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

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

A 'synthetic-sickness' screen for senescence re-engagement targets in mutant cancer backgrounds.

Senescence is a universal barrier to immortalisation and tumorigenesis. As such, interest in the use of senescence-induction in a therapeutic context has been gaining momentum in the past few years; however, senescence and immortalisation remain underserved areas for drug discovery owing to a lack of robust senescence inducing agents and an incomplete understanding of the signalling events underlying this complex process. In order to address this issue we undertook a large-scale morphological siRNA screen for inducers of senescence phenotypes in the human melanoma cell line A375P. Following rescreen and validation in a second cancer cell line, HCT116 colorectal carcinoma, a panel of 16 of the most robust hits were selected for further validation based on significance and the potential to be targeted by drug-like molecules. Using secondary assays for detection of senescence biomarkers p21, 53BP1 and senescence associated beta-galactosidase (SAβGal) in a panel of HCT116 cell lines carrying cancer-relevant mutations, we show that partial senescence phenotypes can be induced to varying degrees in a context dependent manner, even in the absence of p21 or p53 expression. However, proliferation arrest varied among genetic backgrounds with predominantly toxic effects in p21 null cells, while cells lacking PI3K mutation failed to arrest. Furthermore, we show that the oncogene ECT2 induces partial senescence phenotypes in all mutant backgrounds tested, demonstrating a dependence on activating KRASG13D for growth suppression and a complete senescence response. These results suggest a potential mechanism to target mutant KRAS signalling through ECT2 in cancers that are reliant on activating KRAS mutations and remain refractory to current treatments

MiRNA Profile Associated with Replicative Senescence, Extended Cell Culture, and Ectopic Telomerase Expression in Human Foreskin Fibroblasts

Senescence is a highly regulated process that limits cellular replication by enforcing a G1 arrest in response to various stimuli. Replicative senescence occurs in response to telomeric DNA erosion, and telomerase expression can offset replicative senescence leading to immortalization of many human cells. Limited data exists regarding changes of microRNA (miRNA) expression during senescence in human cells and no reports correlate telomerase expression with regulation of senescence-related miRNAs. We used miRNA microarrays to provide a detailed account of miRNA profiles for early passage and senescent human foreskin (BJ) fibroblasts as well as early and late passage immortalized fibroblasts (BJ-hTERT) that stably express the human telomerase reverse transcriptase subunit hTERT. Selected miRNAs that were differentially expressed in senescence were assayed for expression in quiescent cells to identify miRNAs that are specifically associated with senescence-associated growth arrest. From this group of senescence-associated miRNAs, we confirmed the ability of miR-143 to induce growth arrest after ectopic expression in young fibroblasts. Remarkably, miR-143 failed to induce growth arrest in BJ-hTERT cells. Importantly, the comparison of late passage immortalized fibroblasts to senescent wild type fibroblasts reveals that miR-146a, a miRNA with a validated role in regulating the senescence associated secretory pathway, is also regulated during extended cell culture independently of senescence. The discovery that miRNA expression is impacted by expression of ectopic hTERT as well as extended passaging in immortalized fibroblasts contributes to a comprehensive understanding of the connections between telomerase expression, senescence and processes of cellular aging

Induction of Cellular Senescence by Doxorubicin Is Associated with Upregulated miR-375 and Induction of Autophagy in K562 Cells

BACKGROUND: Cellular senescence is a specialized form of growth arrest that is generally irreversible. Upregulated p16, p53, and p21 expression and silencing of E2F target genes have been characterized to promote the establishment of senescence. It can be further aided by the transcriptional repression of proliferation-associated genes by the action of HP1γ, HMGA, and DNMT proteins to produce a repressive chromatin environment. Therefore, senescence has been suggested to functions as a natural brake for tumor development and plays a critical role in tumor suppression and aging. METHODOLOGY/PRINCIPAL FINDINGS: An in vitro senescence model has been established by using K562 cells treated with 50 nM doxorubicin (DOX). Since p53 and p16 are homozygously deleted in the K562 cells, the DOX-induced senescence in K562 cells ought to be independent of p53 and p16-pRb pathways. Indeed, no change in the expression of the typical senescence-associated premalignant cell markers in the DOX-induced senescent K562 cells was found. MicroRNA profiling revealed upregulated miR-375 in DOX-induced senescent K562 cells. Treatment with miR-375 inhibitor was able to reverse the proliferation ability suppressed by DOX (p<0.05) and overexpression of miR-375 suppressed the normal proliferation of K562 cells. Upregulated miR-375 expression was associated with downregulated expression of 14-3-3zeta and SP1 genes. Autophagy was also investigated since DOX treatment was able to induce cells entering senescence and eventually lead to cell death. Among the 24 human autophagy-related genes examined, a 12-fold increase of ATG9B at day 4 and a 20-fold increase of ATG18 at day 2 after DOX treatment were noted. CONCLUSIONS/SIGNIFICANCE: This study has demonstrated that in the absence of p53 and p16, the induction of senescence by DOX was associated with upregulation of miR-375 and autophagy initiation. The anti-proliferative function of miR-375 is possibly exerted, at least in part, by targeting 14-3-3zeta and SP1 genes