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Loss of FOXA1 Drives Sexually Dimorphic Changes in Urothelial Differentiation and Is an Independent Predictor of Poor Prognosis in Bladder Cancer

2015; Elsevier BV; Volume: 185; Issue: 5 Linguagem: Inglês

10.1016/j.ajpath.2015.01.014

1525-2191

Opal L. Reddy, Justin Cates, Lan L. Gellert, Henry Crist, Zhaohai Yang, Hironobu Yamashita, John A. Taylor, Joseph A. Smith, Sam S. Chang, Michael S. Cookson, Chaochen You, Daniel A. Barocas, Magdalena M. Grabowska, Fei Ye, Xue‐Ru Wu, Yajun Yi, Robert J. Matusik, Klaus H. Kaestner, Peter E. Clark, David J. DeGraff,

Epigenetics and DNA Methylation

We previously found loss of forkhead box A1 (FOXA1) expression to be associated with aggressive urothelial carcinoma of the bladder, as well as increased tumor proliferation and invasion. These initial findings were substantiated by The Cancer Genome Atlas, which identified FOXA1 mutations in a subset of bladder cancers. However, the prognostic significance of FOXA1 inactivation and the effect of FOXA1 loss on urothelial differentiation remain unknown. Application of a univariate analysis (log-rank) and a multivariate Cox proportional hazards regression model revealed that loss of FOXA1 expression is an independent predictor of decreased overall survival. An ubiquitin Cre-driven system ablating Foxa1 expression in urothelium of adult mice resulted in sex-specific histologic alterations, with male mice developing urothelial hyperplasia and female mice developing keratinizing squamous metaplasia. Microarray analysis confirmed these findings and revealed a significant increase in cytokeratin 14 expression in the urothelium of the female Foxa1 knockout mouse and an increase in the expression of a number of genes normally associated with keratinocyte differentiation. IHC confirmed increased cytokeratin 14 expression in female bladders and additionally revealed enrichment of cytokeratin 14–positive basal cells in the hyperplastic urothelial mucosa in male Foxa1 knockout mice. Analysis of human tumor specimens confirmed a significant relationship between loss of FOXA1 and increased cytokeratin 14 expression. We previously found loss of forkhead box A1 (FOXA1) expression to be associated with aggressive urothelial carcinoma of the bladder, as well as increased tumor proliferation and invasion. These initial findings were substantiated by The Cancer Genome Atlas, which identified FOXA1 mutations in a subset of bladder cancers. However, the prognostic significance of FOXA1 inactivation and the effect of FOXA1 loss on urothelial differentiation remain unknown. Application of a univariate analysis (log-rank) and a multivariate Cox proportional hazards regression model revealed that loss of FOXA1 expression is an independent predictor of decreased overall survival. An ubiquitin Cre-driven system ablating Foxa1 expression in urothelium of adult mice resulted in sex-specific histologic alterations, with male mice developing urothelial hyperplasia and female mice developing keratinizing squamous metaplasia. Microarray analysis confirmed these findings and revealed a significant increase in cytokeratin 14 expression in the urothelium of the female Foxa1 knockout mouse and an increase in the expression of a number of genes normally associated with keratinocyte differentiation. IHC confirmed increased cytokeratin 14 expression in female bladders and additionally revealed enrichment of cytokeratin 14–positive basal cells in the hyperplastic urothelial mucosa in male Foxa1 knockout mice. Analysis of human tumor specimens confirmed a significant relationship between loss of FOXA1 and increased cytokeratin 14 expression. In the United States, there are approximately 73,000 new cases of urinary bladder cancer (UBC) annually, accounting for approximately 15,000 deaths.1Siegel R. Naishadham D. Jemal A. Cancer statistics, 2013.CA Cancer J Clin. 2013; 63: 11-30Crossref PubMed Scopus (11626) Google Scholar Although the incidence is approximately four times higher in men than in women, women often present with more advanced disease and have worse clinical outcomes.2Fajkovic H. Halpern J.A. Cha E.K. Bahadori A. Chromecki T.F. Karakiewicz P.I. Breinl E. Merseburger A.S. Shariat S.F. Impact of gender on bladder cancer incidence, staging, and prognosis.World J Urol. 2011; 29: 457-463Crossref PubMed Scopus (171) Google Scholar Approximately 90% of UBCs are urothelial carcinomas (UCs), with squamous cell carcinomas (SCCs) and adenocarcinomas largely comprising the remainder. Management of non–muscle-invasive UBC [American Joint Committee on Cancer (AJCC) tumor stages pTa, pTis, and pT1] involves transurethral resection with or without adjuvant intravesical therapy followed by careful surveillance.3Hall M.C. Chang S.S. Dalbagni G. Pruthi R.S. Seigne J.D. Skinner E.C. Wolf Jr., J.S. Schellhammer P.F. Guideline for the management of nonmuscle invasive bladder cancer (stages Ta, T1, and Tis): 2007 update.J Urol. 2007; 178: 2314-2330Abstract Full Text Full Text PDF PubMed Scopus (655) Google Scholar For muscle-invasive UBC (AJCC tumor stage pT2 or higher), radical cystectomy with or without neoadjuvant chemotherapy is the cornerstone of management.4Grossman H.B. Natale R.B. Tangen C.M. Speights V.O. Vogelzang N.J. Trump D.L. deVere White R.W. Sarosdy M.F. Wood Jr., D.P. Raghavan D. Crawford E.D. Neoadjuvant chemotherapy plus cystectomy compared with cystectomy alone for locally advanced bladder cancer.N Engl J Med. 2003; 349: 859-866Crossref PubMed Scopus (2097) Google Scholar Despite these aggressive measures, the disease recurs in approximately 50% of patients, and the 5-year overall survival rate is only 6% for patients who develop or present with distant metastatic disease.5Jemal A. Siegel R. Xu J. Ward E. Cancer statistics, 2010.CA Cancer J Clin. 2010; 60: 277-300Crossref PubMed Scopus (12471) Google Scholar Therefore, there is a significant need for novel approaches geared toward identifying those patients most at risk for disease progression or death, as well as identifying pathways that can act as targets for novel therapeutics. Interestingly, a significant subset (approximately 40%) of UC exhibits squamous differentiation, with some cases showing focal keratinizing squamous metaplasia (KSM) and others developing into predominant or pure SCC. Whereas pure SCC of the bladder has a worse prognosis than UC,6Lynch C.F. Cohen M.B. Urinary system.Cancer. 1995; 75: 316-329Crossref PubMed Scopus (175) Google Scholar whether the presence of squamous differentiation in the background of UC is indeed a poor prognostic indicator is still controversial. Both SCC and UC with squamous differentiation may be associated with resistance to systemic chemotherapy,7Ehdaie B. Maschino A. Shariat S.F. Rioja J. Hamilton R.J. Lowrance W.T. Poon S.A. Al-Ahmadie H.A. Herr H.W. Comparative outcomes of pure squamous cell carcinoma and urothelial carcinoma with squamous differentiation in patients treated with radical cystectomy.J Urol. 2012; 187: 74-79Abstract Full Text Full Text PDF PubMed Scopus (56) Google Scholar which could account for poor overall outcomes. Currently, there is no consensus regarding the extent of squamous differentiation that confers increased clinical risk. Forkhead box A1 (FOXA1) is a member of the forkhead box A subfamily of transcription factors, which includes genes encoding for FOXA1, FOXA2, and FOXA3. FOXA1 protein is expressed in the developing and adult urothelium, whereas FOXA2 protein expression is restricted to early bladder development, where it appears to play a significant role in urothelial development.8Oottamasathien S. Wang Y. Williams K. Franco O.E. Wills M.L. Thomas J.C. Saba K. Sharif-Afshar A.R. Makari J.H. Bhowmick N.A. DeMarco R.T. Hipkens S. Magnuson M. Brock 3rd, J.W. Hayward S.W. Pope JCt Matusik R.J. Directed differentiation of embryonic stem cells into bladder tissue.Dev Biol. 2007; 304: 556-566Crossref PubMed Scopus (85) Google Scholar, 9Gandhi D. Molotkov A. Batourina E. Schneider K. Dan H. Reiley M. Laufer E. Metzger D. Liang F. Liao Y. Sun T.T. Aronow B. Rosen R. Mauney J. Adam R. Rosselot C. Van Batavia J. McMahon A. McMahon J. Guo J.J. Mendelsohn C. Retinoid signaling in progenitors controls specification and regeneration of the urothelium.Dev Cell. 2013; 26: 469-482Abstract Full Text Full Text PDF PubMed Scopus (114) Google Scholar We originally reported that loss of FOXA1 expression is associated with high-grade, late-stage UC, as well as increased tumor cell proliferation and invasion.10DeGraff D.J. Clark P.E. Cates J.M. Yamashita H. Robinson V.L. Yu X. Smolkin M.E. Chang S.S. Cookson M.S. Herrick M.K. Shariat S.F. Steinberg G.D. Frierson H.F. Wu X.R. Theodorescu D. Matusik R.J. Loss of the urothelial differentiation marker FOXA1 is associated with high grade, late stage bladder cancer and increased tumor proliferation.PLoS One. 2012; 7: e36669Crossref PubMed Scopus (68) Google Scholar Furthermore, we found that FOXA2 is expressed in a subset of UC, potentially recapitulating early bladder development. Recently, the Cancer Genome Atlas provided a comprehensive molecular characterization of muscle-invasive UBC.11Cancer Genome Atlas Research NetworkComprehensive molecular characterization of urothelial bladder carcinoma.Nature. 2014; 507: 315-322Crossref PubMed Scopus (2195) Google Scholar In addition to confirming our initial reports regarding the loss of FOXA1 in human UBC, this work extended our original analysis by revealing that FOXA1 is mutated in 5% of UBC. In addition, another recent report found that absence of FOXA1 expression was associated with a basal molecular phenotype that expresses markers associated with squamous differentiation and is more common in female UBC.12Choi W. Porten S. Kim S. Willis D. Plimack E.R. Hoffman-Censits J. Roth B. Cheng T. Tran M. Lee I.L. Melquist J. Bondaruk J. Majewski T. Zhang S. Pretzsch S. Baggerly K. Siefker-Radtke A. Czerniak B. Dinney C.P. McConkey D.J. Identification of distinct basal and luminal subtypes of muscle-invasive bladder cancer with different sensitivities to frontline chemotherapy.Cancer Cell. 2014; 25: 152-165Abstract Full Text Full Text PDF PubMed Scopus (1185) Google Scholar Therefore, in addition to other molecular factors, both FOXA1 and FOXA2 appear to play a central role in bladder development, urothelial differentiation, and malignant progression.9Gandhi D. Molotkov A. Batourina E. Schneider K. Dan H. Reiley M. Laufer E. Metzger D. Liang F. Liao Y. Sun T.T. Aronow B. Rosen R. Mauney J. Adam R. Rosselot C. Van Batavia J. McMahon A. McMahon J. Guo J.J. Mendelsohn C. Retinoid signaling in progenitors controls specification and regeneration of the urothelium.Dev Cell. 2013; 26: 469-482Abstract Full Text Full Text PDF PubMed Scopus (114) Google Scholar, 10DeGraff D.J. Clark P.E. Cates J.M. Yamashita H. Robinson V.L. Yu X. Smolkin M.E. Chang S.S. Cookson M.S. Herrick M.K. Shariat S.F. Steinberg G.D. Frierson H.F. Wu X.R. Theodorescu D. Matusik R.J. Loss of the urothelial differentiation marker FOXA1 is associated with high grade, late stage bladder cancer and increased tumor proliferation.PLoS One. 2012; 7: e36669Crossref PubMed Scopus (68) Google Scholar, 11Cancer Genome Atlas Research NetworkComprehensive molecular characterization of urothelial bladder carcinoma.Nature. 2014; 507: 315-322Crossref PubMed Scopus (2195) Google Scholar However, the prognostic and clinical significance of FOXA1 loss in human UC is currently unknown. To address this issue, we used a tissue microarray (TMA) consisting of >600 bladder tissue samples from 301 patients who underwent cystectomy for UBC to explore the association of FOXA1 loss with clinical outcome in these patients. Although FOXA1 loss is associated with both UC, UC with squamous differentiation, and pure SCC of the urinary bladder in humans, it is unclear whether inactivation of FOXA1 itself is sufficient to drive histologic alterations and/or tumor progression. Therefore, we used an inducible system to genetically ablate Foxa1 in the adult murine urothelium, which enabled us to directly address this question. Human UBC samples were collected by the Translational Pathology Shared Resource, a core facility of the Vanderbilt-Ingram Cancer Center, and later deidentified for testing and analysis. Clinical, pathological, and follow-up data were collected via review of medical records and extracted to the Research Electronic Data Capture database hosted at Vanderbilt University.13Strand D.W. DeGraff D.J. Jiang M. Sameni M. Franco O.E. Love H.D. Hayward W. Lin-Tsai O. Wang A. Cates J.M. Sloane B. Schoenmakers E. Chatterjee K. Matusik R.J. Hayward S. Deficiency in Metabolic Regulators PPARγ and PTEN cooperates to drive keratinizing squamous metaplasia in novel models of human tissue regeneration.Am J Pathol. 2012; 182: 1950-1961Google Scholar This project was approved by the Institutional Review Board at Vanderbilt University. All animal work was approved by and performed in accordance with Institutional Animal Care and Use Committee guidelines. TMAs were generated using 657 bladder tissue cores from 301 patients who underwent cystectomy for UC or SCC of the bladder between January 2000 and May 2010. All patients underwent radical cystectomy, bilateral pelvic lymphadenectomy, and urinary diversion. Patients were subsequently followed up at 3 months, 6 months, and then at increasing intervals based on individual surgeons' practice patterns by physical examination, laboratory studies, and both chest and abdominal imaging. Clinicopathologic data were collected and included patient demographics, such as age at time of surgery, sex, and comorbidity recorded as age-adjusted Charlson comorbidity index,14Charlson M. Szatrowski T.P. Peterson J. Gold J. Validation of a combined comorbidity index.J Clin Epidemiol. 1994; 47: 1245-1251Abstract Full Text PDF PubMed Scopus (4802) Google Scholar and tumor characteristics, such as AJCC tumor stage (seventh edition, 2010 guidelines15Edge S.B. Compton C.C. The American Joint Committee on Cancer: the 7th edition of the AJCC cancer staging manual and the future of TNM.Ann Surg Oncol. 2010; 17: 1471-1474Crossref PubMed Scopus (6473) Google Scholar) and grade. The original hematoxylin and eosin (H&E) slides were reviewed and diagnostic tissue was marked for construction of a TMA using a manual arrayer (Beecher Instruments, Sun Prairie, WI). One to three tissue cores (each 1.5 mm) of representative areas from each of the selected formalin-fixed, paraffin-embedded tissue blocks were used for the array. H&E slides from the TMAs were prepared, and the histopathological diagnosis of tissue samples represented on the TMA were identified for each patient (wherever available), including adjacent benign urothelium, noninvasive papillary urothelial carcinoma (pTa), urothelial carcinoma in situ (pTis), or invasive urothelial carcinoma (pT1-pT4), and were recorded in a Research Electronic Data Capture relational database. Immunohistochemistry (IHC) was performed on human and murine samples. Slides were deparaffinized and rehydrated through a series of graded alcohols and washed in deionized water for 5 minutes. Antigen retrieval was performed by placing slides in 1% antigen unmasking solution (Vector Labs, Burlingame, CA) and heating slides for 25 minutes on high power in a pressure cooker (Cook's Essentials CEPC800). Steam was released in short bursts to prevent boiling and preserve tissue integrity. Slides were cooled to room temperature and washed 3 times for 10 minutes in PBS (pH 7.4). All incubations were performed at room temperature unless otherwise noted. Endogenous peroxidases were blocked by incubation in 1% hydrogen peroxide in methanol for 20 minutes, and slides were again washed 3 times for 10 minutes in phosphate-buffered saline (PBS). Sections were incubated in PBS containing horse serum (Vector Labs) for 30 minutes to reduce nonspecific antibody binding and then incubated overnight with primary antibody at 4°C in a humidified chamber. Antibodies used include goat polyclonal FOXA1 (1:1000; Santa Cruz Biotechnology, Santa Cruz, CA), mouse polyclonal cytokeratin 10 (CK10) (1:200; Dako, Carpinteria, CA), mouse polyclonal cytokeratin 14 (CK14) (1:200; Dako), and mouse polyclonal uroplakin III (UPKIII) (1:5000; Fitzgerald, Acton, MA) all diluted in PBS containing horse serum. Bromodeoxyuridine (BrdU) staining of mouse specimens was stained by the Vanderbilt Experimental Pathology Core. Slides were then washed 3 times for 10 minutes in PBS and sections were incubated in biotinylated secondary antibody diluted in PBS containing horse serum (1:200; Vector Labs) for 1 hour. Specific antibody binding was visualized using Vectastain Elite ABC Peroxidase kit (Vector Labs) according to the manufacturer protocol with diaminobenzidine substrate buffer as the chromogen (Thermo Scientific, Fremont, CA). The intensity and distribution of immunoreactivity for each TMA core were scored by a pathologist (J.M.C.) using the Allred scoring system, where scales of 0 to 3 were used for intensity and 0 to 5 for percentage of positive tumor cells.16Allred D.C. Clark G.M. Elledge R. Fuqua S.A. Brown R.W. Chamness G.C. Osborne C.K. McGuire W.L. Association of p53 protein expression with tumor cell proliferation rate and clinical outcome in node-negative breast cancer.J Natl Cancer Inst. 1993; 85: 200-206Crossref PubMed Scopus (747) Google Scholar An IHC index was assigned to each sample by summation of the intensity score and distribution score. For all analyses, replicate IHC scores from cores of the same pathological stage from the same patient were averaged. Correlations among IHC scores and tumor stage were assessed using Spearman rank coefficients. The extended Mantel-Haenszel test was used to assess correlations between IHC scores after adjusting for tumor stage. For survival analysis and correlation analysis comparing clinical tumor stage with IHC scores, the following exclusion criteria were applied to the original cohort of 301 tumor samples to reduce the potential influence of field effect phenomena. For patients with multiple specimens or different samples from a single specimen represented on the TMA, the sample of the highest pathological stage available was used, and the other samples were excluded. Patient samples recorded as adjacent benign urothelium (n = 25) and cores that could not be histologically assessed (n = 15) were also excluded. Patients with overall tumor stages pT2 or higher were excluded if invasive tumor was not represented on the TMA (n = 17). Because of the low numbers, patients with tumors of low histological grade (n = 8) were also excluded from survival analysis. Two patients had low-grade disease at the time of cystectomy, but we were not able to assess the tissue cores included in the TMAs. This resulted in the use of 234 patients available for survival analysis. Overall survival was defined as the interval from the date of cystectomy to the date of death from any cause, or patients were censored at the date of last follow-up. Univariate analyses were performed using log rank tests and visualized using Kaplan-Meier survival curves. For univariate log rank analyses, age was grouped into quartile distributions, and comorbidity index was grouped into tertile distributions. Tumor stage was grouped by organ-confined disease (AJCC tumor stages pTa, pTis, pT1, and pT2), extravesical disease (pT3 to pT4), and node-positive disease (any pT or N1 to N2). Variables associated with adverse prognosis based on previous studies17Morgan T.M. Barocas D.A. Chang S.S. Phillips S.E. Salem S. Clark P.E. Penson D.F. Smith Jr., J.A. Cookson M.S. The relationship between perioperative blood transfusion and overall mortality in patients undergoing radical cystectomy for bladder cancer.Urol Oncol. 2013; 31: 871-877Abstract Full Text Full Text PDF PubMed Scopus (79) Google Scholar and variables with P < 0.1018Gong Y. Huo L. Liu P. Sneige N. Sun X. Ueno N.T. Lucci A. Buchholz T.A. Valero V. Cristofanilli M. Polycomb group protein EZH2 is frequently expressed in inflammatory breast cancer and is predictive of worse clinical outcome.Cancer. 2011; 117: 5476-5484Crossref PubMed Scopus (59) Google Scholar on univariate analysis were included in a multivariate analysis using a Cox proportional hazards model. The extended Mantel-Haenszel test was used to analyze the correlation between FOXA1 and CK14 expression in human tissue, independent of tumor stage. Fisher's exact test was applied to determine significance of observed phenotypes after Foxa1 knockout in male and female mice. For all tests, P < 0.05 was considered statistically significant. Stata version 12.0 (Stata Corp., College Station, TX) was used for analysis of human clinical data and mouse knockout data. Foxa1loxp mice have been previously described.19Gao N. LeLay J. Vatamaniuk M.Z. Rieck S. Friedman J.R. Kaestner K.H. Dynamic regulation of Pdx1 enhancers by Foxa1 and Foxa2 is essential for pancreas development.Genes Dev. 2008; 22: 3435-3448Crossref PubMed Scopus (232) Google Scholar Globally inducible Foxa1 knockout was achieved by breeding B6.Cg-Tg(UBC-Cre/ERT2)1Ejb/j mice (hereafter simply UBC-Cre/ERT2) to Foxa1loxp/loxp mice, resulting in UBC-Cre/ERT2/Foxa1loxp/loxp mice, respectively. Once sexually mature, male and female UBC-Cre/ERT2/Foxa1loxp/loxp and littermate Foxa1loxp/loxp control mice were injected i.p. with 1 mg/d of tamoxifen for 5 days and separated into three groups. The first group was euthanized immediately after 5 days to determine the efficacy of UBC-Cre/ERT2 to induce ablation of Foxa1 expression. The second and third groups of mice were euthanized 3 and 6 months after tamoxifen injection, respectively, to determine the effect of long-term Foxa1 knockout. All mice were injected with BrdU before euthanasia. A subset of individual bladders were dissected and bisected, with one bladder half being dedicated to formalin fixation and paraffin embedding by standard procedures and the other half being used for microarray studies (see below). Dissected tissue was placed in RNAlater (Qiagen, Venlo, the Netherlands) to stabilize RNA, and RNA was extracted using RNEasy (Qiagen) according to the manufacturer's instructions. Microarray analysis on RNA extracted from bladder tissue dissected from control and experimental mice (four control Foxa1loxp/loxp and three Foxa1 knockout male mice and four control Foxa1loxp/loxp and six Foxa1 knockout female mice) was performed using the Mouse Gene 1.0 ST Array (Affymetrix Corp, Santa Clara, CA) by the Vanderbilt Technologies for Advanced Genomics core. The Mouse Gene 1.0 ST Array contains probes for >28,000 coding transcripts and approximately 7000 noncoding transcripts, including 2000 long intergenic noncoding transcripts. Instrument control and data acquisition were performed using the Affymetrix GeneChip Command Console. Fold change was calculated in a log base 2 space, using the formula FoldChange=2∧(experimental−control) followed by inverting and negating ratios 2 and false discovery rate–adjusted P < 0.01. All statistical analyses were performed in R version 3.0.2 (http://www.r-project.org). Data were analyzed through the use of the Ingenuity Pathway Analysis (IPA; Qiagen, Redwood City, CA) and were used with permission. Clinicopathologic characteristics of the tumors represented on the TMA are listed in Table 1. Primary histological tumor types for the 301 patients in our study included 278 pure UCs (92%) and 23 UCs with squamous differentiation (8%). Four patients (1%) were treated with neoadjuvant chemotherapy. At the time of last follow-up, there were 139 deaths (59%). Median follow-up was 15.1 months (interquartile range, 5.6 to 29.7) for censored patients. Median overall survival duration was 42.1 months (95% CI, 27.4–58.4 months), with a 5-year actuarial estimated overall survival rate of 42.3% (95% CI, 35.6%–48.8%). Confirming our previous finding in an independent cohort,10DeGraff D.J. Clark P.E. Cates J.M. Yamashita H. Robinson V.L. Yu X. Smolkin M.E. Chang S.S. Cookson M.S. Herrick M.K. Shariat S.F. Steinberg G.D. Frierson H.F. Wu X.R. Theodorescu D. Matusik R.J. Loss of the urothelial differentiation marker FOXA1 is associated with high grade, late stage bladder cancer and increased tumor proliferation.PLoS One. 2012; 7: e36669Crossref PubMed Scopus (68) Google Scholar FOXA1 expression was inversely correlated with increasing tumor stage by Spearman rank correlation (P < 0.001; ρ = −0.38; n = 234). We compared the overall survival of patients with low versus high FOXA1 expression. For survival analysis, IHC scores were dichotomized as low or high expression using the median (5.5) as a cutoff value, with values at the median grouped into the high expression category. Low FOXA1 expression was associated with shorter overall survival on univariate log rank analysis (P < 0.001) (Figure 1). Median overall survival for low versus high FOXA1 expression was 22.1 months (95% CI, 16.5–31.9 months) and 68 months (95% CI, 48.2 months to not reached), respectively. Other variables associated with overall survival on univariate analysis include patient age (P < 0.001), Charlson comorbidity index (P < 0.001), and tumor stage (P < 0.001). Sex (P = 0.061) was also included in the multivariate model. After controlling for patient age, tumor stage, sex, and Charlson comorbidity index score, high FOXA1 expression is an independent predictor of higher overall survival (Table 2).Table 1Patient and Tumor Characteristics Included in Tissue MicroarrayCharacteristicFindingAge, years, median (IQR)67 (59–73)Sex, no. (%) Male239 (79) Female62 (21)Race, no. (%)∗Data on race was self-reported by patients. White279 (93) Nonwhite20 (6) Not reported2 (1)Charlson Comorbidity Index, no. (%) 0–151 (17) 2–3124 (41) >4126 (42)T stage, no. (%) Ta7 (2) Tis38 (13) T141 (14) T298 (32) T383 (28) T434 (11)N stage, no. (%) N0231 (77) N1 or greater70 (23)Grade, no. (%) Low grade8 (3) High grade293 (97)IQR, interquartile range.∗ Data on race was self-reported by patients. Open table in a new tab Table 2Multivariate Cox Proportional Hazards Model Predicting Overall Survival after Radical CystectomyVariableOverall survivalHR (95% CI)P valueAge0.99 (0.97–1.02)0.526Charlson Comorbidity Index1.25 (1.11–1.40)<0.001Sex MaleReferent Female0.63 (0.41–0.97)0.037Tumor stage Organ-confined diseaseReferent Extra-vesical disease2.00 (1.27–3.15)0.003 Node-positive disease3.49 (2.31–5.28)<0.001Low FOXA11.49 (1.04–2.13)0.028 Open table in a new tab IQR, interquartile range. To determine the phenotypic effect of genetically ablating Foxa1 expression in murine urothelium, we initiated a breeding program consisting of a tamoxifen-inducible Cre recombinase driven by the ubiquitin promoter (UBC-Cre/ERT2) and our Foxa1loxp/loxp mice, resulting in the creation of UBC-Cre/ERT2/Foxa1loxp/loxp mice. Littermate control Foxa1loxp/loxp mice and experimental UBC-Cre/ERT2/Foxa1loxp/loxp mice were injected with tamoxifen for 5 days and then separated into 3 different groups (Materials and Methods). Histomorphometric analysis of control Foxa1loxp/loxp mice (Figure 2A) and experimental UBC-Cre/ERT2/Foxa1loxp/loxp mice (Figure 2E) after tamoxifen injection for 5 days (Figure 2E) revealed no obvious differences in tissue architecture, whereas IHC analysis of UBC-Cre/ERT2/Foxa1loxp/loxp mice revealed efficient and widespread ablation of urothelial Foxa1 expression; however, control Foxa1loxp/loxp mice retained Foxa1 expression (Figure 2, B and F). Both tamoxifen-treated Foxa1loxp/loxp mice and tamoxifen-treated experimental UBC-Cre/ERT2/Foxa1loxp/loxp expressed UpkIII (Figure 2, C and G), a marker of urothelial differentiation, and were quiescent in regard to proliferation (Figure 2, D and H). These results indicate that the UBC-Cre/ERT2 system efficiently ablates Foxa1 expression in the urothelium of adult mice. Bladder tissue dissected from tamoxifen-injected, genetic control Foxa1loxp/loxp mice exhibited typical murine urothelial morphology, consisting of a superficial umbrella cell layer and a single layer each of intermediate and basal cells (Figure 3A). Bladder tissue dissected from male UBC-Cre/ERT2/Foxa1loxp/loxp mice obtained 3 months after tamoxifen injection revealed basal cell hyperplasia (Figure 3B) and degenerative vacuoles within the umbrella cell layer (Figure 3B). The presence of urothelial hyperplasia was also detected 6 months after tamoxifen-induced Foxa1 knockout (Figure 3, C and D). Nine of the 10 male Foxa1 knockout mice developed urothelial hyperplasia, and the other mouse developed squamous metaplasia. Importantly, urothelial hyperplasia was significantly associated with Foxa1 knockout in male mice (P < 0.0005; Fisher's exact test). We did not detect hyperplasia in any Foxa1loxp/loxp control male mice after tamoxifen injection. Bladder tissue dissected from female Foxa1loxp/loxp control mice (Figure 3E) 6 months after tamoxifen injection appeared identical to that dissected from male Foxa1loxp/loxp control mice. Moreover, analysis of bladder tissue dissected from female UBC-Cre/ERT2/Foxa1loxp/loxp mice obtained 3 months after tamoxifen injection revealed the appearance of degenerative vacuoles within the umbrella cell layer (Figure 3F) similar to those observed in the male mice at 3 months (Figure 3B). Phenotypic alterations consistent with KSM were observed in female UBC-Cre/ERT2/Foxa1loxp/loxp mice after FOXA1 knockout 3 and 6 months after tamoxifen injection (Figure 3, G and H). Seven of 10 female Foxa1 knockout mice developed squamous metaplasia, and one other mouse exhibited areas of cellular hyperplasia. As was the case with urothelial hyperplasia in male Foxa1 knockout mice,