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International audience; Ewing sarcoma is primarily caused by a t(11;22) chromosomal translocation encoding the EWS-FLI1 fusion protein. To exert its oncogenic function, EWS-FLI1 acts as an aberrant transcription factor, broadly altering the gene expression profile of tumor cells. Nuclear factor-kappaB (NFkappaB) is a tightly regulated transcription factor controlling cell survival, proliferation and differentiation, as well as tumorigenesis. NFkappaB activity is very low in unstimulated Ewing sarcoma cells, but can be induced in response to tumor necrosis factor (TNF). We wondered whether NFkappaB activity could be modulated by EWS-FLI1 in Ewing sarcoma. Using a knockdown approach in Ewing sarcoma cells, we demonstrated that EWS-FLI1 has no influence on NFkappaB basal activity, but impairs TNF-induced NFkappaB-driven transcription, at least in part through inhibition of NFkappaB binding to DNA. We detected an in vivo physical interaction between the fusion protein and NFkappaB p65, which could mediate these effects. Our findings suggest that, besides directly controlling the activity of its primary target promoters, EWS-FLI1 can also indirectly influence gene expression in tumor cells by modulating the activity of key transcription factors such as NFkappaB.

International audience; Altered angiogenic response is associated with high-grade cervical dysplasia and with invasive squamous carcinoma of the cervix. Vascular Endothelial Growth Factor (VEGF) is one of the most potent inducers of angiogenesis and is up-regulated in carcinoma of the cervix. Infection by high-risk human papillomavirus and persistent expression of viral oncogene E6 are etiologically linked to the development of cervical cancer. E6 is able to immortalize cells and induce malignant transformation by inactivating p53. In cervical cancer, regulation of VEGF expression is poorly described. Thus, we investigated whether E6 oncoprotein could regulate VEGF expression in HPV18-positive cervical cancer-derived HeLa cells harboring a wild-type p53. The alternative splicing of vegf mRNA renders three major isoforms of 121, 165 and 189 amino-acids in humans. We have designed isoform specific real time QRT-PCR assays to quantitate vegf transcripts and VEGF121 was the predominant isoform. Silencing HPV18 E6 mRNA with specific siRNA reduced VEGF121 expression by at least 50% whereas silencing of p53 did not alter its expression. Treatment with cycloheximide did not inhibit E6-induced VEGF121 expression. Collectively, these results suggest that HPV18 E6 oncoprotein contributes to tumor angiogenesis by inducing VEGF transcription from the promoter in a p53-independent manner.

International audience; 12-Lipoxygenase utilizes arachidonic acid to synthesize 12(S)-hydroperoxyeicosatetraenoic acid, which is converted to the end product 12(S)-hydroxyeicosatetraenoic acid, an eicosanoid that promotes tumorigenesis and metastasis. Increased expression of 12-lipoxygenase has been documented in a number of carcinomas. When overexpressed in human prostate or breast cancer, 12-lipoxygenase promotes tumor angiogenesis and growth in vivo. The present study was undertaken to delineate the mechanisms by which 12-lipoxygenase enhances angiogenesis. Herein we report that nordihydroguaiaretic acid, a pan inhibitor of lipoxygenases and baicalein, a selective inhibitor of 12-lipoxygenase, reduced VEGF expression in human prostate cancer PC-3 cells. Overexpression of 12-lipoxygenase in PC-3 cells resulted in a 3-fold increase in VEGF protein level when compared with vector control cells. An increase in PI 3-kinase activity was found in 12-LOX-transfected PC-3 cells and inhibition of PI 3-kinase by LY294002 significantly reduced VEGF expression. Northern blot and real time PCR analyses revealed an elevated VEGF transcript level in PC-3 cells transfected with a 12-lipoxygenase expression construct. Using a VEGF promoter luciferase construct (-1176/+54), we found a 10-fold increase in VEGF promoter activity in 12-lipoxygenase-transfected PC-3 cells. The region located between -88 and -66 of the VEGF promoter was identified as 12-lipoxygenase responsive using VEGF promoter-based luciferase assays. Further analysis with mutant constructs indicated Sp1 as a transcription factor required for 12-lipoxygenase stimulation of VEGF. Neutralization of VEGF by a function-blocking antibody significantly decreased the ability of 12-lipoxygenase-transfected PC-3 cells to stimulate endothelial cell migration, suggesting VEGF as an important effector for 12-lipoxygenase-mediated stimulation of tumor angiogenesis.