Sylvie Fauconnet

Serum levels of vascular endothelial growth factor as a prognostic factor in bladder cancer.

PURPOSE Vascular endothelial growth factor is an overriding growth factor mediating tumor angiogenesis. We correlated serum vascular endothelial growth factor in patients with bladder cancer with clinical parameters. MATERIALS AND METHODS Serum vascular endothelial growth factor in 58 patients with bladder cancer, including superficial and invasive tumors in 42 and 16, respectively, and 41 healthy controls was measured by sandwich enzyme immunoassay. RESULTS Significant differences in serum vascular endothelial growth factor were observed in healthy controls and patients with bladder cancer (mean 248 versus 100 pg./ml., p <0.001). The serum level was significantly associated with tumor stage (p <0.0001), grade (p <0.002), vascular invasion (p <0.001) and carcinoma in situ (p <0.01). Patients with metastasis had a significantly higher levels than those with localized diseases (mean 582 versus 194 pg./ml., p <0.0001). At a cut-off of 400 pg./ml. the sensitivity and specificity of the test for differentiating patients with and without metastatic diseases was 87.5% and 98%, respectively (p <0.0001). Univariate statistical analysis showed that an increase in serum vascular endothelial growth factor level greater than 400 pg./ml. was significantly related to disease-free survival. On multivariate analysis only stage remained as an independent prognostic factor. CONCLUSIONS Although vascular endothelial growth factor did not remain an independent prognostic factor on multivariate analysis, our data indicate that the level of vascular endothelial growth factor may be a valuable angiogenic marker for identifying metastatic bladder cancer. It may be used as a new predictor of disease.

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.

Regulation of vascular endothelial growth factor expression by insulin-like growth factor-I in endometrial adenocarcinoma cells.

Angiogenesis is crucial for tumor growth and dissemination. Vascular endothelial growth factor (VEGF) is a potent angiogenic factor that promotes endothelial cell proliferation and chemotaxis. VEGF occurs as 5 isoforms, as a result of an alternatively spliced transcript that originates from one gene, of which the 2 majors are the VEGF 121 and 165 isoforms. Our aim was firstly to determine the role of Insulin‐like Growth Factor‐I (IGF‐I) in the regulation of VEGF expression in endometrial adenocarcinoma cells and then the mechanism by which this regulation occurs. IGF‐I treatment of HEC‐1A cells provoked an increase of VEGF mRNA expression that peaked at 48 hr with a 165 isoform mRNA more abundant than the 121 isoform. The IGF‐I action was confirmed at the protein level, whose concentration was increased in the conditioned media. In experiments using transient transfection of VEGF promoter‐luciferase constructs, the IGF‐I failed to increase the activity of the VEGF promoter after a 24‐hr period of IGF‐I treatment, while the addition of Actinomycin D showed an increase of the VEGF mRNA half‐life. Most interestingly, Northern blot analysis showed a different stability of the 2 major VEGF isoform mRNAs (VEGF 121 and 165), of which the 121 isoform was more stable than the 165 isoform. The IGF‐I treatment prolonged the half‐life of both of the VEGF isoform mRNAs. Our results suggest that IGF‐I regulates VEGF expression in endometrial adenocarcinoma cells at the post‐transcriptional level by enhancing the stabilization of the 2 major VEGF isoform mRNAs (VEGF121 and VEGF165). In addition to its proliferative functions, IGF‐I induces VEGF expression and participates in the maintenance of an angiogenic phenotype. Int. J. Cancer 85:117–123, 2000.

Serum-free culture of stromal and functionally polarized epithelial cells of guinea-pig endometrium: a potential model for the study of epithelial-stromal paracrine interactions

Summary— Stromal and glandular epithelial (GE) cells were isolated from guinea‐pig endometrium and grown to near confluency (6–8 days) in primary culture on plastic surfaces in a serum‐supplemented medium (SSM). The stromal cells were subcultured on plastic dishes and maintained for 72 h in SSM. Then SSM was replaced by a chemically defined medium (CDM) and the stromal cells grown to confluency (5–7 days). The GE cells were subcultured in CDM, on a basement membrane matrix (Matrigel) applied to permeable Millicell‐PC filters, and grown to confluency (5 days). Homogeneity of the subcultured endometrial cell populations was ascertained immunocytochemically. The filter‐cultured GE monolayers were polarized morphologically, and displayed epithelialspecific specialized structures. These monolayers had functional tight junctions as verified by a measurable transepithelial resistance. The subcultured cell populations were distinguished by an analysis of their cellular and secretory proteins after labelling with [35S]‐methionine and analysis by polyacrylamide gel electrophoresis. The filter‐cultured GE monolayers allowed identification of the proteins released vectorially in the apical or the basal secretory compartment, thus demonstrating the functional polarization of GE cells in this bicameral culture system. Within the defined conditions of this culture system, the paracrine factors released by the two endometrial cell populations as well as the interplay of stromal‐epithelial interactions and ovarian hormones could be investigated.

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.

Apoptotic effect of the selective PPARβ/δ agonist GW501516 in invasive bladder cancer cells

GW501516 is a selective and high-affinity synthetic agonist of peroxisome proliferator-activated receptor β/δ (PPARβ/δ). This molecule promoted the inhibition of proliferation and apoptosis in few cancer cell lines, but its anticancer action has never been investigated in bladder tumor cells. Thus, this study was undertaken to determine whether GW501516 had antiproliferative and/or apoptotic effects on RT4 and T24 urothelial cancer cells and to explore the molecular mechanisms involved. Our results indicated that, in RT4 cells (derived from a low-grade papillary tumor), GW501516 did not induce cell death. On the other hand, in T24 cells (derived from an undifferentiated high-grade carcinoma), this PPARβ/δ agonist induced cytotoxic effects including cell morphological changes, a decrease of cell viability, a G2/M cell cycle arrest, and the cell death as evidenced by the increase of the sub-G1 cell population. Furthermore, GW501516 triggered T24 cell apoptosis in a caspase-dependent manner including both extrinsic and intrinsic apoptotic pathways through Bid cleavage. In addition, the drug led to an increase of the Bax/Bcl-2 ratio, a mitochondrial dysfunction associated with the dissipation of ΔΨm, and the release of cytochrome c from the mitochondria to the cytosol. GW501516 induced also ROS generation which was not responsible for T24 cell death since NAC did not rescue cells upon PPARβ/δ agonist exposure. For the first time, our data highlight the capacity of GW501516 to induce apoptosis in invasive bladder cancer cells. This molecule could be relevant as a therapeutic drug for high-grade urothelial cancers.

1067 ADIPOCYT-FATTY ACID BINDING PROTEIN, A POTENTIAL PROGNOSTIC MARKER OF BLADDER TRANSITIONAL CELL CARCINOMA

INTRODUCTION AND OBJECTIVES: Few studies provided evidence that loss of adipocyte-fatty acid binding protein (A-FABP) expression is associated with progression of human bladder TCC and suggest that it could be a putative marker of progression of the disease. The re-expression of A-FABP could be a therapeutic approach in early stage bladder cancer to prevent disease progression. The aim of this study is first to determine whether A-FABP is a progression marker by analysing A-FABP mRNA expression within the tumor. Secondly, to demonstrate whether the expression of this potential prognostic marker could be up-regulated by Peroxisome Proliferator-Activated Receptor (PPAR) gamma agonists such as prostaglandins and thiazolidinediones (antidiabetic drugs) known to exhibit anti-neoplastic effects. METHODS: Total RNA was extracted from 26 samples of bladder TCC from clinical stages pTa to pT4 obtained from transurethral resection or cystectomy specimens. The relative A-FABP mRNA expression level was evaluated by RTqPCR. The regulation of AFABP expression was studied by RTqPCR and western-blotting in T24 cells derived from an undifferentiated grade III carcinoma of the bladder. RESULTS: 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. We described for the first time the molecular mechanism by which this marker is up-regulated by PPAR gamma in T24 cells. This effect occurred through a PPARdependent transcriptional mechanism without modifying mRNA stability and interestingly required de novo protein synthesis. CONCLUSIONS: 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 antidiabetic agents already in clinical use open up new perspectives in the treatment of bladder cancer by intravesical instillations of thiazolidinediones in order to inhibit malignant progression of bladder cancer.