Dynamic telomerase gene suppression via network effects of GSK3 inhibition

<b>Background</b>: 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. <b>Methodology/Principal Findings</b>: 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, NFκB, 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. <b>Conclusions/Significance</b>: 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 novel pyrazolopyrimidine ligand of human PGK1 and stress sensor DJ1 modulates the shelterin complex and telomere length regulation

Telomere signaling and metabolic dysfunction are hallmarks of cell aging. New agents targeting these processes might provide therapeutic opportunities, including chemoprevention strategies against cancer predisposition. We report identification and characterization of a pyrazolopyrimidine compound series identified from screens focused on cell immortality and whose targets are glycolytic kinase PGK1 and oxidative stress sensor DJ1. We performed structure–activity studies on the series to develop a photoaffinity probe to deconvolute the cellular targets. In vitro binding and structural analyses confirmed these targets, suggesting that PGK1/DJ1 interact, which we confirmed by immunoprecipitation. Glucose homeostasis and oxidative stress are linked to telomere signaling and exemplar compound CRT0063465 blocked hypoglycemic telomere shortening. Intriguingly, PGK1 and DJ1 bind to TRF2 and telomeric DNA. Compound treatment modulates these interactions and also affects Shelterin complex composition, while conferring cellular protection from cytotoxicity due to bleomycin and desferroxamine. These results demonstrate therapeutic potential of the compound series

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

Regulation of polarized morphogenesis by protein kinase C iota in oncogenic epithelial spheroids.

Protein kinase C iota (PKCι), a serine/threonine kinase required for cell polarity, proliferation and migration, is commonly up- or downregulated in cancer. PKCι is a human oncogene but whether this is related to its role in cell polarity and what repertoire of oncogenes acts in concert with PKCι is not known. We developed a panel of candidate oncogene expressing Madin-Darby canine kidney (MDCK) cells and demonstrated that H-Ras, ErbB2 and phosphatidylinositol 3-kinase transformation led to non-polar spheroid morphogenesis (dysplasia), whereas MDCK spheroids expressing c-Raf or v-Src were largely polarized. We show that small interfering RNA (siRNA)-targeting PKCι decreased the size of all spheroids tested and partially reversed the aberrant polarity phenotype in H-Ras and ErbB2 spheroids only. This indicates distinct requirements for PKCι and moreover that different thresholds of PKCι activity are required for these phenotypes. By manipulating PKCι function using mutant constructs, siRNA depletion or chemical inhibition, we have demonstrated that PKCι is required for polarization of parental MDCK epithelial cysts in a 3D matrix and that there is a threshold of PKCι activity above and below which, disorganized epithelial morphogenesis results. Furthermore, treatment with a novel PKCι inhibitor, CRT0066854, was able to restore polarized morphogenesis in the dysplastic H-Ras spheroids. These results show that tightly regulated PKCι is required for normal-polarized morphogenesis in mammalian cells and that H-Ras and ErbB2 cooperate with PKCι for loss of polarization and dysplasia. The identification of a PKCι inhibitor that can restore polarized morphogenesis has implications for the treatment of Ras and ErbB2 driven malignancies.Cancer Research UK; Royal Marsden/Institute of Cancer Research National Institute for Health Research Biomedical Research Centre (M.L.)

Scoring of senescence signalling in multiple human tumour gene expression datasets, identification of a correlation between senescence score and drug toxicity in the NCI60 panel and a pro-inflammatory signature correlating with survival advantage in peritoneal mesothelioma

Background: Cellular senescence is a major barrier to tumour progression, though its role in pathogenesis of cancer and other diseases is poorly understood in vivo. Improved understanding of the degree to which latent senescence signalling persists in tumours might identify intervention strategies to provoke "accelerated senescence" responses as a therapeutic outcome. Senescence involves convergence of multiple pathways and requires ongoing dynamic signalling throughout its establishment and maintenance. Recent discovery of several new markers allows for an expression profiling approach to study specific senescence phenotypes in relevant tissue samples. We adopted a "senescence scoring" methodology based on expression profiles of multiple senescence markers to examine the degree to which signals of damage-associated or secretory senescence persist in various human tumours. Results: We first show that scoring captures differential induction of damage or inflammatory pathways in a series of public datasets involving radiotherapy of colon adenocarcinoma, chemotherapy of breast cancer cells, replicative senescence of mesenchymal stem cells, and progression of melanoma. We extended these results to investigate correlations between senescence score and growth inhibition in response to similar to 1500 compounds in the NCI60 panel. Scoring of our own mesenchymal tumour dataset highlighted differential expression of secretory signalling pathways between distinct subgroups of MPNST, liposarcomas and peritoneal mesothelioma. Furthermore, a proinflammatory signature yielded by hierarchical clustering of secretory markers showed prognostic significance in mesothelioma. Conclusions: We find that "senescence scoring" accurately reports senescence signalling in a variety of situations where senescence would be expected to occur and highlights differential expression of damage associated and secretory senescence pathways in a context-dependent manner

Theoretical model of complex between phosphoglycerate kinase 1 and DJ1

Telomere signalling and metabolic dysfunction are hallmarks of cell aging, and are associated with a variety of pathologies. Identification of new agents targeting these processes might provide therapeutic opportunities, including chemoprevention strategies against cancer predisposition. Here we report identification and characterization of a pyrazolopyrimidine compound series that emerged from screens focused on cell immortality pathways and whose targets are glycolytic kinase PGK1 and oxidative stress sensor DJ1. We initially performed structure-activity studies on the series to develop an analogue suitable for use as a photoaffinity probe to deconvolute the cellular targets. Biophysical and structural analyses confirmed these targets, pointing to the possibility that PGK1/DJ1 interact, which we confirmed by immunoprecipitation. We additionally performed in silico structural modelling to identify a putative mechanism of complex formation. Glucose homeostasis and oxidative stress have previously been linked to telomere signalling and exemplar compound CRT0063465 was found to block hypoglycaemic telomere shortening. Intriguingly, we find that both PGK1 and DJ1 bind to TRF2 and telomeric DNA. Compound treatment not only modulates these interactions but also affects composition of the Shelterin complex, in addition to conferring cellular protection from cytotoxicity due to bleomycin and desferroxamine. These results highlight the power of chemical approaches in discovery of novel biological mechanisms and demonstrate therapeutic potential of the compound series

Definition of the <i>TERT</i> transcriptional neighbourhood in A2780 cells by transfection screening.

<p>(A), overexpression of <i>TERT</i> activators. A2780 cells were transfected with the luciferase reporters shown on the vertical axis. Each reporter was co-transfected alongside vector control or transcription factor expression plasmid shown in the right hand boxes. Each bar type represents a different expression vector relative to control. 48 h post-transfection, promoter activities were analysed by luciferase assay. (B), overexpression of <i>TERT</i> repressors, transfected as in (A). (C), overexpression of <i>E2F1</i> against the promoter panel, transfected as above. Because of the very strong self-regulatory effect on its own promoter, <i>E2F1</i> is shown on a different scale and separately from the other <i>TERT</i> repressors. Mean ± SEM of 3 experiments (ns: not significant; *: p<0.5; **: p<0.01).</p

Candidate novel interactions included from the screen.

<p>Candidate novel interactions included from the screen.</p

Accession numbers of all human genes from the model.

<p>Accession numbers of all human genes from the model.</p

Topological analysis of the <i>TERT</i> model, and prediction of robust <i>MYC</i> dependent <i>TERT</i> repression.

<p>(A), structure of FFL types I–IV. Structures visualised in Pajek <a href="http://www.ploscompbiol.org/article/info:doi/10.1371/journal.pcbi.1003448#pcbi.1003448-Batagelj1" target="_blank">[103]</a>. Bold lines indicate activation, dashed lines indicate repression. X, Y represent generalised transcription factors, Z represents a regulated gene. (B), activation and repression modules in the <i>TERT</i> transcriptional neighbourhood model. Subnetworks were extracted from the main model and visualised in Pajek <a href="http://www.ploscompbiol.org/article/info:doi/10.1371/journal.pcbi.1003448#pcbi.1003448-Batagelj1" target="_blank">[103]</a>. Extraction was achieved as described in <a href="http://www.ploscompbiol.org/article/info:doi/10.1371/journal.pcbi.1003448#s4" target="_blank">materials and methods</a>. As an indicator of topological importance, node betweenness centralities were calculated and are given in <a href="http://www.ploscompbiol.org/article/info:doi/10.1371/journal.pcbi.1003448#pcbi-1003448-t006" target="_blank">table 6</a>. Additionally, we calculated flow betweenness which is not dependent only on geodesics <a href="http://www.ploscompbiol.org/article/info:doi/10.1371/journal.pcbi.1003448#pcbi.1003448-Freeman1" target="_blank">[77]</a>. (C), Effect of single- and double-node targeting on <i>TERT</i> on-states. Rule-sets for each node were modified in turn individually (black bars) to simulate constitutive repression or activation. For each rule-set change, statespace was derived and the proportion of system states evolving to attractor states with <i>TERT</i> stably on was quantified. The analysis was repeated for each node in the context of double knockouts with <i>MYC</i> also suppressed in each case (grey bars). (D), <i>MYC</i> dependent <i>TERT</i> repression and reversal by <i>AR</i>. A2780 were transfected with 200 nM non-specific control siRNA (Con), 100 nM <i>MYC</i> with 100 nM non-specific (<i>MYC</i>), or 100 nM <i>MYC</i> and 100 nM each specific siRNA. Cells were harvested after 48 h and RNA extracted for analysis of <i>TERT</i> expression normalised to RPS15 by RT-QPCR. Mean ± SEM of <i>TERT</i> expression in treated cells relative to control from three experiments (ns: not significant; *: p<0.05; **: p<0.01). (E), Knockdown of <i>TERT</i> regulatory transcription factors by RNAi. A2780 were transfected with 100 nM each specific siRNA (RNAi) or non-specific control (NS) and harvested after 48 h. 20 µg protein samples were analysed by western blotting against the respective targets. ERK counter-blots were also performed. Each experiment was performed twice. Representative blots are shown.</p