Abstract
Transcriptional reactivation of TERT, the catalytic subunit of telomerase, is necessary for cancer progression in about 90% of human cancers. The recent discovery of two prevalent somatic mutations—C250T and C228T—in the TERT promoter in various cancers has provided insight into a plausible mechanism of TERT reactivation. Although the two hotspot mutations create a similar binding motif for E-twenty-six (ETS) transcription factors, we show that they are functionally distinct, in that the C250T unlike the C228T TERT promoter is driven by non-canonical NF-κB signalling. We demonstrate that binding of ETS to the mutant TERT promoter is insufficient in driving its transcription but this process requires non-canonical NF-κB signalling for stimulus responsiveness, sustained telomerase activity and hence cancer progression. Our findings highlight a previously unrecognized role of non-canonical NF-κB signalling in tumorigenesis and elucidate a fundamental mechanism for TERT reactivation in cancers, which if targeted could have immense therapeutic implications.
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Acknowledgements
We thank the Agency for Science Technology and Research, Singapore (A∗STAR) and IMCB for their financial support of this work. We are grateful to K. C. Low from IMCB for purification of recombinant proteins and cloning of shRNA and TERT promoter constructs. We thank H. C. Tay from SBIC for performing the surgery of mice and the Advanced Molecular Pathology Laboratory (AMPL) of IMCB for the histology work. Y.L. is supported by an A∗STAR fellowship and an IMCB Early Career Researcher (ECR) grant.
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V.T. conceptualized the ideas for this manuscript. Y.L. and V.T. planned and devised the experiments. Y.L., Q.-L.Z. and H.S.C. performed CHIP and real-time PCR experiments along with all biochemical and molecular analysis. W.S. and S.P. performed TACO and all bioinformatics analysis. P.C. and K.-H.C. conducted the orthotopic glioma injections and imaging. M.Lakshmanan and A.R. did the tumour xenograft experiments. Z.Y., J.L. and M.Li genotyped the primary tumours and performed IHC on primary tumours. D.G.T. supported Q.-L.Z.’s studentship. S.-Y.C. provided GBM cell lines. Y.L. wrote the manuscript and V.T. edited it.
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Supplementary Figure 2 GBM cell lines display similar levels of p100 to p52 processing but respond differentially to TWEAK-induced TERT expression according to TERT promoter mutation status.
(a) GBM cell lines were treated with TNF-α (10 ng ml−1) for 1 h and analyzed for TERT, IL-8 and IκBα expression. Plots depict relative fold change in mRNA expression. Data shown represent the mean of 2 independent experiments. (b) Relative Fn14 expression in GBM cell lines carrying either C250T or C228T TERT mutation. Data shown are an average of 2 independent experiments per cell line. Plots depict relative mRNA levels. All raw data are shown in Supplementary Table 2. (c) Cytoplasmic and nuclear fractions of untreated or TWEAK-stimulated C250T and C228T GBM cell lines were analyzed for p52 and RelB activation by western blotting with the indicated antibodies. Data shown is representative of two independent experiments. Unprocessed original scans of blots are found in Supplementary Fig. 7.
Supplementary Figure 3 Canonical NF-κB activation does not induce p65 or p52 recruitment to TERT promoter in GBM cells.
(a) ChIP analysis of control (Ctrl) or TWEAK treated GBM cell lines depicting enrichment of BLC promoter with indicated antibodies. n = 3 independent ChIP assays performed per cell line. Error bars represent S.D. (b) Western blot analysis of untreated or TWEAK-treated C250T-mutant GBM cells transduced with shRNAs targeting p52, RelB, NIK or vector control. Data shown is representative of two independent experiments. (c,d) ChIP was performed in control or TNF-α stimulated GBM cell lines. Enrichment of TERT promoter (c) and NF-κB1A promoter (d) DNA fragments in ChIP DNA were measured by quantitative real-time PCR (ChIP-qPCR) and normalized to DNA input. n = 3 independent ChIP experiments performed for each cell line and error bars represent S.D. ∗P < 0.05; ∗∗P < 0.01; Student’s t-test, twotailed. All raw data are shown in Supplementary Table 2. Unprocessed original scans of blots are found in Supplementary Fig. 7.
Supplementary Figure 4 Lymphotoxin β receptor (LtβR)-mediated activation of non-canonical NF-κB pathway induces recruitment of NF-κB2 p52 and Pol II to C250T TERT promoter, resulting in enhanced TERT transcription and telomerase function.
(a) Cells were treated with agonistic human LTβR antibody for 24 h and total cell extracts were analyzed by western blotting with indicated antibodies. Data shown is representative of three independent experiments. Unprocessed original scans of blots are found in Supplementary Fig. 7. (b) Relative TERT expression of control (Ctrl) or anti-LTβR-treated T98G and U251 cells. Data from one experiment are shown which is representative of 2 independent experiments. (c,d) ChIP was performed in control (Ctrl) or antiLTβR-treated T98G and U251 cells using p52, p65 or Pol II-specific antibodies and IgG as a negative control. Enrichment of TERT promoter DNA (c) and BLC promoter DNA (d) fragments in ChIP DNA was normalized to DNA input. n = 3 independent ChIP experiments per treatment group and cell line. Error bars represent S.D. (e) Proliferation assay of control (Ctrl) or anti-LTβR-treated T98G and U251 cells. Data shown are from 3 independent experiments for each cell line. Error bars represent S.E.M. (f) Relative telomerase activity of T98G and U251 cells that were untreated (Ctrl) or stimulated with LTβR antibody for 1–4 days. Plots represent mean ± S.E.M. Data shown are from 3 independent experiments for each cell line. (g) Relative TERT expression of T98G and U251 cells treated with si-Control (Ctrl), si-NF-κB2 or si-RelB. Data shown represent the mean of 2 independent experiments. All statistical analyses were performed using Student’s t-test (two-tailed): ∗P < 0.05; ∗∗P < 0.01; ∗∗∗P < 0.001. For raw data, refer to Supplementary Table 2.
Supplementary Figure 5 Purified p52 protein binds C250T TERT promoter through its Rel homology domain.
(a) Recombinant GST-tagged p52 and GST proteins were analyzed for binding to HIV-κB and C250T TERT promoter DNA-labeled probes with EMSA (right panel). Coomassie-stained SDSPAGE of recombinant p52 protein (left panel). EMSA shown is representative of three independent experiments. (b) EMSA analysis of recombinant wild-type (WT) and mutant p52 (carrying mutation in 2 amino acid residues of Rel-homology domain) proteins binding to HIV- κB and C250T TERT promoter DNA-labeled probes (right panel). Data shown is representative of two independent experiments. Coomassie-stained SDS-PAGE showing amount of WT and mutant p52 proteins used for EMSA analysis (left panel).
Supplementary Figure 6 Constitutive expression of NF-κB-inducing kinase (NIK) results in transcriptional activation of C250T TERT promoter, which promotes the telomerase activity of GBM cells.
(a) T98G and U251 cells were transfected with vector, human NIK wild-type (WT) or kinase-inactive mutant NIK (KK) expression plasmids and total cell lysates were analyzed by western blotting with the indicated antibodies. Data shown is representative of three independent experiments. Original scans of blots are shown in Supplementary Fig. 7. (b) ChIP was performed in control (Ctrl) or NIK WT-overexpressing T98G and U251 cells using p52, p65 or Pol II-specific antibodies and IgG as a negative control. Enrichment of TERT promoter DNA fragments in ChIP DNA was normalized to input. n = 3 independent ChIP experiments per cell type. Error bars represent S.D. (c) Relative telomerase activity of T98G and U251 cells transfected with vector, NIK WT or NIK KK constructs. Data from one experiment is shown which is representative of 3 and 2 independent experiments for T98G and U251 cells respectively. (d) ChIP analysis of control or NIK WT-overexpressing T98G and U251 cells depicting enrichment of BLC promoter with indicated antibodies. n = 3 independent ChIP experiments per cell type and error bars represent S.D. (e) Luciferase reporter assays were performed in 293T HEK cells that were co-transfected with empty vector or human NIK expression plasmid and the pGL3 basic reporter vector or pGL3 vector containing either the WT TERT promoter region (−340 to −55) or TERT promoter with C250T mutation (−340 to −55). Data shown represent the mean luciferase activity from 2 independent experiments. (f) Luciferase reporter assays were performed in 293T HEK cells that were transfected with pGL3 empty reporter vector or pGL3 vector containing either the WT TERT promoter region or TERT promoter with C250T mutation and subsequently untreated or stimulated with TWEAK (30 ng ml−1) for 1D. Luciferase assay data shown are from one of two independent experiments. (g,h) Vector- or NIK WT-expressing T98G cells were transfected with si-Ctrl, si-RelB or si-NF-κB2 and analyzed for relative TERT expression (average fold change ± S.D.;n = 3 independent experiments). Western blot data is representative of two independent experiments. ∗P < 0.05; ∗∗∗P < 0.001; Student’s t-test, two-tailed. All raw data are shown in Supplementary Table 2. Unprocessed original scans of blots are found in Supplementary Fig. 7.
Supplementary Figure 7 Ectopic NIK expression enhances the in vivo tumorigenicity of C250T GBM cells through p52 activation.
(a) T98G cells were stably transduced with lentiviral expression constructs for vector, NIK WT and NIK WT in combination with shRNA targeting p52 (NIK shp52) and total cell lysates were analyzed by western blotting with the indicated antibodies. Representative image from two independent experiments is shown. Unprocessed original scans of blots are found in Supplementary Fig. 7. (b) 5 NOD-SCID mice were injected subcutaneously with T98G cells expressing vector (red arrows), NIK WT (white arrows) or NIK WT sh-p52 (black arrows) and the tumor growth of these cells in the xenograft mouse model after 10 weeks are depicted. (c) Representative immunohistochemical staining of T98G NIK WT xeonograft tumors with the indicated antibodies. Scale bars, 100 μm. (d) Relative telomerase activity of T98G cells expressing vector, NIK WT or NIK sh-p52. Data shown represent the mean of 3 independent experiments. Error bars represent S.E.M. ∗P < 0.05; Student’s t-test, two-tailed. All raw data are shown in Supplementary Table 2.
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Li, Y., Zhou, QL., Sun, W. et al. Non-canonical NF-κB signalling and ETS1/2 cooperatively drive C250T mutant TERT promoter activation. Nat Cell Biol 17, 1327–1338 (2015). https://doi.org/10.1038/ncb3240
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DOI: https://doi.org/10.1038/ncb3240
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