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Clinical Cancer Research Vol. 12, 2780-2787, May 1, 2006
© 2006 American Association for Cancer Research

Imaging, Diagnosis, Prognosis

N-Cadherin as a Novel Prognostic Marker of Progression in Superficial Urothelial Tumors

Isabelle Lascombe1,2,3, Anne Clairotte4, Sylvie Fauconnet1,2,4, Stéphane Bernardini3, Hervé Wallerand3, Bernadette Kantelip4 and Hugues Bittard3

Authors' Affiliations: 1 Tissue and Cell Biology Engineering; 2 Faculty of Medicine and Pharmacy, University of Franche-Comte; 3 Urology and Andrology Department, St. Jacques University Hospital; 4 Pathologic Cytology and Anatomy Department, Jean Minjoz University Hospital, Besançon, France

Requests for reprints: Isabelle Lascombe, Tissue and Cell Biology Engineering, IFR 133, 240 route de Dole, 25000 Besançon, France. Phone: 33-3-63-08-22-28; E-mail: isabelle.lascombe{at}voila.fr.


   Abstract
Top
 Abstract
Materials and Methods
 Results
 Discussion
 References
 
Purpose: Loss of intercellular adhesion and increased cell motility promote tumor cell invasion and spreading. In bladder cancer, loss or reduced E-cadherin expression has been associated with poor survival, and aberrant expression of N-cadherin has been associated with the invasive phenotype of bladder carcinoma cells. The purpose of this study was to investigate whether N-cadherin expression was associated with the bladder tumor progression.

Experimental Design: E-cadherin and N-cadherin expression was evaluated by immunohistochemistry in 101 tumors (pT1 and pT2-T3) and by reverse transcription-PCR analysis and immunohistochemistry in 28 other fresh frozen tumors (pTa, pT1, and pT2-T3).

Results: N-cadherin expression was absent in normal urothelium, appeared in stage pT1, and increased in pT2-pT3 tumors. In most cases, increased N-cadherin expression in invasive tumors was associated with loss of E-cadherin expression. Progression-free survival and multivariate analyses revealed that N-cadherin expression is an independent prognostic marker for pT1 tumor progression. Analysis of the 28 frozen tumors by immunohistochemistry and reverse transcription-PCR showed a good correlation between protein and gene expression in pT1 and pT2-T3 tumors. Interestingly, in pTa tumors, N-cadherin was not immunodetected, whereas mRNA was present in 50% of cases.

Conclusion: Regulatory defects in the N-cadherin promoter, abnormalities at the translational, or protein processing levels could explain the discrepancies between protein and mRNA expression. Most importantly, this study identified N-cadherin as a novel prognostic marker of progression in superficial urothelial tumors. Clearly, N-cadherin acts in an invasive mode in bladder cancer, but whether it has a primary role in urothelial neoplastic progression has yet to be investigated.

Transitional cell carcinoma of the bladder is the origin of >90% of bladder carcinomas and is classified into superficial (75%) and invasive tumors (25%). The clinical course of superficial (pTa and pT1) papillary urothelial cell carcinoma is characterized by a high risk of recurrence (up to 70%) and a propensity to progress in grade (10-30%) or stage (10-15%; ref. 1). Muscle-invasive tumors (≥pT2) have a poorer prognosis (2) because 50% of these patients will relapse with metastatic disease within 2 years of treatment (3). The cornerstone of diagnosis remains cystoscopy evaluation with transurethral resection. The follow-up policy and treatment of patients with superficial tumors predominantly depends on conventional variables, such as grade and stage, and the associated presence of carcinoma in situ (4, 5). Therefore, extensive research has been carried out to find the reliable molecular markers of progression. However, none of these markers has been accepted into clinical routine.

It is now well established that alterations in the expression and function of cell-cell adhesion molecules, such as cadherins, correlate with the progression to tumor malignancy. Classic cadherins are transmembrane glycoproteins that mediate calcium-dependent cellular adhesive interactions (6) and have been implicated in the invasive process. E-cadherin and N-cadherin, some of the most extensively characterized members of this family, display a unique tissue distribution. Although E-cadherin is expressed in virtually all epithelial tissues (7), N-cadherin is predominantly recovered in neural tissues but is also present in fibroblasts, skeletal muscle, and endothelial cells (8–10).

E-cadherin, which is the most extensively studied member, has been reported to play an invasive suppressor role (11, 12). Loss or reduced expression of E-cadherin was detected in different cancers and, in bladder cancer, is correlated with increased invasive potential and poor survival (13–18). Aberrant expression of N-cadherin has been associated with the invasive phenotype in bladder carcinoma cell lines, meaning that N-cadherin does not play an invasive suppressor role in this pathology (19). This observation has also been reported in breast (20), prostate (21), and melanoma (22, 23) cell lines. A recent study showed that N-cadherin, transfected in bladder carcinoma cell lines expressing E-cadherin, promotes invasion.

Several studies on human cancers have reported the presence of a cadherin switching from E-cadherin to N-cadherin (22, 24). In human breast carcinoma cell lines, E-cadherin and N-cadherin are mutually exclusive where cell lines expressing N-cadherin displayed a fibroblast morphology (20). Transfection of N-cadherin in breast tumor cells induced cell migration, invasion, and metastasis in a dominant fashion because the migratory behavior was elicited even when N-cadherin was coexpressed with E-cadherin (25, 26). Mutual exclusivity of E-cadherin and N-cadherin expression in bladder cancer cell lines is not necessarily the case. Numerous bladder cell lines have been identified to coexpress E-cadherin, P-cadherin, and N-cadherin. This cadherin expression profile has been confirmed to occur in human bladder tumors (27). In an earlier study, we reported that in spite of the decreased expression of E-cadherin in invasive bladder tumors, their cytoplasmic partners (i.e., catenins) still expressed, suggesting the presence of an other cadherin family member, such as N-cadherin (28).

Despite of the accumulation of these results regarding N-cadherin, only a few clinical studies on N-cadherin expression in human cancers were reported and, its true function in human bladder cancer is still unknown. Although no study has clearly shown an independent prognostic use of the cadherin-catenin complex, the loss of membranous expression of one or more of those glycoproteins has been unanimously attributed to an aggressive phenotype of bladder cancer.

Thus, the purpose of this study was to investigate the expression of N-cadherin according to the tumor-node-metastasis stage (1973 WHO and 2004 WHO/International Society of Urological Pathology classifications). To better characterize pT1 tumors for which the clinical outcome is unpredictable, we evaluated, for the first time, the prognostic value of this adhesion molecule within a cohort of 101 patients to identify a new molecular marker involved in bladder cancer progression. In a second series of patients comprising pTa with pT1 and pT2-T3 tumors, we carried out immunodetection and reverse transcription-PCR (RT-PCR) analyses for N-cadherin and E-cadherin expression to evaluate the cellular level of regulation of these molecules. Major data from the present study revealed that N-cadherin was not immunodetected on pTa tumors, whereas in 50% of cases, the mRNA was expressed. Most importantly, N-cadherin expression was a prognostic marker of progression for the pT1 superficial tumors.


   Materials and Methods
Top
 Abstract
 Materials and Methods
Results
 Discussion
 References
 
Tumor samples. Bladder tumor samples from transurethral resection or cystectomy specimens were obtained from two cohort of patients. Thus, N-cadherin and E-cadherin expression was evaluated in 101 cases with transitional cell carcinoma of the bladder by immunodetection and in an independent tumor set of 28 fresh frozen specimens by immunohistochemical and RT-PCR analyses (within the same tumor). Specimens of normal bladder mucosa were obtained from bladder biopsies taken from benign prostatic hyperplasia patients who underwent transurethral surgeries. Histology, grade, stage, and presence of carcinoma in situ were determined by pathologic examination of the transurethral resection or cystectomy and were confirmed by blinded re-review of the original cystoscopic biopsy slides. Tumors were staged according to the 1997 tumor-node-metastasis staging system guidelines (29) and graded according to the 1973 WHO classification and the most recent classification system proposed in 2004 (30), which basically corresponds to the 1998 WHO/International Society of Urological Pathology classification. The median follow-up was 58.1 months (range, 5-188 months) for the cohort of 101 patients. Table 1 summarizes clinicopathologic characteristics of the analyzed samples.


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  		Table 1. Clinicopathologic features of two cohorts of patients with bladder cancer

 
Immunohistochemistry. Tissue samples, obtained by either endoscopic resection or partial cystectomy, were fixed in 4% formalin and paraffin embedded. Then, blocks were cut serially at 4-µm thickness, deparaffinized in toluene, and rehydrated in graded ethanol. Antigen retrieval was achieved by microwave treatment in 0.5 mol/L TBS (pH 6) at 750 W for 30 minutes. Sections were then incubated with primary mouse at room temperature as previously described (28) using an automated immunohistochemical processor (Techmate 500 Plus, DakoCytomation SA, Trappes, France) according to the manufacturer's instructions. The following primary antibodies were used: anti N-cadherin (monoclonal, clone 3B9; Zymed Laboratories, Inc., Montrouge, France; dilution 1:200) and anti E-cadherin (monoclonal, clone HECD-1; Zymed Laboratories; dilution 1:600). Slides were then treated with biotinylated goat anti-mouse IgG (DakoCytomation) and avidin combined in vitro with horseradish peroxidase. Endogenous peroxidase activity was removed by dipping the sections in 5% hydrogen peroxide for 10 minutes at room temperature followed by incubation with streptavidin-horseradish peroxidase for 25 minutes. Finally, peroxidase activity was revealed by 3,3' diaminobenzidine staining (0.9 mg/mL) for 15 minutes. Sections were counterstained with Harris' hematoxylin, dehydrated through alcohol, and mounted using a standard procedure. Negative controls were obtained by omitting the first antibody. The status of N-cadherin and E-cadherin was assessed in a coded manner by a pathologist without knowledge of the clinical or pathologic features of the case. The proportion of stained cells and the cellular localization of immunostaining were used as criteria of evaluation. In normal urothelium, the presence of a functional E-cadherin is visualized by a membranous staining outlining regions of cell-cell contact, which should be seen virtually in all the cells. E-cadherin immunoreactivity in tumor samples was scored according to a classification derived from the work of Shiozaki et al. (31) and Bringuier et al. (13), in which immunostaining was distinguished as normal and abnormal. If the staining pattern was similar to that of normal urothelium (i.e., >90% of the cells with membranous staining), it was evaluated as normal. Abnormal staining was divided into negative staining (<10% of the cells with membranous staining) and heterogenous staining (between 10% and 90% of the cells with membranous staining). Tumors with an abnormal E-cadherin immunostaining, defined as those giving negative staining or heterogeneous staining, were considered one group in statistical analysis. The definition of staining for N-cadherin was as follows: positive, when tumor cells showed immunoreactivity with a membranous staining; negative, when 0% tumor cells showed immunoreactivity. Normal urothelium was used as a negative control for N-cadherin expression because normal bladder does not express this adhesion molecule. Myocardium was used as a positive control tissue in which a positive immunohistochemical detection of N-cadherin was observed. In few cases, a cytoplasmic staining was observed for both E-cadherin and N-cadherin. This was not considered as a staining for a functional protein, and tumor samples were not included in the analysis.

Cell lines. The RT4 and T24 cell lines were purchased from the American Type Culture Collection (Biovalley, Conches, France). The cells were maintained at 37°C in a 5% CO2 atmosphere in McCoy's 5a medium (Invitrogen, Cergy Pontoise, France) supplemented with 10% FCS (Invitrogen), 1% antibiotic antimycotic mixture (10 mg/mL streptomycin, 10,000 units/mL penicillin, 25 µg/mL amphotericin B), 2 mmol/L glutamine, and 15 mmol/L HEPES (Sigma, Saint Quentin Fallavier, France). The cells were tested for the absence of Mycoplasma before total RNA extraction.

Total RNA extraction and RT-PCR. Total RNA was isolated and purified from fresh frozen bladder cancer tissues using the commercially available Qiagen RNA/DNA kit (Qiagen SA, Courtaboeuf, France) and from RT4 and T24 cells using TRIzol reagent purchased from Invitrogen, according to the manufacturer's guidelines. For reverse transcription reaction, 3 µg of the RNA, oligo(dT)12-18 (Invitrogen) and Moloney murine leukemia virus reverse transcriptase (Invitrogen) were used according to the manufacturer's instructions. Subsequent amplifications of the partial cDNA encoding E-cadherin, N-cadherin, and glyceraldehyde-3-phosphate dehydrogenase were done with the following specific oligonucleotide primers: E-cadherin sense, 5'-TCCATTTCTTGGTCTACGCC-3' and antisense, 5'-CACCTTCAGCCAACCTGTTT-3'; N-cadherin sense, 5'-GTGCCATTAGCCAAGGGAATTCAGC-3' and antisense, 5'-GCGTTCCTGTTCCACTCATAGGAGG-3'; glyceraldehyde-3-phosphate dehydrogenase sense, 5'-CCAGCCGAGCCACATCGCTC-3' and antisense, 5'-ATGAGCCCCAGCCTTCTCCAT-3'. PCR conditions (cDNA amount and cycle number) have been perfected and optimized for E-cadherin, N-cadherin, and the glyceraldehyde-3-phosphate dehydrogenase internal control. Finally, the PCR mixtures were subjected to 30 cycles of amplification by denaturation (30 seconds at 94°C), hybridization (30 seconds at 60°C for E-cadherin and glyceraldehyde-3-phosphate dehydrogenase and 30 seconds at 65°C for N-cadherin), and elongation (1 minute at 72°C). PCR cycles are followed by a last elongation step at 72°C for 10 minutes. The PCR products were analyzed by 2% agarose gel electrophoresis with ethidium bromide. The expected sizes of PCR products for E-cadherin, N-cadherin, and glyceraldehyde-3-phosphate dehydrogenase were 361, 373, and 359 bp, respectively. Negative controls for reverse transcription and PCR amplifications were done in the absence of mRNA or directly on mRNA and yielded no detectable band.

Statistical analysis. Statistical analysis was carried out first on the entire group of 101 patients (71 pT1 and 30 pT2-T3). Differences in expression of N-cadherin according to patient and cancer characteristics were assessed by {chi}2 and Fischer exact tests. Second, survival without progression was studied on the pT1 superficial bladder tumor subgroup. According to patient and cancer characteristics, curves were built and compared using the Kaplan-Meier procedure and the log-rank test. A Cox proportional hazards model was then used to select the independent prognostic factors from the variables found to be associated with survival without progression (P < 0.20) in the univariate analysis. The proportional hazards assumption was tested using time-dependent factors (32), and a violation of this assumption was detected for the stage T1a/T1b variable. Therefore, the final Cox proportional hazards model was stratified on this factor. Statistical significance was set at the 5% level. Analyses were done with SYSTAT 10 for windows (SPSS, Inc., Chicago, IL).


   Results
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 Abstract
 Materials and Methods
 Results
Discussion
 References
 
Comparison of results according to the 1973 WHO and 2004 WHO/International Society of Urological Pathology classifications. The essential improvement of the histologic prognosis and the reproducibility of the grading for papillary urothelial neoplasms of the bladder led to introduce a number of classification systems for the grading of these tumors. Indeed, the reproducibility of the grading has been recently discussed. The International Society of Urological Pathology and WHO have proposed new criteria for new classifications in 1998 and 1999, respectively, and very recently in 2004. These classifications have not been approved by all pathologists. Some defended, therefore, the refinement of the 1973 WHO classification for the grading. Thus, despite the absence of detailed defining criteria, the 1973 WHO classification has remained used. In this context, our series of 101 and 28 patients originally diagnosed according to the 1973 WHO classification were regraded by the revised 2004 WHO/International Society of Urological Pathology system. The overall results obtained for N-cadherin and E-cadherin expression were not significantly different according to both classifications.

N-cadherin expression in pT1 superficial tumors compared with muscle-invasive carcinomas. We analyzed the expression of N-cadherin in 71 cases of pT1 tumors and 30 cases of pT2-T3 tumors by immunohistochemistry. Myocardium was used as a positive control tissue, in which a positive immunohistochemical detection was observed (Fig. 1A ). Indeed, as expected, N-cadherin expression was undetected in normal bladder urothelium (Fig. 1B). A summary of the results of N-cadherin staining in tumors is shown in Table 2 . Membranous N-cadherin staining was detected at the cell-cell borders (Fig. 1C) and recorded in 14% pT1 tumors, whereas 86% cases presented no N-cadherin expression (Fig. 1D). The luminal membrane and the parts of the cells in contact with the basement membrane do not react with the N-cadherin antibody. A focal stronger reactivity was detected at the apical functional complexes. Of the invasive tumors, 60% cases were positive. Statistical analysis comparing the N-cadherin expression profile in pT1 and pT2-T3 tumors revealed a significant increased expression in muscle-invasive carcinomas compared with superficial bladder tumors (P < 0.001).


Figure 1
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  		Fig. 1. Immunohistochemical staining of N-cadherin on paraffin-embedded tissue sections from nontumor tissue and from pT1 bladder cancer tissues. A, positive membranous staining in myocarde (positive control). Original magnification, x10. B, normal bladder that does not express N-cadherin. Original magnification, x10. C, transitional cell carcinoma of the bladder with a conserved staining at the cell-cell border. Original magnification, x25. D, negative staining in transitional cell carcinoma of the bladder. All the cells are completely negative. Original magnification, x10.

 

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  		Table 2. N-cadherin expression in transitional cell carcinoma of the bladder according to the pathologic stage and E-cadherin expression status

 
E-cadherin expression in N-cadherin-positive tumors. In tumors for which N-cadherin was detected, E-cadherin expression profile was further explored by immunohistochemistry (Table 2). With respect to N-cadherin positive pT1 tumors, 80% expressed also E-cadherin adhesion molecule. On the contrary, in N-cadherin-positive invasive tumors, most of them presented no E-cadherin expression (61%), and 39% revealed a concomitant expression with E-cadherin. This result suggests that in most cases, the increase of N-cadherin expression in muscle-invasive carcinomas is associated with a loss of E-cadherin expression.

Clinical significance of N-cadherin expression. An univariate statistical analysis was done in pT1 tumors by comparing expression of N-cadherin with that of E-cadherin and with different clinical and anatomic-pathologic variables. Comparison of N-cadherin expression results with classic clinical and histologic data revealed that expression of N-cadherin is not associated with the grade (P = 0.35 with 1973 WHO classification; P = 0.38 with 2004 WHO classification), the presence of carcinoma in situ (P = 0.72), or E-cadherin status (P = 0.41). Considering the subclassification pT1a/pT1b, the results of the expression profile of N-cadherin did not differ significantly according to the invasion of the muscularis mucosae (P = 0.71). N-cadherin expression did not allow to distinguish pT1a and pT1b tumors. Progression-free survival statistical analysis was done using Kaplan-Meier procedure, and differences in the progression-free survival were assessed with the log-rank test. Kaplan-Meier analysis showed that age, sex, grade, and E-cadherin status were not predictive of patient progression-free survival (P = 0.46, P = 0.15, P = 0.10 with 1973 WHO classification; P = 0.69 with 2004 WHO classification; and P = 0.86, respectively). In contrast, a significant effect of clinicopathologic variables, such as the presence of carcinoma in situ and the subclassification pT1a/pT1b, was observed on patient survival without progression (P = 0.02 and P < 0.001, respectively). The progression-free survival curve is plotted according to expression of N-cadherin (Fig. 2 ). Tumors presenting N-cadherin immunoreactivity progressed more rapidly compared with tumors that did not express this adhesion molecule. Multivariate analysis revealed that only stage T1a/T1b and expression of N-cadherin remained significant. The relative risk of progression associated with a membranous expression of N-cadherin was 2.81 (95% confidence interval, 0.99-7.96; P = 0.05). Multivariate analysis is not modified whatever the WHO classification used (1973 or 2004). The evaluation of grade according to both classifications does not change results of progression-free survival. Thus, N-cadherin seemed to be a new independent prognostic marker of pT1 tumor progression.


Figure 2
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  		Fig. 2. Kaplan-Meier curves of progression-free survival in patients with pT1 tumors according to N-cadherin expression status (positive or negative). There was a statistical difference between the two groups (log-rank test, P = 0.17).

 
N-cadherin and E-cadherin expression pattern by immunohistochemical and RT-PCR analyses. We observed N-cadherin expression in 71 patients with pT1 superficial bladder tumors, whereas this molecule is not expressed in normal bladder and showed that this expression was correlated with a significant decrease of the survival without progression. In this context, we carried out a further study on an independent cohort of 28 recent patients (for whom frozen specimens were available) and focused a part of our attention on bladder tumors at an earlier stage (i.e., pTa tumor cases, which are not infiltrating).

N-cadherin and E-cadherin expression status (mRNA and protein) was determined in these pTa tumors and was compared with the expression pattern in pT1 and pT2-T3 tumor cases (Table 3 ). Thus, expression of N-cadherin and E-cadherin was first analyzed by immunohistochemistry, and second, RT-PCR analysis using oligonucleotide primers of N-cadherin and E-cadherin cDNA sequences was carried out to determine whether N-cadherin and E-cadherin protein expression pattern reflected level of N-cadherin and E-cadherin mRNA and to define at which step the regulation occurs (Fig. 3 ). For RT-PCR analysis, two human bladder carcinoma cell lines, RT4 (a well-differentiated cell line) and T24 (a poorly differentiated cell line), were also analyzed. N-cadherin was expressed only in T24 cells. RT4 cells expressed E-cadherin, whereas T24 cells lacked completely E-cadherin expression.


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  		Table 3. Immunohistochemical detection (ID) and RT-PCR analysis of E-cadherin and N-cadherin expression in 28 patients with transitional cell carcinoma of the bladder

 

Figure 3
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  		Fig. 3. RT-PCR analysis of N-cadherin and E-cadherin mRNA expression in RT4 and T24 bladder carcinoma cell lines and in bladder tissue specimens. Glyceraldehyde-3-phosphate dehydrogenase (GAPDH) mRNA was used as a control. Specific cDNAs were synthesized from RNAs using oligo(dT)12-18 in the presence of Moloney murine leukemia virus reverse transcriptase. PCR products were resolved on a 1.5% agarose gel. For details, see Materials and Methods. A gel of representative results.

 
In pTa tumors, E-cadherin protein expression was detected by immunohistochemistry with a membranous immunostaining as in normal bladder. Only one case presented a heterogeneous staining meaning an abnormal expression of E-cadherin. Of eight pT1 tumors, five cases (62.5%) presented a positive staining pattern, similar to normal urothelium, whereas three cases (37.5%) showed heterogeneous E-cadherin expression. No tumor was completely negative. Of these eight cases of pT1 tumors, two cases (25%) expressed E-cadherin (positive and heterogeneous expression) but not N-cadherin, and the other cases (6 of 8, 75%) presented a coexpression of E-cadherin and N-cadherin. In pT2-T3 tumors, membranous E-cadherin-positive staining was recorded in 1 of 8 (12.5%) cases. The other pT2-T3 tumors presented heterogeneous immunostaining. No muscle-invasive carcinoma was completely negative. In this cohort of patients, E-cadherin expression decreased according to the tumor-node-metastasis stage. RT-PCR analysis revealed that all cases expressed the mRNA keeping with protein expression. Obviously, E-cadherin mRNA was detected in tumors with heterogeneous staining because in these tumors, positive and negative areas are present within the same tumor. This result suggests that E-cadherin expression is regulated at the transcriptional level.

On the other hand, in all pTa tumors, N-cadherin expression was undetected by immunohistochemistry similarly to normal bladder mucosa. Its expression appeared only from pT1 tumor stage and was recovered in muscle-invasive carcinomas. RT-PCR analysis revealed that N-cadherin mRNA was detected in 50% of pTa tumors, whereas the protein was not expressed. These results suggest that N-cadherin expression is regulated at the translational or posttranslational level. The number of specimens was too slight to perform a statistical analysis, but results strengthened the benefit of N-cadherin as a predictive marker of progression for superficial urothelial tumors. Indeed, this adhesion molecule was not detected in any pTa tumors by immunohistochemical analysis, whereas it was expressed in pT1 tumors.


   Discussion
Top
 Abstract
 Materials and Methods
 Results
 Discussion
References
 
Cell adhesion molecules of the cadherin superfamily are frequently altered during tumor progression. This process is not limited to the down-regulation of cadherins, such as E-cadherin, for which loss of expression in various epithelial cancers correlates with malignancy and metastasis. Thus, a subset of tumors shows de novo expression or up-regulation of N-cadherin concomitant to their invasive stage. This phenomenon has been reported in melanoma (33), breast (20), prostate (24), pancreas (34), colon (35), squamous cell carcinoma (36), but also in bladder (27). Until now, expression of N-cadherin had been observed in a small number of transitional cell carcinoma of the bladder, but no prognostic value had been recognized (27). As shown in breast cancer (20), the presence of N-cadherin/catenins complexes at the cell surface could promote the adhesion of epithelial cells to surrounding stromal cells that also express N-cadherin. Thus, in bladder tissue, atypical expression of N-cadherin by urothelial carcinoma cells could allow cells to invade vesical muscle or to adhere to endothelium as smooth muscle (37), and endothelial cells (10) are N-cadherin positive. The purpose of the current study was to investigate whether N-cadherin could be a marker of progression for pT1 superficial urothelial tumors, for which the present problem is to distinguish tumors that will recur and those that will progress to a muscle-invasive disease.

As expected, N-cadherin was not expressed in normal urothelium and has been observed only in transitional cell carcinoma of the bladder. In our series of 101 patients, membranous N-cadherin immunostaining was recorded in 14% of pT1 tumors and 60% of pT2-T3 tumors, and this was associated with a full complement of catenins (data not shown). Thus, the expression of this adhesion molecule increased according to the tumor-node-metastasis stage. The evaluation of its prognostic value and a multivariate statistical analysis allowed us to identify, for the first time to our knowledge, N-cadherin as a new independent prognostic marker of pT1 transitional cell carcinoma neoplastic progression. Thus, superficial urothelial pT1 tumors presenting N-cadherin expression could be more aggressive. Our result could lead to a better follow-up of the clinical outcome of the patients with pT1 tumors as the recurrence rate is high (80%), and 40% of them will progress to a poorer prognosis muscle-invasive disease. The N-cadherin/catenins functions are influenced by multiple intracellular and extracellular factors (38). The induction of N-cadherin could involve the expression of transcription factors, such as Snail (39) or twist (40). A positive association between overexpression of epidermal growth factor receptor and high-grade, high-stage bladder cancer has been reported (41, 42). Now, epidermal growth factor is able to down-regulate E-cadherin and subsequently to up-regulate N-cadherin as reported in breast carcinoma cells (43). Physical association between N-cadherin and receptor tyrosine kinases could be involved in bladder cancer as shown in breast cancer cells, resulting in cell motility (44).

Interestingly, in a number of cancer types, the gain of N-cadherin is concomitant to the loss of E-cadherin expression. This process known as the "cadherin switch" is thought to reflect an epithelial-to-mesenchymal transition, whereby tumor cells are released from E-cadherin-dependent cell-cell interactions and acquire a motile phenotype through the induction of N-cadherin. Notably, N-cadherin is able to stimulate tumor cell invasion and metastasis even in the presence of E-cadherin (25, 26), indicating a functional dominance of N-cadherin over the invasion-suppressing properties of E-cadherin (45). In this context, we have explored the expression of E-cadherin and N-cadherin in cancer cell lines and in specimens of bladder tumor tissues. With regard to our cell lines (RT4 and T24), we confirmed the previously published work of Mialhe et al. (46). In poorly differentiated T24 cells, E-cadherin was absent and replaced by N-cadherin. Inversely, in RT4 cells, a well-differentiated cell line, N-cadherin was not expressed, whereas E-cadherin was detected. The exclusive expression of N-cadherin in T24 cells derived from a highly invasive tumor strengthens the concept that this cadherin plays a major role in acquisition of invasive phenotype. In bladder tissue specimens expressing N-cadherin, results were more contrasted. In pT1 tumors, 80% expressed also E-cadherin. In muscle-invasive carcinomas, 39% of cases presented a concomitant expression of both E-cadherin and N-cadherin, which is in agreement with the results of Rieger-Christ et al. (27). On the other hand, 61% of pT2-T3 tumors did not express E-cadherin. This argues for the role of N-cadherin in invasive process of bladder tumors. The discrepancies between in vivo and in vitro studies could be due to a cell clonal selection in cell lines.

On 28 fresh frozen specimens, we analyzed N-cadherin mRNA expression and evaluated the protein expression by immunohistochemistry. We also focused our study on pTa tumors to determine whether N-cadherin was present because these tumors are not infiltrating and are confined to the urothelium without invading the lamina propria. In these pTa superficial tumors, N-cadherin was not detected at the protein level, whereas 50% of cases revealed mRNA expression. Unfortunately, we were too close for a proper view about patients with pTa tumors, and we were unable to determine which tumors progressed. We could hypothesize that pTa tumors expressing N-cadherin at the mRNA level could recur more frequently or could progress to a more invasive stage compared with those that do not express N-cadherin mRNA. The discrepancy between mRNA and protein N-cadherin expression could be due to regulatory defects in the N-cadherin promoter, abnormalities at the translational or protein processing (such as glycosylation ref. 47) levels, and mutations in other parts of the gene that were not investigated by the cDNA analysis (e.g., intronic sequences), which could play a role in causing abnormal processing of the N-cadherin protein. A study done in colon cancer illustrated that a mutation in the expressed DNA of the E-cadherin gene could be a potential mechanism that might disturb the expression or function of E-cadherin in epithelial tumors (48).

Molecular markers for bladder cancer recurrence and progression continue to drive many research programs. Many lines of evidence support the functional implication for N-cadherin in tumor invasion and metastasis. This is the first report linking N-cadherin expression to progression-free survival, thus identifying this adhesion molecule as a predictive factor of pT1 tumor progression. This confirms the relevance of cell adhesion molecule expression to the clinical and biological behavior of superficial bladder tumors. On these bases, further prospective studies may be undertaken to establish whether closer follow-up might be beneficial in patients with T1 superficial bladder tumors that express N-cadherin. For pTa tumors expressing N-cadherin mRNA but not the protein, a complementary study within a large number of cases is needed to evaluate the clinical outcome of the patients and to test whether these tumors are more subjected to recurrence or progression to a later stage. The role of anomalous N-cadherin expression in urothelial cancer cells is not currently understood. It is henceforth necessary to study in more detail the respective role of classic cadherins and their regulation in bladder carcinogenesis. N-cadherin has a potential role to play in bladder cancer, possibly involving migration and invasion of cells.

Research is currently under way to define the role of N-cadherin in urothelial neoplastic progression and identify the cellular signaling pathway used by this adhesion molecule. Large studies are now in progress aiming to validate the use of adhesion molecules as molecular tools for the diagnosis and assessment of bladder cancer. The evaluation of existing or future reagents (such as peptides, antibodies, or other small-molecule drugs) in appropriate preclinical models should reveal whether targeting specific N-cadherin functions represents a suitable strategy for innovative antitumor therapies.


   Acknowledgments
 
We thank technicians from the Service d'Anatomie et Cytologie Pathologiques for their excellent technical assistance for bladder tumor section preparation and automated immunohistochemical procedure and G. Panetton for photographs of immunostaining.


   Footnotes
 
Grant support: Programme régional Hospitalier de Recherche Clinique (Besançon, France) and the Ligue Nationale Contre le Cancer (Comité du Doubs, Besançon, France; Comité du Jura, Lons-Le-Saunier, France).

The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked advertisement in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

Note: I. Lascombe and A. Clairotte contributed equally to this work.

Received 11/ 2/05; revised 2/16/06; accepted 3/ 2/06.


   References
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 Abstract
 Materials and Methods
 Results
 Discussion
 References
 

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E. Baumgart, M. S. Cohen, B. S. Neto, M. A. Jacobs, C. Wotkowicz, K. M. Rieger-Christ, A. Biolo, R. Zeheb, M. Loda, J. A. Libertino, et al.
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