REPORTS

Prognostic Significance of Transcription Factor E2F-1 in Bladder Cancer: Genotypic and Phenotypic Characterization

Farhang Rabbani, Victoria M. Richon, Irene Orlow, Ming-Lan Lu, Marija Drobnjak, Maria Dudas, Elizabeth Charytonowicz, Guido Dalbagni, Carlos Cordon-Cardo

Affiliations of authors: F. Rabbani, G. Dalbagni (Urology Service, Department of Surgery), V. M. Richon (Cell Biology Program), I. Orlow, M.-L. Lu, M. Drobnjak, M. Dudas, E. Charytonowicz, C. Cordon-Cardo (Department of Pathology), Memorial Sloan-Kettering Cancer Center, New York, NY.

Correspondence to: Carlos Cordon-Cardo, M.D., Ph.D., Division of Molecular Pathology, Memorial Sloan-Kettering Cancer Center, Rm. S-801, 1275 York Ave., New York, NY 10021 (e-mail: cordon-c{at}mskcc.org).


    ABSTRACT
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 Abstract
 Introduction
 Methods
 Results
 Discussion
 References
 
BACKGROUND: We sought to identify and characterize potential alterations in E2F-1, a transcription factor that binds to the retinoblastoma protein (pRB), in bladder neoplasms and to elucidate a possible role for E2F-1 as an oncogene or a tumor suppressor gene. METHODS: Tumor samples from 133 evaluable patients with bladder cancer were analyzed for E2F-1 gene mutations by use of polymerase chain reaction-single-strand conformational polymorphism (PCR-SSCP) analysis and DNA sequencing. In addition, tumors were studied for E2F-1 and pRB protein expression by use of immunohistochemistry. Results from the above analyses were correlated with clinicopathologic parameters and outcome. All P values are two-sided. RESULTS: A polymorphism, consisting of a nucleotide change at amino acid codon 393 in exon 7 (GGC->AGC [Gly->Ser]), was identified in seven of 133 case patients, being present in both tumor and corresponding normal tissues. No band-shifts were identified in the nuclear-localization or DNA-binding domains on PCR-SSCP analysis. On immunohistochemical analysis, E2F-1 nuclear reactivity was observed in less than 5% of the cells from 53 tumors and in 5%-75% of the cells from the remaining 80 tumors. The pattern of E2F-1 protein expression was not altered in relation to the identified polymorphism. pRB nuclear reactivity greater than 20% (of tumor cells stained) was present in 66% of the samples. E2F-1 nuclear reactivity correlated inversely with the percentage of cells showing pRB reactivity (Kendall {tau}b = -0.18; P = .019). On multivariate analysis, patients with lower E2F-1 reactivity had statistically significantly increased risks of progression to metastases (P = .001) and death (P = .02). CONCLUSIONS: E2F-1 alterations occur at the phenotypic level, rather than at the genotypic level, in bladder cancer. The adverse outcome for patients whose tumors exhibit low E2F-1 nuclear expression suggests a possible tumor suppressor role for E2F-1 in bladder cancer.



    INTRODUCTION
 Top
 Abstract
 Introduction
 Methods
 Results
 Discussion
 References
 
The retinoblastoma pathway is commonly altered in human cancer, including in those neoplasms arising in the urinary bladder (1). Molecular alterations in this pathway include the following: 1) loss of retinoblastoma (RB) gene function due to mutation or deletion, as well as altered RB protein (pRB) expression in human primary tumors (2-5); 2) pRB inactivation by viral oncoproteins (6-8); and 3) alterations occurring in pRB regulatory proteins, including cyclin D1, cdk4, and p16/INK4A (9-12). Since the activity of transcription factor E2F-1 is regulated, in part, by pRB, alterations in this protein may provide an alternative mechanism for perturbation of the retinoblastoma pathway.

Several lines of evidence support an oncogenic role for E2F-1. Overexpression of E2F-1 in quiescent rat embryo fibroblasts (REFs) induces S phase (13). Furthermore, E2F-1 gene amplification and overexpression have been reported in HEL erythroleukemia cells (14). In addition, it has been reported that complexes of E2F-1 and transcription factor DP-1 cooperate with activated ras oncogene to induce formation of transformed foci in REFs (15). Yamasaki et al. (16) have demonstrated in vivo that inactivation of E2F-1 can reduce the penetrance of the pituitary and thyroid tumors observed in Rb1(+/-) mice and increase the animals' lifespan. Conversely, it has also been documented that loss of the E2F-1 pRB-binding domain is associated with neoplastic transformation and tumorigenesis (15,17). Moreover, neoplastic transformation requires the DNA-binding and transactivation domains (17,18). Although the above studies support an oncogenic role for E2F-1, the tumor-prone phenotype of the murine knockout model for E2F-1 suggests a role as a suppressor gene (19,20). Taken together, these findings suggest that E2F-1 functions as an oncogene or tumor suppressor gene in a tissue-specific manner.

Given the importance of the RB pathway in human bladder cancer, this study was designed to identify and characterize potential E2F-1 alterations in bladder neoplasms. A mutational analysis and expression studies were performed to determine the level at which alterations may occur. E2F-1 expression was correlated with pRB phenotype, as well as clinicopathologic parameters and outcome, to further elucidate the role of E2F-1 as an oncogene or suppressor gene in bladder cancer.


    METHODS
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 Abstract
 Introduction
 Methods
 Results
 Discussion
 References
 
Patient Characteristics and Tissues

Data on 162 patients undergoing radical (n = 159) or partial (n = 3) cystectomy for epithelial bladder cancer at the Memorial Sloan-Kettering Cancer Center between October 1993 and June 1997, inclusive, were entered in a prospective database. Tumor stage was assigned according to the TNM (tumor-node-metastasis) staging system (21) on the basis of the depth of invasion at transurethral resection. Information from patients' charts, imaging studies, and pathology was reviewed and the data were entered in the database. After Institutional Review Board approval and activation of the protocol, normal and tumor tissue samples were obtained from cystectomy specimens. All tissues were embedded in tissue cryopreservative solution (OCT Compound; Miles Laboratories, Elkhart, IN), snap-frozen, and stored at -70 °C. Representative hematoxylin-eosin (H & E)-stained sections of each frozen block were examined microscopically to confirm the presence of tumor, as well as to assess the percentage of tumor cells. Specific areas of normal or tumor tissues were marked on the H & E-stained slide. Unstained slides were aligned by morphology to the stained slide using operating loupes (2.5x magnification), and corresponding areas were microdissected. Tumor samples were examined microscopically to confirm the specificity of dissection. Ten to 20 consecutive, 30-µm sections were cut from each block and DNA was extracted, after microdissection, by use of a nonorganic method (Oncor, Inc., Gaithersburg, MD). Five-micron sections were obtained for immunohistochemical staining. Twenty-three patients with minimal or no tumor in the procured blocks were excluded. Significant inflammation and/or necrosis were present in an additional six patients, who were also excluded. The remaining 133 patients form the subject of this study.

Clinical stage was less than or equal to T1 and greater than or equal to T2 in 34 and 99 patients, respectively. Pathologic stage was less than or equal to pT3a and greater than or equal to pT3b in 33 and 100 patients, respectively. Pathologic grade was G1-G2 in 32 patients and G3 in 101 patients. The histologic type was transitional cell carcinoma in 102 patients, squamous cell carcinoma in 21, adenocarcinoma in four, and other histology in six. Suspicion of vascular invasion was present in 69 (52%) of 133 patients. Nodal status was N0 in 78 patients, N1-N2 in 36, and NX in 19. Neoadjuvant chemotherapy was administered to 12 patients: methotrexate, vinblastine, doxorubicin, and cisplatin in seven patients; ifosfamide, paclitaxel, and cisplatin in two patients; and other regimens in three patients. Adjuvant chemotherapy was administered in 17 patients: methotrexate, vinblastine, doxorubicin, and cisplatin in 14 patients; ifosfamide, paclitaxel, and cisplatin in one patient; and other regimens in two patients. Of the 17 patients who received adjuvant chemotherapy, three had extravesical disease with negative lymph nodes, two had positive lymph nodes but no extravesical disease, and 12 had both extravesical disease and positive lymph nodes. The median follow-up was 16.7 months (range, 0.1-58.6 months). Fifty-five (41%) patients were dead at last follow-up. Median follow-up in patients who were alive at last follow-up was 25.7 months and median survival was 8.2 months in the 55 dead patients.

Molecular Analysis

Mutational analysis of E2F-1 was performed using the polymerase chain reaction-single-strand conformation polymorphism (PCR-SSCP) technique for screening (22), followed by sequencing of the band-shifts identified. The wild-type E2F-1 complementary DNA sequence is numbered as reported by Helin et al. (23). The partial genomic sequence reported by Neuman et al. (24) was used to design primer pairs covering the nuclear-localization domain (I: F 5'-CCTGACCTGCTGCTCTTCG-3', R 5'-GGTCCGTACCGGCGGGC-3'; and II: F 5'-CTGGTTACTGGGCATCCTCCCG-3', R 5'-CCCAGCCCAAGCTCCATAGG-3'); the DNA-binding domain (I: F 5'-GGTGAGGAGTTCCCCAGCCAG-3', R 5'-GGTCTCATAGCGTGACTTCTCC-3'; and II: F 5'-GGAGAAGTCACGCTATGAGACC-3', R 5'-CGGTACCTACAGCCACTGG-3'); and the RB-binding domain (F 5'-TGGAGCAAGAACCGCTGTTGTC-3' and R 5'-AGTCGAAGAGGTCTCTGATGCC-3'). For the PCR reactions, 100 ng of DNA was amplified in the presence of 2 mM MgCl2 (3 mM for the DNA-binding domain II), 200 µM deoxynucleoside triphosphate mix (160 µM for the DNA-binding domain I), 1 µL of 10x buffer, 0.5 U of Taq polymerase (Promega Corp., Madison, WI), 1 µCi of [{alpha}-33P]deoxycytidine triphosphate (NEN Life Science Products, Boston, MA), and 10 pmol of each primer (5 pmol for the DNA-binding domain I) in a final volume of 10 µL. Dimethyl sulfoxide (5%) was added for the reactions involving the nuclear-localization domain I. Samples were incubated at 95 °C for 5 minutes with primer-specific cycling parameters as follows: nuclear-localization domain I [95 °C (30 seconds)-57 °C (30 seconds)-72 °C (40 seconds)] x 30 cycles; nuclear-localization domain II [95 °C (30 seconds)-67 °C (30 seconds)-72 °C (40 seconds)] x two cycles, [95 °C (30 seconds)-66 °C (30 seconds)-72 °C (40 seconds)] x two cycles, [95 °C (30 seconds)-65 °C (30 seconds)-72 °C (40 seconds)] x two cycles, [95 °C (30 seconds)-64 °C (30 seconds)-72 °C (40 seconds)] x two cycles, and [95 °C (30 seconds)-63 °C (30 seconds)-72 °C (40 seconds)] x 22 cycles; DNA-binding domain I [95 °C (30 seconds)-73 °C (30 seconds)-72 °C (40 seconds)] x two cycles, [95 °C (30 seconds)-71 °C (30 seconds)-72 °C (40 seconds)] x two cycles, [95 °C (30 seconds)-69 °C (30 seconds)-72 °C (40 seconds)] x two cycles, [95 °C (30 seconds)-67 °C (30 seconds)-72 °C (40 seconds)] x two cycles, [95 °C (30 seconds)-65 °C (30 seconds)-72 °C (40 seconds)] x two cycles, and [95 °C (30 seconds)-63 °C (30 seconds)-72 °C (40 seconds)] x 20 cycles; DNA-binding domain II [95 °C (30 seconds)-63 °C (30 seconds)-72 °C (40 seconds)] x two cycles, [95 °C (30 seconds)-61 °C (30 seconds)-72 °C (40 seconds)] x two cycles, [95 °C (30 seconds)-59 °C (30 seconds)-72 °C (40 seconds)] x two cycles, and [95 °C (30 seconds)-57 °C (30 seconds)-72 °C (40 seconds)] x 24 cycles; and the pRB-binding domain [95 °C (20 seconds)-63 °C (20 seconds)-72 °C (30 seconds)] x 30 cycles. Final extension was for 10 minutes at 72 °C. The amplified product (2.5 µL) was mixed with denaturing-loading buffer (12 µL). Samples were incubated at 95 °C for 5 minutes, cooled rapidly in dry ice, and loaded onto a nondenaturing acrylamide gel. Human placental DNA was used as a positive control and water without DNA added was used as a negative control. Optimal resolution was obtained with 1x MDETM (mutation detection enhancement) gels (FMC, Rockland, ME) in 0.6x Tris-BorateEDTA (TBE) buffer. For the pRB-binding domain, PCR-SSCP products were also run on 10% polyacrylamide/12% glycerol gels. Gels were dried on filter paper and used to expose x-ray film. Shifted bands were excised from the gel and suspended in 30 µL of distilled water. The DNA was eluted at room temperature for 6 hours and reamplified by PCR for 35 cycles using the same conditions and parameters, with a total reaction volume of 50 µL. Amplified products were separated on a 1.4% agarose gel and purified with the QIAquick Gel Extraction Kit (Qiagen, Chatsworth, CA). The purified PCR products (30-60 ng) were sequenced with both forward and reverse primers using the PRISMTM Ready Dye Terminator Cycle Sequencing kit with AmpliTaq DNA polymerase FS (25) (The Perkin-Elmer Corp./Applied Biosystems Division, Foster City, CA) and a Perkin-Elmer/Applied Biosystems Model 377 DNA Stretch Sequencer.

Antibodies and Immunohistochemistry

Immunohistochemistry was performed using the avidin-biotin immunoperoxidase complex method. Primary antibodies used were the anti-E2F-1 C-20 rabbit purified antiserum (Santa Cruz Biotechnology, CA) at a final concentration of 0.2 µg/mL. We have previously demonstrated the specificity of the anti-E2F-1 C-20 antibody in T24 bladder carcinoma cells (26). For validation of the E2F-1 expression, the KH-95 anti-E2F-1 mouse monoclonal immunoglobulin G (IgG) antibody (Santa Cruz Biotechnology) was used at a final concentration of 5 µg/mL. An anti-pRB murine monoclonal IgG antibody (QED Bioscience, San Diego, CA) was also used in the present study at a final concentration of 1.3 µg/mL.

Analysis of tumor samples was performed after fixing tissue sections with 10% buffered formalin. Endogenous peroxidase activity was blocked in 0.1% H2O2 in phosphate-buffered saline (PBS) for 15 minutes. Endogenous biotin was blocked with an avidin-biotin blocking kit (Vector Laboratories, Inc., Burlingame, CA). To minimize background staining, 10% whole-horse (for pRB and KH-95 anti-E2F-1) or goat (for C-20 anti-E2F-1) serum diluted in 2% bovine serum albumin-PBS was applied for 30 minutes. Primary antibodies were applied for 2-hour (pRB and KH-95) or for 1-hour (C-20) incubation periods. Nonimmune rabbit serum and an irrelevant IgG antibody were used as negative controls. Biotinylated secondary antibodies (Vector Laboratories, Inc.; 3.0 µg/mL for pRB and KH-95 anti-E2F-1 and 1.5 µg/mL for C-20 anti-E2F-1) and avidin-biotin peroxidase complexes (1:25 dilution, Vector Laboratories, Inc.) were sequentially applied for 30 minutes each. Diaminobenzidine (0.05%) was used as the final chromogen and hematoxylin as the nuclear counterstain.

Immunohistochemical staining profiles for E2F-1 and pRB nuclear expression were graded on a four-point scale for intensity (unreactive, weak, moderate, or strong) and a five-point scale for percentage of positive tumor cells (<5%, 5%-25%, 25%-50%, 50%-75%, and 75%-100%).

Statistical Analysis

A total of 133 patients with a pathologic diagnosis of bladder cancer at the Memorial Sloan-Kettering Cancer Center from October 1993 to June 1997, inclusive, were included in the study. Variables included in the data analysis were histology, clinical and pathologic stage, grade, nodal status, suspicion of vascular invasion, use of neoadjuvant chemotherapy, use of adjuvant chemotherapy, pRB reactivity, and E2F-1 reactivity. For analysis as a prognostic variable, pRB reactivity was assessed as a continuous variable. For descriptive purposes, pRB reactivity greater than 20% was considered positive, consistent with our previous reports (27,28).

Correlation of the intensity and percentage of E2F-1 nuclear immunoreactivity with clinical and pathologic variables was performed using the two-sided {chi}2 test or Fisher's exact test when necessary. The Kendall's {tau}b was used as a measure of concordance for ordinal variables. Cohen's {kappa} was used to measure agreement of staining between the two anti-E2F-1 antibodies. All P values are two-sided and statistically significant when less than .05.

There were three end points for analyses: overall survival, metastases, and local or regional (pelvic) recurrence of disease. In the analysis of overall, metastasis-free, and pelvic recurrence-free survival, patients who had the end point of interest were classified as experiencing failure of treatment, and patients who were still alive without these end points or lost to follow-up during the study period were coded as censored. Survival time was defined as the time from date of cystectomy to the end point (death, metastasis, pelvic recurrence, or censoring). Survival times were evaluated using the Kaplan-Meier method (29) and compared using the logrank test (30). Cox proportional hazards analysis was used to obtain maximum likelihood estimates of the hazard ratios and their 95% confidence intervals (CIs) in multivariate analyses (31,32). Validity of the proportional hazards assumption was tested by plotting log-minus-log plots. Statistical analysis was performed using the SPSS statistical package (SPSS Inc., Chicago, IL).


    RESULTS
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 Abstract
 Introduction
 Methods
 Results
 Discussion
 References
 
Molecular Analysis

The mutational analysis by PCR-SSCP revealed no band-shifts for the primer pairs covering the nuclear-localization domain or the DNA-binding domain. However, PCR-SSCP revealed a shifted band in seven of 133 tumor samples, as well as their corresponding normal tissues, for the primer pair covering the RB-binding domain (Fig. 1).Go Sequencing of these shifted bands revealed the same nucleotide change at amino acid 393 in exon 7 [GGC -> AGC (Gly -> Ser)]. This amino acid substitution was present in both tumor and normal tissue samples. For patients whose tissue samples displayed band-shifts, sequencing of all unshifted bands revealed the wild-type sequence.



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Fig. 1. Polymerase chain reaction-single-strand conformation polymorphism analysis that employed a primer pair covering the region of the E2F-1 gene that encodes the pRB (retinoblastoma protein)-binding domain. Human placental DNA was used as the positive (+) control and water without DNA as the negative (-) control. A) Human bladder tumor samples showing shifted bands in samples 1, 5, 21, 29, 55, and 56 (a 10% acrylamide-12% glycerol gel was used). B) A comparison of bladder tumor and corresponding normal tissues for samples with shifted bands (a 10% acrylamide-12% glycerol gel was used). C) A comparison of bladder tumor and corresponding normal tissues for samples with shifted bands (1x MDETM (mutation detection enhancement) gel; FMC, Rockland, ME).

 
Immunohistochemical Analysis

The intensity of E2F-1 immunoreactivity as assessed by the C-20 antibody was classified as follows: unreactive in 34 cases, weak in 72 cases, moderate in 21 cases, and strong in six cases (Fig. 2).Go The percentage of E2F-1-positive tumor cells using the C-20 antibody was found to be less than 5% in 53 cases, 5%-25% in 36 cases, 25%-50% in 20 cases, and 50%-75% in 24 cases. The E2F-1 reactivity was evaluable by both the C-20 and KH-95 anti-E2F-1 antibodies in 125 samples. The classification of the E2F-1 nuclear intensity was identical with the two antibodies in 76 (61%) of 125 patients and within one category of intensity in 123 (98%) of 125 patients. With respect to the percentage of nuclear reactivity, the classification of the E2F-1 nuclear intensity was identical with the two antibodies in 82 (66%) of 125 patients and within one category of reactivity in 118 (94%) of 125 patients. There was good agreement between the two antibodies with respect to percentage of E2F-1 reactivity (Cohen's {kappa} = 0.67). Fig. 2 Goillustrates representative tumor samples with low to undetectable and high levels of E2F-1 nuclear staining. Table 1,Go A, summarizes the clinical and pathologic parameters, correlating these data with the pattern of E2F-1 staining. Squamous cell carcinomas had lower E2F-1 reactivity significantly more frequently than did other histologies (P = .00038). The pRB nuclear reactivity was greater than 20% in 85 (66%) of 128 tumors in which inflammation did not preclude assessment of pRB reactivity. Both E2F-1 nuclear intensity and percentage of positive cells correlated inversely with the percentage of pRB reactivity (Kendall {tau}b = -0.23, P = .002; and Kendall {tau}b = -0.18, P = .019, respectively). Comparison of molecular and immunohistochemical data revealed no statistically significant difference in intensity and percentage of positive E2F-1 nuclear reactivity between those with and without band-shifts.



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Fig. 2. Human bladder tumors with positive (A and B) and negative (C and D) C-20 rabbit polyclonal antibody immunoreactivity to transcription factor E2F-1 (original magnification x400).

 

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Table 1. Relationships between molecular, clinical, and pathologic parameters in bladder cancer

 
The results of the univariate and multivariate analysis of risk factors for locoregional recurrence, progression to metastases, and death are summarized in Tables 1,GoB, and 2,Go respectively. Proportional hazards assumptions were satisfied as demonstrated by parallel curves for different categories of prognostic variables on log-minus-log plots. No variables were statistically significant predictors of locoregional recurrence. Suspicion of vascular invasion and percentage of positive E2F-1 nuclear staining were significant independent predictors of progression to metastases and death. As summarized in Table 2Go, patients with lower E2F-1 reactivity had a statistically significant increase in their risks of progression to metastases (P = .001) and death (P = .02). For progression to metastases, the presence of a suspicion of vascular invasion was associated with a 7.7-fold increased risk (95% CI = 3.5-16.8), E2F-1 reactivity of 5%-25% versus less than 5% with a 2.5-fold decreased risk (hazard ratio [HR] = 0.40; 95% CI = 0.18-0.90), 25%-50% reactivity versus less than 5% with a 7.7-fold decreased risk (HR = 0.13; 95% CI = 0.037-0.47), and 50%-75% reactivity versus less than 5% with a 3.6-fold decreased risk of metastases (HR = 0.28; 95% CI = 0.12-0.70). For overall survival, the presence of a suspicion of vascular invasion was associated with a 4.3-fold increased risk of death (95% CI = 2.3-8.0) and E2F-1 reactivity of 25%-50% versus less than 5% with a 4.5-fold decreased risk (HR = 0.22; 95% CI = 0.075-0.66).


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Table 2. Results of multivariate analysis for predictors of locoregional recurrence, progression to metastases, and overall survival in patients with bladder cancer

 

    DISCUSSION
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 Abstract
 Introduction
 Methods
 Results
 Discussion
 References
 
Our finding that decreased expression of E2F-1 is associated with an increased risk of progression to metastases, and ultimately death, supports the role of E2F-1 as a tumor suppressor gene in bladder cancer. Despite a relatively short follow-up, the substantial number (41%) of deaths, together with the statistical significance of an adverse outcome in patients with low E2F-1 reactivity, suggest that the study is adequately powered.

Our study identified an inverse correlation between E2F-1 nuclear staining and pRB nuclear staining. Since E2F-1 acts in the same pathway as pRB, it is possible that loss of expression of only one is sufficient to eliminate the repressor activity of pRB. Several investigators (4,33) have reported decreased survival in patients with altered pRB expression. In tumors without pRB alterations, decreased E2F-1 expression may be sufficient for loss of pRB-associated repressor activity. In this context, Cote et al. (34) have recently reported that patients whose tumors have high nuclear immunoreactivity for pRB exhibit lower relapse-free and overall survival rates than patients with moderate pRB expression, similar to those in patients whose tumors lacked pRB immunoreactivity. Given that the E2F-1 null phenotype is nonlethal (19), there may be functional redundancy within the E2F family. Since E2F-2 and E2F-3 also bind pRB in vivo (35), pRB/E2F-2 and pRB/E2F-3 complexes may potentially mediate repression in the absence of E2F-1. However, there is a growing body of literature suggesting that members of the E2F family are not functionally redundant (36,37). An alternate explanation for the inverse correlation between E2F-1 expression and pRB expression may be repression of transcription of the E2F-1 gene, an E2F-regulated target, by high levels of pRB/E2F complex resulting from high expression of pRB.

Given the significance of the retinoblastoma pathway in human bladder cancer (3,4,9), alterations of E2F-1 may be an additional mechanism for inactivation of this pathway in bladder neoplasms. Our findings are consistent with the murine knockout model for E2F-1 in which deletion of the DNA-binding domain results in abnormal cellular proliferation and tumor formation, which suggests a role for E2F-1 as a tumor suppressor gene (19,20). Evidence that the repressor activity of pRB does not require E2F binding comes from an experimental construct in which the chimera GAL4/RB represses promoters with GAL4 DNA-binding sites (38-40). The pocket domain of pRB consists two regions termed A and B (41,42), which are separated by a spacer region. This domain's presence is necessary and sufficient for the transcriptional repressor activity of pRB, when the protein is tethered to a promoter in an E2F-independent fashion (38,39). However, the C-terminal region of pRB, along with the A and B domains, is necessary and sufficient for binding of pRB to E2F (43-45) and for the E2F-dependent repressional activity of pRB (38,43-46).

The finding that E2F-1 overexpression mediates apoptosis (20,47-50) suggests an alternate explanation for the poor outcome in patients with low E2F-1 reactivity. Shan and Lee (48) overexpressed E2F-1 in Rat-2 fibroblasts, demonstrating that apoptosis occurs in an E2F-1 dose-dependent fashion. Qin et al. (47) also demonstrated that induction of E2F-1 expression in transfected Rat-1a fibroblasts resulted in S-phase entry and apoptosis. Furthermore, cotransfection of wild-type RB, but not a nonfunctional RB mutant, repressed the ability of E2F-1 to induce apoptosis. A murine E2F-1 knockout model, in which the DNA-binding and dimerization domains were disrupted, identified deficient thymocyte apoptosis (20). Fueyo et al. (50) have demonstrated that E2F-1 overexpression in nude mice harboring subcutaneously implanted gliomas caused growth inhibition or tumor regression as a result of E2F-1-mediated apoptosis as suggested by in vitro experiments. This E2F-1-mediated apoptosis was independent of the endogenous p53, RB, or p16 status; E2F-1 overexpression did not increase Bax expression but did result in activation of the caspase cascade (50). Relative resistance to apoptosis may account for the poor outcome in patients with low E2F-1 reactivity.

Additional mechanisms that may possibly account for our observed results include changes in E2F-1 phosphorylation or alterations in one of the DP proteins. Phosphorylation of E2F-1 by cyclin A-cdk2 or cyclin A-cdc2 can reduce its activity, as demonstrated by reduced binding of E2F-1 to the E2 promoter (51) and facilitation of pRB binding to E2F-1 (52). Alternatively, alterations in one of the DP proteins required for the stable interaction of E2F-1 with pRB (53) may underlie the observation of an adverse outcome with low E2F-1 expression.

Our combined molecular and immunophenotypic analysis did not identify altered E2F-1 expression due to genotypic alterations in the regions studied. Nakamura et al. (54) also failed to detect mutations by PCR-SSCP in the pRB-binding domain of E2F-1 in a variety of carcinomas screened, including lung, pancreatic, stomach, colon, esophageal, and hepatic cancers. We observed a polymorphism in the E2F-1 gene, upstream of the pRB-binding domain, in seven patients; to our knowledge, this represents the first such report. The presence of this polymorphism did not correlate with the level of E2F-1 expression. However, E2F-1 tumor-specific mutations or polymorphisms were not identified in either the nuclear-localization or DNA-binding domain. Since only cases displaying band-shifts were sequenced, some clinically significant mutations may have been missed, given the known false-negative rate of PCR-SSCP in mutation detection (22). Additionally, mutations occurring in the regions not studied or in the sequences used as primer sequences may have been missed.

The preponderance of patients in our series with extravesical disease (75%) accounts for the poor prognosis in this cohort. Our crude survival rate of 59% at a median follow-up of 16.7 months is consistent with the median survival of 1-1.2 years (55,56) and 0.9-2 years (55-57) reported for pathologic stage pT3b and pT4 bladder cancer, respectively. Vascular invasion (58) is a known adverse prognostic factor in bladder cancer as identified in the present series. However, our finding of an adverse prognosis for low E2F-1 nuclear immunoreactivity with respect to progression to metastases and death in patients with bladder cancer has not previously been reported. Since most of our patients had muscle-invasive bladder cancer as selected for by exclusion of patients with no tumor in the procured tissue blocks, the applicability of our findings to superficial bladder cancer may be limited.

While these observations need to be validated in an independent group of patients, they support the role of E2F-1 as a tumor suppressor gene in bladder cancer. Furthermore, these findings suggest that E2F-1 may be useful to the clinician as a marker, better identifying which patients are at risk of relapse after cystectomy and who may benefit from adjuvant therapy.


    NOTES
 
Supported by a grant from the American Foundation for Urologic Disease and Ortho-McNeil Pharmaceuticals (F. Rabbani); by Public Health Service grants CA74823 (V. M. Richon) and CA47538 (C. Cordon-Cardo) from the National Cancer Institute, National Institutes of Health, Department of Health and Human Services; by grants from the Japan Foundation for the Promotion of Cancer Research Fund (V. M. Richon); the DeWitt Wallace Fund for the Memorial Sloan-Kettering Cancer Center (V. M. Richon); and by the American Cancer Society Career Development Award (G. Dalbagni).


    REFERENCES
 Top
 Abstract
 Introduction
 Methods
 Results
 Discussion
 References
 

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Manuscript received October 9, 1998; revised March 12, 1999; accepted March 23, 1999.


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