose-dependent manner. Further investigation showed that Maslinic acid led to the apoptosis of U266 cells. Additionally, the expression of hTERT, BCL-XL was decreased and the expression of BAX was increased in comparison with the control group. Moreover, the expression of c-MYC did not show meaningful change. ConclusionThe results demonstrated that MA induces apoptosis in U266 multiple myeloma cell line. Treatment with MA led to down-regulation of hTERT, BCL-XL and upregulation of BAX in U266 cells. Furthermore, the expression of C-MYC did not change compared with the control group. Further studies may propose this herbal agent as a treatment for multiple myeloma.

ShRNA-mediated silencing of hTERT promote apoptosis and senescence in human ovarian cancer cells

Background: Human telomerase reverse transcriptase (hTERT) is a crucial oncogene and a key factor in cell immortalization. Apart from hematopoiesis stem cell and germ cells, normal cells and tissues almost no expression of this gene. However, the positive expression rate of hTERT in malignant tumors is as high as 80–95%. This study was designed to investigate the mechanism of knockdown of hTERT expression in the regulation of ovarian cancer carcinogenesis in nude mice transplantation model. Methods: We identified the function of hTERT in the treatment of ovarian cancer by constructing a nude mice transplantation tumor model. Then analyzed the mechanism of hTERT in modulating ovarian cancer cell apoptosis and senescence by immunohistochemically staining. Results: Our analysis identified hTERT as a novel regulator of ovarian cancer progression and is one of the most significantly upregulated genes in ovarian cancer. Down-regulation of hTERT inhibits proliferation of ovarian cancer cells by promoting apoptosis and the expression of the senescence gene p53 and p21 . Conclusions: hTERT is a key oncogene in ovarian tumorigenesis and metastasis; the downregulation of hTERT significantly inhibit the proliferation of ovarian cancer and have an effect on treatment of ovarian cancer by activating cell apoptosis and senescence pathway.

ShRNA-mediated silencing of hTERT promote apoptosis and senescence in human ovarian cancer cells.

BackgroundHuman telomerase reverse transcriptase (hTERT) is a crucial oncogene and a key factor in cell immortalization. Apart from hematopoiesis stem cell and germ cells, normal cells and tissues almost no expression of this gene. However, the positive expression rate of hTERT in malignant tumors is as high as 80–95%. This study was designed to investigate the mechanism of knockdown of hTERT expression in the regulation of ovarian cancer carcinogenesis in nude mice transplantation model.MethodsWe identified the function of hTERT in the treatment of ovarian cancer by constructing a nude mice transplantation tumor model. Then analyzed the mechanism of hTERT in modulating ovarian cancer cell apoptosis and senescence by immunohistochemically staining.ResultsOur analysis identified hTERT as a novel regulator of ovarian cancer progression and is one of the most significantly upregulated genes in ovarian cancer. Down-regulation of hTERT inhibits proliferation of ovarian cancer cells by promoting apoptosis and the expression of the senescence gene p53 and p21.ConclusionshTERT is a key oncogene in ovarian tumorigenesis and metastasis; the downregulation of hTERT significantly inhibit the proliferation of ovarian cancer and have an effect on treatment of ovarian cancer by activating cell apoptosis and senescence pathway.

Inhibition of telomerase activity and SK-OV-3/DDP cell apoptosis by rhodium(III) and iron(III) complexes with 4′-(3-thiophenecarboxaldehyde)-2,2′:6′,2″-terpyridine

Two rhodium(III) and iron(III) complexes of 4′-(3-thiophenecarboxaldehyde)-2,2′:6′,2″-terpyridine (tpatpy): [Rh(tpatpy)Cl3]·CH3OH (tpatpy-Rh) and [Fe(tpatpy)Cl3] (tpatpy-Fe) have been prepared and characterized. Tpatpy-Rh (5.03 ± 1.15 μM) and tpatpy-Fe (18.02 ± 0.45 μM) exhibited greater cytotoxicity than the tpatpy ligand (70.11 ± 0.28 μM) and cisplatin (61.21 ± 1.39 μM) against SK-OV-3/DDP cells. In addition, tpatpy-Rh inhibited telomerase by down-regulation of c-myc and hTERT, as well as induced SK-OV-3/DDP cell arrest in the S phase.

Abstract 1469: T-oligo mediates DNA damage responses by modulating telomere associated proteins and telomerase

Abstract T-oligo, a guanine-rich oligonucleotide (GRO) homologous to the 3′ overhang of telomeres, elicits potent DNA-damage responses (DDRs) in cancer cells. However, the detailed molecular mechanism of T-oligo in cancer cells remains elusive. Recent studies confirmed that T-oligo can form intermolecular G-quadruplexes (G4), which are stabilized by hydrogen bonding of guanine residues, and cause disruption of the protective shelterin complex of telomeres. We hypothesize that single-stranded (SS) T-oligo and G4 T-oligo may modulate the shelterin proteins TRF2 and POT2 and thus induce DDRs. In this study, we utilized a pull-down assay using T-oligo, showing that T-oligo is co-localized with TRF2 and POT1 and indicating that T-oligo may interact with these telomeric proteins. We further investigated the modulation of these proteins by western blotting, showing that T-oligo treatment upregulates TRF2 by 2.2 and 3.0-fold (p&amp;lt;0.01) at 48h and 72h, respectively, and POT1 by 3.0-fold (p&amp;lt;0.02) both at 48h and 72h in melanoma cells (MM-AN). Immunofluorescence studies confirmed upregulation of TRF2 (2.4-fold) and POT1 (2.0-fold). Additionally we found that T-oligo can co-localize with telomere binding proteins TRF2 (88.4±4.5%) and POT1 (84.5±8%) using immunofluorescence. Using qRT-PCR, we found that T-oligo inhibited mRNA expression of hTERT, a catalytic subunit of telomerase, by 50%. It has been suggested that JNK activation may lead to downregulation of hTERT, hence we 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. Recently, it has been reported that the novel drug 6-Thio-2'-Deoxyguanosine (6-thio-dG), a nucleoside analogue of the approved drug 6-thioguanine, inhibits growth of cancer cells by its incorporation into the telomere via telomerase followed by subsequent shelterin disruption. Furthermore, this study aims to investigate the potential of 6-thio-dG in combination with T-oligo as an anticancer therapeutic. We first studied the concentrations of 6-thio-dG that are effective in inhibiting the growth of melanoma cells. We found that 1.25μM and 2.5μM 6-thio-dG significantly inhibited growth of melanoma cells by 1.6 and 3.8-fold, respectively (p&amp;lt;0.01). However, treatment of melanoma cells in combination with both 10μM T-oligo and 6-thio-dG (1.25μM or 2.5μM) did not significantly inhibit cell growth in comparison to T-oligo alone. T-oligo may downregulate hTERT, which is required for telomerase activity, and/or disrupt the shelterin complex, both of which are necessary for 6-thio-dG mediated inhibition of growth of melanoma cells. These results indicate that T-oligo and 6-thio-dG may induce their effects by a similar mechanism of action. Citation Format: Nabiha Haleema Khan, Zachary Schrank, Joseph Kellen, Sanjana Singh, Chike Osude, Neelu Puri, Gagan Chhabra. T-oligo mediates DNA damage responses by modulating telomere associated proteins and telomerase [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 1469.

Effects on tubulin polymerization and down-regulation of c-Myc, hTERT and VEGF genes by colchicine haloacetyl and haloaroyl derivatives

Several colchicine analogues in which the N-acetyl residue has been replaced by haloacetyl, cyclohexylacetyl, phenylacetyl and various aroyl moieties have been synthesized. The cytotoxic activities of the synthesized compounds have been measured on three tumor cell lines (HT-29, MCF-7 and A549) and on one non-tumor cell line (HEK-293). These compounds exhibit high antiproliferative activities at the nanomolar level, in many cases with a higher potency than colchicine itself. Some of the compounds, particularly the haloacetyl derivatives, inhibit the polymerization of tubulin in a similar manner as colchicine. As regards the cell cycle, the most active compounds are the chlorobenzoyl and bromobenzoyl derivatives, which cause cell cycle arrest at the G2/M phase when tested at 20 nM, and the bromoacetyl derivative, which arrests the cell cycle at 15 nM. In addition, these colchicine derivatives have shown fairly active downregulating the expression of the c-Myc, hTERT and VEGF genes, as well as VEGF protein secretion, at very low concentrations.

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.

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

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

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

The herbal medicine Melissa officinalis extract effects on gene expression of p53, Bcl-2, Her2, VEGF-A and hTERT in human lung, breast and prostate cancer cell lines

IntroductionEarlier, we verified that Melissa officinalis extract (MOE) elicits potent antiproliferative effects on different human cancer cells. To gain insights into the molecular mechanisms accounting for the cytotoxic effects of MOE, we assessed the expression patterns of several prominent molecules with therapeutic potential in cancer by Quantitative PCR (Q-PCR). MethodsA549, MCF-7 and PC3 cancer cells were grown in complete RPMI 1640 and seeded in 24 well micro plates. After incubation for 72h, 100μg/ml of MOE was added and the cells were further incubated for 72h. Afterwards, the cells were subjected to RNA extraction for the means of Q-PCR. ResultsOur results indicated that in PC3 cancer cells, MOE resulted in a significant downregulation of VEGF-A (0.0004 fold), Bcl-2 (0.001 fold), Her2 (0.02 fold), and hTERT (0.023 fold) compared to the untreated control. In addition, VEGF-A and hTERT mRNA were significantly downregulated in MCF-7 and A549 cancer cells, as well. Notably, high anti-angiogenic activity was closely associated with a high anti-telomerase activity of MOE in studying cancer cells. The decrease in VEGF-A expression was significantly superior than that of hTERT downregulation, as PC3 cancer cells with the highest hTERT down regulation (0.023) presented the highest anti VEGF activity (0.0004 fold), whereas MCF-7 cells with the lowest hTERT inhibition (0.213) showed the lowest VEGF inhibition(0.0435) among the three studied cancer cells. We noticed that the modulation of VEGF-A and hTERT gene expression can be considered as a common target, accounting for the therapeutic potential of MOE on human breast, lung and prostate cancer cells. ConclusionAltogether, it is suggested that the potent antiproliferative activity of the hydroalcoholic extract of Melissa officinalis is somehow explainable by its high potency to inhibit expression of the prominent oncogenes Bcl2, Her2, VEGF-A and hTERT in prostate cancer. In tumors with functional p53, including MCF-7 and A549 cancer cells, the role of p53, Bcl2 and Her2 is less significant. It appears that MOE exerts its antiproliferative effects in these cancer cells partly via concurrent downregulation of VEGF-A and hTERT. Additional studies are needed to clarify the role of other active molecules in cancer cells harboring functional p53.