Oncogene (2004) 23, 1627–1630 & 2004 Nature Publishing Group All rights reserved 0950-9232/04 $25.00 www.nature.com/onc SHORT REPORTS Amplification and overexpression of E2F3 in human bladder cancer Andrew Feber1, Jeremy Clark1, Graham Goodwin1, Andrew R Dodson2, Paul H Smith2, Anne Fletcher1, Sandra Edwards1, Penny Flohr1, Alison Falconer3, Toby Roe1, Gyula Kovacs4, Nening Dennis1, Cyril Fisher5, Richard Wooster6, Robert Huddart3, Christopher S Foster2 and Colin S Cooper*,1 1Section of Molecular Carcinogenesis and Male Urological Cancer Research, Centre, Institute of Cancer Research, Sutton, Surrey SM2 5NG, UK; 2Department of Pathology and Molecular Genetics, University of Liverpool, Duncan Building, Daulby Street, Liverpool L69 3GA, UK; 3Royal Marsden NHS Trust, Downs Road, Sutton, Surrey SM2 5PT, UK; 4Ruprecht-Karls-Universitat, Heidelberg Klinikum, Molekular Onkologie, Im Neuenheimer Feld 365, Heidelberg 69120, Germany; 5Royal Marsden NHS Trust, Fulham Road, London SW3 6JJ, UK; 6Sanger Centre, Wellcome Trust Genome Campus, Hinxton, Cambridge CB10 1SA, UK We demonstrate that, in human bladder cancer, amplifi- H-RAS and FGFR3 (Cappellen et al., 1999; Knowles, cation of the E2F3 gene, located at 6p22, is associated 2001). Comparative genomic hybridization (CGH) and with overexpression of its encoded mRNA transcripts and loss of heterozygosity studies have also identified many high levels of expression of E2F3 protein. Immunohisto- consistent regions of chromosomal gain and loss that chemical analyses of E2F3 protein levels have established may be sites of additional oncogenes and tumour- that around one-third (33/101) of primary transitional cell suppressor genes in bladder cancer (Knowles, 2001). In carcinomas of the bladder overexpress nuclear E2F3 these studies, gains and amplification at 6p22 have been protein, with the proportion of tumours containing over- frequently found in human bladder cancer (Hovey et al., expressed nuclear E2F3 increasing with tumour stage and 1998; Koo et al., 1999; Terracciano et al., 1999; Simon grade. When considered together with the established role et al., 2000) and the presence of this amplicon correlates of E2F3 in cell cycle progression, these results suggest with tumour grade (Richter et al., 1999; Prat et al., 2001) that the E2F3 gene represents a candidate bladder cancer and high tumour cell proliferation (Tomovska et al., oncogene that is activated by DNA amplification and 2001). Previous mapping studies have demonstrated that overexpression. the 6p22 amplicon spans the closely related SOX4, PRL Oncogene (2004) 23, 1627–1630. doi:10.1038/sj.onc.1207274 and E2F3 genes (Bruch et al., 2000; Veltman et al., Published online 29 December 2003 2003). In the current study, we have investigated whether amplification of the E2F3 gene is accompanied Keywords: E2F3 gene; bladder cancer; 6p22 amplicon by its overexpression in human bladder cancer cell lines, and whether overexpression of E2F3 is found in primary human bladder cancer. We initially used CGH onto cDNA microarrays to Urological carcinomas of the bladder, common in screen human bladder cancer cell lines for regions of Western countries, can be classified into two categories genomic gain and amplification. Microarrays were co- based on their histopathology and clinical behaviour. hybridized with bladder cancer cell line DNA labelled Around 70–80% of transitional cell carcinomas of the with Cy5 and normal muscle DNA labelled with Cy3. bladder present as superficial non-muscle invasive Amplification of the E2F3 gene at 6p22 was detected in papillary carcinomas (pTa or pT1) that are associated three (TCCSUP, 5637 and HT1376) bladder cancer cell with a high risk of recurrence (70%) following lines. The result obtained for the TCCSUP cell line is treatment, but low risk (10–20%) of progression to muscle invasion. The remaining 20–30% of bladder shown in Figure 1a. cancers show muscle invasion at the time of diagnosis Amplification of the E2F3 gene in these three cell lines (4T2), have no association with papillary tumours, and was confirmed by Southern analysis (Figure 1b). To are thought to arise from carcinomas in situ (CIS). map this amplicon, CGH studies were carried out onto A variety of genetic changes have been identified in an expanded cDNA microarray containing all the 12 human bladder cancer. The alterations include inactiva- genes within a 6.5 Mb region spanning the E2F3 locus. tion of the RB1, TP53 and INK4A/ARF genes, These experiments (Figure 1c) show that E2F3 maps amplification and overexpression of MDM2, CCND1/ within the peak region of amplification in the TCCSUP, CyclinD1 and ERBB2, and mutational activation of 5637 and HT1376 bladder cancer cell lines, and in two primary transitional cell carcinomas of the bladder (T63 and T110). The PRL and SOX4 genes, which mapped outside the peak region of amplification, had also *Correspondence: CS Cooper; E-mail: [email protected] Received 17 July 2003; revised 29 September 2003; accepted 6 October previously been excluded as candidate oncogenes by 2003 Bruch et al. (2000). They found that the PRL gene was Amplification and overexpression of E2F3 A Feber et al 1628 Figure 1 Amplification and expression of E2F3.(a) CGH onto cDNA microarrays. Duplicate results are presented in blue and red. The results for chromosomes 5 and 6 for the TCCSUP bladder cancer cell line are shown. (b) E2F3 Southern blot. Muscle DNA was used as the normal control. (c) CGH studies were repeated using an expanded cDNA microarray that contained 12 genes within a 6.5 Mb region spanning the E2F3 locus. Mapping positions are taken from the UCSC Human Genome Bioinformatics project April 2002 freeze (http://www.genome.ucsc.edu). Results for the TCCSUP, 5637, HT1376 and HT1197 cell lines, and for two primary bladder tumours designated T63 and T110, are shown. (d) E2F3 Northern blot. RNA from normal bladder was used as a control. Northern blot analysis identified E2F3 transcripts of 4.8 and 4.4 kb that correspond to the known sizes (http://www.genome.ucsc.edu) of the E2F3a (4.74 kb) and E2F3b (4.33 kb) transcripts. (e) E2F3 Western blot. Mouse monoclonal antibodies for Western blot analysis of E2F3 protein were obtained from Upstate Ltd (Milton Keynes, UK), and used at a dilution of 1 : 200. The antibody identified a protein doublet at 58 and 52 kDa. Use of antipeptide antibodies (Santa Cruz Biotechnology Inc., Santa Cruz, CA, USA) corresponding to the common C-terminus of E2F3a and E2F3b and to the N-terminus of E2F3a, together with their competing peptides, allowed these bands to be assigned as E2F3a and E2F3b, respectively. Microarray-CGH studies were carried out as described by Clark et al. (2002). Southern, Northern and Western analyses were performed as described by Sambrook et al. (1989). The probe for Southern and Northern blot analysis was a 0.89 kb clone (IMAGE: 2163947) from exon 7 of the E2F3 gene. All cell lines were obtained from the American Type Culture Collection. All tissue samples were retrieved from the archives of the Department of Pathology, Royal Liverpool UK and the Department of Histopathology, Royal Marsden Hospital NHS Trust, UK. Normal human bladder tissues were obtained from patients in whom there was no history or suggestion of malignancy, either within the bladder or elsewhere not expressed at all in bladder cancer cell lines, and that histochemical analysis of primary transitional cell SOX4 expression was only enhanced in one of four lines carcinoma of the bladder was carried out using tissue containing the 6p22 amplicon. arrays containing triplicate cores of each cancer. In these Northern and Western analyses were used to deter- studies, intense nuclear staining was observed in around mine whether amplification of the E2F3 gene is one-third (33/101) of primary bladder transitional cell accompanied by increased expression of E2F3 mRNA carcinomas (Figures 2d–f), with the proportion of cells and protein. Although E2F3 transcripts were diffuse, containing intense staining varying from 5 to 60%. The when compared to normal bladder, overexpression of proportion of tumours with nuclear E2F3 protein E2F3 mRNA could be observed in the three cell lines increased with tumour grade (G1, 2/16, 12.5%; G2, that exhibited amplification of E2F3 (Figure 1d). These 11/44, 25%; G3, 20/38, 53%) (w2 for trend ¼ 10.24, three cell lines also contained the highest levels of E2F3 DF ¼ 1, P ¼ 0.0014), in agreement with previous reports protein. Relatively high levels of E2F3 protein were also that the presence of the 6p22 amplicon in bladder cancer found in the bladder cancer cell lines J82 and T24 in the correlates with tumour grade (Richter et al., 1999; Prat absence of DNA amplification when compared to the et al., 2001). Our results also suggest a correlation with HT1197, SW780 and SCaBER cell lines (Figure 1e). tumour stage (w2 ¼ 8.48, DF ¼ 1, P ¼ 0.0034), since 56% Immunohistochemical analysis of bladder cancer cell (18/32) of tumours with evidence of muscle invasion lines demonstrates that overexpressed E2F3 is present (pT2-4) exhibited nuclear staining compared to 18% predominantly in cell nuclei (Figure 2a), while cell lines (15/59) of superficial non-muscle invasive cancers (pTa showing low levels of E2F3 (Figure 2b) exhibit faint and pT1). cytoplasmic staining. Immunohistochemical screening Several lines of evidence highlight the importance of of a series of normal bladders (n ¼ 20) failed to identify E2F3 in cell cycle progression and proliferation. E2F3À/À nuclear E2F3 staining in any but some terminally mouse embryo fibroblasts have a proliferative and cell differentiated umbrella cells at the surface of the cycle defect when compared to their wild-type counter- urothelium (Figure 2c): in no case was nuclear E2F3 parts, and a critical threshold level of one or more E2F3- staining found in the subsurface epithelium. Immuno- regulated genes appears to determine the timing of the Oncogene Amplification and overexpression of E2F3 A Feber et al 1629 Figure 3 Control of the pRB pathway.