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Dynamic telomerase gene suppression via network effects of GSK3 inhibition.

Bilsland AE, Hoare S, Stevenson K, Plumb J, Gomez-Roman N, Cairney C, Burns S, Lafferty-Whyte K, Roffey J, Hammonds T, Keith WN - PLoS ONE (2009)

Bottom Line: Better understanding of upstream pathways is critical for effective anti-telomerase therapeutics and may reveal new targets to inhibit hTERT expression.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.

View Article: PubMed Central - PubMed

Affiliation: Centre for Oncology and Applied Pharmacology, University of Glasgow, Cancer Research UK Beatson Laboratories, Garscube Estate, Bearsden, Glasgow, United Kingdom.

ABSTRACT

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.

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Network topology and dynamic behaviour.(A) Network motif analysis. Motifs in figure 5(B) were identified in MetaCore. Representative examples are shown. Green arrows: positive regulation; red arrows: negative regulation (reaction mechanisms not shown). (B) BIO regulates the repression module. A2780 were treated for 16 h with DMSO or 5 µM BIO prior to ChIP with p53 antibody and QPCR detection of indicated promoters. Mean±SEM of three experiments (*: p<0.05; **: p<0.01). (C) Dynamic regulation of network transcription factor expression levels under long term BIO treatment. Expression of network transcription factors in 20 µg protein samples from each time point of the 25 week time course were analysed by western blotting (C, Control; B, BIO treatment). Two independent treatments were analysed. Representative blots are shown. (D) Densitometry of (C): expression relative to control (c-Jun not shown due to scale). Mean±SEM of three measurements of each band. (E) Dynamic regulation of hTERT and TP53 promoters by Sp1. A2780 were treated for 16 h with 5 µM BIO or 21 days with 2.5 µM prior to ChIP with Sp1 antibody and QPCR detection of the hTERT and TP53 promoters. Mean±SEM of three experiments (ns: not significant; *: p<0.5; **: p<0.01).
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pone-0006459-g007: Network topology and dynamic behaviour.(A) Network motif analysis. Motifs in figure 5(B) were identified in MetaCore. Representative examples are shown. Green arrows: positive regulation; red arrows: negative regulation (reaction mechanisms not shown). (B) BIO regulates the repression module. A2780 were treated for 16 h with DMSO or 5 µM BIO prior to ChIP with p53 antibody and QPCR detection of indicated promoters. Mean±SEM of three experiments (*: p<0.05; **: p<0.01). (C) Dynamic regulation of network transcription factor expression levels under long term BIO treatment. Expression of network transcription factors in 20 µg protein samples from each time point of the 25 week time course were analysed by western blotting (C, Control; B, BIO treatment). Two independent treatments were analysed. Representative blots are shown. (D) Densitometry of (C): expression relative to control (c-Jun not shown due to scale). Mean±SEM of three measurements of each band. (E) Dynamic regulation of hTERT and TP53 promoters by Sp1. A2780 were treated for 16 h with 5 µM BIO or 21 days with 2.5 µM prior to ChIP with Sp1 antibody and QPCR detection of the hTERT and TP53 promoters. Mean±SEM of three experiments (ns: not significant; *: p<0.5; **: p<0.01).

Mentions: We identified multiple potential reciprocal repression (toggle switch) and feedback oscillator motifs as in the examples in figure 7A, which may provide substantial scope for dynamic network behaviour. We also identified several types of coherent feed forward motifs. In particular, three distinct sub-networks form candidate activation and repression “modules”. The activation module links all positive regulators of hTERT via densely overlapping coherent type 1 motifs, while the repression module comprises several coherent type 2 motifs organised by p53 and AR which inhibit hTERT and its activators. Both motif types are reported to reduce noise in gene expression networks [21].


Dynamic telomerase gene suppression via network effects of GSK3 inhibition.

Bilsland AE, Hoare S, Stevenson K, Plumb J, Gomez-Roman N, Cairney C, Burns S, Lafferty-Whyte K, Roffey J, Hammonds T, Keith WN - PLoS ONE (2009)

Network topology and dynamic behaviour.(A) Network motif analysis. Motifs in figure 5(B) were identified in MetaCore. Representative examples are shown. Green arrows: positive regulation; red arrows: negative regulation (reaction mechanisms not shown). (B) BIO regulates the repression module. A2780 were treated for 16 h with DMSO or 5 µM BIO prior to ChIP with p53 antibody and QPCR detection of indicated promoters. Mean±SEM of three experiments (*: p<0.05; **: p<0.01). (C) Dynamic regulation of network transcription factor expression levels under long term BIO treatment. Expression of network transcription factors in 20 µg protein samples from each time point of the 25 week time course were analysed by western blotting (C, Control; B, BIO treatment). Two independent treatments were analysed. Representative blots are shown. (D) Densitometry of (C): expression relative to control (c-Jun not shown due to scale). Mean±SEM of three measurements of each band. (E) Dynamic regulation of hTERT and TP53 promoters by Sp1. A2780 were treated for 16 h with 5 µM BIO or 21 days with 2.5 µM prior to ChIP with Sp1 antibody and QPCR detection of the hTERT and TP53 promoters. Mean±SEM of three experiments (ns: not significant; *: p<0.5; **: p<0.01).
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Related In: Results  -  Collection

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getmorefigures.php?uid=PMC2714081&req=5

pone-0006459-g007: Network topology and dynamic behaviour.(A) Network motif analysis. Motifs in figure 5(B) were identified in MetaCore. Representative examples are shown. Green arrows: positive regulation; red arrows: negative regulation (reaction mechanisms not shown). (B) BIO regulates the repression module. A2780 were treated for 16 h with DMSO or 5 µM BIO prior to ChIP with p53 antibody and QPCR detection of indicated promoters. Mean±SEM of three experiments (*: p<0.05; **: p<0.01). (C) Dynamic regulation of network transcription factor expression levels under long term BIO treatment. Expression of network transcription factors in 20 µg protein samples from each time point of the 25 week time course were analysed by western blotting (C, Control; B, BIO treatment). Two independent treatments were analysed. Representative blots are shown. (D) Densitometry of (C): expression relative to control (c-Jun not shown due to scale). Mean±SEM of three measurements of each band. (E) Dynamic regulation of hTERT and TP53 promoters by Sp1. A2780 were treated for 16 h with 5 µM BIO or 21 days with 2.5 µM prior to ChIP with Sp1 antibody and QPCR detection of the hTERT and TP53 promoters. Mean±SEM of three experiments (ns: not significant; *: p<0.5; **: p<0.01).
Mentions: We identified multiple potential reciprocal repression (toggle switch) and feedback oscillator motifs as in the examples in figure 7A, which may provide substantial scope for dynamic network behaviour. We also identified several types of coherent feed forward motifs. In particular, three distinct sub-networks form candidate activation and repression “modules”. The activation module links all positive regulators of hTERT via densely overlapping coherent type 1 motifs, while the repression module comprises several coherent type 2 motifs organised by p53 and AR which inhibit hTERT and its activators. Both motif types are reported to reduce noise in gene expression networks [21].

Bottom Line: Better understanding of upstream pathways is critical for effective anti-telomerase therapeutics and may reveal new targets to inhibit hTERT expression.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.

View Article: PubMed Central - PubMed

Affiliation: Centre for Oncology and Applied Pharmacology, University of Glasgow, Cancer Research UK Beatson Laboratories, Garscube Estate, Bearsden, Glasgow, United Kingdom.

ABSTRACT

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.

Show MeSH
Related in: MedlinePlus