Dynamic Telomerase Gene Suppression via Network Effects of GSK3 Inhibition

Abstract

BackgroundTelomerase 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 FindingsIn 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.Conclusions/SignificanceOur 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.

High-Throughput RNAi Screening Reveals Novel Regulators of Telomerase

“…One such study identified GSK3B as a regulator of telomerase expression and showed that targeting this protein strongly affected cell proliferation as well as telomere length, providing a link between the Wnt pathway and telomere maintenance (50). Our results complement and extend this previous study by analyzing the effects of modulating kinase activity on TA and ultimately telomerase function at the telomeres.…”

“…We do note that other studies have used RNAi screening with the same siRNA platform to identify telomerase regulators, most notably by looking at modulators of telomerase expression rather than activity (50). One such study identified GSK3B as a regulator of telomerase expression and showed that targeting this protein strongly affected cell proliferation as well as telomere length, providing a link between the Wnt pathway and telomere maintenance (50).…”

Comparative Large Scale Characterization of Plant versus Mammal Proteins Reveals Similar and Idiosyncratic N-α-Acetylation Features

“…After few washes in PBS, the cells were lysed adding in flask Nonidet P-40 lysis buffer (150 mM NaCl, 1% Nonidet P-40, 50 mM Tris, pH 8, and EDTA-free protease inhibitor). The cells were scraped off the walls, and the lysate was centrifuged to remove debris (54). The supernatant was stored at Ϫ20°C.…”

XAV939 promotes apoptosis in a neuroblastoma cell line via telomere shortening

“…Studies have shown that targeted inhibition of GSK-3β protein greatly affected cell proliferation and telomere length, suggesting a relationship between telomere maintenance and Wnt signaling pathways (31). Our previous study also demonstrated that XAV939 induced the apoptosis of NB cells as detected by Annexin V and Hoechst 33342 staining as well as decreased expression of Bcl-2 (14).…”