JunD regulates genes involved in antioxidant defence. We took advantage of the chronic oxidative stress resulting from junD deletion to examine the role of reactive oxygen species (ROS) in tumour development. In a model of mammary carcinogenesis, junD inactivation increased tumour incidence and revealed an associated reactive stroma. junD-inactivation in the stroma was sufficient to shorten tumour-free survival rate and enhance metastatic spread. ROS promoted conversion of fibroblasts into highly migrating myofibroblasts through accumulation of the hypoxia-inducible factor (HIF)-1α transcription factor and the CXCL12 chemokine. Accordingly, treatment with an antioxidant reduced the levels of HIF and CXCL12 and numerous myofibroblast features. CXCL12 accumulated in the stroma of HER2-human breast adenocarcinomas. Moreover, HER2 tumours exhibited a high proportion of myofibroblasts, which was significantly correlated to nodal metastases. Interestingly, this subset of tumours exhibited a significant nuclear exclusion of JunD and revealed an associated oxido-reduction signature, further demonstrating the relevance of our findings in human cancers. Collectively, our data uncover a new mechanism by which oxidative stress increases the migratory properties of stromal fibroblasts, which in turn potentiate tumour dissemination.

The pathophysiological mechanism involved in side effects of radiation therapy, and especially the role of the endothelium remains unclear. Previous results showed that plasminogen activator inhibitor-type 1 (PAI-1) contributes to radiation-induced intestinal injury and suggested that this role could be driven by an endothelium-dependent mechanism. We investigated whether endothelial-specific PAI-1 deletion could affect radiation-induced intestinal injury. We created a mouse model with a specific deletion of PAI-1 in the endothelium (PAI-1KOendo) by a Cre-LoxP system. In a model of radiation enteropathy, survival and intestinal radiation injury were followed as well as intestinal gene transcriptional profile and inflammatory cells intestinal infiltration. Irradiated PAI-1KOendo mice exhibited increased survival, reduced acute enteritis severity and attenuated late fibrosis compared with irradiated PAI-1flx/flx mice. Double E-cadherin/TUNEL labeling confirmed a reduced epithelial cell apoptosis in irradiated PAI-1KOendo. High-throughput gene expression combined with bioinformatic analyses revealed a putative involvement of macrophages. We observed a decrease in CD68+cells in irradiated intestinal tissues from PAI-1KOendo mice as well as modifications associated with M1/M2 polarization. This work shows that PAI-1 plays a role in radiation-induced intestinal injury by an endothelium-dependent mechanism and demonstrates in vivo that the endothelium is directly involved in the progression of radiation-induced enteritis.

BackgroundEndothelial dysfunction has been implicated in the pathogenesis of diverse pathologies ranging from vascular and immune diseases to cancer. TNF-α is one of the mediators of endothelial dysfunction through the activation of transcription factors, including NF-κB. While HUVEC (macrovascular cells) have been largely used in the past, here, we documented an NF-κB gene signature in TNFα-stimulated microvascular endothelial cells HMEC often used in tumor angiogenesis studies.Methodology/Principal FindingsWe measured mRNA expression of 55 NF-κB related genes using quantitative RT-PCR in HUVEC and HMEC. Our study identified twenty genes markedly up-regulated in response to TNFα, including adhesion molecules, cytokines, chemokines, and apoptosis regulators, some of them being identified as TNF-α-inducible genes for the first time in endothelial cells (two apoptosis regulators, TNFAIP3 and TNFRSF10B/Trail R2 (DR5), the chemokines GM-CSF/CSF2 and MCF/CSF1, and CD40 and TNF-α itself, as well as NF-κB components (RELB, NFKB1or 50/p105 and NFKB2 or p52/p100). For eight genes, the fold induction was much higher in HMEC, as compared to HUVEC. Most importantly, our study described for the first time a connection between NF-κB activation and the induction of most, if not all, of these genes in HMEC as evaluated by pharmacological inhibition and RelA expression knock-down by RNA interference.Moreover, since TNF-α is highly expressed in tumors, we further applied the NF-κB gene signature documented in TNFα-stimulated endothelial cells to human breast tumors. We found a significant positive correlation between TNF and the majority (85 %) of the identified endothelial TNF-induced genes in a well-defined series of 96 (48 ERα positive and 48 ERα negative) breast tumors.Conclusion/SignificanceTaken together these data suggest the potential use of this NF-κB gene signature in analyzing the role of TNF-α in the endothelial dysfunction, as well as in breast tumors independently of the presence of ERα.

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Background & AimsRadiation therapy in the pelvic area is associated with side effects that impact the quality of life of cancer survivors. Interestingly, the gastrointestinal tract is able to adapt to significant changes in oxygen availability, suggesting that mechanisms related to hypoxia sensing help preserve tissue integrity in this organ. However, hypoxia-inducible factor (HIF)-dependent responses to radiation-induced gut toxicity are unknown. Radiation-induced intestinal toxicity is a complex process involving multiple cellular compartments. Here, we investigated whether epithelial or endothelial tissue-specific HIF-1α deletion could affect acute intestinal response to radiation.MethodsUsing constitutive and inducible epithelial or endothelial tissue-specific HIF-1α deletion, we evaluated the consequences of epithelial or endothelial HIF-1α deletion on radiation-induced enteritis after localized irradiation. Survival, radiation-induced tissue injury, molecular inflammatory profile, tissue hypoxia, and vascular injury were monitored.ResultsSurprisingly, epithelium-specific HIF-1α deletion does not alter radiation-induced intestinal injury. However, irradiated VECad-Cre+/-HIF-1αFL/FL mice present with lower radiation-induced damage, showed a preserved vasculature, reduced hypoxia, and reduced proinflammatory response compared with irradiated HIF-1αFL/FL mice.ConclusionsWe demonstrate in vivo that HIF-1α impacts radiation-induced enteritis and that this role differs according to the targeted cell type. Our work provides a new role for HIF-1α and endothelium-dependent mechanisms driving inflammatory processes in gut mucosae. Results presented show that effects on normal tissues have to be taken into account in approaches aiming to modulate hypoxia or hypoxia-related molecular mechanisms.

Background:Vascular endothelial growth factor (VEGF) is a multifunctional cytokine that has important roles in angiogenesis. Our knowledge of the significance of VEGF isoforms in human cancer remains incomplete.Methods:Bioluminescence imaging and transcriptomic analysis were used to study the colonisation capacity of the human breast cancer cells MDA-MB-231 controlling or overexpressing the VEGF165 or VEGF189 isoform (named cV-B, V165-B and V189-B, respectively) in nude mice.Results:When injected into the bloodstream, V189-B cells induced less metastasis in the lungs and bone than V165-B and cV-B control cells, consistent with longer survival of these mice and delay in tumour uptake in the mice injected with a V189-B clone. Histological analysis confirmed that there were less αSMA-positive cells in the lungs of the mice injected with V189-B. In vitro V189-B cells decreased both cell invasion and survival. Using transcriptomic analysis, we identified a subset of 18 genes expressed differentially between V189 and V165 cell lines and in 120 human breast tumours. V165 was associated with poor prognosis, whereas V189 was not, suggesting a complex regulation by VEGF isoforms. Our results showed a negative correlation between the expression pattern of VEGF189 and the levels of expression of seven genes that influence metastasis.Conclusion:Our findings provide the first evidence that VEGF isoforms have different effects on breast cancer cell line colonisation in vivo.

Exposure to low doses of environmental estrogens such as bisphenol A and genistein (G) alters mammary gland development. The effects of environmental anti-androgens, such as the fungicide vinclozolin (V), on mammary gland morphogenesis are unknown. We previously reported that perinatal exposure to G, V, and the GV combination causes histological changes in the mammary gland during the peripubertal period, suggesting alterations to the peripubertal hormone response. We now investigate whether perinatal exposure to these compounds alters the gene expression profiles of the developing glands to identify the dysregulated signaling pathways and the underlying mechanisms. G, V, or GV (1 mg/kg body weight per day) was added to diet of Wistar rats, from conception to weaning; female offspring mammary glands were collected at postnatal days (PNDs) 35 and 50. Genes displaying differential expression and belonging to different functional categories were validated by quantitative PCR and immunocytochemistry. At PND35, G had little effect; the slight changes noted were in genes related to morphogenesis. The changes following exposure to V concerned the functional categories associated with development (Cldn1, Krt17, and Sprr1a), carbohydrate metabolism, and steroidogenesis. The GV mixture upregulated genes (Krt17, Pvalb, and Tnni2) involved in muscle development, indicating effects on myoepithelial cells during mammary gland morphogenesis. Importantly, at PND50, cycling females exposed to GV showed an increase in the expression of genes (Csn2, Wap, and Elf5) related to differentiation, consistent with the previously reported abnormal lobuloalveolar development previously described. Thus, perinatal exposure to GV alters the mammary gland hormone response differently at PND35 (puberty) and in animals with established cycles.

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