Isabelle Lascombe

Differential regulation of vascular endothelial growth factor expression by peroxisome proliferator-activated receptors in bladder cancer cells

The growth of any solid tumor depends on angiogenesis. Vascular endothelial growth factor (VEGF) plays a prominent role in vesical tumor angiogenesis regulation. Previous studies have shown that the peroxisome proliferator-activated receptor γ (PPARγ) was involved in the angiogenesis process. Here, we report for the first time that in two different human bladder cancer cell lines, RT4 (derived from grade I tumor) and T24 (derived from grade III tumor), VEGF (mRNA and protein) is differentially up-regulated by the three PPAR isotypes. Its expression is increased by PPARα, β, and γ in RT4 cells and only by PPARβ in T24 cells via a transcriptional activation of theVEGF promoter through an indirect mechanism. This effect is potentiated by an RXR (retinoid-X-receptor), selective retinoid LG10068 providing support for a PPAR agonist-specific action on VEGF expression. While investigating the downstream signaling pathways involved in PPAR agonist-mediated up-regulation of VEGF, we found that only the MEK inhibitor PD98059 reduced PPAR ligand-induced expression of VEGF. These data contribute to a better understanding of the mechanisms by which PPARs regulate VEGF expression. They may lead to a new therapeutic approach to human bladder cancer in which excessive angiogenesis is a negative prognostic factor.

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.

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.

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.

Cell death and restoration of TRAIL-sensitivity by ciglitazone in resistant cervical cancer cells

Known activators of the Peroxisome Proliferator-Activated Receptor γ (PPARγ), thiazolidinediones (TZD) induce apoptosis in a variety of cancer cells through dependent and/or independent mechanisms of the receptor. We tested a panel of TZD (Rosiglitazone, Pioglitazone, Ciglitazone) to shed light on their potential therapeutic effects on three cervical cancer cell lines (HeLa, Ca Ski, C-33 A). In these cells, only ciglitazone triggered apoptosis through PPARγ-independent mechanisms and in particular via both extrinsic and intrinsic pathways in Ca Ski cells containing Human PapillomaVirus (HPV) type 16. It also inhibits cervical cancer xenograft development in nude mice. Ciglitazone kills cervical cancer cells by activating death receptor signalling pathway, caspase cascade and BH3 interacting-domain death agonist (Bid) cleavage through the up-regulation of Death Receptor 4 (DR4)/DR5 and soluble and membrane-bound TNF related apoptosis inducing ligand (TRAIL). Importantly, the drug let TRAIL-resistant Ca Ski cells to respond to TRAIL through the downregulation of cellular FLICE-Like Inhibitory Protein (c-FLIP) level. For the first time, we revealed that ciglitazone is able to decrease E6 viral oncoprotein expression known to block TRAIL pathway and this was associated with cell death. Our results highlight the capacity of ciglitazone to restore TRAIL sensitivity and to prevent E6 blocking action to induce apoptosis in cervical cancer cells.

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.