Ischémie et angiogénèse tumorale : effets des carences en glucose et en acides aminés sur l'expression du VEGF-A par les cellules tumorales et implication de la réponse (« Unfolded Protein Response »).

Dans les tumeurs solides, les cellules soumises à des conditions ischémiques induisent des voies de signalisation distinctes contribuant à la commutation angiogénique et au développement tumoral. La composante hypoxique de l'ischémie induit l'expression du VEGF-A par la voie de signalisation dépendante de HIF-1. Par contre, la contribution des carences en glucose ou en glutamine est peu connue. En utilisant un milieu sans sérum, nous montrons que l'activation et la signalisation dépendante d'IRE1 sont des événements liant les réponses dépendantes de l'hypoxie et des carences en glucose à la surexpression du VEGF-A dans divers modèles cellulaires. Des cellules tumorales exprimant un dominant négatif de IRE1 et des cellules MEF IRE1-/- n'induisent pas l'expression du VEGF-A en conditions d'hypoxie ou de carences en glucose. Ces stress mènent à l'induction du VEGF-A par des mécanismes en partie distincts. Ces données sont corrélées avec une réduction de l'angiogenèse et de la croissance tumorale in vivo et démontrent le rôle essentiel joué par IRE1 en réponse à l'ischémie. En absence de glutamine, les ARNm du VEGF-A sont surexprimés indépendamment de l'accumulation des protéines HIF et de la transcription des gènes dépendants de la réponse UPR. De plus, l'expression protéique du VEGF-A est diminuée en corrélation avec la réduction de la synthèse protéique globale. Ces résultats suggèrent que les carences en glutamine n'ont aucun effet sur le processus angiogénique induit par le VEGF-A dans certaines tumeurs. Nous avons également recherché l'implication des carences en glutamine et en glucose dans les réponses métaboliques des cellules tumorales. Ces stress augmentent rapidement le processus d'apoptose, d'une manière plus importante en absence de glutamine qu'en absences de glucose. La spectrométrie FTIR a permis d'évaluer le statut métabolique des cellules tumorales ischémiques et a corrélé leur phénotype hyper-glycolytique avec leur statut prolifératif et agressif.In solid tumors, cells subjected to ischemic conditions trigger distinct signaling pathways which contribute to the angiogenic switch and tumor development. The hypoxic component of ischemia leads to the expression of VEGF-A by the HIF-1-dependent signaling pathways. Alternatively, the contribution of glucose or glutamine deprivation is still not well understood. Using a serum-free medium, we demonstrate that IRE1 activation and signaling is a common molecular determinant linking hypoxia- and hypoglycemia-dependent responses to the upregulation of VEGF-A in various cell models. Indeed, tumor cells expressing a dominant negative IRE1 transgene as well as IRE1-/- MEF were unable to trigger VEGF-A upregulation upon either hypoxic or glucose deprivation conditions. We showed that these various cellular stresses lead to the induction of VEGF-A by partly distinct mechanisms. These data correlated with a reduction of tumor angiogenesis and growth in vivo and demonstrated the essential role played by IRE1 in response to tumor ischemia. Under glutamine deprivation, VEGF-A mRNA was up-regulated and this effect was neither associated to HIF proteins accumulation nor to the transcriptional induction of UPR-dependent genes. Besides, VEGF-A protein expression was down-regulated in correlation to the global decrease of protein synthesis. These results suggested that amino-acids deprivation has no effect on the VEGF-A-driven angiogenic process in a number of tumors. We then questioned the implication of glucose and glutamine deprivations in the metabolic responses of tumor cells. This stress rapidly increase the apoptotic response of the cells, and to a much greater extent in the absence of glutamine than under glucose deprivation. FTIR spectrometry was used to evaluate the metabolic status of tumor cells and correlated the hyper-glycolytic phenotype with their proliferative and aggressive status

Inhibition of DDR1 enhances in vivo chemosensitivity in KRAS-mutant lung adenocarcinoma

Platinum-based chemotherapy in combination with immune-checkpoint inhibitors is the current standard of care for patients with advanced lung adenocarcinoma (LUAD). However, tumor progression evolves in most cases. Therefore, predictive bioma ricers are needed for better patient stratification and for the identification of new therapeutic strategies, including enhancing the efficacy of chemotoxic agents. Here, we hypothesized that discoidin domain receptor 1 (DDR1) may be both a predictive factor for chemoresistance in patients with LUAD and a potential target positively selected in resistant cells. By using biopsies from patients with LUAD, KRAS-mutant LUAD cell lines, and in vivo genetically engineered KRAS-driven mouse models, we evaluated the role of DDR1 in the context of chemotherapy treatment. We found that DORT is upregulated during chemotherapy both in vitro and in viva. Moreover, analysis of a cohort of patients with LUAD suggested that high DOR1 levels in pretreatment biopsies correlated with poor response to chemotherapy. Additionally, we showed that combining DORI inhibition with chemotherapy prompted a synergistic therapeutic effect and enhanced cell death of KRAS-mutant tumors in vivo. Collectively, this study suggests a potential role for DDR1 as both a predictive and prognostic biomarker, potentially improving the chemotherapy response of patients with LUAD

Efficient mouse transgenesis using Gateway-compatible ROSA26 locus targeting vectors and F1 hybrid ES cells

The ability to rapidly and efficiently generate reliable Cre/loxP conditional transgenic mice would greatly complement global high-throughput gene targeting initiatives aimed at identifying gene function in the mouse. We report here the generation of Cre/loxP conditional ROSA26-targeted ES cells within 3–4 weeks by using Gateway® cloning to build the target vectors. The cDNA of the gene of interest can be expressed either directly by the ROSA26 promoter providing a moderate level of expression or by a CAGG promoter placed in the ROSA26 locus providing higher transgene expression. Utilization of F1 hybrid ES cells with exceptional developmental potential allows the production of germ line transmitting, fully or highly ES cell-derived mice by aggregation of cells with diploid embryos. The presented streamlined procedures accelerate the examination of phenotypical consequences of transgene expression. It also provides a unique tool for comparing the biological activity of polymorphic or splice variants of a gene, or products of different genes functioning in the same or parallel pathways in an overlapping manner

Histological mapping of biochemical changes in solid tumors by FT-IR spectral imaging.

Fourier-transform infrared (FT-IR) spectral imaging was used for analyzing biochemical changes in tumor cells. Metabolic parameters of human lung A549/8 adenocarcinoma and U87 glioma cells were compared under stress conditions in culture along with tumor progression after cell implantation onto the chick embryo chorio-allantoic membrane. In cell culture, glucose consumption and lactic acid release were higher in U87 cells. A549/8 cells were less sensitive to oxidative stress as observed through changes in fatty acyl chains. In vivo biochemical mapping of highly (U87) vs. poorly (A549/8) angiogenic tumors provided results comparable to culture models. Therefore, FT-IR imaging allows detecting subtle chemical changes in tumors, which might be useful for diagnosis.Journal ArticleResearch Support, Non-U.S. Gov'tinfo:eu-repo/semantics/publishe

Beta-actin is involved in modulating erythropoiesis during development by fine-tuning gata2 expression levels

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Acute L-glutamine deprivation compromises VEGF-a upregulation in A549/8 human carcinoma cells.

Tumor ischemia participates in angiogenesis and cancer progression through cellular responses to hypoxia and nutrient deprivation. However, the contribution of amino acids limitation to this process remains poorly understood. Using serum-free cell culture conditions, we tested the impact of L-glutamine deprivation on metabolic and angiogenic responses in A549/8 carcinoma cells. In these cells, lowering glutamine concentration modified the cell cycle distribution and significantly induced apoptosis/necrosis. Although glutamine deprivation led to a HIF-independent increase in VEGF-A mRNA, the corresponding protein level remained low and correlated with the inhibition of protein synthesis and activation of the GCN2/eIF2alpha pathway. Limitation of glutamine availability also hampers hypoxia- and hypoglycemia-induced VEGF-A protein upregulation. Thus, glutamine deprivation may have no direct effect on VEGF-dependent angiogenesis, compared to hypoxia or to glucose deprivation, and may instead be detrimental to cancer progression by antagonizing ischemia-induced stresses.Journal ArticleResearch Support, Non-U.S. Gov'tFLWINinfo:eu-repo/semantics/publishe

Different Levels of Twist1 Regulate Skin Tumor Initiation, Stemness, and Progression

Twist1 promotes epithelial-to-mesenchymal transition (EMT), invasion, metastasis, and cancer stem cell (CSC) properties. However, it remains unclear whether Twist1 is also required for tumor initiation and whether Twist1-induced cancer stemness and EMT are functionally linked. Using a conditional deletion of Twist1 at different stages of skin carcinogenesis, we show that Twist1 is required for skin tumor initiation and progression in a gene-dosage-dependent manner. Moreover, conditional ablation of Twist1 in benign tumors leads to increased apoptosis, reduced cell proliferation, and defective tumor maintenance and propagation independently of its EMT-inducing abilities. Concomitant deletion of Twist1 and p53 rescues the apoptotic response, but not the cell proliferation and propagation defects. These results reveal that Twist1 is required for tumor initiation and maintenance in a p53-dependent and -independent manner. Importantly, our findings also indicate that tumor stemness and EMT can be regulated by distinct mechanisms.SCOPUS: ar.jSCOPUS: ar.jinfo:eu-repo/semantics/publishe

IRE1 signaling is essential for ischemia-induced vascular endothelial growth factor-A expression and contributes to angiogenesis and tumor growth in vivo.

In solid tumors, cancer cells subjected to ischemic conditions trigger distinct signaling pathways contributing to angiogenic stimulation and tumor development. Characteristic features of tumor ischemia include hypoxia and glucose deprivation, leading to the activation of hypoxia-inducible factor-1-dependent signaling pathways and to complex signaling events known as the unfolded protein response. Here, we show that the activation of the endoplasmic reticulum stress sensor IRE1 is a common determinant linking hypoxia- and hypoglycemia-dependent responses to the up-regulation of vascular endothelial growth factor-A (VEGF-A). Tumor cells expressing a dominant-negative IRE1 transgene as well as Ire1alpha-null mouse embryonic fibroblasts were unable to trigger VEGF-A up-regulation upon either oxygen or glucose deprivation. These data correlated with a reduction of tumor angiogenesis and growth in vivo. Our results therefore suggest an essential role for IRE1-dependent signaling pathways in response to ischemia and identify this protein as a potential therapeutic target to control both the angiogenic switch and tumor development.Journal ArticleResearch Support, N.I.H. ExtramuralResearch Support, Non-U.S. Gov'tinfo:eu-repo/semantics/publishe

Beta-actin binds to specific regions of the <i>Gata2</i> gene.

<p>(A) Immunoprecipitation with the anti beta-actin and anti gamma-actin antibodies followed by qRT-PCR with 9 specific primers for the <i>Gata2</i> promotor region yielded 2 loci of interest: amplicon 3 and 8. Error bars represent mean ±SEM; *P<.05, **P<.01. (B) Genomic alignment with multiple species showing that amplicon 3 is partly overlapping with a highly conserved region in the <i>Gata2</i> promotor, especially in mammals. Also the localization of the specific amplicons relative to the mouse <i>Gata2</i> gene is shown. Amplicon8 is located between exons 1 and 2. Figure was made using UCSC genome bioinformatics software (<a href="http://genome.ucsc.edu/" target="_blank">http://genome.ucsc.edu/</a>).</p