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The source tissue for biomarkers mRNA expression profiling of tumors has traditionally been fresh-frozen tissue. The adaptation of formalin-fixed, paraffin-embedded (FFPE) tissues for routine mRNA profiling would however be invaluable in view of their abundance and the clinical information related to them. However, their use in the clinic remains a challenge due to the poor quality of RNA extracted from such tissues. Here, we developed a method for the selection of melanoma archival paraffin-embedded tissues that can be reliably used for transcript biomarker profiling. For that, we used qRT-PCR to conduct a comparative study in matched pairs of frozen and FFPE melanoma tissues of the expression of 25 genes involved in angiogenesis/tumor invasion and 15 housekeeping genes. A classification method was developed that can select the samples with a good frozen/FFPE correlation and identify those that should be discarded on the basis of paraffin data for four reference genes only. We propose therefore a simple and inexpensive assay which improves reliability of mRNA profiling in FFPE samples by allowing the identification and analysis of ''good'' samples only. This assay which can be extended to other genes would however need validation at the clinical level and on independent tumor series. [ABSTRACT FROM AUTHOR]

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 proangiogenic and survival factors. [ABSTRACT FROM AUTHOR]

EMMPRIN's 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. [ABSTRACT FROM AUTHOR]

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. Methodology/Principal Findings: The present study aimed to identify molecular biomarkers associated with the response to sunitinib, a Tyrosine kinase inhibitor. To evaluate this relationship, primary tumors from 23 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. Nine of the 23 patients (39%) responded to sunitinib. Among transcripts analyzed, only the levels of vascular endothelial growth factor (VEGF) soluble isoforms (VEGF121 and VEGF165) were associated with the response to sunitinib (P = 0.04 for both). Furthermore, the ratio of VEGF soluble isoforms (VEGF121/ VEGF165) was significantly associated with prognosis (P = 0.02). Conclusions: This preliminary study provides a promising tool that might help in the management of metastatic RCC, and could be extended to other tumors treated by TKI. [ABSTRACT FROM AUTHOR]

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. [ABSTRACT FROM AUTHOR]