Abstract
E2F3 and CDKAL1 are candidate genes from the 6p22 region frequently amplified in bladder cancer. Expression of E2F3 isoforms (E2F3a and b) and CDKAL1 were examined and modulated in 6p22-amplified bladder cell lines. Eight lines with amplification showed overexpression of both E2F3 isoforms and CDKAL1. shRNA-mediated knockdown of CDKAL1 had no effect on proliferation. Knockdown of E2F3a or E2F3b alone induced antiproliferative effects, with the most significant effect on proliferation being observed when both isoforms were knocked down together. As E2Fs interact with the Rb tumour suppressor protein, Rb expression was analysed. There was a striking relationship between 6p22.3 amplification, E2F3 overexpression and lack of Rb expression. This was also examined in primary bladder tumours. Array-CGH detected 6p22.3 amplification in 8/91 invasive tumours. Five were studied in more detail. Four showed 13q14.2 loss (including RB1) and expressed no Rb protein. In the fifth, 13q was unaltered but the CDKN2A locus was deleted. This tumour was negative for p16 and positive for Rb protein. As p16 is a negative regulator of the Rb pathway, its loss represents an alternative mechanism for inactivation. Indeed, a phospho-specific Rb antibody showed much Rb protein in a hyperphosphorylated (inactive) form. We conclude that inactivation of the Rb pathway is required in addition to E2F3 overexpression in this subset of bladder tumours.
This is a preview of subscription content, access via your institution
Access options
Subscribe to this journal
Receive 50 print issues and online access
$259.00 per year
only $5.18 per issue
Buy this article
- Purchase on Springer Link
- Instant access to full article PDF
Prices may be subject to local taxes which are calculated during checkout
Similar content being viewed by others
References
Aaboe M, Birkenkamp-Demtroder K, Wiuf C, Sorensen FB, Thykjaer T, Sauter G et al. (2006). SOX4 expression in bladder carcinoma: clinical aspects and in vitro functional characterization. Cancer Res 66: 3434–3442.
Aslanian A, Iaquinta PJ, Verona R, Lees JA . (2004). Repression of the Arf tumor suppressor by E2F3 is required for normal cell cycle kinetics. Genes Dev 18: 1413–1422.
Benedict WF, Lerner SP, Zhou J, Shen X, Tokunaga H, Czerniak B . (1999). Level of retinoblastoma protein expression correlates with p16 (MTS-1/INK4A/CDKN2) status in bladder cancer. Oncogene 18: 1197–1203.
Blais A, Dynlacht BD . (2004). Hitting their targets: an emerging picture of E2F and cell cycle control. Curr Opin Genet Dev 14: 527–532.
Blaveri E, Brewer JL, Roydasgupta R, Fridlyand J, DeVries S, Koppie T et al. (2005). Bladder cancer stage and outcome by array-based comparative genomic hybridization. Clin Cancer Res 11: 7012–7022.
Bruch J, Schulz WA, Haussler J, Melzner I, Bruderlein S, Moller P et al. (2000). Delineation of the 6p22 amplification unit in urinary bladder carcinoma cell lines. Cancer Res 60: 4526–4530.
Bruch J, Wohr G, Hautmann R, Mattfeldt T, Bruderlein S, Moller P et al. (1998). Chromosomal changes during progression of transitional cell carcinoma of the bladder and delineation of the amplified interval on chromosome arm 8q. Genes Chromosomes Cancer 23: 167–174.
Chapman EJ, Harnden P, Chambers P, Johnston C, Knowles MA . (2005). Comprehensive analysis of CDKN2A status in microdissected urothelial cell carcinoma reveals potential haploinsufficiency, a high frequency of homozygous co-deletion and associations with clinical phenotype. Clin Cancer Res 11: 5740–5747.
Chapman EJ, Hurst CD, Pitt E, Chambers P, Aveyard JS, Knowles MA . (2006). Expression of hTERT immortalises normal human urothelial cells without inactivation of the p16/Rb pathway. Oncogene 25: 5037–5045.
Chatterjee SJ, George B, Goebell PJ, Alavi-Tafreshi M, Shi SR, Fung YK et al. (2004). Hyperphosphorylation of pRb: a mechanism for RB tumour suppressor pathway inactivation in bladder cancer. J Pathol 203: 762–770.
Christian BJ, Loretz LJ, Oberley TD, Reznikoff CA . (1987). Characterization of human uroepithelial cells immortalized in vitro by simian virus 40. Cancer Res 47: 6066–6073.
Cooper CS, Nicholson AG, Foster C, Dodson A, Edwards S, Fletcher A et al. (2006). Nuclear overexpression of the E2F3 transcription factor in human lung cancer. Lung Cancer 54: 155–162.
Cordon-Cardo C, Wartinger D, Petrylak D, Dalbagni G, Fair WR, Fuks Z et al. (1992). Altered expression of the retinoblastoma gene product: prognostic indicator in bladder cancer. J Natl Cancer Inst 84: 1251–1256.
Cote RJ, Dunn MD, Chatterjee SJ, Stein JP, Shi SR, Tran QC et al. (1998). Elevated and absent pRb expression is associated with bladder cancer progression and has cooperative effects with p53. Cancer Res 58: 1090–1094.
DeGregori J, Leone G, Miron A, Jakoi L, Nevins JR . (1997). Distinct roles for E2F proteins in cell growth control and apoptosis. Proc Natl Acad Sci USA 94: 7245–7250.
Dimova DK, Dyson NJ . (2005). The E2F transcriptional network: old acquaintances with new faces. Oncogene 24: 2810–2826.
Dyson N . (1998). The regulation of E2F by pRB-family proteins. Genes Dev 12: 2245–2262.
Evans AJ, Gallie BL, Jewett MA, Pond GR, Vandezande K, Underwood J et al. (2004). Defining a 0.5-mb region of genomic gain on chromosome 6p22 in bladder cancer by quantitative-multiplex polymerase chain reaction. Am J Pathol 164: 285–293.
Feber A, Clark J, Goodwin G, Dodson AR, Smith PH, Fletcher A et al. (2004). Amplification and overexpression of E2F3 in human bladder cancer. Oncogene 23: 1627–1630.
Fiegler H, Carr P, Douglas EJ, Burford DC, Hunt S, Smith J et al. (2003). DNA microarrays for comparative genomic hybridization based on DOP-PCR amplification of BAC and PAC clones. Genes Chromosomes Cancer 36: 361–374.
Frolov MV, Huen DS, Stevaux O, Dimova D, Balczarek-Strang K, Elsdon M et al. (2001). Functional antagonism between E2F family members. Genes Dev 15: 2146–2160.
He Y, Armanious MK, Thomas MJ, Cress WD . (2000). Identification of E2F-3B, an alternative form of E2F-3 lacking a conserved N-terminal region. Oncogene 19: 3422–3433.
Humbert PO, Verona R, Trimarchi JM, Rogers C, Dandapani S, Lees JA . (2000). E2f3 is critical for normal cellular proliferation. Genes Dev 14: 690–703.
Hurst CD, Fiegler H, Carr P, Williams S, Carter NP, Knowles MA . (2004). High-resolution analysis of genomic copy number alterations in bladder cancer by microarray-based comparative genomic hybridization. Oncogene 23: 2250–2263.
Kallioniemi A, Kallioniemi O-P, Citro G, Sauter G, DeVries S, Kerschmann R et al. (1995). Identification of gains and losses of DNA sequences in primary bladder cancer by comparative genomic hybridisation. Genes Chromosomes Cancer 12: 213–219.
Lazzerini Denchi E, Attwooll C, Pasini D, Helin K . (2005). Deregulated E2F activity induces hyperplasia and senescence-like features in the mouse pituitary gland. Mol Cell Biol 25: 2660.
Le Frere-Belda MA, Gil Diez de Medina S, Daher A, Martin N, Albaud B, Heudes D et al. (2004). Profiles of the 2 INK4a gene products, p16 and p14ARF, in human reference urothelium and bladder carcinomas, according to pRb and p53 protein status. Hum Pathol 35: 817.
Lees JA, Saito M, Vidal M, Valentine M, Look T, Harlow E et al. (1993). The retinoblastoma protein binds to a family of E2F transcription factors. Mol Cell Biol 13: 7813–7825.
Leone G, DeGregori J, Yan Z, Jakoi L, Ishida S, Williams RS et al. (1998). E2F3 activity is regulated during the cell cycle and is required for the induction of S phase. Genes Dev 12: 2120–2130.
Leone G, Nuckolls F, Ishida S, Adams M, Sears R, Jakoi L et al. (2000). Identification of a novel E2F3 product suggests a mechanism for determining specificity of repression by Rb proteins. Mol Cell Biol 20: 3626–3632.
Logothetis CJ, Xu H-J, Ro JY, Hu S-X, Sahin A, Ordonez N et al. (1992). Altered expression of retinoblastoma protein and known prognostic variables in locally advanced bladder cancer. J Natl Cancer Inst 84: 1256–1261.
Oeggerli M, Schraml P, Ruiz C, Bloch M, Novotny H, Mirlacher M et al. (2006). E2F3 is the main target gene of the 6p22 amplicon with high specificity for human bladder cancer. Oncogene 25: 6538–6543.
Oeggerli M, Tomovska S, Schraml P, Calvano-Forte D, Schafroth S, Simon R et al. (2004). E2F3 amplification and overexpression is associated with invasive tumor growth and rapid tumor cell proliferation in urinary bladder cancer. Oncogene 23: 5616–5623.
Olsson AY, Feber A, Edwards S, Te Poele R, Giddings I, Merson S et al. (2007). Role of E2F3 expression in modulating cellular proliferation rate in human bladder and prostate cancer cells. Oncogene 26: 1028–1037.
Orlic M, Spencer CE, Wang L, Gallie BL . (2006). Expression analysis of 6p22 genomic gain in retinoblastoma. Genes Chromosomes Cancer 45: 72–82.
Paulie S, Hansson Y, Lundblad ML, Perimann P . (1983). Lectins as probes for identification of tumor-associated antigens on urothelial and colonic carcinoma cell lines. Int J Cancer 31: 297–303.
Paulson QX, McArthur MJ, Johnson DG . (2006). E2F3a stimulates proliferation, p53-independent apoptosis and carcinogenesis in a transgenic mouse model. Cell Cycle 5: 184–190.
Prat E, Bernues M, Caballin MR, Egozcue J, Gelabert A, Miro R . (2001). Detection of chromosomal imbalances in papillary bladder tumors by comparative genomic hybridization. Urology 57: 986–992.
Saxena R, Voight BF, Lyssenko V, Burtt NP, de Bakker PI, Chen H et al. (2007). Genome-wide association analysis identifies loci for type 2 diabetes and triglyceride levels. Science 316: 1331–1336.
Scott LJ, Mohlke KL, Bonnycastle LL, Willer CJ, Li Y, Duren WL et al. (2007). A genome-wide association study of type 2 diabetes in Finns detects multiple susceptibility variants. Science 316: 1341–1345.
Steinthorsdottir V, Thorleifsson G, Reynisdottir I, Benediktsson R, Jonsdottir T, Walters GB et al. (2007). A variant in CDKAL1 influences insulin response and risk of type 2 diabetes. Nat Genet 39: 770–775.
Stevens C, La Thangue NB . (2004). The emerging role of E2F-1 in the DNA damage response and checkpoint control. DNA Repair (Amst) 3: 1071–1079.
Stott FJ, Bates S, James MC, McConnell BB, Starborg M, Brookes S et al. (1998). The alternative product from the human CDKN2A locus, p14(ARF), participates in a regulatory feedback loop with p53 and MDM2. EMBO J 17: 5001–5014.
Tomlinson DC, Hurst CD, Knowles MA . (2007). Knockdown by shRNA identifies S249C mutant FGFR3 as a potential therapeutic target in bladder cancer. Oncogene 26: 5889–5899.
Tomovska S, Richter J, Suess K, Wagner U, Rozenblum E, Gasser TC et al. (2001). Molecular cytogenetic alterations associated with rapid tumor cell proliferation in advanced urinary bladder cancer. Int J Oncol 18: 1239–1244.
Trimarchi JM, Lees JA . (2002). Sibling rivalry in the E2F family. Nat Rev Mol Cell Biol 3: 11–20.
Veltman JA, Fridlyand J, Pejavar S, Olshen AB, Korkola JE, DeVries S et al. (2003). Array-based comparative genomic hybridization for genome-wide screening of DNA copy number in bladder tumors. Cancer Res 63: 2872–2880.
Wu Q, Hoffmann MJ, Hartmann FH, Schulz WA . (2005). Amplification and overexpression of the ID4 gene at 6p22.3 in bladder cancer. Mol Cancer 4: 16.
Xu HJ, Cairns P, Hu SX, Knowles MA, Benedict WF . (1993). Loss of RB protein expression in primary bladder cancer correlates with loss of heterozygosity at the RB locus and tumor progression. Int J Cancer 53: 781–784.
Yeager T, Stadler W, Belair C, Puthenveettil J, Olopade O, Reznikoff C . (1995). Increased p16 levels correlate with pRB alterations in human urothelial cells. Cancer Res 55: 493–497.
Zeggini E, Weedon MN, Lindgren CM, Frayling TM, Elliott KS, Lango H et al. (2007). Replication of genome-wide association signals in UK samples reveals risk loci for type 2 diabetes. Science 316: 1336–1341.
Ziebold U, Lee EY, Bronson RT, Lees JA . (2003). E2F3 loss has opposing effects on different pRB-deficient tumors, resulting in suppression of pituitary tumors but metastasis of medullary thyroid carcinomas. Mol Cell Biol 23: 6542–6552.
Ziebold U, Reza T, Caron A, Lees JA . (2001). E2F3 contributes both to the inappropriate proliferation and to the apoptosis arising in Rb mutant embryos. Genes Dev 15: 386–391.
Acknowledgements
This work was supported by a programme grant from Cancer Research UK (C6228/A5433). We would like to thank Jo Brown and Filomena Esteves for their help with tissue collection and immunohistochemistry. U6 promoter and pRetroSuper constructs were kind gifts from Dr D Takai and Dr R Agami, respectively.
Author information
Authors and Affiliations
Corresponding author
Rights and permissions
About this article
Cite this article
Hurst, C., Tomlinson, D., Williams, S. et al. Inactivation of the Rb pathway and overexpression of both isoforms of E2F3 are obligate events in bladder tumours with 6p22 amplification. Oncogene 27, 2716–2727 (2008). https://doi.org/10.1038/sj.onc.1210934
Received:
Revised:
Accepted:
Published:
Issue Date:
DOI: https://doi.org/10.1038/sj.onc.1210934
Keywords
This article is cited by
-
mRNA–miRNA bipartite networks reconstruction in different tissues of bladder cancer based on gene co-expression network analysis
Scientific Reports (2022)
-
γ-tubulin as a signal-transducing molecule and meshwork with therapeutic potential
Signal Transduction and Targeted Therapy (2018)
-
MiR-377 targets E2F3 and alters the NF-kB signaling pathway through MAP3K7 in malignant melanoma
Molecular Cancer (2015)
-
Molecular biology of bladder cancer: new insights into pathogenesis and clinical diversity
Nature Reviews Cancer (2015)
-
Selective roles of E2Fs for ErbB2- and Myc-mediated mammary tumorigenesis
Oncogene (2015)