Hugues Bittard

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

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.

Relationship between expansion of the CAG repeat in exon 1 of the androgen receptor gene and idiopathic male infertility

OBJECTIVE To determine whether expansion of CAG repeats in exon 1 of the androgen receptor is correlated with impaired spermatogenesis in patients with male idiopathic infertility. DESIGN A retrospective study. SETTING Medical school in Besançon, France. PARTICIPANT(S) Thirty-seven infertile patients with azoospermia or oligospermia and 50 fertile controls. INTERVENTION(S) History, physical, hormonal assays, semen analysis, and collection of blood samples in order to study the androgen receptors gene. MAIN OUTCOME MEASURE(S) Blood samples were collected from each infertile patient and control. The length of the CAG repeat segment was evaluated by using polymerase chain reaction (PCR) electrophoresis in exon 1 and PCR single-strand conformation polymorphism in exons 2-8. RESULT(S) The mean length of the CAG repeats was significantly different between infertile and fertile patients (23.91 +/- 0.5 vs. 22.20 +/- 0.4). No mutation was detected in exons 2-8 of the androgen receptor gene in infertile patients. CONCLUSION(S) Expansion of the CAG repeat segment of the androgen receptor is correlated with male idiopathic infertility. The number of CAG repeats may therefore have a modulatory effect on normal androgen receptor function.

The expression of Twist has an impact on survival in human bladder cancer and is influenced by the smoking status

OBJECTIVES Twist is considered as transcription factor that regulates epithelial mesenchymal transition (EMT) by at least inhibition of E-cadherin expression. EMT is a key event in the tumor invasion process. The purpose of this study is to investigate the expression of Twist but also those of E- and N-cadherin in human primary bladder tumor and to evaluate its prognostic value. As smoking cigarettes is a strong bladder cancer risk factor, we tried to evaluate the impact of the tobacco status on these molecular abnormalities. MATERIALS AND METHODS To delineate on the oncogenic role for Twist in human bladder cancer, we evaluated the E- and N-cadherin but also Twist expression (n = 70) by immunohistochemistry. We evaluated the prognostic value of these expressions. Moreover, we tried to correlate these protein expressions to the smoking status of the patients. Overall survival (OS) and progression-free survival (PFS) were evaluated using the Kaplan-Meier method, and multivariate analysis was performed using the Cox proportional hazard analysis. RESULTS Of the 70 bladder tumors, 28 (40%) cases were positive for Twist expression, 16 (23%) cases were negative for E-cadherin expression, and 12 (17%) were positive for N-cadherin expression. When categorized into negative vs. positive expression, Twist was associated with the stage (P = 0.001), the grade (P < 0.001), the progression (P = 0.02), and the E-cadherin expression (P = 0.01). Moreover, positive Twist expression clearly predicted poorer PFS (P = 0.02). In the multivariate analysis, both positive Twist expression and loss of E-cadherin expression were independent prognostic factors for PFS (P = 0.046 and P = 0.001, respectively) and only loss of E-cadherin expression for the OS (P < 0.001). We also demonstrated that almost 60% (16/28) of patients with Twist-positive expression were current smokers at the time of the diagnosis, corroborating the fact that smoking modulates the expression of EMT markers including Twist. CONCLUSION Positive Twist expression may be a useful prognostic marker for patients with bladder cancer. Its expression seems to be correlated to the tobacco status of the patients.

Expression of e-cadherin and α-, β-, γ-catenins in patients with bladder cancer : Identification of γ-catenin as a new prognostic marker of neoplastic progression in T1 superficial urothelial tumors

Loss of intercellular adhesion facilitates tumor invasion. To clarify the relation between altered expression of cell adhesion molecules and progression of T1 superficial bladder tumors, 101 cases (71 T1 tumors, 30 T2/T3 tumors) were examined immunohistochemically for E-cadherin and α-, β-, and γ-catenins. A highly significant correlation was observed between the decreased expression of all molecules and increased TNM stage (P < .001). Univariate analysis, performed in cases of T1 tumors, revealed association of abnormal E-cadherin with βcatenin diminution. Survival curves were established with the Kaplan-Meier method and analyzed according to clinical and histopathologic parameters using the log-rank test. Cox multivariate analysis revealed only γcatenin as an independent predictor of progression-free survival in patients with stage T1 bladder urothelial tumors. The characterization of T1 tumors that will progress could lead to the identification of patients who might benefit from surgery to avoid vesical muscle invasion and, consequently, metastasis. Urothelial carcinoma is the most common solid malignancy of the bladder. It is distinguished as muscle-invasive carcinoma or superficial bladder tumor. Muscle-invasive carcinomas generally are associated with poor prognosis, whereas the clinical outcome of superficial bladder tumors is relatively unpredictable. The recurrence rate of T1 superficial bladder tumors is high (80%), and 40% of them will progress to a poorer prognosis muscle-invasive disease. Stage and grade, based on histopathologic criteria, are classic prognostic variables that permit evaluating recurrence or progression of the disease, which is useful for clinical purposes. 1 The identification of new prognostic markers allowing improvement of the biologic assessment of the T1 superficial tumors could be of great clinical value for disease management. Tumor progression is accompanied by altered expression of cell adhesion molecules such as cadherins. E-cadherin, one subtype of transmembrane glycoprotein that mediates calciumdependent adhesion of cells, is expressed specifically in epithelia and is involved in maintenance of their phenotype. Its cytoplasmic domain associates with cytoplasmic proteins termed catenins. βCatenin or γ-catenin binds directly, whereas α-catenin links the bound β-catenin or γ-catenin to the actin microfilament network of the cellular cytoskeleton. This binding is essential for stable cell-to-cell adhesion. 2 Reduction in expression of these molecules has been reported in human cancers. 3-5 In the bladder, studies have shown that decreased E-cad

A‐FABP, a candidate progression marker of human transitional cell carcinoma of the bladder, is differentially regulated by PPAR in urothelial cancer cells

Superficial pT1 bladder tumors are characterized by a high risk of recurrence and progression in grade and stage. Few studies provided evidence that loss of adipocyte‐fatty acid binding protein (A‐FABP) expression was associated with bladder cancer progression. A‐FABP is a lipid binding protein playing a role in intracellular lipid transport and metabolism, as well as in signal transduction. We reported from bladder tumors that decrease of A‐FABP transcript level significantly correlated to tumor stage and to histologic grade (p < 0.05). Namely, in poor prognosis high grade pT1 tumors there was a loss of A‐FABP expression compared to good prognosis tumors suggesting that re‐expression of A‐FABP could be a therapeutic approach in early stage bladder cancer to prevent disease progression. We demonstrated for the first time that this marker is upregulated by Peroxisome Proliferator‐Activated Receptor (PPAR) α, β and γ in T24 cells (derived from an undifferentiated grade III carcinoma) and only by PPARβ in RT4 cells (derived from a well differentiated grade I papillary tumor). This effect occurred through a PPAR‐dependent transcriptional mechanism without modifying mRNA stability and interestingly required de novo protein synthesis. Data as a whole suggest a prognostic significance of A‐FABP in bladder cancer outcome and the potential utility of overexpression of this protein by PPAR agonists open up new perspectives in the treatment of bladder cancer.

Phospho-Akt pathway activation and inhibition depends on N-cadherin or phospho-EGFR expression in invasive human bladder cancer cell lines

OBJECTIVES A particular interest in epithelial-mesenchymal transition (EMT), which takes place during embryonic development, provided potential mechanisms involved in the progression of many epithelial tumors, including bladder cancer (BC). The phospho-Akt signaling pathway is supposed to be involved in invasion and progression of human tumors, including BC. Moreover, it has been demonstrated in bladder cancer cell lines that N-cadherin or phospho-epithelial growth factor receptor (EGFR) expression are correlated to tumor progression. Our objectives were to evaluate the potential phospho-Akt pathway involvement in N-cadherin and/or phospho-EGFR positive BC cell lines and to evaluate the prognostic value of E- and N-cadherin expression in patients undergoing cystectomy for invasive BC. MATERIALS AND METHODS We screened a panel of invasive and noninvasive BC cell lines for E- and N-cadherin, phospho-EGFR, and phospho-Akt expression using the Western blot technique (WB). The potential role of N-cadherin in invasion was assessed by Matrigel assays with and without the N-cadherin blocking monoclonal antibody GC-4. Then we used the Affymetrix microarray technique to evaluate the prognostic value of E- and N-cadherin expression in 30 patients undergoing a cystectomy for invasive BC. RESULTS N-cadherin and phospho-EGFR expression are associated with Akt activation and with invasive behavior modulation. Even if Akt activation is sufficient in promoting invasion, its inactivation by LY294002 (PI-3 kinase inhibitor) is less efficient on invasion than inhibition of N-cadherin and phospho-EGFR by GC-4 (monoclonal antibody) and gefitinib (anti-tyrosine kinase), respectively. N-cadherin and phospho-EGFR inhibition decreased phospho-Akt activation but also caused restoration and reinforcing of E-cadherin expression, respectively, while phospho-Akt inhibition did not have any impact on E-cadherin expression. In a group of high-risk bladder tumors (T(1)G(3)), N- and E-cadherin expression could be considered as a prognostic marker. In a group of patients with invasive BC (pT(2)-T(4)) undergoing cystectomy, we showed a shorter overall survival when BC expressed N-cadherin (P = 0.0064) and when E-cadherin expression was down-regulated (P = 0.00165). The N (positive) /E (negative) profile has the worst prognosis (P = 0.00153). CONCLUSIONS We confirmed the partial responsibility of p-Akt activation in invasion of some BC cell lines expressing N-cadherin or p-EGFR and also the potential role of N-cadherin and p-EGFR as target in cancer therapy. N/E- cadherin expression profile has a significant prognostic value in invasive BC.

Ureterorenoscopy with Holmium-Yttrium-Aluminum-Garnet Fragmentation Is a Safe and Efficient Technique for Stone Treatment in Patients with a Body Mass Index Superior to 30 kg/m2

PURPOSE The aim of the study was to analyze results and morbidity after flexible ureterorenoscopy in patients with a body mass index (BMI) >30 kg/m(2) and to compare with results obtained in a large cohort of nonobese patients. PATIENTS AND METHODS We conducted a retrospective study including all flexible ureterorenoscopy performed for stone retrieval in our institution between January 2004 and December 2008. During the study period, 224 procedures were performed, of which 18 had to be excluded because of missing BMI data. Thus, a total of 206 procedures were included in the final analysis (34 in 29 obese patients, 172 in 149 nonobese patients). Characteristics of the patients (age, BMI, previous treatment), stones (nature, location, number), and procedures (operating time, morbidity, outcome) were analyzed. Success was defined as clear imaging (completely stone free) on renal tomography and ultrasonography at 1, 3, and 6 months follow-up. RESULTS Mean BMI was 34±0.6 kg/m(2) in obese patients (OP) and 24±0.2 kg/m(2) in nonobese patients (NOP). Mean stone size, location, and composition were not significantly different between groups. Operative time was also similar in OP and NOP (102.5±6.1 min vs 103±3.4 min, P=NS). The rate of minor complications (fever, hematuria, flank pain) was similar in OP (11.8%) and NOP (11.4%). No major complication necessitating prolonged hospital stay or new surgical procedure was observed. The overall stone-free rate was not significantly different between OP (79.4%) and NOP (70%). CONCLUSION Flexible ureterorenoscopy is an appropriate treatment for use in obese patients and achieves excellent stone-free rates with low morbidity.

The antidiabetic drug ciglitazone induces high grade bladder cancer cells apoptosis through the up-regulation of TRAIL.

Background Ciglitazone belongs to the thiazolidinediones class of antidiabetic drug family and is a high-affinity ligand for the Peroxisome Proliferator-Activated Receptor γ (PPARγ). Apart from its antidiabetic activity, this molecule shows antineoplastic effectiveness in numerous cancer cell lines. Methodology/Principal Findings Using RT4 (derived from a well differentiated grade I papillary tumor) and T24 (derived from an undifferentiated grade III carcinoma) bladder cancer cells, we investigated the potential of ciglitazone to induce apoptotic cell death and characterized the molecular mechanisms involved. In RT4 cells, the drug induced G2/M cell cycle arrest characterized by an overexpression of p53, p21waf1/CIP1 and p27Kip1 in concomitance with a decrease of cyclin B1. On the contrary, in T24 cells, it triggered apoptosis via extrinsic and intrinsic pathways. Cell cycle arrest and induction of apoptosis occurred at high concentrations through PPARγ activation-independent pathways. We show that in vivo treatment of nude mice by ciglitazone inhibits high grade bladder cancer xenograft development. We identified a novel mechanism by which ciglitazone kills cancer cells. Ciglitazone up-regulated soluble and membrane-bound TRAIL and let TRAIL-resistant T24 cells to respond to TRAIL through caspase activation, death receptor signalling pathway and Bid cleavage. We provided evidence that TRAIL-induced apoptosis is partially driven by ciglitazone-mediated down-regulation of c-FLIP and survivin protein levels through a proteasome-dependent degradation mechanism. Conclusions/Significance Therefore, ciglitazone could be clinically relevant as chemopreventive or therapeutic agent for the treatment of TRAIL-refractory high grade urothelial cancers.

Protein kinase C signalling pathway is involved in the regulation of vascular endothelial growth factor expression in human bladder transitional carcinoma cells

Vascular endothelial growth factor (VEGF) is a potent angiogenic factor associated with the growth and metastasis of various cancers and plays a prominent role in vesical angiogenesis regulation. In this study, we investigated the effect of the phorbol 12-myristate 13-acetate (PMA) on the expression of VEGF in human bladder transitional carcinoma cells (RT4). RT4 cells expressed three VEGF isoforms (VEGF(189), VEGF(165), VEGF(121)). PMA increased VEGF mRNA expression time-dependently with a peak at 4 h. PMA increased the half-life of VEGF mRNA. The amount of VEGF protein in conditioned media was increased by PMA in a dose-dependent manner with a maximal effect at 10(-7) M. Staurosporine and calphostin C (PKC inhibitors) decreased PMA-induced VEGF mRNA expression as opposed to protein kinase A or cyclic nucleotide-dependent protein kinase inhibitors. Thus, in RT4 cells, VEGF expression is up-regulated by PMA via the PKC signalling pathway and according to a posttranscriptional mechanism.

Expression in bladder transitional cell carcinoma by real‐time quantitative reverse transcription polymerase chain reaction array of 65 genes at the tumor suppressor locus 9q34.1‐2: Identification of 5 candidates tumor suppressor genes

Frequent deletions on 9q34.1‐2 were reported in bladder transitional cell carcinoma. High deletion mapping studies delimited a critical interval between markers D9S61 and D9S66, which is highly susceptible to contain a tumor suppressor gene. Expression level of the 65 genes localized in this region was analyzed by real‐time quantitative RT‐PCR, comparing tumor to normal urothelium. Five genes exhibited a significantly reduced expression level: C9orf9, KIAA0625, ABL1, LAMC3 and KIAA1857‐netrin‐G2, which exhibited the most significant downregulation (p=0.0007). KIAA1857‐netrin‐G2 belongs to the netrins and might then be a tumor suppressor gene in bladder cancer, as netrin1 receptor DCC has been implicated in tumorigenesis.