Reactivation Of TERT Research Articles

Extended replicative lifespan of primary resting T cells by CRISPR/dCas9-based epigenetic modifiers and transcriptional activators

Extension of the replicative lifespan of primary cells can be achieved by activating human telomerase reverse transcriptase (hTERT) to maintain sufficient telomere lengths. In this work, we utilize CRISPR/dCas9-based epigenetic modifiers (p300 histone acetyltransferase and TET1 DNA demethylase) and transcriptional activators (VPH and VPR) to reactivate the endogenous TERT gene in unstimulated T cells in the peripheral blood mononuclear cells (PBMCs) by rewiring the epigenetic marks of the TERT promoter. Importantly, we have successfully expanded resting T cells and delayed their cellular senescence for at least three months through TERT reactivation, without affecting the expression of a T-cell marker (CD3) or inducing an accelerated cell division rate. We have also demonstrated the effectiveness of these CRISPR tools in HEK293FT and THP-1-derived macrophages. TERT reactivation and replicative senescence delay were achieved without inducing malignancy transformation, as shown in various cellular senescence assays, cell cycle state, proliferation rate, cell viability, and karyotype analyses. Our chromatin immunoprecipitation (ChIP)-qPCR data together with TERT mRNA and protein expression analyses confirmed the specificity of CRISPR-based transcription activators in modulating epigenetic marks of the TERT promoter, and induced telomerase expression. Therefore, the strategy of cell immortalization described here can be potentially adopted and generalized to delay cell death or even immortalize any other cell types.

Extra-nuclear TERT counteracts oxidative stress and promotes progression in papillary thyroid carcinoma

The reactivation of TERT is associated with poor outcome in papillary thyroid cancer (PTC). Extra-telomeric functions of TERT were reported, with a protective role against oxidative stress (OS). The aim of the present study was to explore the extra-nuclear TERT localization in PTC and its role in cancer progression.TERT nuclear export under OS were analyzed in K1 PTC cell line. We investigated the role of different TERT localizations using specific TERT constructs that limit its localization to the nucleus or to the mitochondria. The effect of SRC kinase inhibitor PP2, which reduces TERT nuclear export, was investigated as well. Moreover, TERT localization was analyzed in 39 PTC tissues and correlated with the genetic profile and the level of OS, DNA damage and apoptosis in the tumors and with the clinical characteristics of the patients.We demonstrated that TERT is exported from the nucleus in response to OS induced either from H2O2 or the BRAF inhibitor PLX4720. We proved that extra-nuclear TERT reduces mitochondrial OS and induces mitochondrial fragmentation. Moreover, limiting mitochondrial TERT localization reduced proliferation, migration, AKT phosphorylation and glycolysis and increased DNA damage and p21 expression. Finally, in PTC tissues the fraction of mitochondrial/nuclear TERT resulted inversely correlated with OS and p21 expression and associated with tumor persistence.In conclusion, our data indicate that extra-nuclear TERT is involved in reducing the effect of excessive OS, thus promoting cancer cell survival. Extra-nuclear TERT may thus represent a marker of cancer progression and a possible therapeutic target in PTC.

TERT accelerates BRAF mutant-induced thyroid cancer dedifferentiation and progression by regulating ribosome biogenesis.

TERT reactivation occurs frequently in human malignancies, especially advanced cancers. However, in vivo functions of TERT reactivation in cancer progression and the underlying mechanism are not fully understood. In this study, we expressed TERT and/or active BRAF (BRAF V600E) specifically in mouse thyroid epithelium. While BRAF V600E alone induced papillary thyroid cancer (PTC), coexpression of BRAF V600E and TERT resulted in poorly differentiated thyroid carcinoma (PDTC). Spatial transcriptome analysis revealed that tumors from mice coexpressing BRAF V600E and TERT were highly heterogeneous, and cell dedifferentiation was positively correlated with ribosomal biogenesis. Mechanistically, TERT boosted ribosomal RNA (rRNA) expression and protein synthesis by interacting with multiple proteins involved in ribosomal biogenesis. Furthermore, we found that CX-5461, an rRNA transcription inhibitor, effectively blocked proliferation and induced redifferentiation of thyroid cancer. Thus, TERT promotes thyroid cancer progression by inducing cancer cell dedifferentiation, and ribosome inhibition represents a potential strategy to treat TERT-reactivated cancers.

TPP1 promoter mutations cooperate with TERT promoter mutations to lengthen telomeres in melanoma.

Overcoming replicative senescence is an essential step during oncogenesis, and the reactivation of <i>TERT</i> through promoter mutations is a common mechanism. <i>TERT</i> promoter mutations are acquired in about 75% of melanomas but are not sufficient to maintain telomeres, suggesting that additional mutations are required. We identified a cluster of variants in the promoter of <i>ACD</i> encoding the shelterin component TPP1. <i>ACD</i> promoter variants are present in about 5% of cutaneous melanoma and co-occur with <i>TERT</i> promoter mutations. The two most common somatic variants create or modify binding sites for E-twenty-six (ETS) transcription factors, similar to mutations in the <i>TERT</i> promoter. The variants increase the expression of <i>TPP1</i> and function together with TERT to synergistically lengthen telomeres. Our findings suggest that <i>TPP1</i> promoter variants collaborate with <i>TERT</i> activation to enhance telomere maintenance and immortalization in melanoma.

P70.17 Molecular Characteristics and Prognosis TERT Mutations in East Asian Non-Small-Cell Lung Cancer Patients

Mutations in the TERT promoter (TERTp) are a common mechanism of TERT reactivation in many solid cancers, The aim of this study is to investigate mutations and prognosis of non-small cell lung cancer (NSCLC) harboring TERT promoter mutations. A total of 429 patients with non-small-cell lung cancer were recruited between July 2012 and December 2018. The status of TERT promoter mutations and other genes were detected by next generation sequencing. TERT gene promoter mutation rate was 3.73% (16/429) in non-small cell lung cancer, including c.-58-u5858A>T plus c.-58-u6149C>T(1 patient), c.-58-u6149C>T(1 patient), c.-58-u533T>G(1 patient), c.58-u66C>A plus c.-58-u3358G>A(1 patient), c.-58-u3363G>T(1 patient), c.-58-u2785C>T plus c.-58-u2257C>T(1 patient), c.-58-u760T>A(1 patient), c.-58-u4217G>C plus c.-58-u2692G>A plus c.-58-u1597G>A(1 patient), c.-58-u1324T>C(1 patient), c.-58-u900C>T(1 patient), c.-59-u5461C>G plus c.-58-u5438C>G plus c.-58-u5427C>T(1 patient), c.-58-u1160C>T(1 patient), c.-58-u6544G>A(2 patients), and c.-58-u66C>T(2 patients), and median overall survival (OS) for these patients was 18.5 months. Among them, all patients were TERT gene with co-occurring mutations. Briefly, patients with (n=7) or without (n=9) co-occurring TP53 mutations had a median OS of 8.0 months and 18.0 months respectively (P=0.04); patients with (n=3) or without (n=13) co-occurring PTPRT mutations had a median OS of not up to now and 18.0 months respectively (P=0.67); patients with (n=3) or without (n=13) co-occurring STK11 mutations had a median OS of not up to now and 18.0 months respectively (P=0.90); patients with (n=3) or without (n=13) co-occurring ARID1A mutations had a median OS of 12.0 months and 18.0 months respectively (P=0.30). TERT gene promoter mutation represent a distinct subset of NSCLC. Next generation sequencing showed that TERT mutations commonly co-existed with other driver genes. Our finding expands the mutant spectrum of TERT gene and adds new understanding of the phenotype.

Synergistic activation of mutant TERT promoter by Sp1 and GABPA in BRAFV600E-driven human cancers

The activating TERT promoter mutations and BRAFV600E mutation are well-established oncogenic alterations in human cancers. Coexistence of BRAFV600E and TERT promoter mutations is frequently found in multiple cancer types, and is strongly associated with poor patient prognosis. Although the BRAFV600E-elicited activation of ERK has been demonstrated to contribute to TERT reactivation by maintaining an active chromatin state, it still remains to be addressed how activated ERK is selectively recruited to mutant TERT promoter. Here, we report that transcription factor GABPA mediates the regulation of BRAFV600E/MAPK signaling on TERT reactivation by selectively recruiting activated ERK to mutant TERT promoter, where activated ERK can phosphorylate Sp1, thereby resulting in HDAC1 dissociation and an active chromatin state. Meanwhile, phosphorylated Sp1 further enhances the binding of GABPA to mutant TERT promoter. Taken together, our data indicate that GABPA and Sp1 synergistically activate mutant TERT promoter, contributing to tumorigenesis and cancer progression, particularly in the BRAFV600E-driven human cancers. Thus, our findings identify a direct mechanism that bridges two frequent oncogenic alterations together in TERT reactivation.

Telomere length is key to hepatocellular carcinoma diversity and telomerase addiction is an actionable therapeutic target

Telomerase activation is the earliest event in hepatocellular carcinoma (HCC) development. Thus, we aimed to elucidate the role of telomere length maintenance during liver carcinogenesis. Telomere length was measured in the tumor and non-tumor liver tissues of 1,502 patients (978 with HCC) and integrated with TERT alterations and expression, as well as clinical and molecular (analyzed by genome, exome, targeted and/or RNA-sequencing) features of HCC. The preclinical efficacy of anti-TERT antisense oligonucleotides (ASO) was assessed in vitro in 26 cell lines and in vivo in a xenograft mouse model. Aging, liver fibrosis, male sex and excessive alcohol consumption were independent determinants of liver telomere attrition. HCC that developed in livers with long telomeres frequently had wild-type TERT with progenitor features and BAP1 mutations. In contrast, HCC that developed on livers with short telomeres were enriched in the non-proliferative HCC class and frequently had somatic TERT promoter mutations. In HCCs, telomere length is stabilized in a narrow biological range around 5.7 kb, similar to non-tumor livers, by various mechanisms that activate TERT expression. Long telomeres are characteristic of very aggressive HCCs, associated with the G3 transcriptomic subclass, TP53 alterations and poor prognosis. In HCC cell lines, TERT silencing with ASO was efficient in highly proliferative and poorly differentiated cells. Treatment for 3 to 16 weeks induced cell proliferation arrest in 12 cell lines through telomere shortening, DNA damage and activation of apoptosis. The therapeutic effect was also obtained in a xenograft mouse model. Telomere maintenance in HCC carcinogenesis is diverse, and is associated with tumor progression and aggressiveness. The efficacy of anti-TERT ASO treatment in cell lines revealed the oncogenic addiction to TERT in HCC, providing a preclinical rationale for anti-TERT ASO treatment in HCC clinical trials. Telomeres are repeated DNA sequences that protect chromosomes and naturally shorten in most adult cells because of the inactivation of the TERT gene, coding for the telomerase enzyme. Here we show that telomere attrition in the liver, modulated by aging, sex, fibrosis and alcohol, associates with specific clinical and molecular features of hepatocellular carcinoma, the most frequent primary liver cancer. We also show that liver cancer is dependent on TERT reactivation and telomere maintenance, which could be targeted through a novel therapeutic approach called antisense oligonucleotides.

Abstract 3105: Oncopig carcinoma cell lines: A foundation for co-clinical trials

Abstract The Oncopig cancer model (OCM) was developed to support a broad range of solid and liquid tumor malignancies while providing the appropriate anatomical and physiological models for preclinical and co-clinical trials to expedite human clinical trials. The OCM develops tumors following Cre recombinase induced expression of KRASG12D and TP53R167H transgenes in a temporally and spatially controlled manner. To date, in vitro transformation and in vivo tumor formation of soft tissue sarcomas (STS), hepatocellular carcinoma (HCC), and pancreatic ductal adenocarcinoma (PDAC) have been demonstrated. However, the OCM was designed to generate cancers of all tissue origins. To demonstrate the potential importance of the OCM, we have successfully isolated and transformed multiple OCM cell types, each a progenitor of clinically relevant human disease. Cell type specific cell isolations were performed, and cells cultured under appropriate conditions. Within the first 48 hours, isolated cells were treated with adenoviral vector encoding Cre recombinase (AdCre) at 200-400 MOI for 5 hours in low serum (5%) medium. Both control and transformed (AdCre treated) cells underwent basic histopathologic screening (H&amp;E staining, immunohistochemical staining for vimentin and cytokeratin), and verification of transgene expression (RT-PCR) and tumorigenicity (xenograft into SCID mice). Cell types isolated include fibroblasts, hepatocytes, pancreatic ductal cells, dermal epithelial cells, splenocytes, ovarian surface epithelial cells, fallopian tube secretory epithelial cells, renal proximal tubule epithelial cells, urinary bladder epithelial cells, bone marrow (without specific cell isolation), and testis (no specific cell isolation). Each cell type was successfully transformed, as shown by in vitro phenotypic changes and tumor formation in vivo. Additional gene expression analysis (RNA-Seq) of the STS and HCC cell lines verified the OCM transformed cells recapitulate transcriptional hallmarks of human cancers. Consistent with human STS, OCM STS cell lines display altered TP53 signaling, Wnt signaling activation, and evidence of epigenetic reprogramming. OCM HCC cell lines show transcriptional evidence of TERT reactivation, apoptosis evasion, angiogenesis activation, and Wnt signaling activation, all similar to human HCC. While not an exhaustive list of Oncopig cell types, we have not encountered cells resistant to malignant transformation through Cre recombinase exposure. These results support the unprecedented potential for modeling of additional cancer types, including colorectal, ovarian, fallopian tube, uterine, renal, bladder, skin, and hematological cancers to support and expand preclinical, translational, and co-clinical investigations. Citation Format: Laurie A. Rund, Kyle M. Schachtschneider, Ron C. Gaba, Daniel R. Principe, Paul Grippo, Regina M. Schwind, Howard Ozer, Lawrence B. Schook. Oncopig carcinoma cell lines: A foundation for co-clinical trials [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 3105.

A validated, transitional and translational porcine model of hepatocellular carcinoma.

Difficult questions are confronting clinicians attempting to improve hepatocellular carcinoma (HCC) outcomes. A large animal model with genetic, anatomical, and physiological similarities to humans is required to transition from mouse models to human clinical trials to address unmet clinical needs. To validate our previously reported inducible porcine cancer model (Oncopig) as a transitional HCC model, Oncopig hepatocyte cultures were transformed using Cre recombinase. The resulting porcine HCC cells (pHCC) expressed oncogenic TP53R167H and KRASG12D, and displayed nuclear pleomorphisms with pale to granular cytoplasm arranged in expanded plates similar to human HCC histopathology. Human HCC transcriptional hallmarks were detected in pHCC cells using RNA-seq, including TERT reactivation, apoptosis evasion, angiogenesis activation, and Wnt signaling activation. Master regulators of gene expression were conserved across Oncopig and 18 human HCC cell lines. pHCC injection into SCID mice resulted in tumors recapitulating human HCC characteristics, including thick trabeculae formation, pseudoacini patterning, and sheets of well-vascularized stroma. Finally, autologous injection of pHCC cells subcutaneously yielded a tumor histologically characterized as Edmondson Steiner (HCC nuclear grade assessment system) grade 2 HCC with trabecular patterning and T-lymphocyte infiltration. These data demonstrate the Oncopig HCC model's utility for improving detection, treatment, and biomarker discovery relevant to human HCC.

Long-Range Chromatin Interactions Drive Mutant TERT Promoter Activation.

This study identifies a key mechanism by which cancer-specific mutant TERT promoters cause reactivation of TERT Because the mechanism uncovered here is not utilized by promoters that drive TERT in normal cells, this mechanism could be exploited to make inhibitors which have the potential to block telomerase function and hence the progression of up to 90% of human cancers. Cancer Discov; 6(11); 1276-91. ©2016 AACR.See related commentary by Min and Shay, p. 1212This article is highlighted in the In This Issue feature, p. 1197.

HTERT Suppression via Small Interference RNA in Cervical Cancer Cells

Telomerase (TERT) functions to extend the telomeric repeat terminus of each chromosome allowing embryonic cells to proliferate into an adult organism. The TERT gene is subsequently inactivated following maturation, consequently conveying a finite lifespan to every adult cell line, as shortened chromosome arms trigger cell apoptosis. This process ensures that older cells lines, which are invariably accumulating mutations, are eliminated from the body and replaced by stem cells containing founding DNA. One of the defining attributes of a cancer cell is the ability to divide perpetually. This capability to divide continually is often due to the reactivation of TERT. Therefore, the abolishment of TERT activity presents a promising avenue for cancer treatment. Here, we demonstrate through qRT-PCR and ELISA techniques that although small interference RNA (siRNA) results in a transcription knockdown of ninety-seven percent the actual protein activity reduction is far less dramatic. KEYWORDS: Telomerase; Small Interference RNA; Cancer; Tumor; hTERT; qRT-PCR; RNA Interference; ELISA

Reactivation of Tert in the medial prefrontal cortex and hippocampus rescues aggression and depression of Tert(-/-) mice.

The role of telomerase reverse transcriptase (TERT) has been extensively investigated in the contexts of aging and cancer. Interestingly, Tert−/− mice exhibit additional but unexpected aggressive and depressive behaviors, implying the potential involvement of TERT function in mood control. Our conditional rescue experiments revealed that the depressive and aggressive behaviors of Tert−/− mice originate from Tert deficiency in two distinct brain structures. Reactivation of Tert in the hippocampus was sufficient to normalize the depressive but not the aggressive behaviors of Tert−/− mice. Conversely, re-expression of Tert in the medial prefrontal cortex (mPFC) reversed the aggressive but not the depressive behavior of Tert−/− mice. Mechanistically, decreased serotonergic signaling and increased nitric oxide (NO) transmission in the hippocampus transduced Tert deficiency into depression as evidenced by our observation that the infusion of a pharmacological agonist for serotonin receptor 1a (5-HTR1A) and a selective antagonist for neuronal NO synthase into the hippocampus successfully normalized the depressive behavior of Tert−/− mice. In addition, increased serotonergic transmission by the 5-HTR1A agonist in the mPFC was sufficient to rescue the aggressive behavior of Tert−/− mice. Thus, our studies revealed a novel function of TERT in the pathology of depression and aggression in a brain structure-specific manner, providing direct evidence for the contribution of TERT to emotional control.

Reactivation of telomerase in cancer.

Activation of telomerase is a critical step in the development of about 85 % of human cancers. Levels of Tert, which encodes the reverse transcriptase subunit of telomerase, are limiting in normal somatic cells. Tert is subjected to transcriptional, post-transcriptional and epigenetic regulation, but the precise mechanism of how telomerase is re-activated in cancer cells is poorly understood. Reactivation of the Tert promoter involves multiple changes which evolve during cancer progression including mutations and chromosomal re-arrangements. Newly described non-coding mutations in the Tert promoter region of many cancer cells (19 %) in two key positions, C250T and C228T, have added another layer of complexity to telomerase reactivation. These mutations create novel consensus sequences for transcription factors which can enhance Tert expression. In this review, we will discuss gene structure and function of Tert and provide insights into the mechanisms of Tert reactivation in cancers, highlighting the contribution of recently identified Tert promoter mutations.

NFκB drives TERT promoter reactivation in cancer

NFκB drives TERT promoter reactivation in cancer

CBIO-32MUTANT IDH EXPRESSION DRIVES hTERT REACTIVATION AS PART OF THE CELLULAR TRANSFORMATION PROCESS

IDH1 mutations in lower grade glioma result in the production of 2-hydroxyglutarate (2HG), disrupted patterns of histone methylation, altered gene expression, and cellular transformation. IDH1 mutations, however, are also linked to mutations in the ATRX gene and the hTERT promoter, suggesting that IDH1 mutation contributes to events that stabilize telomeres and help drive the cellular immortalization and transformation processes. To determine if and how IDH1 mutation influences telomeric stabilization and cellular immortalization, pRb/p53-deficient normal human astrocytes (E6/E7 NHA) were lentivirally infected with constructs encoding WT (IDH1 WT) or R132H mutant IDH 1 (IDH1mut). Following selection, the cells were then monitored for 2HG levels, histone modifications, hTERT promoter mutations, ATRX and telomerase expression, and telomere length as they progressed through their lifespan. Introduction of IDH1mut, but not IDH1 WT, resulted in 2HG production and alterations in histone methylation within 20 population doublings (PD). Neither IDH1mut nor IDH1 WT expression, however, had any direct effect on telomerase expression or telomere length, and cells expressing either IDH1 WT or IDH1mut entered a telomere-induced crisis at PD 70. While cells expressing IDH1 WT failed to reactivate telomerase and escape from crisis, cells expressing IDH1mut emerged from crisis after 51 days, grew indefinitely in culture, and formed colonies in soft agar and tumors in vivo. The transformed IDH1mut cells did not exhibit the ALT phenotype or hTERT promoter mutations, but rather exhibited telomerase expression and activity, and shortened but stabilized telomeres. These results show that while IDH1 mutations do not directly contribute to ATRX gene mutation, hTERT promoter mutation, or TERT reactivation, they are permissive for the events that indirectly drive the TERT reactivation that contributes to both the immortalization and transformation of astrocytic cells.

Non-canonical NF-κB signalling and ETS1/2 cooperatively drive C250T mutant TERT promoter activation

Transcriptional reactivation of TERT, the catalytic subunit of telomerase, is necessary for cancer progression in about 90% of human cancers. The recent discovery of two prevalent somatic mutations-C250T and C228T-in the TERT promoter in various cancers has provided insight into a plausible mechanism of TERT reactivation. Although the two hotspot mutations create a similar binding motif for E-twenty-six (ETS) transcription factors, we show that they are functionally distinct, in that the C250T unlike the C228T TERT promoter is driven by non-canonical NF-κB signalling. We demonstrate that binding of ETS to the mutant TERT promoter is insufficient in driving its transcription but this process requires non-canonical NF-κB signalling for stimulus responsiveness, sustained telomerase activity and hence cancer progression. Our findings highlight a previously unrecognized role of non-canonical NF-κB signalling in tumorigenesis and elucidate a fundamental mechanism for TERT reactivation in cancers, which if targeted could have immense therapeutic implications.

Telomerase regulates MYC-driven oncogenesis independent of its reverse transcriptase activity.

Constitutively active MYC and reactivated telomerase often coexist in cancers. While reactivation of telomerase is thought to be essential for replicative immortality, MYC, in conjunction with cofactors, confers several growth advantages to cancer cells. It is known that the reactivation of TERT, the catalytic subunit of telomerase, is limiting for reconstituting telomerase activity in tumors. However, while reactivation of TERT has been functionally linked to the acquisition of several "hallmarks of cancer" in tumors, the molecular mechanisms by which this occurs and whether these mechanisms are distinct from the role of telomerase on telomeres is not clear. Here, we demonstrated that first-generation TERT-null mice, unlike Terc-null mice, show delayed onset of MYC-induced lymphomagenesis. We further determined that TERT is a regulator of MYC stability in cancer. TERT stabilized MYC levels on chromatin, contributing to either activation or repression of its target genes. TERT regulated MYC ubiquitination and proteasomal degradation, and this effect of TERT was independent of its reverse transcriptase activity and role in telomere elongation. Based on these data, we conclude that reactivation of TERT, a direct transcriptional MYC target in tumors, provides a feed-forward mechanism to potentiate MYC-dependent oncogenesis.

TERT Promoter Mutations Reactivate Telomerase in Urothelial Carcinoma

Abstract TERT promoter mutations correlate with high TERT and telomerase reactivation in urothelial cancer.

Combined analysis of TMPRSS2–ERG and TERT for improved prognosis of biochemical recurrence in prostate cancer

Prostate cancer (PCa) is a heterogeneous disease with diverse clinical outcomes. TMPRSS2-ERG is the most common gene fusion in PCa, whereas activation of telomerase is a common feature of various malignancies. The aim of our study was to explore the combined utility of these and some other biomarkers in predicting biochemical recurrence after radical prostatectomy. Prostate specimens and urine sediments from 179 previously untreated patients with pT2-pT3 stage PCa were analyzed for expression of telomerase (TERT and TR) and the TMPRSS2-ERG fusion gene by means of reverse transcription PCR. Real-time PCR was used for quantification of ERG and SPINK1 expression. In total, 74% (117/158) of the prostate adenocarcinomas were positive for the TMPRSS2-ERG and/or TERT expression. Noninvasively, these transcripts were identified in 31% (19/61) of catheterized urine specimens. Significantly higher expression of ERG was detected in TMPRSS2-ERG-positive tumors (P<0.0001), whereas more intense expression of SPINK1 was characteristic for the TMPRSS2-ERG-negative tumors (P=0.003). TERT-positive cases also had elevated levels of ERG (P=0.016), suggesting a possible link between aberrant expression of ERG and reactivation of TERT in prostate tumors. The cases negative for both transcripts, TMPRSS2-ERG and TERT, rarely recurred (P=0.014) and showed significantly longer biochemical recurrence-free period (P=0.022) as compared to the TMPRSS2-ERG and/or TERT-positive cases. The results of our study suggest that combined analysis of TMPRSS2-ERG and TERT expression can be a valuable tool for early prediction of biochemical recurrence of PCa after radical prostatectomy.

The Protein Subunit of Telomerase Is Downregulated with Normal Myeloid Differentiation, but Reactivated in Leukemic Progenitors.

Telomeres are comprised of TTAGGG nucleotide repeats and a large protein complex that together protect chromosomal ends. Telomeres shorten progressively with cell division in the absence of telomerase, a reverse transcriptase that synthesizes telomere repeats. Critical telomere shortening has potent effects on cancer development, either blocking cancer progression by impairing proliferation and survival or promoting cancer by enhancing chromosomal instability. Although telomerase is upregulated in the vast majority of human cancers, where it is thought to endow cancer cells with unlimited proliferative capacity, the regulation of telomerase during the course of normal lineage development or during leukemogenesis remains unclear. Here, we show that expression of TERT, the protein catalytic subunit of telomerase, is tightly regulated during myeloid development in both human and mouse. Real time PCR analysis of RNA from purified human stem cell and progenitor cell populations showed that TERT mRNA was highest in human HSCs (100%, values normalized to HSC). TERT mRNA remained high in common myeloid progenitors (CMP, 47%), but was dramatically decreased with differentiation to granulocyte-monocyte progenitors (GMP, 3%) or megakaryocyte-erythroid progenitors (MEP, 0.2%). TERT was similarly down regulated during normal myeloid development in mouse bone marrow. In marked contrast, TERT was reactivated in leukemic GMP in a transgenic Faslpr/lprhMRP8BCL2 mouse model of acute myelogenous leukemia. Notably, transplantation experiments demonstrated that leukemic GMP provided the most robust leukemic transplantation potential compared with HSC or blasts suggesting that TERT reactivation may provide an important final step in leukemic pathogenesis by enhancing the replicative life-span of leukemic progenitors. Currently, we are in the process of assessing TERT regulation in human leukemic progenitors isolated from patients with CML in chronic, accelerated and blast phases. Together, these data indicate that TERT is most highly expressed in stem cells and multipotent progenitors. This pattern of TERT expression may reflect a need for efficient telomere maintenance in these stem/progenitor compartments. Furthermore, these data are consistent with our recent data showing novel, telomere length-independent functions for TERT in stem/progenitor cell compartments.