Mathematical model of a telomerase transcriptional regulatory network developed by cell-based screening: analysis of inhibitor effects and telomerase expression mechanisms.

Cancer cells depend on transcription of telomerase reverse transcriptase (TERT). Many transcription factors affect TERT, though regulation occurs in context of a broader network. Network effects on telomerase regulation have not been investigated, though deeper understanding of TERT transcription requires a systems view. However, control over individual interactions in complex networks is not easily achievable. Mathematical modelling provides an attractive approach for analysis of complex systems and some models may prove useful in systems pharmacology approaches to drug discovery. In this report, we used transfection screening to test interactions among 14 TERT regulatory transcription factors and their respective promoters in ovarian cancer cells. The results were used to generate a network model of TERT transcription and to implement a dynamic Boolean model whose steady states were analysed. Modelled effects of signal transduction inhibitors successfully predicted TERT repression by Src-family inhibitor SU6656 and lack of repression by ERK inhibitor FR180204, results confirmed by RT-QPCR analysis of endogenous TERT expression in treated cells. Modelled effects of GSK3 inhibitor 6-bromoindirubin-3'-oxime (BIO) predicted unstable TERT repression dependent on noise and expression of JUN, corresponding with observations from a previous study. MYC expression is critical in TERT activation in the model, consistent with its well known function in endogenous TERT regulation. Loss of MYC caused complete TERT suppression in our model, substantially rescued only by co-suppression of AR. Interestingly expression was easily rescued under modelled Ets-factor gain of function, as occurs in TERT promoter mutation. RNAi targeting AR, JUN, MXD1, SP3, or TP53, showed that AR suppression does rescue endogenous TERT expression following MYC knockdown in these cells and SP3 or TP53 siRNA also cause partial recovery. The model therefore successfully predicted several aspects of TERT regulation including previously unknown mechanisms. An extrapolation suggests that a dominant stimulatory system may programme TERT for transcriptional stability.

Telomere maintenance mechanisms in malignant peripheral nerve sheath tumors: expression and prognostic relevance.

This study investigated the prevalence and the prognostic relevance of the 2 known telomere maintenance mechanisms (TMMs), telomerase activity (TA) and alternative lengthening of telomeres (ALT), in malignant peripheral nerve sheath tumors (MPNST). In 57 specimens from 49 patients with MPNST (35 sporadic, 14 neurofibromatosis type 1-related), TA was determined using the telomeric repeat amplification protocol, and ALT was detected by assaying ALT-associated promyelocytic leukemia bodies (APB) and terminal restriction fragment (TRF) length distribution. TA or ALT (defined on the basis of APB) alone was found in 24.6% or 26.3% of the lesions, respectively, whereas 6 cases (10.5%) were TA+/ALT+. A concordance between APB and TRF results in defining the ALT status was observed in 44 of 57 cases (77.2%; P < .0001). TA was more frequently expressed in samples from patients with neurofibromatosis type 1 than in those with sporadic disease (60% vs 29.4%, P = 0.087). In the overall series, TA proved to be prognostic for 5-year disease-specific death (hazard ratio, 3.78; 95% confidence interval [CI], 1.60-8.95; P = .002), even when adjusted for the presence of neurofibromatosis type 1 (hazard ratio, 4.22; 95% CI, 1.804-9.874; P = .001) and margin status after surgery (hazard ratio, 5.78; 95% CI, 2.19-15.26; P < .001). Conversely, ALT did not significantly affect clinical outcome of MPNST using either APB expression (hazard ratio, 1.25; 95% CI 0.54-2.89; P = 0.605) or TRF distribution (hazard ratio, 0.57; 95% CI, 0.17-1.96; P = .375) as the detection approach. Our results indicate for the first time that both TMMs, TA and ALT, are present in MPNST and differentially affect patient prognosis.

Malignancy without immortality? Cellular immortalization as a possible late event in melanoma progression.

Cell senescence is a permanent growth arrest following extended proliferation. Cultured cancer cells including metastatic melanoma cells often appear immortal (proliferate indefinitely), while uncultured benign nevi (moles) show senescence markers. Here, with new explantation methods, we investigated which classes of primary pigmented lesions are typically immortal. Nevi yielded a few proliferating cells, consistent with most nevus cells being senescent. No nevus culture (0/28) appeared immortal. Some thin and thick melanoma cultures proved immortal under these conditions, but surprisingly few (4/37). All arrested cultures displayed three senescence markers in some cells: ß-galactosidase, nuclear p16, and heterochromatic foci/aggregates. However, melanoma cultures also showed features of telomeric crisis (arrest because of ultrashort telomeres). Moreover, crisis markers including anaphase bridges were frequent in uncultured vertical growth-phase (VGP) melanomas. Conversely, all immortal melanoma cultures expressed telomerase reverse transcriptase and telomerase, showing aneuploidy. The findings suggest that primary melanomas are typically precrisis, with immortalization/telomere maintenance as a late event.

TCEAL7 inhibition of c-Myc activity in alternative lengthening of telomeres regulates hTERT expression.

Replicative senescence forms a major barrier to tumor progression. Cancer cells bypass this by using one of the two known telomere maintenance mechanisms: telomerase or the recombination-based alternative lengthening of telomeres (ALT) mechanism. The molecular details of ALT are currently poorly understood. We have previously shown that telomerase is actively repressed through complex networks of kinase, gene expression, and chromatin regulation. In this study, we aimed to gain further understanding of the role of kinases in the regulation of telomerase expression in ALT cells. Using a whole human kinome small interfering RNA (siRNA) screen, we highlighted 106 kinases whose expression is linked to human telomerase reverse transcriptase (hTERT) promoter activity. Network modeling of transcriptional regulation implicated c-Myc as a key regulator of the 106 kinase hits. Given our previous observations of lower c-Myc activity in ALT cells, we further explored its potential to regulate telomerase expression in ALT. We found increased c-Myc binding at the hTERT promoter in telomerase-positive compared with ALT cells, although no expression differences in c-Myc, Mad, or Max were observed between ALT and telomerase-positive cells that could explain decreased c-Myc activity in ALT. Instead, we found increased expression of the c-Myc competitive inhibitor TCEAL7 in ALT cells and tumors and that alteration of TCEAL7 expression levels in ALT and telomerase-positive cells affects hTERT expression. Lower c-Myc activity in ALT may therefore be obtained through TCEAL7 regulation. Thus, TCEAL7 may present an interesting novel target for cancer therapy, which warrants further investigation.

Dynamic telomerase gene suppression via network effects of GSK3 inhibition.

BACKGROUND: Telomerase controls telomere homeostasis and cell immortality and is a promising anti-cancer target, but few small molecule telomerase inhibitors have been developed. Reactivated transcription of the catalytic subunit hTERT in cancer cells controls telomerase expression. Better understanding of upstream pathways is critical for effective anti-telomerase therapeutics and may reveal new targets to inhibit hTERT expression. METHODOLOGY/PRINCIPAL FINDINGS: In a focused promoter screen, several GSK3 inhibitors suppressed hTERT reporter activity. GSK3 inhibition using 6-bromoindirubin-3'-oxime suppressed hTERT expression, telomerase activity and telomere length in several cancer cell lines and growth and hTERT expression in ovarian cancer xenografts. Microarray analysis, network modelling and oligonucleotide binding assays suggested that multiple transcription factors were affected. Extensive remodelling involving Sp1, STAT3, c-Myc, NFkappaB, and p53 occurred at the endogenous hTERT promoter. RNAi screening of the hTERT promoter revealed multiple kinase genes which affect the hTERT promoter, potentially acting through these factors. Prolonged inhibitor treatments caused dynamic expression both of hTERT and of c-Jun, p53, STAT3, AR and c-Myc. CONCLUSIONS/SIGNIFICANCE: Our results indicate that GSK3 activates hTERT expression in cancer cells and contributes to telomere length homeostasis. GSK3 inhibition is a clinical strategy for several chronic diseases. These results imply that it may also be useful in cancer therapy. However, the complex network effects we show here have implications for either setting.

A role for NANOG in G1 to S transition in human embryonic stem cells through direct binding of CDK6 and CDC25A.

In this study, we show that NANOG, a master transcription factor, regulates S-phase entry in human embryonic stem cells (hESCs) via transcriptional regulation of cell cycle regulatory components. Chromatin immunoprecipitation combined with reporter-based transfection assays show that the C-terminal region of NANOG binds to the regulatory regions of CDK6 and CDC25A genes under normal physiological conditions. Decreased CDK6 and CDC25A expression in hESCs suggest that both CDK6 and CDC25A are involved in S-phase regulation. The effects of NANOG overexpression on S-phase regulation are mitigated by the down-regulation of CDK6 or CDC25A alone. Overexpression of CDK6 or CDC25A alone can rescue the impact of NANOG down-regulation on S-phase entry, suggesting that CDK6 and CDC25A are downstream cell cycle effectors of NANOG during the G1 to S transition.

Downregulation of multiple stress defense mechanisms during differentiation of human embryonic stem cells.

Evolutionary theory predicts that cellular maintenance, stress defense, and DNA repair mechanisms should be most active in germ line cells, including embryonic stem cells that can differentiate into germ line cells, whereas it would be energetically unfavorable to keep these up in mortal somatic cells. We tested this hypothesis by examining telomere maintenance, oxidative stress generation, and genes involved in antioxidant defense and DNA repair during spontaneous differentiation of two human embryonic stem cell lines. Telomerase activity was quickly downregulated during differentiation, probably due to deacetylation of histones H3 and H4 at the hTERT promoter and deacetylation of histone H3 at hTR promoter. Telomere length decreased accordingly. Mitochondrial superoxide production and cellular levels of reactive oxygen species increased as result of increased mitochondrial biogenesis. The expression of major antioxidant genes was downregulated despite this increased oxidative stress. DNA damage levels increased during differentiation, whereas expression of genes involved in different types of DNA repair decreased. These results confirm earlier data obtained during mouse embryonic stem cell differentiation and are in accordance with evolutionary predictions.

Telomerase promoter reprogramming and interaction with general transcription factors in the human mesenchymal stem cell.

The human adult mesenchymal stem cell (hMSC) does not express telomerase and has been shown to be the target for neoplastic transformation after transduction with hTERT. These findings lend support to the stem cell hypothesis of cancer development but by supplying hTERT, the molecular events required to upregulate hTERT expression in cancer development are missed. Therefore, the hMSC is ideal for the identification of molecular mechanisms regulating telomerase gene expression in stem cells. This study shows that the repression of hTERT expression in hMSC is chromatin based and that modifications of the chromatin environment lead to reactivation of telomerase gene expression. It is shown that repression of hTERT expression in hMSCs is due to promoter-specific histone hypoacetylation coupled with low Pol II and TFIIB trafficking. This repression is overcome by treatment with Trichostatin A (TSA), an HDAC inhibitor, concomitant with increases in promoter-specific histone acetylation and increases in Pol II and TFIIB tracking. hTR expression is also increased in TSA-treated hMSCs, concomitant with changes in Pol II and TFIIB dynamics.

Lack of telomerase gene expression in alternative lengthening of telomere cells is associated with chromatin remodeling of the hTR and hTERT gene promoters.

The presence of active telomere maintenance mechanisms in immortal cells allows the bypass of senescence by maintaining telomere length. In most immortal cell lines and tumors, telomere maintenance is attributable to telomerase reactivation. However, a number of immortal cell lines and tumors can achieve telomere maintenance in the absence of detectable telomerase activity by the alternative lengthening of telomere (ALT) mechanism. Epigenetic mechanisms have been implicated in the regulation of telomerase expression. We show that specific modifications within the chromatin environment of the hTR and hTERT promoters correlate with expression of hTR and hTERT in ALT, normal and telomerase-positive tumor cell lines. Lack of expression of hTR and hTERT in ALT cell lines is associated with histone H3 and H4 hypoacetylation and methylation of Lys9 histone H3. Conversely, hTR and hTERT expression in telomerase-positive cell lines is associated with hyperacetylation of H3 and H4 and methylation of Lys4 H3. Methylation of Lys20 H4 was not linked to gene expression but instead was specific to the hTR and hTERT promoters of ALT cells. This may provide an insight into the differences between ALT and telomerase-positive cells as well as a novel marker for the ALT phenotype. Treatment of normal and ALT cells with 5-azadeoxycytidine in combination with Trichostatin A caused chromatin remodeling of both promoters and reactivation of hTR and hTERT expression in ALT and normal cell lines. This data establishes a definite link between the chromatin environment of the telomerase gene promoters and transcriptional activity.

The hTERT and hTERC telomerase gene promoters are activated by the second exon of the adenoviral protein, E1A, identifying the transcriptional corepressor CtBP as a potential repressor of both genes.

Telomerase plays a role in the unlimited replicative capacity of the majority of cancer cells and provides a potential anticancer target. The regulation of telomerase is complex but transcriptional control of its two essential components, hTERC (RNA component) and hTERT (reverse transcriptase component), is of major importance. To investigate this further, we have used the adenoviral protein, E1A, as a tool to probe potential pathways involved in the control of telomerase transcription. The second exon of the adenoviral protein E1A activates both telomerase gene promoters in transient transfections. The corepressor, C terminal binding protein, is one of only two proteins known to bind to this region, and we propose that E1A activates both promoters by sequestering CtBP, thereby relieving repression. Activation by exon 2 of E1A involves the SP1 sites in both promoters, and consistent with this, SP1 and CtBP interact in coimmunoprecipitation studies. Modulation of the chromatin environment has been implicated in the regulation of hTERT transcription and appears to involve the SP1 sites. CtBP can be found within a histone-modifying complex and it is possible that a CtBP complex, associating with the SP1 sites, represses transcription from the telomerase promoters by modifying chromatin structure.

Involvement of NF-Y and Sp1 binding sequences in basal transcription of the human telomerase RNA gene.

The proximal promoter of the telomerase RNA gene, hTR, contains four Sp1 sites and one CCAAT box. We have carried out a functional analysis of the role of these sequence elements. Two Sp1 sites downstream of the CCAAT box mediated negative regulation, while the other two Sp1 sites were positive regulators with the strongest effect mediated by the negative regulatory Sp1 site closely flanking the CCAAT box. Basal transcriptional activity is maintained via the CCAAT box even when all four Sp1 sites are mutated, suggesting nuclear factor-Y (NF-Y) is a fundamental regulator of hTR promoter function. Chromatin immunoprecipitation revealed binding of NF-Y, Sp1 and TFIIB to the promoter in vivo. Thus the interaction of NF-Y at the CCAAT box is pivotal to hTR gene transcription and surrounding sequence elements may provide an environment for the regulation of activity through recruitment of additional protein complexes.