Samia Mourah

Assessment of microsatellite instability in urine in the detection of transitional-cell carcinoma of the bladder

Loss of heterozygosity (LOH) and alterations in microsatellite DNA markers have been reported in bladder‐cancer tumors. We have studied, in a blinded fashion, using PCR‐based microsatellite analysis, genetic alterations of cells exfoliated in urine of 59 Caucasian patients and control patients; 31 with initially confirmed bladder transitional‐cell carcinoma (TCC), 17 with signs and symptoms suggestive of bladder cancer, 6 control patients who underwent renal transplantation, and 5 control patients with urolithiasis. Microsatellite analysis of cells exfoliated in the urine allowed the diagnosis of 83% (10/12) of patients with bladder TCC recurrence confirmed by cystoscopy, while 100% of patients followed up for transitional‐cell carcinoma of the bladder for up to 12 months without evidence of tumor recurrence upon routine cystoscopy showed no microsatellite alterations. None of the patients without neoplasia (negative controls) had any microsatellite alterations, whereas all patients who underwent renal transplantation had additional new alleles corresponding to contamination with donors renal and urothelial cells (positive controls). No control patients had any evidence of transitional‐cell carcinoma by cystoscopy. Our results provide objective evidence that non‐invasive molecular detection of bladder TCC by microsatellite analysis is reproducible with a sensitivity of 83% and a specificity of 100% in Caucasian patients. This non‐invasive procedure represents a potential clinical tool for the detection and the screening of bladder TCC. Int. J. Cancer (Pred. Oncol.) 79:629–633, 1998.

β‐Catenin and NF‐κB cooperate to regulate the uPA/uPAR system in cancer cells

Expression of the urokinase plasminogen activator (uPA) and urokinase plasminogen activator receptor (uPAR) has recently been shown to be directly regulated by the Wnt/β‐catenin signaling pathway in colon cancer cells, through β‐catenin binding to T‐cell factor binding element motifs present in their gene promoters. In our study, we present evidence that inhibition of β‐catenin causes upregulation of uPA/uPAR gene expression enhancing invasive potential. Using MCF‐7, MDA‐MB‐231 (breast cancer cells) and SW480 (colon cancer cells), we found that siRNA‐mediated silencing of β‐catenin increased uPA, uPAR and plasminogen activator inhibitor‐1 (PAI‐1) expression at the mRNA and protein levels. This increase was responsible for the observed enhanced invasive capacity of MDA‐MB‐231 and SW480 cancer cells. In addition, β‐catenin stabilization and accumulation by lithium chloride treatment, a well‐known inhibitor of glycogen synthase kinase‐3β (GSK‐3β), or by β‐catenin/T‐cell factor‐4 expression vectors transfection led to a decrease in uPA, uPAR and PAI‐1 mRNA expression in the studied cancer models. Treatment of β‐catenin siRNA‐transfected cells with a specific inhibitor of nuclear factor‐kappaB (NF‐κB), SN50, significantly reduced enhancement of uPA, uPAR and PAI‐1 expression and cancer cell invasion, observed in β‐catenin siRNA‐transfected cells. Furthermore, β‐catenin siRNA‐treated cells exhibited NF‐κB nuclear accumulation. These data suggest that β‐catenin regulates the uPA/uPAR system in cooperation with NF‐κB transcription factor, which constitutes a novel mechanism of regulation.

VEGF and VEGFR-1 are coexpressed by epithelial and stromal cells of renal cell carcinoma.

Tumor angiogenesis is a dynamic process that plays a major role in cancer progression. Vascular endothelial growth factor (VEGF) and its receptors play a pivotal role in angiogenesis. The expression of VEGF and its receptors VEGFR‐1 and VEGFR‐2 in renal cell carcinoma (RCC) was investigated in the perspective of anti‐VEGF treatments.

Invading Basement Membrane Matrix Is Sufficient for MDA-MB-231 Breast Cancer Cells to Develop a Stable In Vivo Metastatic Phenotype

Introduction The poor efficacy of various anti-cancer treatments against metastatic cells has focused attention on the role of tumor microenvironment in cancer progression. To understand the contribution of the extracellular matrix (ECM) environment to this phenomenon, we isolated ECM surrogate invading cell populations from MDA-MB-231 breast cancer cells and studied their genotype and malignant phenotype. Methods We isolated invasive subpopulations (INV) from non invasive populations (REF) using a 2D-Matrigel assay, a surrogate of basal membrane passage. INV and REF populations were investigated by microarray assay and for their capacities to adhere, invade and transmigrate in vitro, and to form metastases in nude mice. Results REF and INV subpopulations were stable in culture and present different transcriptome profiles. INV cells were characterized by reduced expression of cell adhesion and cell-cell junction genes (44% of down regulated genes) and by a gain in expression of anti-apoptotic and pro-angiogenic gene sets. In line with this observation, in vitro INV cells showed reduced adhesion and increased motility through endothelial monolayers and fibronectin. When injected into the circulation, INV cells induced metastases formation, and reduced injected mice survival by up to 80% as compared to REF cells. In nude mice, INV xenografts grew rapidly inducing vessel formation and displaying resistance to apoptosis. Conclusion Our findings reveal that the in vitro ECM microenvironment per se was sufficient to select for tumor cells with a stable metastatic phenotype in vivo characterized by loss of adhesion molecules expression and induction of pro-angiogenic and survival factors.

Distinct expression patterns of the E3 ligase SIAH-1 and its partner Kid/KIF22 in normal tissues and in the breast tumoral processes.

SIAH proteins are the human members of an highly conserved family of E3 ubiquitin ligases. Several data suggest that SIAH proteins may have a role in tumor suppression and apoptosis. Previously, we reported that SIAH-1 induces the degradation of Kid (KIF22), a chromokinesin protein implicated in the normal progression of mitosis and meiosis, by the ubiquitin proteasome pathway. In human breast cancer cells stably transfected with SIAH-1, Kid/KIF22 protein level was markedly reduced whereas, the Kid/KIF22 mRNA level was increased. This interaction has been further elucidated through analyzing SIAH and Kid/KIF22 expression in both paired normal and tumor tissues and cell lines. It was observed that SIAH-1 protein is widely expressed in different normal tissues, and in cells lines but showing some differences in western blotting profiles. Immunofluorescence microscopy shows that the intracellular distribution of SIAH-1 and Kid/KIF22 appears to be modified in human tumor tissues compared to normal controls. When mRNA expression of SIAH-1 and Kid/KIF22 was analyzed by real-time PCR in normal and cancer breast tissues from the same patient, a large variation in the number of mRNA copies was detected between the different samples. In most cases, SIAH-1 mRNA is decreased in tumor tissues compared to their normal counterparts. Interestingly, in all breast tumor tissues analyzed, variations in the Kid/KIF22 mRNA levels mirrored those seen with SIAH-1 mRNAs. This concerted variation of SIAH-1 and Kid/KIF22 messengers suggests the existence of an additional level of control than the previously described protein-protein interaction and protein stability regulation. Our observations also underline the need to re-evaluate the results of gene expression obtained by qRT-PCR and relate it to the protein expression and cellular localization when matched normal and tumoral tissues are analyzed.

EMMPRIN Promotes Melanoma Cells Malignant Properties through a HIF-2alpha Mediated Up-Regulation of VEGF-Receptor-2

EMMPRINs expression in melanoma tissue was reported to be predictive of poor prognosis. Here we demonstrate that EMMPRIN up-regulated VEGF receptor-2 (VEGFR-2) in two different primary melanoma cell lines and consequently increased migration and proliferation of these cells while inhibiting their apoptosis. SiRNA inhibition of VEGFR-2 expression abrogated these EMMPRIN effects. EMMPRIN regulation of VEGFR-2 was mediated through the over-expression of HIF-2α and its translocation to the nucleus where it forms heterodimers with HIF-1β. These results were supported by an in vivo correlation between the expression of EMMPRIN with that of VEGFR-2 in human melanoma tissues as well as with the extent of HIF-2α localization in the nucleus. They demonstrate a novel mechanism by which EMMPRIN promotes tumor progression through HIF-2α/VEGFR-2 mediated mechanism, with an autocrine role in melanoma cell malignancy. The inhibition of EMMPRIN in cancer may thus simultaneously target both the VEGFR-2/VEGF system and the matrix degrading proteases to block tumor cell growth and invasion.

Inhibition of the proprotein convertases represses the invasiveness of human primary melanoma cells with altered p53, CDKN2A and N-Ras genes.

Background Altered tumor suppressor p53 and/or CDKN2A as well as Ras genes are frequently found in primary and metastatic melanomas. These alterations were found to be responsible for acquisition of invasive and metastatic potential through their defective regulatory control of metalloproteinases and urokinase genes. Methodology/Principal Findings Using primary human melanoma M10 cells with altered p53, CDKN2A and N-Ras genes, we found that inhibition of the proprotein convertases (PCs), enzymes involved in the proteolytic activation of various cancer-related protein precursors resulted in significantly reduced invasiveness. Analysis of M10 cells and their gastric and lymph node derived metastatic cells revealed the presence of all the PCs found in the secretory pathway. Expression of the general PCs inhibitor α1-PDX in these cells in a stable manner (M10/PDX) had no effect on the mRNA expression levels of these PCs. Whereas, in vitro digestion assays and cell transfection experiments, revealed that M10/PDX cells display reduced PCs activity and are unable to process the PCs substrates proIGF-1R and proPDGF-A. These cells showed reduced migration and invasion that paralleled decreased gelatinase MMP-2 activity and increased expression and secretion of tissue inhibitor of metalloproteinase-1 (TIMP-1) and TIMP-2. Furthermore, these cells showed decreased levels of urokinase-type plasminogen activator receptor (uPAR) and increased levels of plasminogen activator inhibitor-1 (PAI-1). Conclusions Taken together, these data suggest that inhibition of PCs activity results in decreased invasiveness of primary human melanoma cells despite their altered p53, CDKN2A and N-Ras genes, suggesting that PCs may serve as novel therapeutic targets in melanoma.

Identification of a novel biomarker signature associated with risk for bone metastasis in patients with renal cell carcinoma.

When renal cell carcinoma (RCC) metastasizes to bone (a frequent site of systemic spread of this cancer) it becomes highly resistant to radiation therapy and chemotherapy. A better understanding of the biology of bone metastasis in RCC may permit to identify biomarkers for early detection of subclinical disease and better stratification of patients prior to treatment. We therefore investigated in this study, using a multiplex real-time RT-PCR assay, the expression of a panel of 16 biomarkers involved in angiogenesis and tumor invasion; the panel was applied to primary tumors and normal tissues obtained from clear-cell RCC patients with and without bone metastases. We identified a novel combination of biomarkers associated with the risk of bone metastasis. Among the transcripts of the genes studied, VEGFR-1, VEGFR-2, HIF-1alpha, uPA , and PA I-1 overexpression in tumor tissues was significantly associated with the presence of bone metastasis (p=0.02, p=0.02, p<0.0001, p=0.04, and p=0.03, respectively). No differences were found in the expression of these transcripts in the corresponding normal tissues. This preliminary study provides a promising tool that may help in the management of RCC patients with bone metastasis. Indeed, these predictive markers could be useful to identify subclinical disease, improve staging, and guide treatment decisions.

Translational Research: Precision Medicine, Personalized Medicine, Targeted Therapies: Marketing or Science?

Personalized medicine is based on: 1) improved clinical or non-clinical methods (including biomarkers) for a more discriminating and precise diagnosis of diseases; 2) targeted therapies of the choice or the best drug for each patient among those available; 3) dose adjustment methods to optimize the benefit-risk ratio of the drugs chosen; 4) biomarkers of efficacy, toxicity, treatment discontinuation, relapse, etc. Unfortunately, it is still too often a theoretical concept because of the lack of convenient diagnostic methods or treatments, particularly of drugs corresponding to each subtype of pathology, hence to each patient. Stratified medicine is a component of personalized medicine employing biomarkers and companion diagnostics to target the patients likely to present the best benefit-risk balance for a given active compound. The concept of targeted therapy, mostly used in cancer treatment, relies on the existence of a defined molecular target, involved or not in the pathological process, and/or on the existence of a biomarker able to identify the target population, which should logically be small as compared to the population presenting the disease considered. Targeted therapies and biomarkers represent important stakes for the pharmaceutical industry, in terms of market access, of return on investment and of image among the prescribers. At the same time, they probably represent only the first generation of products resulting from the combination of clinical, pathophysiological and molecular research, i.e. of translational research.

Use of soluble isoforms of vascular endothelial growth factor to predict response to sunitinib in metastatic renal cell carcinomas.

e13508 Background: Angiogenesis is the target of several agents in the treatment of malignancies, including renal cell carcinoma (RCC). There is a real need for surrogate biomarkers that can predict selection of patients who may benefit from antiangiogenic therapies, prediction of disease outcome and which may improve the knowledge regarding mechanism of action of these treatments. Tyrosine kinase inhibitors (TKI) have proven efficacy in metastatic RCC (mRCC). However, the molecular mechanisms underlying the clinical response to these drugs remain unclear. The present study aimed to identify molecular biomarkers associated with the response to sunitinib, a TKI. Methods: To evaluate this relationship, primary tumors from 23 clear-cell metastatic RCC patients treated by sunitinib were analyzed for a panel of 16 biomarkers involved in tumor pathways targeted by sunitinib, using real-time quantitative reverse- transcriptase PCR. Results: Eighteen of the 23 patients (78%) responded to sunitinib (9 responses an...