Nouveaux marqueurs des glioblastomes (valeur pronostique, profil d'expression, implication dans la vascularisation et la résistance aux antiangiogéniques)

L angiogenèse est une composante majeure de l agressivité des tumeurs malignes comme le glioblastome (GBM). Pourtant le traitement des patients par l Avastin, un anticorps bloquant du VEGF ne leur confère qu une augmentation limitée de la durée de survie sans progression. Les mécanismes de récurrence tumorale sont extrêmement complexes. Les glioblastomes sont en effet des tumeurs particulièrement hétérogènes sur le plan génétique, il existe très peu de marqueurs moléculaires d expression fiables. La contribution à la récidive des potentiels angiogéniques, infiltrants, et souches est difficile à modéliser. Notre laboratoire s intéresse à la caractérisation de nouveaux modulateurs de l angiogenèse, dont certains pourraient contribuer à la croissance tumorale, indépendamment ou en aval du VEGF chez les patients traités par l Avastin. Nous avons étudié l'expression de deux gènes candidats, surexprimés de façon significative dans les GBM et dont l'expression semble liée à l'angiogenèse tumorale : DPY19L1 et KIF20A. Nous avons identifié DPY19L1 comme marqueur pronostique du GBM. Ce gène est exprimé dans les cellules musculaires lisses, où il pourrait participer à la résistance de la tumeur aux anti-angiogéniques, en interagissant avec la voie thrombospondine/TGFb. KIF20A quant à lui est exprimé dans les cellules souches tumorales et semble impliqué dans la vascularisation et la résistance tumorale. Dans un second temps, nous avons étudié la façon dont les GBM échappent aux traitements anti-angiogéniques, tel que l Avastin, par la mise en place d'un système d'étude in vitro et in vivo, basé sur l utilisation de cellules de patients atteints de GBM, ayant la capacité de pousser sous forme de neurosphères. Les cellules xénogreffées chez la souris immunodéfisciente permettent le développement d une tumeur très invasive, co-optive et insensible aux traitements anti-angiogéniques. Ces tumeurs vont donc permettre d étudier ce mode de vascularisation participant activement à la récidive de la tumeur chez les patients traités avec l Avastin afin de développer des traitements contrecarrant ce mécanisme. Dans ce modèle, seul l un des gènes candidats définis au début de ce travail, DPY19L1, semble participer à la croissance tumorale.Angiogenesis is a major element driving malignancy of tumors like glioblastoma (GBM). However, Avastin,a neutralizing antibody directed against VEGF, provides only a limited therapeutical benefit in terms ofprogression free survival. The mechanisms of recurrence are complex due to extreme heterogeneity ofglioblastoma at the genetic and tissular levels. There is a lack of diagnosis and prognosis markers for GBM.The relative contribution of the angiogenic, infiltrative, and stem potentials to tumor relapse is difficult tomodel. Our laboratory aims at characterizing new modulators of tumor vascularization, some of whichcould contribute to the tumor growth and resistance, independently or downstream VEGF in patientstreated with Avastin. We have studied the expression of two candidate genes, significantly overexpressedin GBM and which expression seems to be linked to tumor vascularization: DPY19L1 and KIF20A. Weidentified DPY19L1 as a prognosis marker of GBM. This gene is expressed in smooth muscle cellsspecifically in tumoral tissue, where it could participate to tumor resistance to anti-angiogenics, byinteracting with the thrombospondin/TGFb pathway. KIF20A is expressed in glioma stem cells and seemsto be implicated in the vascularization and tumor resistance. Next, we have studied the way by whichGBM resist to anti-angiogenics such as Avastin, by the development of an in vitro and in vivo modelsystem, based on GBM cells cultured as neurospheres. When xenografted in immunodeficient mice, thesecells induce the growth of very invasive, co-optive tumors which are insensitive to angiogenesis inhibitors.These tumors will allow investigating alternative modes of vascularization which are actively involved intumor recurrence in patients treated with Avastin, namely co-option and transdifferentiation and theirmolecular regulation. In this model, one of the candidate genes defined at the beginning of this study,DPY19L1, seems to be implied in tumor growth and specifically labels tumor cells with co-optive andtransdifferenciating properties.BORDEAUX1-Bib.electronique (335229901) / SudocSudocFranceF

FBXW7/hCDC4 controls glioma cell proliferation in vitro and is a prognostic marker for survival in glioblastoma patients

BACKGROUND: In the quest for novel molecular mediators of glioma progression, we studied the regulation of FBXW7 (hCDC4/hAGO/SEL10), its association with survival of patients with glioblastoma and its potential role as a tumor suppressor gene in glioma cells. The F-box protein Fbxw7 is a component of SCF(Fbxw7), a Skp1-Cul1-F-box E3 ubiquitin ligase complex that tags specific proteins for proteasome degradation. FBXW7 is mutated in several human cancers and functions as a haploinsufficient tumor suppressor in mice. Any of the identified targets, Cyclin E, c-Myc, c-Jun, Notch1/4 and Aurora-A may have oncogenic properties when accumulated in tumors with FBXW7 loss. RESULTS: We tested the expression of FBXW7 in human glioma biopsies by quantitative PCR and compared the transcript levels of grade IV glioma (glioblastoma, G-IV) with those of grade II tumors (G-II). In more than 80% G-IV, expression of FBXW7 was significantly reduced. In addition, levels of FBXW7 were correlated with survival indicating a possible implication in tumor aggressiveness. Locus 4q31.3 which carries FBXW7 was investigated by in situ hybridization on biopsy touchprints. This excluded allelic loss as the principal cause for low expression of FBXW7 in G-IV tumors. Two targets of Fbxw7, Aurora-A and Notch4 were preferentially immunodetected in G-IV biopsies. Next, we investigated the effects of FBXW7 misregulation in glioma cells. U87 cells overexpressing nuclear isoforms of Fbxw7 lose the expression of the proliferation markers PCNA and Ki-67, and get counterselected in vitro. This observation fits well with the hypothesis that Fbxw7 functions as a tumor suppressor in astroglial cells. Finally, FBXW7 knockdown in U87 cells leads to defects in mitosis that may promote aneuploidy in progressing glioma. CONCLUSION: Our results show that FBXW7 expression is a prognostic marker for patients with glioblastoma. We suggest that loss of FBXW7 plays an important role in glioma malignancy by allowing the accumulation of multiple oncoproteins and that interfering with Fbxw7 or its downstream targets would constitute a new therapeutic advance

The Dct−/− Mouse Model to Unravel Retinogenesis Misregulation in Patients with Albinism

We have recently identified encoding dopachrome tautomerase (DCT) as the eighth gene for oculocutaneous albinism (OCA). Patients with loss of function of suffer from eye hypopigmentation and retinal dystrophy. Here we investigate the eye phenotype in mice. We show that their retinal pigmented epithelium (RPE) is severely hypopigmented from early stages, contrasting with the darker melanocytic tissues. Multimodal imaging reveals specific RPE cellular defects. Melanosomes are fewer with correct subcellular localization but disrupted melanization. RPE cell size is globally increased and heterogeneous. P-cadherin labeling of newborn RPE reveals a defect in adherens junctions similar to what has been described in tyrosinase-deficient embryos. The first intermediate of melanin biosynthesis, dihydroxyphenylalanine (L-Dopa), which is thought to control retinogenesis, is detected in substantial yet significantly reduced amounts in postnatal mouse eyecups. L-Dopa synthesis in the RPE alone remains to be evaluated during the critical period of retinogenesis. The mouse should prove useful in understanding the molecular regulation of retinal development and aging of the hypopigmented eye. This may guide therapeutic strategies to prevent vision deficits in patients with albinism.Approches de génétique moléculaire et fonctionnelle pour déchiffrer les mécanismes physiopathologiques de l'albinisme oculocutané

Dopachrome tautomerase variants in patients with oculocutaneous albinism

Purpose: Albinism is a clinically and genetically heterogeneous condition. Despite analysis of the 20 known genes, ~30% patients remain unsolved. We aimed to identify new genes involved in albinism. Methods: We sequenced a panel of genes with known or predicted involvement in melanogenesis in 230 unsolved albinism patients. Results: We identified variants in the Dopachrome tautomerase (DCT) gene in two patients. One was compound heterozygous for a 14-bp deletion in exon 9 and c.118T>A p.(Cys40Ser). The second was homozygous for c.183C>G p.(Cys61Trp). Both patients had mild hair and skin hypopigmentation, and classical ocular features. CRISPR-Cas9 was used in C57BL/6J mice to create mutations identical to the missense variants carried by the patients, along with one loss-of-function indel. When bred to homozygosity the three mutations revealed hypopigmentation of the coat, milder for Cys40Ser compared with Cys61Trp or the frameshift mutation. Histological analysis identified significant hypopigmentation of the retinal pigmented epithelium (RPE) indicating that defective RPE melanogenesis could be associated with eye and vision defects. DCT loss of function in zebrafish embryos elicited hypopigmentation both in melanophores and RPE cells. Conclusion: DCT is the gene for a new type of oculocutaneous albinism that we propose to name OCA8

Correlating Global Gene Regulation to Angiogenesis in the Developing Chick Extra-Embryonic Vascular System

International audienceBACKGROUND: Formation of blood vessels requires the concerted regulation of an unknown number of genes in a spatial-, time- and dosage-dependent manner. Determining genes, which drive vascular maturation is crucial for the identification of new therapeutic targets against pathological angiogenesis. METHOLOGY/PRINCIPAL FINDINGS: We accessed global gene regulation throughout maturation of the chick chorio-allantoic membrane (CAM), a highly vascularized tissue, using pan genomic microarrays. Seven percent of analyzed genes showed a significant change in expression (>2-fold, FDR<5%) with a peak occurring from E7 to E10, when key morphogenetic and angiogenic genes such as BMP4, SMO, HOXA3, EPAS1 and FGFR2 were upregulated, reflecting the state of an activated endothelium. At later stages, a general decrease in gene expression occurs, including genes encoding mitotic factors or angiogenic mediators such as CYR61, EPAS1, MDK and MYC. We identified putative human orthologs for 77% of significantly regulated genes and determined endothelial cell enrichment for 20% of the orthologs in silico. Vascular expression of several genes including ENC1, FSTL1, JAM2, LDB2, LIMS1, PARVB, PDE3A, PRCP, PTRF and ST6GAL1 was demonstrated by in situ hybridization. Up to 9% of the CAM genes were also overexpressed in human organs with related functions, such as placenta and lung or the thyroid. 21-66% of CAM genes enriched in endothelial cells were deregulated in several human cancer types (P<.0001). Interfering with PARVB (encoding parvin, beta) function profoundly changed human endothelial cell shape, motility and tubulogenesis, suggesting an important role of this gene in the angiogenic process. CONCLUSIONS/SIGNIFICANCE: Our study underlines the complexity of gene regulation in a highly vascularized organ during development. We identified a restricted number of novel genes enriched in the endothelium of different species and tissues, which may play crucial roles in normal and pathological angiogenesis

La réponse du gliome aux traitements anti-angiogéniques (de la modélisation à l'analyse morphologique et transcriptomique)

Les glioblastomes multiformes sont les tumeurs cérébrales les plus agressives. Elles se caractérisent par une néovascularisation très dense et elles présentent une résistance accrue à la plupart des chimiothérapies. L angiogenèse tumorale définit le mécanisme de vascularisation essentiel à la croissance et la dissémination des tumeurs. La thérapie antiangiogénique constitue une nouvelle stratégie prometteuse dans le traitement des glioblastomes. Nous avons modélisé la réponse des glioblastomes aux traitements antiangiogéniques en réalisant le knockdown d un facteur clé de l angiogenèse, le VEGF par la technique de l interférence à l ARN dans des cellules de gliome humain greffées sur la membrane chorioallantoïdienne de poulet comme modèle principal d étude in vivo. Nous avons déterminé les voies de signalisation régulées en réponse à un traitement anti-angiogénique et identifié le CHI3L1 et l elafin/PI3 comme nouveaux marqueurs pronostiques de survie dans les glioblastomes.. Nous avons également montré que la thérapie combinatoire anti-Interleukine-6/anti-VEGF présente un effet synergique dans le traitement du glioblastome expérimental. Cependant nous avons observé que ces traitements provoquent également la surexpression de gènes protumoraux, en particulier codant pour des facteurs proinvasifs. Nos résultats suggèrent que la combinaison de la thérapie antiangiogénique avec des inhibiteurs de la survie et/ou de l invasion pourrait être une thérapie prometteuse pour le traitement des glioblastomes. Enfin, nous avons montré pour la première fois, qu une nouvelle métalloprotéase extraite du venin de serpent, appelée l atragin a un effet antiangiogénique potentiel.Glioblastoma multiforme are the most aggressive type of primary brain tumor. They are characterized by very dense neovascularization and they exhibit profound resistance to chemotherapy. Tumor angiogenesis defines the vascularization mechanism essential for tumor growth and spread. The antiangiogenic therapy is a promising new strategy in the treatment of glioblastoma. We have modelized the glioblastoma response to antiangiogenic treatment by knockdown of the key regulator of angiogenesis, VEGF using RNA interference on tumor cells grafted on the chicken chorioallantoic membrane model. We showed that the antiangiogenesis in experimental glioma drives expression of critical genes which relate to disease aggressiveness in glioblastoma patients. In particular, CHI3L1 and PI3/elafin expression profiles are inversely correlated with the survival of patients. These two genes may be useful as new prognostic markers and new therapeutic targets. We have also shown that that experimental glioma treated with a combination of Interleukin-6 and VEGF inhibitors do benefit of synergistic effect but still contain viable tumor cells which up-regulate major pathways of cell survival, proliferation and cell invasiveness. These results suggest that combining antiangiogenic therapy with inhibitors of invasion/survival pathways should improve clinical management of patients with glioblastoma. For the first time, we have determined the antiangiogenic effect of a new snake venom metalloprotease, called the atragin. Atragin may be useful as potential antiangiogenic drug and may as well reveal new therapeutic target in cancer biology.BORDEAUX1-BU Sciences-Talence (335222101) / SudocSudocFranceF

Hagedorn M: Inhibition of angiogenesis and the angiogenesis/invasion shift

Abstract Angiogenesis has become a major target in cancer therapy. However, current therapeutic strategies have their limitations and raise several problems. In most tumours, anti-angiogenesis treatment targeting VEGF (vascular endothelial growth factor) has only limited overall survival benefit compared with conventional chemotherapy alone, and reveals several specific forms of resistance to anti-VEGF treatment. There is growing evidence that anti-VEGF treatment may induce tumour cell invasion by selecting highly invasive tumour cells or hypoxia-resistant cells, or by up-regulating angiogenic alternative pathways such as FGFs (fibroblast growth factors) or genes triggering new invasive programmes. We have identified new genes upregulated during glioma growth on the chick CAM (chorioallantoic membrane). Our results indicate that antiangiogenesis treatment in the experimental glioma model drives expression of critical genes which relate to disease aggressiveness in glioblastoma patients. We have identified a molecular mechanism in tumour cells that allows the switch from an angiogenic to invasive programme. Furthermore, we are focusing our research on alternative inhibitors that act, in part, independently of VEGF. These are endogenous molecules that play a role in the control of tumour growth and may constitute a starting point for further development of novel therapeutic or diagnostic tools