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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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Related in: MedlinePlus

Whole-kinome RNAi screen of the hTERT promoter in A2780.(A) hTERT-luciferase was cotransfected with 50 nM siRNA in triplicate. 48 h post-transfection, luciferase assays were performed. 3 independent siRNA per target were assessed. Hit criterion was >2-fold change in promoter activity by at least 2/3 siRNA. 235 hit IDs were analysed in MetaCore using the “direct interactions” algorithm limited to phosphorylation interactions. Green arrows: positive regulation; red: negative regulation. Blue circles: siRNA repressed the hTERT promoter. Circle shading intensity indicates fold change (minimum 2-fold). Average derived from all independent hit siRNA is shown. (B) Relative repression of the hTERT promoter by siRNA against targets in the network model. Luciferase activities were calculated relative to control (non-specific) for each siRNA. Figure shows mean±SEM derived from all independent hit siRNA for each target.
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pone-0006459-g008: Whole-kinome RNAi screen of the hTERT promoter in A2780.(A) hTERT-luciferase was cotransfected with 50 nM siRNA in triplicate. 48 h post-transfection, luciferase assays were performed. 3 independent siRNA per target were assessed. Hit criterion was >2-fold change in promoter activity by at least 2/3 siRNA. 235 hit IDs were analysed in MetaCore using the “direct interactions” algorithm limited to phosphorylation interactions. Green arrows: positive regulation; red: negative regulation. Blue circles: siRNA repressed the hTERT promoter. Circle shading intensity indicates fold change (minimum 2-fold). Average derived from all independent hit siRNA is shown. (B) Relative repression of the hTERT promoter by siRNA against targets in the network model. Luciferase activities were calculated relative to control (non-specific) for each siRNA. Figure shows mean±SEM derived from all independent hit siRNA for each target.

Mentions: We searched MetaCore for direct phosphorylation interactions between hits and network transcription factors (figure 8A; blue circles represent mean fold promoter repression with hTERT promoter activity values for each network target given in figure 8B). At least 54 hit kinases participate in upstream pathways and 38 hits directly phosphorylate one or more of the transcription factors. Critical divergence hubs with respect to the network transcription factors are GSK3 itself, p90-RSK, several PKC isoforms, PKA, JNK and p38. These hubs are predicted to be important modifiers of hTERT suppression with GSK3 inhibitors and preferred targets for combinatorial inhibition.


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)

Whole-kinome RNAi screen of the hTERT promoter in A2780.(A) hTERT-luciferase was cotransfected with 50 nM siRNA in triplicate. 48 h post-transfection, luciferase assays were performed. 3 independent siRNA per target were assessed. Hit criterion was >2-fold change in promoter activity by at least 2/3 siRNA. 235 hit IDs were analysed in MetaCore using the “direct interactions” algorithm limited to phosphorylation interactions. Green arrows: positive regulation; red: negative regulation. Blue circles: siRNA repressed the hTERT promoter. Circle shading intensity indicates fold change (minimum 2-fold). Average derived from all independent hit siRNA is shown. (B) Relative repression of the hTERT promoter by siRNA against targets in the network model. Luciferase activities were calculated relative to control (non-specific) for each siRNA. Figure shows mean±SEM derived from all independent hit siRNA for each target.
© Copyright Policy
Related In: Results  -  Collection

Show All Figures
getmorefigures.php?uid=PMC2714081&req=5

pone-0006459-g008: Whole-kinome RNAi screen of the hTERT promoter in A2780.(A) hTERT-luciferase was cotransfected with 50 nM siRNA in triplicate. 48 h post-transfection, luciferase assays were performed. 3 independent siRNA per target were assessed. Hit criterion was >2-fold change in promoter activity by at least 2/3 siRNA. 235 hit IDs were analysed in MetaCore using the “direct interactions” algorithm limited to phosphorylation interactions. Green arrows: positive regulation; red: negative regulation. Blue circles: siRNA repressed the hTERT promoter. Circle shading intensity indicates fold change (minimum 2-fold). Average derived from all independent hit siRNA is shown. (B) Relative repression of the hTERT promoter by siRNA against targets in the network model. Luciferase activities were calculated relative to control (non-specific) for each siRNA. Figure shows mean±SEM derived from all independent hit siRNA for each target.
Mentions: We searched MetaCore for direct phosphorylation interactions between hits and network transcription factors (figure 8A; blue circles represent mean fold promoter repression with hTERT promoter activity values for each network target given in figure 8B). At least 54 hit kinases participate in upstream pathways and 38 hits directly phosphorylate one or more of the transcription factors. Critical divergence hubs with respect to the network transcription factors are GSK3 itself, p90-RSK, several PKC isoforms, PKA, JNK and p38. These hubs are predicted to be important modifiers of hTERT suppression with GSK3 inhibitors and preferred targets for combinatorial inhibition.

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