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Distinct Expression Profiles of p63 Variants during Urothelial Development and Bladder Cancer Progression

2011; Elsevier BV; Volume: 178; Issue: 3 Linguagem: Inglês

10.1016/j.ajpath.2010.11.061

ISSN

1525-2191

Autores

Orit Karni-Schmidt, Mireia Castillo-Martín, Tian Shen, Nataliya Gladoun, Josep Domingo-Domènech, Marta Sänchez‐Carbayo, Yingchun Li, Scott W. Lowe, Carol Prives, Carlos Cordon‐Cardo,

Tópico(s)

RNA modifications and cancer

Resumo

The TP63 gene, a member of the TP53 tumor suppressor gene family, can be expressed as at least six isoforms due to alternative promoter use and alternative splicing. The lack of p63 isoform–specific antibodies has limited the analysis of the biological significance of p63. We report a novel set of well-defined antibodies to examine p63 isoforms in mouse and human urothelium during embryogenesis and tumor progression, respectively. We provide evidence that basal and intermediate urothelial cells express p63 isoforms, with the TAp63 variant the first to be detected during development, whereas umbrella cells are characterized by a p63-negative phenotype. Notably, we report that p63-null mice develop a bladder with an abnormal urothelium, constituted by a single layer of cells that express uroplakin II and low molecular weight cytokeratins, consistent with an umbrella cell phenotype. Finally, analysis of 202 human bladder carcinomas revealed a new categorization of invasive tumors into basal-like (positive for ΔNp63 and high molecular weight cytokeratins and negative for low molecular weight cytokeratins) versus luminal-like (negative for ΔNp63 and high molecular weight cytokeratins and positive for low molecular weight cytokeratins) phenotypes, with ΔNp63 expression associated with an aggressive clinical course and poor prognosis. This study highlights the relevance of p63 isoforms in both urothelial development and bladder carcinoma progression, with ΔNp63 acting as an oncogene in certain invasive bladder tumors. The TP63 gene, a member of the TP53 tumor suppressor gene family, can be expressed as at least six isoforms due to alternative promoter use and alternative splicing. The lack of p63 isoform–specific antibodies has limited the analysis of the biological significance of p63. We report a novel set of well-defined antibodies to examine p63 isoforms in mouse and human urothelium during embryogenesis and tumor progression, respectively. We provide evidence that basal and intermediate urothelial cells express p63 isoforms, with the TAp63 variant the first to be detected during development, whereas umbrella cells are characterized by a p63-negative phenotype. Notably, we report that p63-null mice develop a bladder with an abnormal urothelium, constituted by a single layer of cells that express uroplakin II and low molecular weight cytokeratins, consistent with an umbrella cell phenotype. Finally, analysis of 202 human bladder carcinomas revealed a new categorization of invasive tumors into basal-like (positive for ΔNp63 and high molecular weight cytokeratins and negative for low molecular weight cytokeratins) versus luminal-like (negative for ΔNp63 and high molecular weight cytokeratins and positive for low molecular weight cytokeratins) phenotypes, with ΔNp63 expression associated with an aggressive clinical course and poor prognosis. This study highlights the relevance of p63 isoforms in both urothelial development and bladder carcinoma progression, with ΔNp63 acting as an oncogene in certain invasive bladder tumors. The TP63 gene is a member of the TP53 gene family that also includes TP73, which encodes at least six isoform proteins due to alternative promoter use and alternative splicing.1Augustin M. Bamberger C. Paul D. Schmale H. Cloning and chromosomal mapping of the human p53-related KET gene to chromosome 3q27 and its murine homolog Ket to mouse chromosome 16.Mamm Genome. 1998; 9: 899-902Crossref PubMed Scopus (56) Google Scholar, 2Osada M. Ohba M. Kawahara C. Ishioka C. Kanamaru R. Katoh I. Ikawa Y. Nimura Y. Nakagawara A. Obinata M. Ikawa S. Cloning and functional analysis of human p51, which structurally and functionally resembles p53.Nat Med. 1998; 4: 839-843Crossref PubMed Scopus (474) Google Scholar, 3Yang A. Kaghad M. Caput D. McKeon F. On the shoulders of giants: p63, p73 and the rise of p53.Trends Genet. 2002; 18: 90-95Abstract Full Text Full Text PDF PubMed Scopus (452) Google Scholar, 4Yang A. Kaghad M. Wang Y. Gillett E. Fleming M.D. Dotsch V. Andrews N.C. Caput D. McKeon F. p63, a p53 homolog at 3q27-29, encodes multiple products with transactivating, death-inducing, and dominant-negative activities.Mol Cell. 1998; 2: 305-316Abstract Full Text Full Text PDF PubMed Scopus (1840) Google Scholar The revelation of an internal promoter within this family was first attributed to TP63.4Yang A. Kaghad M. Wang Y. Gillett E. Fleming M.D. Dotsch V. Andrews N.C. Caput D. McKeon F. p63, a p53 homolog at 3q27-29, encodes multiple products with transactivating, death-inducing, and dominant-negative activities.Mol Cell. 1998; 2: 305-316Abstract Full Text Full Text PDF PubMed Scopus (1840) Google Scholar TP63 has a domain topology similar to TP53, with an N-terminal transactivation domain, a central sequence–specific DNA-binding domain, and a C-terminal oligomerization domain. The two main groups of p63 isoforms generated by alternative promoter use include the TA isoforms, in which the N-terminal transactivation domain is retained, and the ΔN isoforms, which lack the TA domain.4Yang A. Kaghad M. Wang Y. Gillett E. Fleming M.D. Dotsch V. Andrews N.C. Caput D. McKeon F. p63, a p53 homolog at 3q27-29, encodes multiple products with transactivating, death-inducing, and dominant-negative activities.Mol Cell. 1998; 2: 305-316Abstract Full Text Full Text PDF PubMed Scopus (1840) Google Scholar Although TAp63 has been shown to transactivate p53 target genes, ΔNp63 can act both as dominant negative factor toward TAp63, p73, and p53,5Deyoung M.P. Ellisen L.W. p63 and p73 in human cancer: defining the network.Oncogene. 2007; 26: 5169-5183Crossref PubMed Scopus (214) Google Scholar, 6Finlan L.E. Hupp T.R. p63: the phantom of the tumor suppressor.Cell Cycle. 2007; 6: 1062-1071Crossref PubMed Scopus (43) Google Scholar and also possess transactivation activity.7Dohn M. Zhang S. Chen X. p63alpha and DeltaNp63alpha can induce cell cycle arrest and apoptosis and differentially regulate p53 target genes.Oncogene. 2001; 20: 3193-3205Crossref PubMed Scopus (257) Google Scholar, 8Helton E.S. Zhu J. Chen X. The unique NH2-terminally deleted (DeltaN) residues, the PXXP motif, and the PPXY motif are required for the transcriptional activity of the DeltaN variant of p63.J Biol Chem. 2006; 281: 2533-2542Crossref PubMed Scopus (90) Google Scholar In contrast to TP53, which plays critical roles in regulating multiple cellular pathways and is mutated in many human cancers (reviewed in references 9Lane D. Levine A. p53 research: the past thirty years and the next thirty years.Cold Spring Harb Perspect Biol. 2010; 2: a000893Crossref Scopus (312) Google Scholar, 10Olivier M. Hollstein M. Hainaut P. TP53 mutations in human cancers: origins, consequences, and clinical use.Cold Spring Harb Perspect Biol. 2010; 2: a001008Crossref Scopus (1155) Google Scholar, 11Vousden K.H. Prives C. Blinded by the light: the growing complexity of p53.Cell. 2009; 137: 413-431Abstract Full Text Full Text PDF PubMed Scopus (2315) Google Scholar), TP63 is rarely mutated,12Guo X. Mills A.A. p63, cellular senescence and tumor development.Cell Cycle. 2007; 6: 305-311Crossref PubMed Scopus (29) Google Scholar, 13Moll U.M. Slade N. p63 and p73: roles in development and tumor formation.Mol Cancer Res. 2004; 2: 371-386PubMed Google Scholar and its role in cancer is not yet clarified. Data from mouse models are also inconclusive: although some studies show that p63+/− mice are not tumor prone,14Keyes W.M. Vogel H. Koster M.I. Guo X. Qi Y. Petherbridge K.M. Roop D.R. Bradley A. Mills A.A. p63 heterozygous mutant mice are not prone to spontaneous or chemically induced tumors.Proc Natl Acad Sci U S A. 2006; 103: 8435-8440Crossref PubMed Scopus (80) Google Scholar other groups reported that p63+/− mice developed spontaneous tumors, including squamous cell carcinoma and sarcomas.15Flores E.R. Sengupta S. Miller J.B. Newman J.J. Bronson R. Crowley D. Yang A. McKeon F. Jacks T. Tumor predisposition in mice mutant for p63 and p73: evidence for broader tumor suppressor functions for the p53 family.Cancer Cell. 2005; 7: 363-373Abstract Full Text Full Text PDF PubMed Scopus (421) Google Scholar Despite the structural and partly functional identity among the TP53 family members, studies in knockout mice demonstrate significant functional differences among them. For example, studies in mice suggest a unique role for p63 in development, including its role in epidermis formation,5Deyoung M.P. Ellisen L.W. p63 and p73 in human cancer: defining the network.Oncogene. 2007; 26: 5169-5183Crossref PubMed Scopus (214) Google Scholar, 6Finlan L.E. Hupp T.R. p63: the phantom of the tumor suppressor.Cell Cycle. 2007; 6: 1062-1071Crossref PubMed Scopus (43) Google Scholar, 13Moll U.M. Slade N. p63 and p73: roles in development and tumor formation.Mol Cancer Res. 2004; 2: 371-386PubMed Google Scholar maintenance of the proliferative potential of basal regenerative cells in stratified epithelium,16McKeon F. p63 and the epithelial stem cell: more than status quo?.Genes Dev. 2004; 18: 465-469Crossref PubMed Scopus (177) Google Scholar, 17Senoo M. Pinto F. Crum C.P. McKeon F. p63 Is essential for the proliferative potential of stem cells in stratified epithelia.Cell. 2007; 129: 523-536Abstract Full Text Full Text PDF PubMed Scopus (697) Google Scholar and serving as a molecular switch for initiation of an epithelial stratification program.18Koster M.I. Kim S. Mills A.A. DeMayo F.J. Roop D.R. p63 is the molecular switch for initiation of an epithelial stratification program.Genes Dev. 2004; 18: 126-131Crossref PubMed Scopus (552) Google Scholar Normal bladder is coated by a mucosa termed urothelium, a pseudo-stratified epithelium composed of basal, intermediate, and superficial umbrella cells. There are up to seven layers in human bladder urothelium and only up to three layers in the mouse bladder urothelium.19Castillo-Martin M. Domingo-Domenech J. Karni-Schmidt O. Matos T. Cordon-Cardo C. Molecular pathways of urothelial development and bladder tumorigenesis.Urol Oncol. 2010; 28: 401-408Abstract Full Text Full Text PDF PubMed Scopus (207) Google Scholar It is well documented that the basal and intermediate cell layers are characterized by a nuclear p63-positive phenotype, along with the expression of high molecular weight cytokeratins (CKs), such as CK5 and CK14. In contrast, umbrella cells of the superficial layer display a distinct phenotype characterized by lack of p63 expression and presence of certain low molecular weight CKs, such as CK20 and CK18, as well as uroplakins [eg, uroplakin II (UPII)19Castillo-Martin M. Domingo-Domenech J. Karni-Schmidt O. Matos T. Cordon-Cardo C. Molecular pathways of urothelial development and bladder tumorigenesis.Urol Oncol. 2010; 28: 401-408Abstract Full Text Full Text PDF PubMed Scopus (207) Google Scholar, 20Moll R. Divo M. Langbein L. The human keratins: biology and pathology.Histochem Cell Biol. 2008; 129: 705-733Crossref PubMed Scopus (967) Google Scholar]. Our group and others previously found that p63 is essential for urothelial differentiation21Cheng W. Jacobs W.B. Zhang J.J. Moro A. Park J.H. Kushida M. Qiu W. Mills A.A. Kim P.C. DeltaNp63 plays an anti-apoptotic role in ventral bladder development.Development. 2006; 133: 4783-4792Crossref PubMed Scopus (81) Google Scholar and that p63-null mice developed a nontransitional default simple cubical epithelium.22Urist M.J. Di Como C.J. Lu M.L. Charytonowicz E. Verbel D. Crum C.P. Ince T.A. McKeon F.D. Cordon-Cardo C. Loss of p63 expression is associated with tumor progression in bladder cancer.Am J Pathol. 2002; 161: 1199-1206Abstract Full Text Full Text PDF PubMed Scopus (222) Google Scholar In addition, Signoretti et al23Signoretti S. Pires M.M. Lindauer M. Horner J.W. Grisanzio C. Dhar S. Majumder P. McKeon F. Kantoff P.W. Sellers W.R. Loda M. p63 regulates commitment to the prostate cell lineage.Proc Natl Acad Sci U S A. 2005; 102: 11355-11360Crossref PubMed Scopus (129) Google Scholar reported that the urothelium of 18.5-day p63-null embryos contain a single layer that resembles umbrella cells and expresses uroplakin III (UPIII). We now report, by analyzing the histological and immunophenotypic features of the mucosa in p63-null mice, that it is constituted of exclusively umbrella cells. Approximately 90% of malignant neoplasms that occur in the urinary bladder are of epithelial origin,24Cordon-Cardo C. Molecular alterations associated with bladder cancer initiation and progression.Scand J Urol Nephrol Suppl. 2008; 218: 154-165Crossref PubMed Scopus (104) Google Scholar and most of them correspond histologically to urothelial carcinomas (UCs). Because of a dearth of effective p63-specific antibodies, most reported studies have measured TP63 mRNA expression in human bladder tumors by RT-PCR using isoform-specific primers.22Urist M.J. Di Como C.J. Lu M.L. Charytonowicz E. Verbel D. Crum C.P. Ince T.A. McKeon F.D. Cordon-Cardo C. Loss of p63 expression is associated with tumor progression in bladder cancer.Am J Pathol. 2002; 161: 1199-1206Abstract Full Text Full Text PDF PubMed Scopus (222) Google Scholar, 25Park B.J. Lee S.J. Kim J.I. Lee C.H. Chang S.G. Park J.H. Chi S.G. Frequent alteration of p63 expression in human primary bladder carcinomas.Cancer Res. 2000; 60: 3370-3374PubMed Google Scholar, 26Di Como C.J. Urist M.J. Babayan I. Drobnjak M. Hedvat C.V. Teruya-Feldstein J. Pohar K. Hoos A. Cordon-Cardo C. p63 expression profiles in human normal and tumor tissues.Clin Cancer Res. 2002; 8: 494-501PubMed Google Scholar, 27Comperat E. Bieche I. Dargere D. Ferlicot S. Laurendeau I. Benoit G. Vieillefond A. Verret C. Vidaud M. Capron F. Bedossa P. Paradis V. p63 gene expression study and early bladder carcinogenesis.Urology. 2007; 70: 459-462Abstract Full Text Full Text PDF PubMed Scopus (18) Google Scholar The caveat of using this method is that one cannot be conclusive regarding protein expression levels. Previous studies dealing with p63 expression in bladder cancer reported that there was a correlation with tumor grade.22Urist M.J. Di Como C.J. Lu M.L. Charytonowicz E. Verbel D. Crum C.P. Ince T.A. McKeon F.D. Cordon-Cardo C. Loss of p63 expression is associated with tumor progression in bladder cancer.Am J Pathol. 2002; 161: 1199-1206Abstract Full Text Full Text PDF PubMed Scopus (222) Google Scholar, 27Comperat E. Bieche I. Dargere D. Ferlicot S. Laurendeau I. Benoit G. Vieillefond A. Verret C. Vidaud M. Capron F. Bedossa P. Paradis V. p63 gene expression study and early bladder carcinogenesis.Urology. 2007; 70: 459-462Abstract Full Text Full Text PDF PubMed Scopus (18) Google Scholar, 28Comperat E. Camparo P. Haus R. Chartier-Kastler E. Bart S. Delcourt A. Houlgatte A. Francois R. Capron F. Vieillefond A. Immunohistochemical expression of p63, p53 and MIB-1 in urinary bladder carcinoma: a tissue microarray study of 158 cases.Virchows Arch. 2006; 448: 319-324Crossref PubMed Scopus (53) Google Scholar Controversy has arisen from the analyses of ΔNp63 because some groups found that loss of ΔNp63 was associated with an aggressive phenotype,29Koga F. Kawakami S. Fujii Y. Saito K. Ohtsuka Y. Iwai A. Ando N. Takizawa T. Kageyama Y. Kihara K. Impaired p63 expression associates with poor prognosis and uroplakin III expression in invasive urothelial carcinoma of the bladder.Clin Cancer Res. 2003; 9: 5501-5507PubMed Google Scholar, 30Koga F. Kawakami S. Kumagai J. Takizawa T. Ando N. Arai G. Kageyama Y. Kihara K. Impaired Delta Np63 expression associates with reduced beta-catenin and aggressive phenotypes of urothelial neoplasms.Br J Cancer. 2003; 88: 740-747Crossref PubMed Scopus (67) Google Scholar, 31Fukushima H. Koga F. Kawakami S. Fujii Y. Yoshida S. Ratovitski E. Trink B. Kihara K. Loss of DeltaNp63alpha promotes invasion of urothelial carcinomas via N-cadherin/Src homology and collagen/extracellular signal-regulated kinase pathway.Cancer Res. 2009; 69: 9263-9270Crossref PubMed Scopus (43) Google Scholar whereas others have reported that ΔNp63 overexpression could act as an oncogene in bladder cancer and other tumor types by promoting cell survival and proliferation.32He Y. Wu X. Tang W. Tian D. Luo C. Yin Z. Du H. Impaired delta NP63 expression is associated with poor tumor development in transitional cell carcinoma of the bladder.J Korean Med Sci. 2008; 23: 825-832Crossref PubMed Scopus (10) Google Scholar, 33Hibi K. Trink B. Patturajan M. Westra W.H. Caballero O.L. Hill D.E. Ratovitski E.A. Jen J. Sidransky D. AIS is an oncogene amplified in squamous cell carcinoma.Proc Natl Acad Sci U S A. 2000; 97: 5462-5467Crossref PubMed Scopus (440) Google Scholar, 34Patturajan M. Nomoto S. Sommer M. Fomenkov A. Hibi K. Zangen R. Poliak N. Califano J. Trink B. Ratovitski E. Sidransky D. DeltaNp63 induces beta-catenin nuclear accumulation and signaling.Cancer Cell. 2002; 1: 369-379Abstract Full Text Full Text PDF PubMed Scopus (177) Google Scholar Moreover, ΔNp63 overexpression has also been reported in squamous cell carcinoma and linked to 3q27-29 genomic amplification, an amplicon that includes the TP63 gene.33Hibi K. Trink B. Patturajan M. Westra W.H. Caballero O.L. Hill D.E. Ratovitski E.A. Jen J. Sidransky D. AIS is an oncogene amplified in squamous cell carcinoma.Proc Natl Acad Sci U S A. 2000; 97: 5462-5467Crossref PubMed Scopus (440) Google Scholar, 35Taniere P. Martel-Planche G. Saurin J.C. Lombard-Bohas C. Berger F. Scoazec J.Y. Hainaut P. TP53 mutations, amplification of P63 and expression of cell cycle proteins in squamous cell carcinoma of the oesophagus from a low incidence area in Western Europe.Br J Cancer. 2001; 85: 721-726Crossref PubMed Scopus (49) Google Scholar Using novel and commercially available anti-p63 isoform–specific antibodies, we analyzed the presence of different p63 variants expressed in the urothelium of mice during development, normal human urothelium, and different stages of human bladder cancer. Our results suggest a crucial role for the distinct expression of p63 isoforms in bladder tumor progression. Therefore, we propose a new categorization of invasive bladder cancer based on ΔNp63 expression, which has critical prognostic implications. J82 invasive bladder cancer cell line was grown and maintained in minimal essential medium supplemented with 10% fetal bovine serum and 1% nonessential amino acids. Transfections were performed using this cell line because J82 cells do not express p63 intrinsically,22Urist M.J. Di Como C.J. Lu M.L. Charytonowicz E. Verbel D. Crum C.P. Ince T.A. McKeon F.D. Cordon-Cardo C. Loss of p63 expression is associated with tumor progression in bladder cancer.Am J Pathol. 2002; 161: 1199-1206Abstract Full Text Full Text PDF PubMed Scopus (222) Google Scholar, 24Cordon-Cardo C. Molecular alterations associated with bladder cancer initiation and progression.Scand J Urol Nephrol Suppl. 2008; 218: 154-165Crossref PubMed Scopus (104) Google Scholar, 25Park B.J. Lee S.J. Kim J.I. Lee C.H. Chang S.G. Park J.H. Chi S.G. Frequent alteration of p63 expression in human primary bladder carcinomas.Cancer Res. 2000; 60: 3370-3374PubMed Google Scholar with Lipofectamine 2000 reagent (Invitrogen Corp, Carlsbad, CA) in accordance with the manufacturer's protocols. Expression vectors derived from p3xFLAG-CMV-7.1 were TAp63α, TAp63β, TAp63γ, ΔNp63α, ΔNp63β, ΔNp63γ, and a backbone vector as a control. To detect different isoforms of p63, we assembled a panel of antibodies that was further validated in tissue samples. These include mouse monoclonal anti-p63 [4A4 (Santa-Cruz Biotechnology Inc, Santa Cruz, CA)] to detect all p63 variants, rabbit polyclonal purified anti-ΔNp63, rabbit polyclonal anti-p63α [H-129 (Santa-Cruz)], mouse monoclonal anti-p63α, mouse monoclonal anti-p63α/β, and anti-Flag (Sigma Aldrich Co., St. Louis, MO). Mouse (pantropic) monoclonal p53 antibody clone PAb1801 (Calbiochem-EMD Chemicals, Gibbstown, NJ) was used to recognize p53, and a rabbit polyclonal actin antibody (Sigma) was used as a loading control for immunoblotting studies (see as follows). For immunoblotting, cells were washed twice with PBS and then collected in cold extraction buffer [10 mmol/L Tris (pH 7.5), 1 mmol/L EDTA, 400 mmol/L NaCl, 10% glycerol, 0.5% NP40, 5 mmol/L NaF, 1 mmol/L dithiothreitol, and 0.1 mmol/L phenylmethylsulfonyl fluoride] followed by centrifugation at 13,000 × g for 10 minutes at 4°C. Supernatants were resuspended in 6× protein sample buffer [30% glycerol, 2M β-mercaptoethanol, 12% SDS, 500 mmol/L Tris (pH 6.8), and 0.5 mg/ml of Bromophenol Blue] and boiled for 10 minutes at 95°C. The samples were loaded onto SDS-polyacrylamide gels and transferred by a semi-dry transfer to nitrocellulose for 1 to 2 hours. SDS–polyacrylamide gel electrophoresis and immunoblotting procedures were conducted under standard conditions. Cells were plated on coverslips in 60-mm culture dishes, and after 48 hours they were washed twice with PBS followed by incubation with 4% paraformaldehyde (Sigma) for 15 minutes. Cells were then washed three times with PBS, incubated with PBS/0.5% Triton X-100 for 1½ minutes, and then blocked with 0.5% bovine serum albumin (Sigma) in PBS for 30 minutes at room temperature before the treatment with 50 μL of the diluted primary antibodies (as described in the Antibodies and Immunoblotting section) for 1 hour at room temperature. The coverslips were then washed three times with PBS and double stained by incubation with 50 μL of diluted (1:100) Alexa Fluor 594 donkey anti-mouse IgG antibody and Alexa Fluor 488 donkey anti-rabbit IgG secondary antibodies (Invitrogen) for 1 hour. Coverslips were then washed three times with PBS and mounted with Vectashield mounting medium containing DAPI. The immunofluorescence images were collected and analyzed using a Nikon Intensilight C-HGFI microscope (Nikon Gmbh, Dusseldorf, Germany) and SPOT Advanced software (SPOT Imaging, Sterling Heights, MI). Immunohistochemical and immunofluorescence analyses were performed on formalin-fixed paraffin-embedded tissue sections from normal and p63-null mouse bladder samples. Normal human bladder and tissue microarrays built from superficial and invasive human bladder carcinomas (under institutional review board–approved protocols) were also used for the present study. We analyzed four tissue microarrays of 0.6-mm-diameter tissue cores arrayed in triplicate on a paraffin block.36Hoos A. Urist M.J. Stojadinovic A. Mastorides S. Dudas M.E. Leung D.H. Kuo D. Brennan M.F. Lewis J.J. Cordon-Cardo C. Validation of tissue microarrays for immunohistochemical profiling of cancer specimens using the example of human fibroblastic tumors.Am J Pathol. 2001; 158: 1245-1251Abstract Full Text Full Text PDF PubMed Scopus (342) Google Scholar Tissue microarrays included tissues from 202 bladder cancer patients, distributed as follows: 147 non–muscle invasive UCs (31 pTa and 116 pT1) and 55 invasive UCs (22 pT2, 23 pT3, and 10 pT4). Follow-up clinical data for correlation with marker expression were available from 183 of the 202 bladder cancer patients included in this study. Pathological stage and grade of these 183 carcinomas were distributed as follows: 27 pTa carcinomas (20 low grade and 7 high grade), 109 pT1 high-grade carcinomas, 18 pT2 carcinomas, 20 pT3 carcinomas, and 9 pT4 carcinomas. We also studied 10 human normal bladder tissue samples from young patients with no history of bladder cancer (n = 3) and adjacent normal mucosa from patients with a diagnosis of bladder cancer (n = 7). Immunohistochemical procedures were conducted using 5-μm deparaffinized tissue sections, submitted to antigen retrieval by steam treatment for 15 minutes in 10 mmol/L citrate buffer (pH 6.0). Slides were then treated with 10% normal serum for 30 minutes, followed by primary antibody (as described in the Antibodies and Immunoblotting section) incubation overnight at 4°C. Antibodies specifically used for immunofluorescence in tissue samples include CK18 (rabbit polyclonal CK18, Abcam, Cambridge, MA), UPII (mouse monoclonal N-18, Santa Cruz), and CK5 (mouse monoclonal AF-138, Covance Inc, Princeton, NJ). Slides were then incubated with Alexa Fluor 488 and Alexa Fluor 594 IgG secondary antibodies (Invitrogen) for 30 minutes and were then mounted with Vectashield mounting medium containing DAPI. For enzyme-based immunohistochemistry, slides were incubated with biotinylated immunoglobulins for 30 minutes (Vector Laboratories Inc, Burlingame, CA) followed by avidin-biotin complexes (Vector Laboratories) for 30 minutes. Diaminobenzidine was used as chromogen and hematoxylin as the nuclear counterstain. The immunohistochemistry images were collected using a standard light microscope, and the immunofluorescence images were collected and analyzed using a Nikon microscope and SPOT Advanced software. A positive result for the different p63 isoforms or p53 was considered when more than 10% of the tumor cells displayed a nuclear expression of the protein by immunohistochemistry or immunofluorescence. Mice used in this study were wild-type strain C57BL6 and p63-null strain B6.129S7-Trp63 (Jackson Laboratories, Bar Harbor, ME). All procedures were performed in accordance with the guidelines of the Institutional Animal Care and Use Committee. After sacrifice, bladders were removed and fixed in 10% buffered formalin for further paraffin embedding and immunostaining. A total of 202 patients diagnosed as having bladder cancer were included in this study for analysis of the association among p63 isoforms, p53, and clinicopathological variables. The associations of protein expression patterns with overall, disease-free, and recurrence-free survival, defined as the time elapsed between the date of diagnosis and last follow-up date or progression date, respectively, were assessed in 183 patients from whom all required data were available. The log-rank test and survival curves were plotted using the standard Kaplan-Meier method. Significant associations among different antibodies were assessed using χ2 test and Fisher's t-test. P < 0.05 was considered statistically significant. Statistical analyses were performed using SPSS v16.0 software (SPSS Inc, Chicago, IL). To investigate the expression of specific p63 protein isoforms, we first evaluated a panel of anti-p63 antibodies for their specificity. We transfected J82, a bladder cancer cell line from an invasive UC lacking detectable levels of p63 protein, with TAp63α, TAp63β, TAp63γ, ΔNp63α, ΔNp63β, or ΔNp63γ and a control backbone vector. Several isoform-specific p63 antibodies were tested by both immunoblotting and immunofluorescence assays, including newly generated ΔNp63, p63α, and p63α/β antibodies (Figure 1). Schematic illustration of p63 variants showing the transactivation domain, DNA binding domain, oligomerization domain, sterile α motif, and the antibodies that recognize the indicated p63 isoforms are shown in Figure 1A. Expression of TAp63β was substantially low; therefore, we detected a weaker band of this isoform probing with anti-Flag, p63 (4A4), and p63α/β antibodies (Figure 1B). As previously reported, p63 protein expression was localized to the nucleoplasm of the transfected cells4Yang A. Kaghad M. Wang Y. Gillett E. Fleming M.D. Dotsch V. Andrews N.C. Caput D. McKeon F. p63, a p53 homolog at 3q27-29, encodes multiple products with transactivating, death-inducing, and dominant-negative activities.Mol Cell. 1998; 2: 305-316Abstract Full Text Full Text PDF PubMed Scopus (1840) Google Scholar, 26Di Como C.J. Urist M.J. Babayan I. Drobnjak M. Hedvat C.V. Teruya-Feldstein J. Pohar K. Hoos A. Cordon-Cardo C. p63 expression profiles in human normal and tumor tissues.Clin Cancer Res. 2002; 8: 494-501PubMed Google Scholar (Figure 1C). To date, we have not been able to generate a TAp63-specific antibody. Therefore, for the purpose of this study, we assume that the TAp63 isoform is expressed when a nuclear positivity is observed with the total p63 antibody (4A4), but there is no ΔNp63 expression. To investigate the expression of p63 during urothelial development, we designed a strategy that allowed the analysis of protein levels of p63 variants in wild-type mice during18Koster M.I. Kim S. Mills A.A. DeMayo F.J. Roop D.R. p63 is the molecular switch for initiation of an epithelial stratification program.Genes Dev. 2004; 18: 126-131Crossref PubMed Scopus (552) Google Scholar embryogenesis, as well as in newborn and adult mice. Mouse urothelium displays histological features similar to human urothelium despite having only up to three layers in the mucosa,19Castillo-Martin M. Domingo-Domenech J. Karni-Schmidt O. Matos T. Cordon-Cardo C. Molecular pathways of urothelial development and bladder tumorigenesis.Urol Oncol. 2010; 28: 401-408Abstract Full Text Full Text PDF PubMed Scopus (207) Google Scholar which comprise a basal, an intermediate, and a superficial layer of umbrella cells. Basal and intermediate urothelial cells displayed nuclear positivity for all p63 antibodies analyzed, which validated the use of these antibodies in mouse tissue samples (Figure 2A). It was of interest to assess which p63 isoforms were implicated during embryogenesis and urothelial development. We were surprised to discover that the first isoform to be expressed was TAp63 because it could be detected in urothelium as early as at the embryonic age of 16.5 days (Figure 2B). Only later after birth, approximately 24 hours after delivery, did we detect expression of ΔNp63. Furthermore, we observed distinct expression of p63α before and after birth, indicating that the first isoform to be expressed is actually TAp63α (Figure 2C). We cannot rule out the existence of ΔNp63 protein below the levels of detection by the antibody during embryogenesis. However, even if that was the case, its level of expression significantly increased after birth to reach adult mice concentration. These results ar

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