Elsevier

Urology

Volume 63, Issue 6, June 2004, Pages 1198-1204
Urology

Basic science
c-FLIP expression in bladder urothelial carcinomas: its role in resistance to Fas-mediated apoptosis and clinicopathologic correlations

https://doi.org/10.1016/j.urology.2004.01.007Get rights and content

Abstract

Objectives

To investigate the incidence of Fas (exon 9) mutations and the expression of Fas, Fas-Fas ligand (FasL) system, and cellular FLICE-like inhibitory protein (c-FLIP) in relation to standard clinicopathologic parameters and patient outcome in bladder carcinoma. Disruption of apoptotic cell death has been implicated in tumor aggressiveness in bladder urothelial carcinomas. The FasL system is involved in the execution of apoptosis induced by the immune system. c-FLIP protein constitutes an important endogenous inhibitor of Fas and other death receptor-mediated apoptosis.

Methods

The expression of Fas, FasL, and c-FLIP was quantified immunohistochemically in paraffin-embedded tissues from 53 patients for whom clinical information was available. DNA extracted from the same samples was screened for mutations in Fas exon 9 by single-strand conformation polymorphism and sequencing. The effect of Fas, FasL, and c-FLIP on clinical outcome was assessed by univariate and multivariate analyses.

Results

Positive immunostaining was detected for Fas, FasL, and c-FLIP in 72%, 66%, and 81% of cases, respectively. Concurrent expression of Fas and FasL was seen in 27 samples (51%), of which 22 (81.5%) also displayed c-FLIP positivity. FasL and c-FLIP expression increased with advancing stage but was absent from normal urothelium. None of the 53 urothelial carcinoma samples analyzed showed evidence of mutations by polymerase chain reaction single-strand conformation polymorphism and direct sequencing. Survival analysis demonstrated that although both FasL and c-FLIP expression adversely affected survival, only c-FLIP remained statistically significant on multivariate analysis.

Conclusions

The frequent expression and coexpression of Fas, FasL, and c-FLIP in urothelial carcinomas implicates c-FLIP as an inhibitor of the Fas-FasL-induced death pathway in these tumors. Moreover, c-FLIP conveys independent prognostic information in the presence of classical prognosticators.

Section snippets

Patients

Fifty-three consecutive patients with primary invasive (N0M0) UC presenting at Asklepeion Voula Hospital in Athens between 1985 and 1995, for whom sufficient paraffin-embedded tissue was available, were enrolled in the present investigation. Of the 53 patients, 45 were men and 8 were women (median age 72 years, range 35 to 90). By the time this study was undertaken, 15 patients had died of their disease after a median survival of 12 months (range 7 to 39) and 2 patients had been lost to

Fas gene mutation detection

Using PCR-SSCP, a normal banding pattern was obtained for the vast majority of the samples (48 of 53; 91%). In 5 cases in which an aberrant banding pattern was observed, direct sequencing revealed a normal coding sequence of Fas exon 9. Therefore, none of the 53 UC samples tested showed evidence of mutations by PCR-SSCP and direct sequencing.

Fas, FasL, and c-FLIP expression

Immunoreactivity (defined as expression in 10% or more of neoplastic cells) was detected for Fas, FasL, and c-FLIP in 72%, 66%, and 81% of cases,

Comment

In vitro studies have indicated that despite Fas expression in UC cell lines, poorly differentiated cells are insensitive to either recombinant Fas ligand or agonistic apoptosis-inducing monoclonal antibody against Fas,12 raising the idea that bladder cancer cells must have acquired effective mechanisms to neutralize Fas signaling. The high frequency of Fas and FasL coexpression, an apparently counterintuitive combination of factors with deleterious effects on cell survival, documented in the

Conclusions

The results of the present investigation demonstrated that Fas, FasL, and c-FLIP are frequently expressed and coexpressed in bladder carcinoma, supporting the suspected involvement of c-FLIP as a mechanism to counteract apoptosis driven by Fas/FasL coexpression. c-FLIP gained further importance in this regard because we were unable to demonstrate structural alterations of the Fas death domain in our samples. More importantly, c-FLIP emerged for the first time as an independent prognostic

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