[CANCER RESEARCH 58. 1291-1297. March 15. 19981 Uroplakin II Gene Is Expressed in Transitional Cell Carcinoma But Not in Bilharzial Bladder Squamous Cell Carcinoma: Alternative Pathways of Bladder Epithelial Differentiation and Tumor Formation1 Ren-Long Wu,2 Imán Osman,2 Xue-Ru Wu, Ming-Lan Lu, Zuo-Feng Zhang, Feng-Xia Liang, Reda Hamza, Howard Scher, Carlos Cordon-Cardo, and Tung-Tien Sun3 Epithelial Biology Unii ¡R-L.W., F-X. L, T-T. S.I, The Ronald Perelman Department of Dermatology, and Departments of Urology ¡X-R.W.. T-T. S.¡,Mii-nihinltigy ¡X-R.W.¡, and Pharmacology, Kaplan Cancer Center [T-T, S.}, New York University Medical School, New York, New York 10016; Memorial Sloun-Ki'llei'inx Cancer Center, New York, New York 10021 [I. O.. M-L L, Z-F. 7... H. S.. C. C-C.¡; Veteran Administration Medical Center, New York, New York 10010 [X-R. W./: and National Cancer Institute, Fom El- khalig, Cairo, Egypt ¡R.H.\ ABSTRACT However, the earlier survey did not include Schistosoma infection- related bilharzial bladder neoplasms. Bladder tumors in Schisiosnma- Uroplakins (UPs) are integral membrane proteins that are synthesized infected patients are distinct from the uninfected population, in that as the major differentiation products of mammalian urothelium. We have cloned the human UP-II gene and localized it on chromosome Ilq23. A the former are predominantly SCCs (over 70%) rather than the usual survey of 50 transitional cell carcinomas (TCCs) revealed a UP-II poly TCCs (16, 17). Because schistosomiasis is particularly prevalent in morphism but no tumor-specific mutations. Immunohistochemical stain the Middle East and in African countries, bilharzial bladder cancer is ing using rabbit antisera against a synthetic peptide of UP-II and against the most common malignancy in this region, accounting for 28% of all total UPs showed UP reactivity in 39.5% (17 of 43 cases) of conventional cancers (39 and 11% of cancers in males and females, respectively; TCCs, 12.8% (5 of 39) of bilharzial-related TCCs, and 2.7% (1 of 36) of Ref. 17). The mechanism by which Schistosoma induces bladder bilharzial-related squamous cell carcinomas (SCCs). The finding that cancer is unclear, but it has been postulated that the infection may lead fewer bilharzial TCCs express UPs than conventional TCCs (12.8 versus to malignancy through local tissue damage, mechanical irritation, 40%) raised the possibility that the former are heterogeneous, expressing bilharzial toxins, or even secondary bacterial infection (17, 18). Vi SCC features to varying degrees. Our data strongly support the hypoth tamin A deficiency, which can cause dramatic squamous metaplasia esis that urothelium can undergo at least three pathways of differentia tion: (a) urothelium-type pathway,- (h) epidermis-type pathway; and 10 of the bladder urothelium (19), may contribute to the frequent forma glandular-type pathway, characterized by the production of UPs, K1/K10 tion of SCC (17). The biological behavior of the SCC is significantly keratins, and secreted glycoproteins, respectively. Vitamin A deficiency different from that of the TCC (17. 18, 20. 21) in terms of macro and mesenchymal factors may play a role in determining the relative scopic type (SCC and TCC are mostly bulky invasive solid tumors contributions of these pathways to urothelial differentiation as well as to and papillary lesions, respectively), age of onset (fifth versus sixth to the formation of TCC, SCC, and adenocarcinoma, or a mixture thereof. seventh decades), differentiation status (more versus less differenti ated as defined by squamous features; see "Discussion"), nodal mé tastases (10-15 versus 40%), distant métastases(lessversus more INTRODUCTION frequent), and radio- and chemosensitivity (relatively resistant versus The apical surface of terminally differentiated urothelial cell is sensitive). In addition, the spectrum of p53 gene mutation in bilharzial covered with numerous rigid-looking plaques that contain a highly bladder tumors was found to be distinguishable from that of the specialized plasma membrane termed the AUM4 (1-4). This mem conventional TCC, suggesting a unique etiological background (22- brane consists of protein particles arranged in an ordered hexagonal 24). Thus, a clear differential diagnosis and a better understanding of lattice with p6 symmetry and a lattice constant of 16 nm (5-9). We the interrelationship between these two main types of bladder cancer have shown previously that mammalian urothelial plaques contain can be clinically significant. four major integral membrane proteins: (a) UP-Ia (27 kDa); (b) UP-Ib Unlike UP-Ia and UP-Ib. which have four putative transmembrane (28 kDa); (c) UP-II (15 kDa); and (</)UP-III (47 kDa; Refs. 10-13). domains (13), UP-II and UP-III have only one transmembrane domain Most of the hydrophilic domains of UPs project luminally (11-13), (11-13). Recent data indicate that UP-II and UP-III are preferentially forming the 16-nm particle seen above the lipid bilayer (7-9). cross-linkedto UP-Ia and UP-Ib,respectively,suggestingthe existenceof Through their binding to the cytoskeleton, these plaques may stabilize two types of 16-nm AUM particles consisting of UP-Il/UP-Ia and UP- the urothelial luminal surface, preventing it from rupturing during III/UP-Ib (25). The cDNA-derived amino acid sequences of mouse and bladder distention (4, 14). A significant proportion of urothelial- bovine UP-II are 83% identical, suggesting a high degree of structural derived tumors still express UP, because antibodies to UPs can stain and possibly functionalconservation (26). To determine whether genetic- 88% of papillary noninvasive TCCs, 53% of invasive TCCs, and 66% defects in UPs can contribute to any of the human bladder diseases, we of TCC métastasesbut not any of the nonurothelial carcinomas (15). set out to clone and characterizehuman UP genes. Because mouse UP-II These data suggest that UP can serve as a unique marker for the is the smallest UP, in terms of both its gene (5 exons spanning 2 kb; Ref. positive identification of urothelial-derived TCCs (15). 27) and its proteinsize (85 and 100amino acid residuesfor the prepro and mature sequences, respectively; Refs. 10 and 27), we decided to focus Received 9/10/97; accepted 1/15/98. initially on the human UP-II gene. The costs of publication of this article were defrayed in part by the payment of page In this paper, we report the isolation, chromosomal localization, and charges. This article must therefore be hereby marked advertisement in accordance with detection of a polymorphism of the human UP-II gene. In addition, we 18 U.S.C. Section 1734 solely to indicate this fact. 1Supported by NIH Grants DK39753. DK47529, and DK49469 (to T-T. S.) and show that whereas UPs are expressed in many TCCs, UP-II and other CA47538 (to C. C-C). UPs are down-regulated in bilharzial-related SCC. Our results suggest 2 R-L. W. and I. O. contributed equally to this work. 3 To whom requests for reprints should be addressed, at Department of Dermatology. that bilharzialTCC may be heterogeneous,containing some UP-negative New York University Medical School. 550 First Avenue, New York, NY 10016. SCC components. Furthermore, we postulate that urothelium can un 4 The abbreviations used are: AUM, asymmetric unit membrane; SCC. squamous cell carcinoma; SCCP, single-strand conformational polymorphism; TCC, transitional cell dergo alternative pathways of differentiation,each characterized by the carcinoma; UP, uroplakin. expression of specific differentiation markers, and that these alternative 1291 Downloaded from cancerres.aacrjournals.org on September 26, 2021. © 1998 American Association for Cancer Research. ALTERNATIVE PATHWAYS Oí-UROTHIÃœ.IAL II MOR IORMATION differentiationpathways may contributeto the formation of various types activity and incubated with normal goat serum (diluted 1:10 in 2% BSA), of bladder neoplasm including TCC, SCC, and adenocarcinoma. followed by overnight incubation at 4°Cwith various antisera to UPs (diluted 1:10,000 in 2% BSA/PBS). Biotinylated goat antirabbit IgGs were applied for l h at 25°C(Vector Laboratories, Burlingame, CA; 1:800 dilution), followed by avidin-biotin peroxidase complexes for 30 min (Vector Laboratories; 1:25 MATERIALS AND METHODS dilution). Diaminobenzidine and hematoxylin were used as the final chromo- Genomic Cloning and SSCP. A human genomic library in A Fix-II phage gen and nuclear counterstain, respectively. The immunoreactivities were clas (Stratagene, La Jolla, CA) was screened (28) using a 32P-labeled bovine UP-II sified as positive if more than 20% tumor cells showed positive membrane cDNA (12). PCR-SSCP assays were performed on a subset of 57 bladder and/or cytoplasmic staining. tumors using a modification (29) of the method by Orila et al. (30). The Statistical Methods. The associations between the UP expression patterns sequences of the five sets of PCR primers used to amplify exons 1-5 of the and clinicopathological parameters, including tumor stage, grade, and tumor human UP-II gene, as shown in Fig. 4, are as follows: Exon 1, 5'-CTGCCAG- type, were assessed by Fisher's exact test (34); the two-tailed Ps were used to CACCTATTCCACCTC-3' and 5'-CCTGCCAGAGATGGAAGAGC-3'; assess the significance level. For variables with more than two categories, the exon 2,5'-CCATCGGAGCTCCCTCTGC-3' and 5'-GGGACTAGAGGGAT- dose-response relationship was assessed by the trend test using the Mantel- GCCTTG-3'; exon 3, 5'-GAAACTTGACCCAGTCTTCC-3' and 5'-CTTC- Haenszel method (35). The FREQ procedure in SAS was used for analyzing CCTAGGTGCCTCAGG-3'i exon 4, 5'-CTCTTCCTGTAAGTCCCAAT- the data (36). AC-3' and 5'-GAATGGTCAG GGAAGCGTTTG-3'; and exon 5. 5'-CCA- CAGTGGTCTCCCCTCTC-3' and 5'-CTGGAGAAGCTGCTGCTCCG-3'. Each PCR reaction mixture contained 100 ng of tumor genomic DNA in 10 RESULTS H\ of 10 ITIMTris-HCl (pH 8.3), 50 mM KC1, 2.5 mM MgC2, 250 JIM each of cold deoxynucleotide-5'-triphosphate, 1.5 /LIMeach PCR primer, 0.5 unit of Isolation and Characterization of Human UP-II Gene.