Uroplakin II Gene Is Expressed in Transitional Cell

[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 ImÃ¡n 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 Â¡R-L.W., F-X. L, T-T. S.I, The Ronald Perelman Department of Dermatology, and Departments of Urology Â¡X-R.W.. T-T. S.Â¡,Mii-nihinltigy Â¡X-R.W.Â¡, 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.Â¡; Veteran Administration Medical Center, New York, New York 10010 [X-R. W./: and National Cancer Institute, Fom El- khalig, Cairo, Egypt Â¡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 mÃ© tastases (10-15 versus 40%), distant mÃ©tastases(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 (diseases, we of TCC mÃ©tastasesbut 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 OÃ-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 4Â°Cwith various antisera to UPs (diluted 1:10,000 in 2% BSA/PBS). Biotinylated goat antirabbit IgGs were applied for l h at 25Â°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. A human Tag DNA polymerase, and 0.1 /Â¿Iof ["P]dCTP (DuPont New England genomic library in A Fix-II phage (Stratagene) was screened with a 32P-labeledbovine UP-II cDNA (12), yielding two positive clones. The Nuclear. Cambridge, MA). PCR reactions were performed using a thermal cycler (Perkin-Elmer Corp.. Foster City, CA) by denaturation at 94Â°Cfor 3 one with a longer 5'-flanking sequence was further characterized. Two min, followed by 40 cycles of amplification (45 s at 94Â°C,45 s at 65Â°C,and Soci fragments (3.3 and 5 kb), which togethercontainedthe entire coding 45 s at 72Â°C),and were then kept at 72Â°Cfor 3 min. The PCR products were region of the human UP-II gene, were subcloned and sequenced (Fig. 1). denatured and loaded onto a denaturing 8% mutation detection enhancement The gene contained 5 exons spanning approximately 2 kb, similar to the polyacrylamide gel (J. T. Baker, Phillipsburg, NJ) and electrophoresed at room mouse gene (27). Its deduced amino acid sequence is 79% identical to temperature for 14-18 h. After electrophoresis, the gels were dried and exposed to X-ray film at 70Â°Covernight. Mobility-shifted DNA bands were those of the mouse (26, 27) and bovine (12) analogues, consisting of a hydrophobic NH-,-terminalsignal peptide (~25 amino acid residues), a reamplified and directly sequenced by the dideoxy method (31). prosequence (â€”59amino acid residues) harboring three potential N- Fluorescence in SiiÂ«Hybridization. A human UP-II genomic fragment was labeled with digoxigenin-dUTP by nick translation (Genome Systems. St. glycosylation sites and ending with a RGRR cleavage site for furin that Louis, MO). The labeled probes were combined with sheared human DNA and may be involved in UP-II processing/maturation,and a mature protein hybridized to normal metaphase chromosomes derived from phytohemagglu- (100 residues) with a COOH-terminal hydrophobic potential transmem tinin-stimulated peripheral blood lymphocytes from a male donor in a solution brane domain (â€”25residues;Fig. 2). containing 50% formamide, 10% dextran sulfate, and 2X SSC. Specific signals Chromosomal Localization of the Human UP-II Gene. A 21-kb for one-color experiments were detected by incubating the hybridized speci fragment of the human UP-II gene containing all five exons was labeled mens with tluoresceinated antidigoxigenin antibodies followed by counter- staining with 4',6-diamidino-2-phenylindole. Two-color experiments were with digoxigenin and used as a probe to hybridize to metaphase chro mosomes of human lymphocytes. The initial experiment resulted in conducted using the same protocol, except for the inclusion of biotin-labeled specificlabelingof the long arm of the group C chromosome, which was first probe followed by Texas Red-labeled avidin (32). believedto be chromosome 11on the basis of 4',6-diamidino-2-phenylin- Tissues. A cohort of 118 patients with primary bladder tumors was eval uated. Seventy-five cases were bilharzial bladder cancer; these samples were dole staining. A second experiment was conducted in which a biotin- obtained from the Pathology Department at the National Cancer Institute in labeled probe from the cyclin Dl locus, which has previously been Cairo, Egypt. Schistosomiasis infection was confirmed in all 75 of these cases mapped to 1Ipl3, was cohybridized with the UP-II probe. This experi by the presence of ova on histolÃ³gica! sections. Thirty-nine of these cases were ment resulted in the specific labelingof the proximal and distal long arm TCCs, whereas the remaining 36 were SCCs. An additional 43 cases of of chromosome 11(Fig. 3a). A total of 80 metaphasecells were analyzed, conventional TCC were obtained from the Pathology Department at the Me with 59 exhibiting specific labeling. Measurements of 10 specifically morial-Sloan Kettering Cancer Center in New York. H&E-stained sections of hybridizedchromosome 11s demonstrated that the UP-II gene is located all cases were examined to evaluate the pathological type, tumor grade, and at a position that is 82% of the distance from the centromere to the stage. Tumors were staged as Pis (carcinoma in situ, 1 case), PI (17 cases), P2 (4 cases), P3 (89 cases), and P4 (7 cases). Twenty-five tumors were classified telomere of the long arm of chromosome 11 to the region corresponding as low grade (grade 1), 68 were classified as intermediate grade (grade 2), and to band Ilq23 (Fig. 3Â¿>). 25 were classified as high grade (grade 3). Detection of a Polymorphism in the UP-II Gene. To examine Antibodies and Immunohistochemical Techniques. Three rabbit antisera possible polymorphism and potential tumor-specific mutations of the against UP proteins or peptides were used in this study: (a) a rabbit antiserum human UP-II gene, we performed SSCP assays on genomic DNA was raised against a synthetic peptide corresponding to amino acid residues from 57 cases of human TCCs. PCRs were done using five pairs of 7-19 (DSGSGFTVTRLLA) of the mature bovine UP (12); (b) a rabbit anti- primers corresponding to intron sequences that flanked exons 1-5 serum was generated against total UPs of highly purified bovine AUM, and (Fig. 4o). Only one major mobility-shifted band was found in exon 2 this antiserum reacts strongly with the 47-kDa UP-III, moderately with the of the UP-II gene in one tumor (case 265; Fig. 4b). This was due to 27-kDa UP Ib, and weakly with the 15-kDa UP-II (11, 26); and (c) an anti-47-kDa antibody was affinity-purified from the above antitotal UPs a G to A substitution at position 114 (Fig. 4c); this mutation is silent, against (he electrophoretically purified 47-kDa UP band (10, 11). The first and however, because both CCG and CCA encode proline. third antibodies were shown to be monospecific for the 15-kDa UP-II and the UPs Are Not Expressed in Bilharzial-relatedSCCs of the Urinary 47-kDa UP-III, respectively, by Â¡mmunoblotting. Bladder. To determinewhether UP-II is differentiallyexpressed in The avidin-biotin immunohistochemical staining was conducted as de TCCs versus SCCs, we conducted immunohistochemicalstaining assays scribed previously (33). Briefly, deparaffinized tissue sections were treated on these two types of bladder tumors using a rabbit antiserum to a with 1% hydrogen peroxide in PBS to block the endogenous peroxidase synthetic peptide corresponding to amino acid residues 7-19, located at 1292

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45 LVALPPCHLTGGNATLMVRRANDSK 811 901 991 VTSSFVVPPCRGRRELVSVVDSGAGFTVT 1081 100 RLSAYQVTNLVPGTKFY I_ ^_ 3 1171 1261 1351 GGTTCTCTACTTTCTCCCAAACCACAAAAGCATTTCCTACCTAGTGAAGAAGGGGACAGCCACTGAGTCCAGCAGAGAGATCCCAATGTC 117 3 ^ I ISYLVKKGTATESSREIPMS 1441 137 T L P RL 1531 1621 1711

141 ^i l R N M

144 ESIGLGMARTGGMVVITVLLSVAMFLLVLG 1981 174 FIIALALGSRK 2071 2161 2251 2341 2431 CACAGCCCA

Fig. 1. The deoxynucleotide and deduced amino acid sequences of the human UP-II gene. Arrows bracket the four introns; an asterisk marks the first base of the TA A stop codon: and a 3' potential polyadenylation sue (AATAAA} is underlined. the NH2 terminus of mature human/bovine UP-II. This antibody has positive. Only 1 of the 32 SCC cases (2.7%) was stained; reexami- previously been shown to be monospecific for UP-II by immunoblotting nation of the histology of this case revealed, however, that the staining (11, 12). Our results showed that most of the TCC specimens were UP-II was due to the presence of some TCC-like components in this tumor. positive (Fig. 5, A and B), thus confirming the earlier observation by Moll The difference between the UP staining of the bilharzial TCCs versus et al. (15). Interestingly, however, we found that bilharzial-related SCCs bilharzial SCCs was therefore highly significant (P = O.tXXHI. were not immunoreactive (Fig. 5C). A large panel of bladder cancer specimens consisting of 118 pa tients (43, 39, and 36 cases of conventional TCC, bilharzial TCC, and bilharzial SCC, respectively) were next surveyed using a polyclonal aMBaHBH rabbit antiserum to total UPs. Immunoblotting assays showed that this MASTL PVQTLPLILI LLAVLAPG.T ADFNISSLSG antiserum strongly recognized the 47-kDa UP-III and moderately WLVALPPCHLTMAS P PVWTLSWILIGGÃ‘ATLMVRRLLAVLVPGAAAÃ‘DSKWTSSADFNISSLSG85 recognized the 28-kDa UP-Ib, with relatively little reactivity toward UP-Ia and UP-II (11, 26). Like the UP-II antibody, this antiserum FWPPCRGRR strongly decorated the terminally differentiated umbrella cells of LLSPALTESL LIALPPCHLT GGNATLMVRR ANDSKWKSD FWPPCRGRR FWPFCRGRP,i50TATESSRE1P normal human urothelium; cells underneath the umbrella cells stained LLSPVMTESL1 LVALPPCHLTGFTVTRLSAYGGNATLTVRRQVTNL'.PGTKANDSKWRSSFYISYLVKKG only very weakly, if at all (Fig. 6/4). In addition to the strong staining of the superficial cells, papillary bladder carcinoma cells showed ELVSWDSGA MB36LLSPALTESLELVSWDSGS GYTVTRLSAY QVTNLTPGTK YYISYRVQKG TSTESSPETP irregular staining of the cytoplasm and cell borders of many cell layers ELVSWDSGS1MAPLLGFTVTRLSAYSPPIRTLPLILIQVTNLAPGTKLLALL5PG.AÃ•YISYLVTKG35ADFNISSLSGASTESSREIP within the tumor (Fig. 6ÃŸ).The invasive TCCs showed heterogeneous 51 100 staining with intense reactivity in some of the cells (Fig. 6C). In sharp H MSTLPRRMME SIGLGMARTG GMWITVLLS VAMFLLVLGF IIALALGSRK contrast, these antibodies did not stain the bilharzial SCCs analyzed. M MSTLPRKMME SIGLGMARTG GMWITVLLS VAMFLLWGL IVALHWDARK Even in its earliest stage of formation, histologically corresponding to B MSTFPRRKAE SIGLAMARTG GMWITVLLS VAMFLLVLGL IIALALGARK TMD dysplastic squamous metaplasia, UP staining was not discernible (Fig. Fig. 2. A comparison of the amino acid sequeiKOs of human, mouse, and bovine UP-II. 6D). Superficially invasive SCCs (Fig. 6Â£T)anddeep invasive lesions Rows H. M. and H denote the deduced amino acid sequences of human (this study), mouse with squamous features (Fig. 6F) were consistently UP negative. (26. 27). and bovine ( 12) UP-II. respectively. Note the positions of the signal peptide (SP). Our results indicate that 39.5% ( 17 of 43 cases) of the conventional the single COOH-terminal putative transmembrane domain (TMD). potential W-glycosylation siles (asterisks), and the putative furin consensus sequence (underlined} and TCC specimens were UP positive. However, in the case of the cleavage site (arrow) that precedes the NH2-terminal sequence (NH;-liLVSV. . . . ) of the bilharzial-related tumors, only 12.8% (5 of 39) of the TCCs were UP mature UP-II. 1293

Fig. 3. Localization of the human UP-II gene on chromosome 11 by the fluorescence in xitu hybridi/atinn technique. A, the simultaneous localization of UP-II gene (green) and the reference cyclin DI gene (rcd\ see "Materials and Methods"). B, an ideogram of human chromosome 11. Arrow, the position of the UP-II gene at location 1Iq23.

DISCUSSION replication error (40). Our initial study revealed a polymorphism but did not reveal any tumor-specific defects in the UP-II gene (Fig. 4). UP-II Gene in Bladder Cancers. The present study was under More studies are needed to test the possible occurrence of UP-II taken to assess whether detects in UPs may contribute to the patho- mutations in other clinical entities and to isolate and characterize the genesis of bladder diseases, specifically neoplastic processes. As a other human UP genes. prerequisite tor such analyses, we first isolated and characterized the Clinical Significance of UP Staining of Bladder Cancers. Our human UP-II gene (Figs. 1-3). We have previously mapped the UP-II data indicate that ~40% of the conventional TCCs were stained by an gene to mouse chromosome 9 and cattle chromosome 15. which antiserum to total UPs. a value reasonably close to the 88 and 53% predicted a human chromosome location of llq (37). Our current staining of the papillary and invasive TCC, respectively, as reported mapping of this gene to human chromosome 11q23 is therefore by Moll el til. (15), who used the same immunological reagent. We entirely consistent with our earlier data. found, however, that this antiserum stained only 13% of the bilharzial- Because UPs are the main urothelial-specialized products, defective related TCCs (5 of 39 cases), indicating that even the so-called TCCs UPs could potentially result in a greater turnover rate of umbrella of the bilharzial patients may not be equivalent to the conventional cells, leading to urothelial hyperplasia. Bladder epithelial hyperplasia TCCs seen in the Western world. It is possible that the TCCs of the caused by freezing (38) and uracil feeding (39) has been shown to bilharzial patients, many of whom suffer from vitamin A deficiency to induce bladder tumor formation, presumably due to increased DNA some degree (41), may exhibit various degrees of SCC features including, as we showed here, a reduced level of UP expression (see below). This interpretation is consistent with our histologicul finding that bilharzial-related bladder cancers tend to be somewhat heteroge 5' 3' l IV V neous, consisting of areas with TCC-like and SCC-like components. "Ã•97 Ã•29 199 216 Ã•9Ã•I bp An important corollary of this interpretation is that bilharzial-related bladder neoplasms may represent a continuous spectrum of lesions B ranging from pure TCC to pure SCC, with most of the specimens containing both tumor types. It would be interesting to determine whether the ratio of TCC:SCC contributions could be correlated with the serum vitamin A levels of the bilharzial patients. Regardless of the mechanism underlying the formation of bilharzial bladder SCC, it is important to note that the optimal treatment of bilharzial SCC differs from that of TCC. Thus, chemotherapy using 8 9 M-VAC has been found to be quite effective for treating invasive TCC but is relatively ineffective for treating bladder SCC and adenocarcinoma (42, 43). Immunohistochemical staining of bilharzial- o related bladder tumors using antibodies to UPs and other urothelial TÂ» markers provides a way to identify the TCC elements of both bilhar zial and conventional bladder tumors. Finally, our findings raise the fundamental question as to how the UP genes are suppressed in SCC. Future studies on how UP genes are regulated during normal urothe GATC GATC lial differentiation and squamous metaplasia may yield new insights Fig. 4. SSCP analysis of the UP-11 gene. A, genomic organization of the human UP-II into the molecular mechanism of squamous metaplasia and some gene and design of PCR primers for SSCP analysis. Open boxes and thin lines of the tup bar represent exons and introns. respectively. The lower ahorl lint's denote the analyzed initial events of bilharzial SCC formation. PCR products covering Ihe five exons; their lengths are indicated nehm: B. the gel Are SCCs More Differentiated Than TCCs? Because SCC elab electrophoretic pattern of the exon 2 PCR products of a normal individual (Lane /) and orates eosinophilic cornified cells that are easily discernible histolog- 8 TCCs (Lanes 2-9}. Note the presence of a mobility-shifted band (arrow) in one bladder cancer specimen (Lane 2; case 265). C. a direct sequencing gel of the normal (left) versus ically, whereas the differentiated products of urothelium and TCC, the mobility-shifted band (right). Note the C to T substitution (arrows). i.e., the 0.1-0.6-/MIT1 urothelial plaques, are invisible by light micros- 1294

Fig. 5. Immunohistochemical siaining of UP-II in human hilhar/ial TCCs and SCCs. Paraffin tissue sections were stained using a rabbit antiscrum raised against a synthetic oligopeptide corresponding to amino acid residues 7-19 located nearlhe NH, terminus of UP-II (II. 12, 26). A. invasive bilhar/ial bladder TCC: B. a conventional TCC: C. an invasive sec. copy, one can easily get an impression that SCC is more differentiated the contraction/expansion of the micturition cycle. As the major than TCC. Our data clearly demonstrate, however, that TCC expresses protein subunits of the urothelial plaques. UPs are excellent markers abundant levels of urothelial-specific differentiation marker, i.e., the of this normal or default pathway of urothelial differentiation. Several UP proteins, whereas SCC fails to express these differentiation mark other less-characterized AUM-associated antigens, including urohin- ers (Fig. 5 and 6). Therefore, judging the degree of urothelial differ gin (45), may also be useful markers of this pathway. The fact that entiation based purely on light microscopy can be misleading (see TCCs frequently retain UP expression (Ref. 15; Figs. 5 and 6) below for a discussion on the distinct differentiation pathways of SCC strongly suggests that they are derived from urothelial cells undergo and TCC). ing this line of differentiation. Multiple Pathways of Urothelial Differentiation and Their Pos Urothelium can also undergo keratini/.ation. which becomes the pre sible Contribution to Several Urothelial Malignancies. An enig dominant urothelial feature during vitamin A deficiency. Although it has matic feature of urothelium is its capacity to yield at least three rather frequently been said that vitamin A deficiency can induce many stratified diverse types of neoplasm, i.e., TCC, SCC, and adenocarcinoma. This squamous epithelia in the body to undergo keratini/ation. we have shown diversity becomes easily comprehensible, however, if one considers earlier that most of these epithelia undergo relatively limited extents of the possibility that mammalian urothelial cells can undergo at least keratinization, as judged histologically and biochemically (46). The ke- three pathways of differentiation, i.e., the urothelial type, the epider ratinizing rabbit corneal, conjunctiva!, and esophageal epithelia. for ex mal type, and the glandular type, that correlate with the three major ample, do not really histologically resemble the keratinized epidermis, types of urothelial neoplasm (Fig. 7). and they synthesize only limited amounts of keratins Kl and KIO (46). Under normal conditions, terminally differentiated urothelial cells molecular markers for keratinization (46, 47). In this regard, bladder undergo membrane specialization, synthesizing as their major differ epithelium is exceptional, in that vitamin A deficiency can induce its entiation products the L'P proteins that form numerous rigid-looking transformation to a fully keratinized epithelium with the formation of plaques covering the apical surface of the umbrella cells (10, 26. 44). well-formed spinous. granular, and comified cell layers (19). Such a These superficial cells apparently fulfill the function of forming a keratinizing urothelium synthesizes not only KI and KIO keratinization highly effective and durable permeability barrier that can withstand markers but also K6 and K16 keratin markers characteristic of hyper-

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Fig. 6. Immunohistochemical staining of UP-Ib/UP-III in human bilhar/ial TCCs and SCCs. Staining was performed using a rabbit antiserum to total UPs; this anliserum reacts strongly with UP-III, moderately with UP-Ib. and weakly with UP-Ia and UP-II (10, 11). A. normal human urothelium; fl. a papillary superficial noninvasive TCC; C an invasive high-grade TCC; D. a metaplastic squamous urothelium: E. an intermediate-stage SCC producing papillary fronts: F, the invasive edge of a high-grade SCC. Note the strong staining of the umbrella cells of normal urothelium in A. the diffuse and disorganized staining pattern in ÃŸand C. and the lack of staining in SCC specimens. 1295

through the sharing of a common pool of (relatively) undifferen- Pathways of Urothelial Differentiation tiated basal cells; some of them may fall into the category of stem cells (60-62). These urothelial basal cells are by definition pluri of potent, because they are capable of undergoing several pathways of Differentiated differentiation in responding to appropriate environmental signals Cells__ (Fig. 7). This situation is somewhat different from that of the keratinocytes of skin, cornea, and esophagus, which are unipotent, Vit-r cellâ€”Cornified capable of differentiating along one of the major keratinocyte pathways (the so-called skin-type, cornea-type, and esophagus- â€¢otheliumâ€”KmbryoPhenotypecellâ€”Umbrella type pathways that are marked by the synthesis of Kl/KIO, K3/ Glandular epith.TumorSCCTCCAdeno-CAMarkerKl/KIOUroplakinsPSA/PSM?K12, and K4/K13 keratin pairs; Refs. 47, 49, and 63). UGSM Concluding Remarks. Although it has been known for some time Fig. 7. A schematic diagram illustrating the alternative pathways of urothelial differenti that urothelium can give rise to a wide range of neoplasms, the cellular ation. Vitamin A deficiency promotes urothelial keratini/ation accompanied by the expression origin of and interrelationship among these tumors have not been ad of keratins Kl/KIO. markers for keratini/ation (46. 471; it is hypothesized that urothelial cells undergoing this differentiation pathway will give rise to SCC. Normal urothelium expresses dressed in detail. We have presented here a hypothesis (Fig. 7) that takes UPs as its major differentiation products; such cells form TCC. Embryonic urogenital sinus into account not only our observation that bilharzial SCC fails to express mesenchyrne or other mesenchymal signals can induce urothelium to /mm-differentiate into UPs (Figs. 5 and 6), but also many enigmatic and puzzling properties of a glandular epithelium secreting specific proteins such as prostate-specific antigen: such urothelial cells are hypothesized to yield adenocarcinoma. The terminally differentiated (and mammalian urothelium. These include its capability to undergo squa suprabasally locatedl cells of all these pathways most likely originate from a common pool of mous (19) and glandular metaplasia (51, 55-59) and to yield diverse pluripotent basal cells (see the text). forms of carcinoma including TCC, SCC, and adenocarcinoma (20, 21, 52, 54). More data are needed to test this hypothesis by studying the plastic keratinocytes (19, 48. 49). This raises the question of whether potential of urothelial differentiation, preferably using clonal populations vitamin A deficiency plays a role in promoting squamous metaplasia, of urothelial cells (64, 65). and the molecular regulation of genes encod which, with additional bilharzial factors, eventually yields SCC. This ing UPs as well as markers of the postulated SCC and glandular path possibility is supported by the finding that epithelial metaplastic changes ways. Data already exist demonstrating the in vivo convertibility of TCC are commonly noted and apparently represent early changes in the de to a glandular tumor in response to appropriate mesenchymal induction velopment of bilharzial SCC; thus bilharzial SCC of the bladder and (59), thus raising the interesting possibility that one may be able to cause ureter seems to progress from squamous metaplasia to in situ carcinoma in vivo interconversion between other types of bladder neoplasm, such as and finally to infiltrating carcinoma (50). In addition, El-Aaser et al. (41) conversion from SCC to TCC or vice versa, as modulated by the vitamin found that bilharzial bladder cancer patients with SCC, the most prevalent A level. The conversion of SCC to TCC, which is more amenable to type in Egypt, had significantly lower serum levels of vitamin A than currently available radiotherapies and chemotherapies (42, 43), can have normal male subjects. In contrast, bilharzial bladder cancer patients with major biological implications as well as clinical ramifications. TCC had levels that were not significantly different from those of normal male subjects (41). REFERENCES The third type of differentiation that can be adopted by a postnatal Porter, K. R.. and Bonneville. M. A. An Introduction to the Fine Structure of Cells mammalian urothelium is that of glandular epithelium. Of particular and Tissues. New York: Lea and Febiger, 1963. interest is the proven capability of postnatal bladder epithelium to be Hicks, R. M. The fine structure of the transitional epithelium of rat ureter. J. Cell Biol.. 26: 25-48. 1965. converted to a prostatic epithelium (51). Thus, postnatal rat bladder Koss. L. G. The asymmetric unit membranes of the epithelium of the urinary bladder epithelium, when combined with embryonic urogenital sinus mesen- of the rat. 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A hexagonal arrangement of frequent than SCC and TCC (24, 52-54). involve the activation of this subunits in membrane of mouse urinary bladder. J. Mol. Biol.. 46: 593-596. 1969. Brisson. A., and Wade. R. H. Three-dimensional structure of luminal plasma mem particular pathway of urothelial differentiation. Consistent with this pos brane protein from urinary bladder. J. Mol. Biol., 166: 21-36, 1983. sibility, a recent survey indicated that a subset of locally advanced and Taylor. K. A., and Robertson. J. D. Analysis of the three-dimensional structure of the metastatic TCCs is prostate-specific antigen positive (55). Colonie epi urinary bladder epithelial cell membranes. J. Ullrastruct. Res., 87: 23-30, 1984. Walz. T., Haner, M., Wu, X-R.. Henn, C, Engel. A.. Sun. T-T, and Aebi. U. Towards thelial metaplasia can also occur in bladder epithelium (56-58). More the molecular architecture of the asymmetric unit membrane of the mammalian over, Fujii et al. 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16. Kitinya, J. N.. Lauren. P. A., Eshleman. L. L. Paljarvi, L.. and Tanaka. K. The 42. Loehrer. P. J.. Einhorn. L. H.. Elson, P. J.. el al. A randomized comparison of incidence of squamous and transitional cell carcinomas of the urinary bladder in cisplatin alone or in combination with methotrexate. vinblastine. and doxoruhicin in northern Tanzania in areas of high and low levels of endemic Schi.

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Downloaded from cancerres.aacrjournals.org on September 26, 2021. © 1998 American Association for Cancer Research. 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 Formation

Ren-Long Wu, Iman Osman, Xue-Ru Wu, et al.

Cancer Res 1998;58:1291-1297.

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