Signaling pathway inhibitor library screening reveals b catenin TCF4 as a novel telomerase regulator in cancer cell lines

SIGNALING PATHWAY INHIBITOR LIBRARY SCREENING REVEALS β-CATENIN/TCF4 AS A NOVEL TELOMERASE REGULATOR IN CANCER CELL LINES JOELLE TOH LING LING (M.SC.), NUS A THESIS SUBMITTED FOR THE DEGREE OF MASTER OF SCIENCE DEPARTMENT OF BIOCHEMISTRY NATIONAL UNIVERSITY OF SINGAPORE 2013 I Declaration page I hereby declare that the thesis is my original work and it has been written by me in its entirety I have duly acknowledged all the sources of information which have been used in the thesis This thesis has also not been submitted for any degree in any university previously _ Joelle Toh Ling Ling January 2013 II Acknowledgements I would like to express my sincere gratitude to Dr Wang Xueying (my supervisor) and Dr Zhang Yong for their scientific discussions and guidance in the project I would like to thank Institute of Molecular and Cell Biology for the sponsorship of my study, and my thesis committee: Dr Thilo Hagen, Dr Wu Qiang, Dr Liu Jianhua and Dr Luo Yan for their insightful feedback in our thesis community meetings I would also like to thank A/P Tan Tin Wee, the former acting head of the department of Biochemistry when I was a student, for taking his precious time to give me advice during my graduating years despite his busy schedule Last but not least, I would like to thank my fellow labmate Ling Li and Dr Zhang for their technical support in the telomere length assay (Figure 3.20) and Zoey for her encouragement III Table of Contents No 1.1 1.1.1 1.1.2 1.2 1.2.1 1.2.2 1.2.3 1.3 1.4 1.4.1 1.4.1.1 1.4.1.2 1.4.1.3 1.4.1.4 1.4.1.5 1.4.1.6 1.4.1.7 1.4.2 1.4.3 1.4.4 1.5 1.5.1 1.5.2 1.5.3 1.5.4 Content Title Declaration Page Acknowledgement Table of contents Summary List of Tables List of Figures List of publications for the project Introduction Telomere Telomeric DNA Telomeric protein (Shelterin) Telomerase Telomerase RNA (TER) Telomerase reverse transcriptase (TERT) Dyskerin Telomerase in human cancer hTERT regulation hTERT transcriptional regulation by diverse transcription factors and signaling pathways hTERT transcriptional regulation by Myc/Max/Mad network hTERT transcriptional regulation by Sp1 hTERT transcriptional regulation by AP-1 and Ap-2 hTERT transcriptional regulation by HIF-1 hTERT transcriptional regulation by Ets Proteins hTERT transcriptional regulation by STAT hTERT transcriptional regulation by Estrogen hTERT transcriptional regulation by epigenetic modifications hTERT regulation by alternative splicing hTERT regulation by post-translational modification Extra-telomeric functions of hTERT hTERT regulates DNA repair hTERT regulates mitochondrial functions under oxidative stress hTERT regulates apoptotic pathway hTERT regulates cell growth and proliferation Page I II III IV VII IX X XII 1 10 11 12 16 16 17 20 22 23 24 26 27 28 29 30 31 32 32 33 36 IV 1.6 1.7 1.7.1 1.7.2 2.1 2.1.1 2.1.2 2.1.3 2.1.4 2.1.5 2.2 2.2.1 2.2.2 2.2.3 2.2.4 2.2.5 2.2.6 2.2.7 2.2.8 2.2.9 2.2.10 2.2.11 2.2.12 2.2.13 3.1 3.1.1 3.1.2 3.1.3 3.1.4 3.2 3.2.1 3.2.2 3.3 3.3.1 3.3.2 3.3.3 Telomerase related screen Wnt signaling pathway Canonical Wnt pathway Non-canonical Wnt pathway Material and Methods Material Cell line Inhibitor library Plasmid List of primers List of antibodies Methods Cell Culture Wnt-3a treatment Generation of stable cell lines Real-time PCR–based version of telomere repeat amplification protocol (qTRAP) Telomerase inhibitor screen RT-qPCR Western blot β-catenin/Tcf4 complex consensus binding sequence Generation of mutants Luciferase assays Telomere length assay Electrophoretic mobility shift assay (EMSA) Chromatin immunoprecipitation (ChIP) assays Results Inhibitors screening Verification of screening platform using STAT III and IV inhibitors Wnt pathway inhibitor library screening EGFR pathway inhibitors library screening JAK/STAT pathway inhibitor library screening Validation of the positive hits from the screening experiments in MCF7 and AGS Verification for Wnt inhibitors Verification for EGFR inhibitors Mechanistic study of β-catenin/TCF on hTERT Effect of FH535 (β-catenin/TCF inhibitor) on mRNA expression of essential components of telomerase (hTERT, hTER and DKC1) and c-Myc Co-relation of TA and β-catenin expression in SW480 and SW620 Canonical Wnt/β-catenin signaling regulates TA in cancer cell via hTERT transcription regulation 38 55 55 60 61 61 61 61 64 66 67 68 68 69 69 70 71 72 73 73 74 74 75 76 76 78 78 79 81 85 89 91 91 94 96 96 99 101 V 3.3.3.1 Effect of Wnt-3a treatment on TA and mRNA level of mRNA level of essential components of telomerase (hTERT, hTER and DKC1) 3.3.3.2 Effect of lithium chloride treatment on TA and mRNA level of hTERT, hTERT 3.3.3.3 Effect of Wnt-3a and lithium chloride treatment on hTERT promoter luciferase activity 3.3.3.4 Effect of knocking down of β-catenin on TA, hTERT mRNA and hTERT (949bp) in cancer cell lines 3.3.3.5 Effect of knocking down of β-catenin on telomere length in cancer cell lines 3.3.4 Effect of over expression of β-catenin on TA, hTERT mRNA and hTERT (949bp) in cancer cell lines 3.3.5 β-catenin regulate the hTERT promoter through intereaction with TCF4 3.3.5.1 TCF is involved in β-catenin dependent hTERT transcription regulation in cancer cell lines 3.3.5.2 β-catenin/TCF4 regulate hTERT promoter in cancer cell lines 3.3.5.3 Characterization of the distal TCF4 binding site (TBE) in the human hTERT promoter 3.3.5.4 In vitro and in vivo occupancy of TCF4 on TBE in hTERT promoter Discussion References 102 105 109 111 115 120 124 124 127 132 139 144 149 VI Summary Telomerase is a ribonucleoprotein complex consisting of a reverse transcriptase protein unit (TERT) and a RNA unit (TER) Telomerase activity (TA) has been observed in ~85% of all human tumors, implying that immortality conferred by telomerase, play a key role in malignant transformation (Shay and Bacchetti 1997) Inhibition of telomerase has been shown to result in telomere-shortening, subsequent growth arrest and senescence in a wide range of tumor cell lines (Hahn et al 1999; Zhang et al 1999) The almost universal presence of telomerase in human cancers and cancer stem cells, together with its near-absence in most normal tissues make telomerase an attractive therapeutic target However mechanism of telomerase activation in cancer has yet been documented in detail and insight into the mechanism will definitely improve telomerase base cancer therapy Well-defined signaling pathway (Wnt, EGFR and JAK/STAT) inhibitors that are known to play important roles in cancer progression were screened to identify new telomerase regulators Hits from the inhibitors libraries were verified in a wide range of cancer cell lines (stomach adenocarcinoma: AGS, breast cancer: MCF7, colorectal cancer: HCT116/LS174T) and are therefore expected to be general TA inhibitors for some of the major types of cancer β-catenin/TCF4 complex was identified as a novel TA regulator from the screen and was later found to inhibit TA via transcription regulation of hTERT (human TERT) Activation of the Wnt pathway either by Wnt ligand (Wnt-3a) or LiCl (activates VII Wnt signaling by inhibiting GSK-3β) treatment as well as overexpression of a constitutively active form of β-catenin (Δ-N β-catenin) up regulated hTERT mRNA expression and telomerase activity (TA) in cancer cell lines On the other hand, knocking down of endogenous β-catenin via shRNA reduces hTERT mRNA expression and TA In addition, a β-catenin/TCF4 consensus binding sequence from -659bp to -653 bp (5’-TGCAAAG-3’) upstream of transcription start site in hTERT promoter was also found and evidences from promoter studies, electrophoretic mobility shift assay, and chromatin immunoprecipitation assay, showed that β-catenin/TCF4 bind to hTERT promoter in vivo and in vitro Taken together, this is the first study has shown that Wnt signaling regulates telomerase via the transcription regulation of hTERT VIII List of Tables
 Table no 1.1 1.2 2.1 2.2 2.3 2.4 2.5 3.1 3.2 3.3 Content List of current screening strategies published List of approaches in telomerase cancer therapy List of Wnt inhibitors examined in the screen List of EGFR inhibitors examined in the screen List of JAK/STAT inhibitors examined in the screen List of primers used in the project List of antibodies used in the project A summary of positive hits from Wnt pathway inhibitors library screening in HCT116 and LS174T List of positive hits from the EGFR pathway inhibitors library Changes on the mean telomere length of HCT116, MCF7, MCF10A and 293T cell lines in scramble shRNA or β-catenin RNA lent virus treatment IX List of Figures
 Figure no 1.1 1.2 2.1 3.1 3.2 3.3 3.4 3.5 3.6 3.7 3.8 3.9 3.10 3.11 3.12 3.13 3.14 3.15 3.16 Content Diagrammatic representation of a T-loop and D-loop structure of a telomere Representation of transcription factors and their binding sites on hTERT promoter hTERT plasmids that were generated STAT III and V inhibitors were able to reduce TA in HCT116 cells by 60% and 40% respectively Effect of TA by Wnt pathway inhibitors compounds A to O in HCT116 at (A) 1x IC50 and (B) 5x IC50 (except inhibitor C that was at 2.5x IC50) Effect of TA by Wnt pathway inhibitors compounds A to O in LS174T at (A) 1x IC50 and (B) 5x IC50 (except inhibitor C that was at 2.5x IC50) Effect of TA by EGFR pathway inhibitors compounds A to M in HCT116 at (A) 1x IC50 and (B) 5x IC50 Effect of TA by EGFR pathway inhibitors compounds A to M in LS174T at (A) 1x IC50 and (B) 5x IC50 Effect of TA by JAK/STAT pathway inhibitors A to D in HTC116 at 5x IC50 (A) Effect of TA by Wnt pathway inhibitors compounds B, C, F and K on MCF7 at a dose of 5x IC50 (2.5x IC50 for C) (B) Concentration dependent effect of inhibitor C (FH535) on MCF7 Effect of TA by inhibitor C (FH535) at 2.5x IC50 on AGS Effect of TA by (A) EGFR pathway inhibitor B, C, G and M on MCF7 at a dose of 5x IC50 Effect of FH535 at 2.5x IC50 on mRNA expression of core components of telomerase in (A) MCF7 (B) HCT116 (A) TA and (B) β-catenin protein levels in SW480 and SW620 cell lines Figure 3.12 Wnt3a treatment increased TA in MCF7, HCT116 and 293T (n = 3; *P < 0.05, t-test) Figure 3.13 Effect of Wnt3a on hTERT, hTER and DKC1 mRNA level in (A) MCF7 (B) HCT116 and (C) 293T cell lines (n = 3; *P < 0.05, t-test) 15mM of LiCl treatment increased TA in MCF7, HCT116 and 293T (n = 3; *P < 0.05, t-test) Figure 3.15 Effect of 15mM of LiCl on (A) hTERT , hTERT and DKC1 mRNA level in 293T, (B) hTERT mRNA on MCF7, HCT116, BJ and MCF10A (n = 3; *P < 0.05, t-test) Figure 3.16 Effect of (A) Wnt-3a and (B) 15mM LiCl treatment on 949bp hTERT promoter activity in MCF7, HCT116 and 293T (n = 3; *P < 0.05, t-test) X Jiang XR, Jimenez G, Chang E, Frolkis M, Kusler B, Sage M, Beeche M, Bodnar AG, Wahl GM, Tlsty TD, Chiu CP Telomerase expression in human somatic cells does not induce changes associated with a transformed phenotype Nat Genet 1999; 21: 111-4 Jenuwein T, Allis CD Translating the histone code Science 2001; 293: 1074–80 Jeppesen P, Mitchell A, Turner B, Perry P Antibodies to defined histone epitopes reveal variations in chromatin conformation and underacetylation of centric heterochromatin in human metaphase chromosomes Chromosoma 1992; 101; 322–32 Kang HJ, Choi YS, Hong SB, Kim KW, Woo RS, et al Ectopic expression of the catalytic subunit of telomerase protects against brain injury resulting from ischemia and NMDA-induced neurotoxicity J Neurosci 2004; 24: 1280 – Kanaya T, Kyo S, Takakura M, Ito H, Namiki M, Inoue M hTERT is a critical determinant of telomerase activity in renal-cell carcinoma Int J Cancer 1998; 78: 539-43 Kanaya T, Kyo S, Hamada K, Takakura M, Kitagawa Y, Harada H, Inoue M Adenoviral expression of p53 represses telomerase activity through downregulation of human telomerase reverse transcriptase transcription Clin Cancer Res 2000; 6: 1239-47 Kang SS, Kwon T, Kwon DY, Do SI Akt protein kinase enhances human telomerase activity through phosphorylation of telomerase reverse transcriptase subunit J Biol Chem 1999; 274: 13085-90 Kanoh J, Ishikawa F spRap1 and spRif1, recruited to telomeres by Taz1, are essential for telomere function in fission yeast Curr Biol 2001; 11: 1624-30 Karlseder J, Broccoli D, Dai Y, Hardy S, de Lang T p53- and ATMdependent apoptosis induced by telomeres lacking TRF2 Science 1999; 283: 1321-1325 Karlseder J, Hoke K, Mirzoeva OK, Bakkenist C, Kastan MB, Petrini JH, de Lange T 2004 The telomeric protein TRF2 binds the ATM kinase and can inhibit the ATM-dependent DNA damage response PLoS Biol 2004; 2: e240 Karner CM, Chirumamilla, R, Aoki S, Igarashi P, Wallingford JB., Carroll T.J Wnt9b signaling regulates planar cell polarity and kidney tubule morphogenesis Nat Genet 2009; 41: 793–9 160 Kawano Y, Kypta R Secreted antagonists of the Wnt signalling pathway J Cell Sci 2003; 116: 2627-34 Khalil AM, Boyar FZ, Driscoll DJ Dynamic histone modifications mark sex chromosome inactivation and reactivation during mammalian spermatogenesis Proc Natl Acad Sci U S A 2004; 101: 16583–7 Khalil AM, Wahlestedt C Epigenetic mechanisms of gene regulation during mammalian spermatogenesis Epigenetics 2004; 3; 21–8 Kilian A, Bowtell DD, Abud HE, Hime GR, Venter DJ, Keese PK, Duncan EL, Reddel RR, Jefferson RA Isolation of a candidate human telomerase catalytic subunit gene, which reveals complex splicing patterns in different cell types Hum Mol Genet 1997; 6: 2011-9 Kimmins S, Sassone-Corsi P Chromatin remodelling and epigenetic features of germ cells Nature 2005; 434; 583–9 Kim NW, Piatyszek MA, Prowse KR, Harley CB, West MD, Ho PL, Coviello GM, Wright WE, Weinrich SL, Shay JW Specific association of human telomerase activity with immortal cells and cancer Science 1994; 266: 20115 Kim SH, Han S, You YH, Chen DJ, Campisi J The human telomereassociated protein TIN2 stimulates interactions between telomeric DNA tracts in vitro EMBO Rep 2003; 4: 685-691 Kim SH, Beausejour C, Davalos, AR, Kaminker P, Heo SJ, Campisi J TIN2 mediates functions of TRF2 at human telomeres J Biol Chem 2004; 279: 43799-804 Kirkpatrick KL, Ogunkolade W, Elkak AE, Bustin S, Jenkins P, Ghilchick M, Newbold RF, Mokbel K hTERT expression in human breast cancer and noncancerous breast tissue: correlation with tumour stage and c-Myc expression Breast Cancer Res Treat 2003; 77: 277-84 Kirwan M, Dokal I Dyskeratosis congenita: a genetic disorder of many faces Clin Genet 2008, 73, 103–12 Kiss AM, Jády BE, Darzacq X, Verheggen C, Bertrand E, Kiss T A Cajal body-specific pseudouridylation guide RNA is composed of two box H/ACA snoRNA-like domains Nucleic Acids Res 2002; 30: 4643-9 Ko LJ, Prives C p53: puzzle and paradigm Genes Dev 1996; 10: 1054-72 161 Konnikova L, Simeone MC, Kruger MM, Kotecki M, Cochran BH Signal transducer and activator of transcription (STAT3) regulates human telomerase reverse transcriptase (hTERT) expression in human cancer and primary cells Cancer Res 2005; 65: 6516-20 Koshiji M, Kageyama Y, Pete EA, Horikawa I, Barrett JC, Huang LE HIF1alpha induces cell cycle arrest by functionally counteracting Myc EMBO J 2004; 23: 1949-56 Kramer KM, Haber JE New telomeres in yeast are initiated with a highly selected subset of TG1-3 repeats Genes Dev 1993; 7: 2345-56 Kraus C, Liehr T, Hülsken J, Behrens J, Birchmeier W, Grzeschik KH, Ballhausen WG Localization of the human β-catenin gene (CTNNB1) to 3p21: a region implicated in tumor development Genomics 1994; 23: 272–4 Kusumoto M, Ogawa T, Mizumoto K, Ueno H, Niiyama H, Sato N, Kyo S, Takakura M, Fujiwara T, Inoue M Understanding and exploiting hTERT promoter regulation for diagnosis and treatment of human cancers Cancer Sci 2008; 99:1528-38 Kyo S, Kanaya T, Takakura M, Tanaka M, Inoue M Human telomerase reverse transcriptase as a critical determinant of telomerase activity in normal and malignant endometrial tissues Int J Cancer 1999 ; 80: 60-3 Kyo S, Takakura M, Inoue M Telomerase activity in cancer as a diagnostic and therapeutic target Histol Histopathol 2000; 15: 813-24 Kyte JA Cancer vaccination with telomerase peptide GV1001 Expert Opin Investig Drugs 2009; 18: 687-94 de Lange T Telomere capping one strand fits all Science 2001; 292: 10751076 de Lange T Shelterin: the protein complex that shapes and safeguards human telomeres Genes Dev 2005; 19: 2100-10 de Lange T How telomeres solve the end-protection problem Science 2009; 326: 948-52 Lansdorp PM, Verwoerd NP, Vanderijke FM, Dragowska V, Little MT, Dirks RW, Raap AL, Tanke HJ Heterogeneity in telomere length of human chromosomes Hum Mol Genet 1996; 5: 685– 91 162 Latrick CM, Cech TR POT1-TPP1 enhances telomerase processivity by slowing primer dissociation and aiding translocation EMBO J 2010; 29: 92433 Lee J, Sung YH, Cheong C, Choi YS, Jeon HK, Sun W, Hahn WC, Ishikawa F, Lee HW TERT promotes cellular and organismal survival independently of telomerase activity Oncogene 2008; 27: 3754–60 Lee JH, Chung IK Curcumin inhibits nuclear localization of telomerase by dissociating the Hsp90 co-chaperone p23 from hTERT Cancer Lett 2010; 290: 76-86 Lei M, Podell ER, Cech TR Structure of human POT1 bound to telomeric single-stranded DNA provides a model for chromosome end-protection Nat Struct Mol Biol 2004; 11: 1223-1229 Lei M, Zaug, AJ, Podell, ER, Cech TR Switching human telomerase on and off with hPOT1 protein in vitro J Biol Chem 2005; 280: 20449-56 Levanon D, Goldstein RE, Bernstein Y, Tang H, Goldenberg D, Stifani S, Paroush Z, Groner Y Transcriptional repression by AML1 and LEF-1 is mediated by the TLE/Groucho corepressors Proc Natl Acad Sci USA 1998; 95: 11590–5 Le S, Sternglanz R, Greider CW Identification of two RNA-binding proteins associated with human telomerase RNA Mol Biol Cell 2000; 11: 999-1010 Levy DE, Darnell JE Jr Stats: transcriptional control and biological impact Nat Rev Mol Cell Biol 2002; 3: 651–62 Levy L, Neuveut C, Renard CA, Charneau P, Branchereau S, Gauthier F, Van Nhieu JT, Cherqui D, Petit-Bertron AF, Mathieu D, Buendia MA Transcriptional activation of interleukin-8 by beta-catenin-Tcf4 J Biol Chem 2002; 277: 42386–93 Li H, Zhao L, Yang Z, Funder JW, Liu JP Telomerase is controlled by protein kinase Calpha in human breast cancer cells J Biol Chem 1998; 273: 33436-42 Lindsey J, McGill NI, Lindsey LA, Green DK, Cooke HJ In vivo loss of telomeric repeats with age in humans Mutat Res 1991; 256: 45-8 163 Lingner J, Cooper JP, Cech TR Telomerase and DNA end replication: no longer a lagging strand problem? Science 1995; 269:1533–4 Lin SY, Elledge SJ Multiple tumor suppressor pathways negatively regulate telomerase Cell 2003; 113: 881-9 Liu F, van den Broek O, Destrée O, Hoppler S Distinct roles for Xenopus Tcf/Lef genes in mediating specific responses to Wnt/beta-catenin signalling in mesoderm development Development 2005; 132: 5375–85 Liu JP, Chen W, Schwarer AP, Li H Telomerase in cancer immunotherapy Biochim Biophys Acta 2010; 1805: 35-42 Liu K, Hodes RJ, Weng Np Cutting edge: telomerase activation in human T lymphocytes does not require increase in telomerase reverse transcriptase (hTERT) protein but is associated with hTERT phosphorylation and nuclear translocation J Immunol 2001; 166: 4826-30 Loayza D, Parsons, H, Donigian, J, Hoke K, de Lange T DNA binding features of human POT1: A nonamer 5′-TAGGGTTAG-3′ minimal binding site, sequence specificity, and internal binding to multimeric sites J Biol Chem 2004; 279: 13241-8 Love JJ, Li X, Case DA, Giese K, Grosschedl R, Wright PE Structural basis for DNA bending by the architectural transcription factor LEF-1 Nature 1995; 376: 791–5 Maida Y, Kyo S, Kanaya T, Wang Z, Yatabe N, Tanaka M, Nakamura M, Ohmichi M, Gotoh N, Murakami S, Inoue M Direct activation of telomerase by EGF through Ets-mediated transactivation of TERT via MAP kinase signaling pathway Oncogene 2002; 21: 4071-9 Makarov VL, Hirose Y, Langmore JP Long G tails at both ends of human chromosomes suggest a C strand degradation mechanism for telomere shortening Cell 1997; 88: 657-666 Martín-Rivera L, Herrera E, Albar JP, Blasco MA Expression of mouse telomerase catalytic subunit in embryos and adult tissues Proc Natl Acad Sci USA 1998; 95:10471–6 Maser RS, DePinho RA Keeping telomerase in its place Nat Med 2002; 8: 934 – Masutomi K, Possemato R, Wong JM, Currier JL, Tothova Z, Manola JB, Ganesan S, Lansdorp PM, Collins K, Hahn WC The telomerase reverse 164 transcriptase regulates chromatin state and DNA damage responses Proc Natl Acad Sci U S A 2005; 102: 8222-7 Mavroudis D, Bolonakis I, Cornet S, Myllaki G, Kanellou P, Kotsakis A, Galanis A, Nikoloudi I, Spyropoulou M, Menez J, Miconnet I, Niniraki M, Cordopatis P, Kosmatopoulos K, Georgoulias V A phase I study of the optimized cryptic peptide TERT(572y) in patients with advanced malignancies Oncology 2006; 70: 306-14 Meier UT The many facets of H/ACA ribonucleoproteins Chromosoma 2005; 114: 1–14 Meyerson M, Counter CM, Eaton EN, Ellisen LW, Steiner P, Caddle SD, Ziaugra L, Beijersbergen RL, Davidoff MJ, Liu Q, Bacchetti S, Haber DA, Weinberg RA hEST2, the putative human telomerase catalytic subunit gene, is up-regulated in tumor cells and during immortalization Cell 1997; 90: 78595 Miller JR, Moon RT Signal transduction through beta-cate- nin and speciation of cell fate during embryogenesis Genes Dev 1996; 10: 2527-39 Misiti S, Nanni S, Fontemaggi G, Cong YS, Wen J, Hirte HW, Piaggio G, Sacchi A, Pontecorvi A, Bacchetti S, Farsetti A Induction of hTERT expression and telomerase activity by estrogens in human ovary epithelium cells Mol Cell Biol 2000; 20: 3764-71 Mitchell JR, Cheng J, Collins K A box H/ACA small nucleolar RNA-like domain at the human telomerase RNA 3′ end Mol Cell Biol 1999; 19: 56776 Mitchell JR, Collins K Human telomerase activation requires two independent interactions between telomerase RNA and telomerase reverse transcriptase Mol Cell 2000; 6: 361-71 Mitchell JR, Wood E, Collins K A telomerase component is defective in the human disease dyskeratosis congenita Nature 1999; 402: 551-5 Morin GB The human telomere terminal transferase enzyme is a ribonucleoprotein that synthesizes TTAGGG repeats Cell 1989; 59: 521-9 Morin PJ, Sparks AB, Korinek V, Barker N, Clevers H, Vogelstein B, Kinzler KW Activation of beta- catenin-Tcf signaling in colon cancer by mutations in beta- catenin or APC Science 1997; 275: 1787-90 165 Morales CP, Holt SE, Ouellette M, Kaur KJ, Yan Y, Wilson KS, White MA, Wright WE, Shay JW Absence of cancer-associated changes in human fibroblasts immortalized with telomerase Nat Genet 1999; 21: 115-8 Myers E, Hill AD, Kelly G, McDermott EW, O'Higgins NJ, Buggy Y, Young LS Associations and interactions between Ets-1 and Ets-2 and coregulatory proteins, SRC-1, AIB1, and NCoR in breast cancer Clin Cancer Res 2005; 11: 2111-22 Myung K, Datta A, Kolodner RD Suppression of spontaneous chromosomal rearrangements by S phase checkpoint functions in Saccharomyces cerevisiae Cell 2001; 104: 397-408 Nabetani A, Ishikawa F Alternative lengthening of telomeres pathway: recombination-mediated telomere maintenance mechanism in human cells J Biochem 201l; 149: 5-14 Nateri AS, Spencer-Dene B, Behrens A Interaction of phosphorylated c-Jun with TCF4 regulates intestinal cancer development Nature 2005; 437: 281–5 Nakamura M, Tanaka M Adenovirus-mediated p53 gene transduction inhibits telomerase activity independent of its effects on cell cycle arrest and apoptosis in human pancreatic cancer cells Clin Cancer Res 1999; 5: 2140-7 Neznanov N, Man AK, Yamamoto H, Hauser CA, Cardiff RD, Oshima RG A single targeted Ets2 allele restricts development of mammary tumors in transgenic mice Cancer Res 1999; 59: 4242-6 Nishi H, Nakada T, Kyo S, Inoue M, Shay JW, Isaka K Hypoxia-inducible factor mediates upregulation of telomerase (hTERT) Mol Cell Biol 2004; 24: 6076-83 Noordermeer J, Klingensmith, J, Perrimon, N, Nusse, R Dishevelled and armadillo act in the wingless signalling pathway in Drosophil Nature 1994; 367: 80–3 Nakamura TM, Morin GB, Chapman KB et al Telomerase catalytic subunit homologs from fission yeast and human Science 1997; 277: 955-9 Oh H, Taffet GE, Youker KA, Entman ML, Overbeek PA, Michael LH, Schnei- der MD Telomerasereversetranscriptasepromotescardiacmus- cle cell proliferation, hypertrophy, and survival Proc Natl Acad Sci USA 2001; 98: 10308 –13 166 Oh S, Song YH, Yim J, Kim TK Identification of Mad as a repressor of the human telomerase (hTERT) gene Oncogene 2000; 19: 1485-90 O'Sullivan RJ, Karlseder J Telomeres: protecting chromosomes against genome instability Nat Rev Mol Cell Biol 2010; 11: 171-81 Pardue M -L, de Baryshe PG Telomeres and telomerase: more than the end of the line Chromosoma 1999; 108: 73–82 Parkhurst MR, Riley JP, Igarashi T, Li Y, Robbins PF, Rosenberg SA Immunization of patients with the hTERT:540-548 peptide induces peptidereactive T lymphocytes that not recognize tumors endogenously expressing telomerase Clin Cancer Res 2004; 10: 4688-98 Park JI, Venteicher AS, Hong JY, Choi J, Jun S, Shkreli M, Chang W, Meng Z, Cheung P, Ji H, McLaughlin M, Veenstra TD, Nusse R, McCrea PD, Artandi SE Telomerase modulates Wnt signalling by association with target gene chromatin Nature 2009; 460: 66-72 Pascolo E, Wenz C, Lingner J, Hauel N, Priepke H, Kauffmann I, GarinChesa P, Rettig WJ, Damm K, Schnapp A Mechanism of human telomerase inhibition by BIBR1532, a synthetic, non-nucleosidic drug candidate J Biol Chem 2002; 277: 15566-72 Peifer M, Rauskolb C, Williams M, Riggleman B, Wieschaus E "The segment polarity gene armadillo interacts with the wingless signaling pathway in both embryonic and adult pattern formation" Development 1991; 111: 1029-43 Peifer M, Berg S, Reynolds AB (March 1994) "A repeating amino acid motif shared by proteins with diverse cellular roles" Cell 1944; 76: 789–91 Piatyszek MA, Kim NW, Weinrich SL, Hiyama K, Hiyama E, Wright WE, Shay JW Detection of telomerase activity in human cells and tumors by a telomeric repeat amplification protocol (TRAP) Methods Cell Sci 1995; 1: 1– 15 Pogacic VFD, Filipowicz W Human H/ACA small nucleolar ribonucleoproteins and telomerase share evolutionarily conserved proteins NHP2 and NOP10 Mol Cell Biol 2000; 20: 9028-40 Polakis P Wnt signaling and cancer Genes Dev 2000; 14: 1837–51 Rahman R, Latonen L, Wiman KG hTERT antagonizes p53- induced apoptosis independently of telomerase activity Oncogene 2005; 24: 1320 – 167 Rao YK, Kao TY, Wu MF, Ko JL, Tzeng YM Identification of small molecule inhibitors of telomerase activity through transcriptional regulation of hTERT and calcium induction pathway in human lung adenocarcinoma A549 cells Bioorg Med Chem 2010; 18: 6987-94 Rubinfeld B, Robbins P, El-Gamil M, Albert I, Porri E, Polakis P Stabilization of beta-catenin by genetic defects in melanoma cell lines Science 1997; 275: 1790-2 Roose J, Molenaar M, Peterson J, Hurenkamp J, Brantjes H, Moerer P, van de Wetering M, Destree O, Clevers H The Xenopus Wnt effector XTcf-3 interacts with Groucho-related transcriptional repressors Nature 1998; 395: 608–12 Saneyoshi T, Kume S, Amasaki Y, Mikoshiba K The Wnt/calcium pathway activates NF-AT and promotes ventral cell fate in Xenopus embryos Nature 2002; 417: 295–299 Sarin KY, Cheung P, Gilison D, Lee E, Tennen RI Conditional telomerase induction causes proliferation of hair follicle stem cells Nature 2005; 436: 1048 – 52 Sfeir AJ, Chai, W, Shay JW, Wright WE Telomere-end processing the terminal nucleotides of human chromosomes Mol Cell 2005; 18: 131-8 Shats I, Milyavsky M, Tang X, Stambolsky P, Erez N, Brosh R, Kogan I, Braunstein I, Tzukerman M, Ginsberg D, Rotter V p53-dependent downregulation of telomerase is mediated by p21waf1 J Biol Chem 2004; 279: 50976-85 Shay JW, Wright WE The reactivation of telomerase activity in cancer progression Trends Genet 1996; 12: 129-31 Shay JW, Bacchetti S A survey of telomerase activity in human cancer Eur J Cancer 1997; 33 : 787-91 Shay, J.W Telomerase in cancer: diagnostic, prognostic and therapeutic implications Cancer J Sci Am 1998; 4: S26– S34 Shay JW, Wright WE Haylick, his limit, and cellular aging NatureRev Mol Cell Biol 2000; 1: 72– Shay JW, Keith WN Targeting telomerase for cancer therapeutics Br J Cancer 2008; 98: 677-83 168 Shore D RAP1: A protean regulator in yeast Trends Genet 1994; 10: 408412 Shulewitz M, Soloviev I, Wu T, Koeppen H, Polakis P, Sakanaka C Repressor roles for TCF-4 and Sfrp1 in Wnt signaling in breast cancer Oncogene 2006; 25: 4361–9 Smith LL, Coller HA, Roberts JM Telomerase modulates expression of growth-controlling genes and enhances cell proliferation Nature Cell Biol 2003; 5: 474-9 Sikand K, Kaul D, Varma N Receptor Ck-dependent signaling regulates hTERT gene transcription BMC Cell Biol 2006; 7: Smith LL, Coller HA, Roberts JM Telomerase modulates ex- pression of growth-controlling genes and enhances cell proliferation Nat Cell Biol 2003; 5: 474 – 79 Song N, Liu J, An S, Nishino T, Hishikawa Y, Koji T Immunohistochemical Analysis of Histone H3 Modifications in Germ Cells during Mouse Spermatogenesis Acta Histochem Cytochem 2011; 44: 183-90 Sprung CN, Reynolds GE, Jasin M, Murnane JP Chromosome healing in mouse embryonic stem cells Proc Natl Acad Sci U S A 1999; 96: 6781-6 Stambolic V, Ruel L, Woodgett JR Lithium inhibits glycogen synthase kinase-3 activity and mimics wingless signalling in intact cells Curr Biol 1996; 6: 1664-8 Stampfer MR, Garbe J, Levine G, Lichtsteiner S, Vasserot AP, Yaswen P Expression of the telomerase catalytic subunit, hTERT, induces resistance to transforming growth factor beta growth inhibition in p16INK4A(-) human mammary epithelial cells Proc Natl Acad Sci USA 2001; 98: 4498–503 Stansel RM, de Lange T, Griffith JD T-loop assembly in vitro involves binding of TRF2 near the 3′ telomeric overhang EMBO J 2001; 20: E553240 Stellwagen AE, Haimberger ZW, Veatch JR, Gottschling DE Ku interacts with telomerase RNA to promote telomere addition at native and broken chromosome ends Genes Dev 2003; 17: 2384-95 Stewart SA, Hahn WC, O'Connor BF, Banner EN, Lundberg AS, Modha P, Mizuno H, Brooks MW, Fleming M, Zimonjic DB, Popescu NC, Weinberg 169 RA Telomerase contributes to tumorigenesis by a telomere lengthindependent mechanism Proc Natl Acad Sci USA 2002; 99: 12606–11 Strahl BD, Allis CD The language of covalent histone modifications Nature 2000; 403: 41–5 van Steensel B, Smogorzewska A, and de Lange T TRF2 protects human telomeres from end-to-end fusions Cell 1998; 92: 401-413 Vaziri H, Benchimol S From telomere loss to p53 induction and activation of a DNA-damage pathway at senescence: The telomere loss/DNA damage model of cell aging Exp Gerontol 1996; 31: 295–301 van Steensel B, de Lange T Control of telomere length by the human telomeric protein TRF1 Nature 1997; 385: 740-3 Struewing I, Boyechko T, Barnett C, Beildeck M, Byers SW, Mao CD The balance of TCF7L2 variants with differential activities in Wnt-signaling is regulated by lithium in a GSK3beta-independent manner Biochem Biophys Res Commun 2010; 399: 245–50 Su Z, Dannull J, Yang BK, Dahm P, Coleman D, Yancey D, Sichi S, Niedzwiecki D, Boczkowski D, Gilboa E, Vieweg J Telomerase mRNAtransfected dendritic cells stimulate antigen-specific CD8+ and CD4+ T cell responses in patients with metastatic prostate cancer J Immunol 2005; 174: 3798-807 Takakura M, Kyo S, Kanaya T, Tanaka M, Inoue M Expression of human telomerase subunits and correlation with telomerase activity in cervical cancer Cancer Res 1998; 58: 1558-61 Takakura M, Kyo S, Kanaya T, Hirano H, Takeda J, Yutsudo M, Inoue M, Cloning of human telomerase catalyticsubunit (hTERT) gene promoter and identification of proximal corepromoter sequences essential for transcriptional activation inimmortalized and cancer cells Cancer Res 1999; 59: 551-7 Takakura M, Kyo S, Sowa Y, Wang Z, Yatabe N, Maida Y, Tanaka M, Inoue M Telomerase activation by histone deacetylase inhibitor in normal cells Nucleic Acids Res 2001; 29: 3006-11 Takai H, Smogorzewska A, de Lange T DNA damage foci at dysfunctional telomeres Curr Biol 2003; 13: 1549-56 Tesmer VM, Ford LP, Holt SE, Frank BC, Yi X, Aisner DL, Ouellette M, Shay JW, Wright WE Two inactive fragments of the integral RNA cooperate 170 to assemble active telomerase with the human protein catalytic subunit (hTERT) in vitro Mol Cell Biol 1999; 19: 6207-16 Toaldo C, Pizzimenti S, Cerbone A, Pettazzoni P, Menegatti E, Daniela B, Minelli R, Giglioni B, Dianzani MU, Ferretti C, Barrera G PPARgamma ligands inhibit telomerase activity and hTERT expression through modulation of the Myc/Mad/Max network in colon cancer cells J Cell Mol Med 2010; 14: 1347-57 Ulaner GA, Hu JF, Vu TH, Oruganti H, Giudice LC, Hoffman AR Regulation of telomerase by alternate splicing of human telomerase reverse transcriptase (hTERT) in normal and neoplastic ovary, endometrium and myometrium Int J Cancer 2000; 85: 330-5 Ulaner GA, Hu JF, Vu TH, Giudice LC, Hoffman AR Telomerase activity in human development is regulated by human telomerase reverse transcriptase (hTERT) transcription and by alternate splicing of hTERT transcripts Cancer Res 1998; 58: 4168-72 Vaziri H, Benchimol S 1998 Reconstitution of telomerase activity in normal human cells leads to elongation of telomeres and extended repli- cative life span Curr Biol 1998; 8: 279–282 Vonderheide RH, Hahn WC, Schultze JL, Nadler LM The telomerase catalytic subunit is a widely expressed tumor-associated antigen recognized by cytotoxic T lymphocytes Immunity 1999; 10: 673-9 Vulliamy T, Marrone A, Goldman F, Dearlove A, Bessler M, Mason PJ, Dokal I The RNA component of telomerase is mutated in autosomal dominant dyskeratosis congenita Nature 2001; 413: 432– 35 Wang CW, Yao DS, Horng SG, Chiu HC, Chen CK, Lee CL, Huang HY, Wang HS, Soong YK, Pao CC Feasibility of human telomerase reverse transcriptase mRNA expression in individual blastomeres as an indicator of early embryo development J Assist Reprod Genet 2004; 21: 163-8 Wan, J, Xie LY, Allan S, Beach D, and Hannon GJ Myc activates telomerase Genes Dev 1998; 12: 1769 – 74 Weinrich SL, Pruzan R, Ma L, Ouellette M, Tesmer VM, Holt SE, Bodnar AG, Lichtsteiner S, Kim NW, Trager JB, Taylor RD, Carlos R, Andrews WH, Wright WE, Shay JW, Harley CB, Morin GB Reconstitution of human telomerase with the template RNA component hTR and the catalytic protein subunit hTRT Nat Genet 1997; 17: 498-502 171 Wellinger RJ, Sen D The DNA structures at the ends of eukaryotic chromosomes Eur J Cancer 1997; 33:735–49 Wen J, Cong YS, and Bacchetti S Reconstitution of wild-type or mutant telomerase activity in telomerase-negative immortal human cells Hum Mol Genet.1998; 7: 1137–41 Wyatt HD, West SC, Beattie TL InTERTpreting telomerase structure and function Nucleic Acids Res 2010; 38: 5609-22 Wu KJ, Grandori C, Amacker M, Simon-Vermot N, Polack A, Lingner J, Dalla-Favera R Direct activation of TERT transcription by c-MYC Nat Genet 1999; 21: 220– Xi L, Chen G, Zhou J, Xu G, Wang S Inhibition of telomerase enhances apoptosis induced by sodium butyrate via mitochondrial pathway Apoptosis 2006; 11: 789 – 98 Xin H, Liu D, Wan M, Safari A, Kim H, Sun W, O'Connor MS, Songyang Z TPP1 is a homologue of ciliate TEBP-beta and interacts with POT1 to recruit telomerase Nature 2007; 445: 559-62 Xu D, Gruber A, Björkholm M, Peterson C, Pisa P Suppression of telomerase reverse transcriptase (hTERT) expression in differentiated HL-60 cells: regulatory mechanisms Br J Cancer 1999; 80: 1156-61 Xu D, Wang Q, Gruber A, Björkholm M, Chen Z, Zaid A, Selivanova G, Peterson C, Wiman KG, Pisa P Downregulation of telomerase reverse transcriptase mRNA expression by wild type p53 in human tumor cells Oncogene 2000; 19: 5123-33 Xu D, Popov N, Hou M, Wang Q, Björkholm M, Gruber A, Menkel AR, Henriksson M Switch from Myc/Max to Mad1/Max binding and decrease in histone acetylation at the telomerase reverse transcriptase promoter during differentiation of HL60 cells Proc Natl Acad Sci U S A 2001; 98: 3826-31 Xu D, Dwyer J, Li H, Duan W, Liu JP Ets2 maintains hTERT gene expression and breast cancer cell proliferation by interacting with c-Myc J Biol Chem 2008; 283: 23567-80 Xu Y, Ayrapetov MK, Xu C, Gursoy-Yuzugullu O, Hu Y, Price BD Histone H2A.Z Controls a Critical Chromatin Remodeling Step Required for DNA Double-Strand Break Repair Mol Cell 2012; 12: S1097-2765 172 Yang B, Yu RL, Tuo S, Tuo CW, Liu QZ, Zhang N, Lu XC, Chi XH, Lv SB, Cai LL Antisense oligonucleotide against hTERT (Cantide) inhibits tumor growth in an orthotopic primary hepatic lymphoma mouse model PLoS One 2012; 7: e41467 Yang C, Przyborski S, Cooke MJ, Zhang X, Stewart R, Anyfantis G, Atkinson SP, Saretzki G, Armstrong L, Lako M A key role for telomerase reverse transcriptase unit in modulating human embryonic stem cell proliferation, cell cycle dynamics, and in vitro differentiation Stem Cells 2008; 26: 850–63 Yang Q, Zheng YL, Harris CC POT1 and TRF2 cooperate to maintain telomeric integrity Mol Cell Biol 2005; 25: 1070-80 Yatabe N, Kyo S, Maida Y, Nishi H, Nakamura M, Kanaya T, Tanaka M, Isaka K, Ogawa S, Inoue M HIF-1-mediated activation of telomerase in cervical cancer cells Oncogene 2004; 23: 3708-15 Yeo M, Rha SY, Jeung HC, Shen XH, Yang SH, An SW, Roh JK, Chung HC Different role of functional domains of hTR in DNA binding to telomere and telomerase reconstruction FEBS Lett 2005; 579: 127-32 Yi F, Pereira L, Hoffman JA, Shy BR, Yuen CM, Liu DR, Merrill BJ Opposing effects of Tcf3 and Tcf1 control Wnt stimulation of embryonic stem cell self-renewal Nat Cell Biol 2011; 13: 762–70 Yi X, White DM, Aisner DL, Baur JA, Wright WE, Shay JW An alternate splicing variant of the human telomerase catalytic subunit inhibits telomerase activity Neoplasia 2000; 2: 433-40 Zaug AJ, Linger J, Cech TR Method for determining RNA 3' ends and application to human telomerase RNA Nucleic Acids Res 1996; 24: 532-3 Zaug AJ, Podell ER, Nandakumar J, Cech TR Functional interaction between telomere protein TPP1 and telomerase Genes Dev 2010; 24: 613-22 Zhang X, Mar V, Zhou W, Harrington L, Robinson MO Telomere shortening and apoptosis in telomerase-inhibited human tumor cells Genes Dev 1999; 13: 2388-99 Zhang L, Zhou F, Li Y, Drabsch Y, Zhang J, van Dam H, ten Dijke P Fasassociated factor is a scaffold protein that promotes β-transducin repeatcontaining protein (β-TrCP)-mediated β-catenin ubiquitination and degradation J Biol Chem 2012; 287: 30701-10 173 Zhao Y, Abreu E, Kim J, Stadler G, Eskiocak U, Terns MP, Terns RM, Shay JW, Wright WE Processive and distributive extension of human telomeres by telomerase under homeostatic and nonequilibrium conditions Mol Cell 2011; 42: 297-307 Zhu H, Fu W, Mattson MP The catalytic subunit of telomerase protects neurons against amyloid b-peptide-induced apoptosis J Neurochem 2000; 75: 117 – 24 Zhu XD, Niedernhofer L, Kuster, B, Mann, M, Hoeijmakers JH., and de Lange T 2003 ERCC1/XPF removes the 3′ overhang from uncapped telomeres and represses formation of telomeric DNA-containing double minute chromosomes Mol Cell 2003; 12: 1489-98 Zvereva MI, Shcherbakova DM, Dontsova OA Telomerase: structure, functions, and activity regulation Biochemistry (Mosc) 2010; 75: 1563-83 174 ... (ChIP) assays Results Inhibitors screening Verification of screening platform using STAT III and IV inhibitors Wnt pathway inhibitor library screening EGFR pathway inhibitors library screening JAK/STAT... Telomerase related screen Wnt signaling pathway Canonical Wnt pathway Non-canonical Wnt pathway Material and Methods Material Cell line Inhibitor library Plasmid List of primers List of antibodies... the inhibition of ataxia telangiectasia mutated (ATM) and ATM and Rad3-related (ATR) protein kinases though detail mechanism has yet being elucidated (d''Adda di Fagagna et al 2003; Takai et al