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PDF file 1160K, AM is a growth factor survival for LECs

PDF file 114K, described the legends of all the supplementary figures

Purpose: To study the role of the adrenomedullin system [adrenomedullin and its receptors (AMR), CLR, RAMP2, and RAMP3] in prostate cancer androgen-independent growth.Experimental Design: Androgen-dependent and -independent prostate cancer models were used to investigate the role and mechanisms of adrenomedullin in prostate cancer hormone-independent growth and tumor-associated angiogenesis and lymphangiogenesis.Results: Adrenomedullin and AMR were immunohistochemically localized in the carcinomatous epithelial compartment of prostate cancer specimens of high grade (Gleason score >7), suggesting a role of the adrenomedullin system in prostate cancer growth. We used the androgen-independent Du145 cells, for which we demonstrate that adrenomedullin stimulated cell proliferation in vitro through the cAMP/CRAF/MEK/ERK pathway. The proliferation of Du145 and PC3 cells is decreased by anti-adrenomedullin antibody (αAM), supporting the fact that adrenomedullin may function as a potent autocrine/paracrine growth factor for prostate cancer androgen-independent cells. In vivo, αAM therapy inhibits the growth of Du145 androgen-independent xenografts and interestingly of LNCaP androgen-dependent xenografts only in castrated animals, suggesting strongly that adrenomedullin might play an important role in tumor regrowth following androgen ablation. Histologic examination of αAM-treated tumors showed evidence of disruption of tumor vascularity, with depletion of vascular as well as lymphatic endothelial cells and pericytes, and increased lymphatic endothelial cell apoptosis. Importantly, αAM potently blocks tumor-associated lymphangiogenesis, but does not affect established vasculature and lymphatic vessels in normal adult mice.Conclusions: We conclude that expression of adrenomedullin upon androgen ablation in prostate cancer plays an important role in hormone-independent tumor growth and in neovascularization by supplying/amplifying signals essential for pathologic neoangiogenesis and lymphangiogenesis. Clin Cancer Res; 19(22); 6138–50. ©2013 AACR.

PDF file 97K, AM induced activation of MAPK pathway and Normal Vascularization is not disrupted by {alpha}AM treatment

Synergistic drug combinations are an attractive anticancer strategy but prove challenging to identify. Here we present a stepwise approach consisting in revealing core cancer vulnerabilities and exploiting them through drug combination screen to uncover synergistic treatments for glioblastoma patients.MethodsWe established an innovative method, based on high-throughput screening, target deconvolution and functional genomics, to reveal core vulnerabilities in glioblastoma. Combination drug screen targeting these vulnerabilities was then designed to unveil synergistic associations. The therapeutic potential of the top drug combination was validated in two different clinically-relevant models: an organotypicex vivomodel and a syngeneic orthotopic mouse model of glioblastoma.ResultsLarge-scale monotherapy drug screening identified 83 potent anti-glioblastoma compounds. Target deconvolution using public chemoinformatic databases uncovered 1,100 targets and interactors of the hit compounds. Screening of a focused siRNA library targeting the top 292 drug interactors revealed 22 targetable vulnerabilities, 9 of which were confirmed as core glioblastoma vulnerabilities by mining the CRISPR screen cohort data from the online Cancer Dependency Map portal. Six selective inhibitors of the core vulnerabilities were then screened in combination with a custom-made library of 88 compounds and synergies amongst the 528 tested pairwise combinations were predicted. The combinations of CHK1 / MEK and AURKA / BET inhibitors were highlighted and validated in 3D tumor spheroids. Using an organotypicex vivomodel and a syngeneic orthotopic mouse model, we definitively ascertained the efficacy of dual AURKA / BET inhibition in glioblastoma.ConclusionsCollectively, we uncovered that dual inhibition of BET proteins and aurora kinase A is highly synergistic against GBM. Moreover, our study indicates that our approach to exploit drug poly-pharmacology for the rational design of drug combination screens represent a valuable strategy to discover synergistic treatments against refractory cancers.

L’objet d’etude de cette recherche est la « zone de rencontre » ou se construit et s’etablit le cœur de la fonction enseignante : la transmission des savoirs. Une transmission qui se deploie dans le cadre des rites d’interaction et dans la gestion des situations d’enseignement. Une triangulation des donnees est utilisee afin de construire une « validite locale et contextuelle ». Ainsi, les questionnaires, les observations de type ethnographique et l’etude de cas empruntee au cadre de l’ergonomie de l��activite font emerger l’existence d’un espace interactionnel entre l’enseignant d’Education Physique et Sportive et ses eleves. Cet espace que nous appelons « zone de rencontre » est cree dans l’action. Elle revele dans la relation didactique, d’une part la construction de l’identite enseignante et d’autre part elle est le lieu de la transmission des savoirs. Des profils emergent et devoilent une dialectique entre la construction du sujet et celle des rencontres ritualisees avec ses eleves. De meme, une analyse epistemologique de la notion de savoirs renvoie a un corpus de connaissances et de valeurs construites a l’interface entre un ordre social et un ordre personnel. Ainsi, cette recherche nous a permis d’explorer et d’approfondir le processus de transmission afin d’en extraire un potentiel de formation pour la professionnalisation des enseignants : ce processus se realise pendant des phases dites d’interaction et dans des zones circonscrites et co-construites par les acteurs : la « zone de rencontre ».

A2B5+ cells isolated from human glioblastomas exhibit cancer stem cell properties. The A2B5 epitope belongs to the sialoganglioside family and is synthetized by the ST8 alpha-N-acetyl-neuraminidase &alpha, 2,8-sialyltransferase 3 (ST8SIA3) enzyme. Glycolipids represent attractive targets for solid tumors, therefore, the aim of this study was to decipher A2B5 function in glioblastomas. To this end, we developed cell lines expressing various levels of A2B5 either by genetically manipulating ST8SIA3 or by using neuraminidase. The overexpression of ST8SIA3 in low-A2B5-expressing cells resulted in a dramatic increase of A2B5 immunoreactivity. ST8SIA3 overexpression increased cell proliferation, migration, and clonogenicity in vitro and tumor growth when cells were intracranially grafted. Conversely, lentiviral ST8SIA3 inactivation in low-A2B5-expressing cells resulted in reduced proliferation, migration, and clonogenicity in vitro and extended mouse survival. Furthermore, in the shST8SIA3 cells, we found an active apoptotic phenotype. In high-A2B5-expressing cancer stem cells, lentiviral delivery of shST8SIA3 stopped cell growth. Neuraminidase treatment, which modifies the A2B5 epitope, impaired cell survival, proliferation, self-renewal, and migration. Our findings prove the crucial role of the A2B5 epitope in the promotion of proliferation, migration, clonogenicity, and tumorigenesis, pointing at A2B5 as an attractive therapeutic target for glioblastomas.

In glioblastomas, apoptosis inhibitor proteins (IAPs) are involved in apoptotic and nonapoptotic processes. We previously showed that IAP inhibition induced a loss of stemness and glioblastoma stem cells differentiation by activating nuclear factor-κB under normoxic conditions. Hypoxia has been shown to modulate drug efficacy. Here, we investigated how IAPs participate in glioblastoma stem-like cell maintenance and fate under hypoxia. We showed that in a hypoxic environment, IAPs inhibition by GDC-0152, a small-molecule IAPs inhibitor, triggered stem-like cell apoptosis and decreased proliferation in four human glioblastoma cell lines. We set up a three-dimensional glioblastoma spheroid model in which time-of-flight secondary ion mass spectrometry analyses revealed a decrease in oxygen levels between the periphery and core. We observed low proliferative and apoptotic cells located close to the hypoxic core of the spheres and glial fibrillary acidic protein+ cells at their periphery. These oxygen-dependent GDC-0152 antitumoral effects have been confirmed on human glioblastoma explants. Notably, serine–threonine kinase activation analysis revealed that under hypoxic conditions, IAP inhibition activated ataxia telangiectasia and Rad3-related protein signaling. Our findings provide new insights into the dual mechanism of action of IAP inhibitors that depends on oxygen level and are relevant to their therapeutic application in tumors. Stem Cells 2019;37:731–742

Glioblastoma (GBM) is characterized by highly aggressive growth and invasive behavior. Due to the highly lethal nature of GBM, new therapies are urgently needed and repositioning of existing drugs is a promising approach. We have previously shown the activity of Proscillaridin A (ProA), a cardiac glycoside inhibitor of the Na(+)/K(+) ATPase (NKA) pump, against proliferation and migration of GBM cell lines. ProA inhibited tumor growth in vivo and increased mice survival after orthotopic grafting of GBM cells. This study aims to decipher the mechanism of action of ProA in GBM tumor and stem-like cells. ProA displayed cytotoxic activity on tumor and stem-like cells grown in 2D and 3D culture, but not on healthy cells as astrocytes or oligodendrocytes. Even at sub-cytotoxic concentration, ProA impaired cell migration and disturbed EB1 accumulation at microtubule (MT) plus-ends and MT dynamics instability. ProA activates GSK3β downstream of NKA inhibition, leading to EB1 phosphorylation on S155 and T166, EB1 comet length shortening and MT dynamics alteration, and finally inhibition of cell migration and cytotoxicity. Similar results were observed with digoxin. Therefore, we disclosed here a novel pathway by which ProA and digoxin modulate MT-governed functions in GBM tumor and stem-like cells. Altogether, our results support ProA and digoxin as potent candidates for drug repositioning in GBM.

International audience; Glioblastomas (GBM) are lethal primitive brain tumours characterized by a strong intra-tumour heterogeneity. We observed in GBM tissues the coexistence of functionally divergent micro-territories either enriched in more differentiated and non-mitotic cells or in mitotic undifferentiated OLIG2 positive cells while sharing similar genomic abnormalities. Understanding the formation of such functionally divergent micro-territories in glioblastomas (GBM) is essential to comprehend GBM biogenesis, plasticity and to develop therapies. Here we report an unexpected anti-proliferative role of beta-catenin in non-mitotic differentiated GBM cells. By cell type specific stimulation of miR-302, which directly represses cyclin D1 and stemness features, beta-catenin is capable to change its known proliferative function. Nuclear beta-catenin accumulation in non-mitotic cells is due to a feed forward mechanism between DOCK4 and beta-catenin, allowed by increased GSK3-beta activity. DOCK4 over expression suppresses selfrenewal and tumorigenicity of GBM stem-like cells. Accordingly in the frame of GBM median of survival, increased level of DOCK4 predicts improved patient survival.

e13535 Background: GB are highly aggressive tumors which systematically relapse. Our objective was to identify disease progression mechanisms and genomic drivers of GB treatment resistance. Methods: Ten paired frozen tumors from initial and recurrent surgery after RTCT were screened by CGH Array. Next, NGS of the selected genes was performed on 19 paired tumors (38 samples). Molecular alterations were correlated with patient data. TCGA was used to characterize the molecular profile of MPDZ. Results: Nineteen IDHwt GB patients with a median age of 54.5 years (37.2-72.8) were included. Using CGH array, unsupervised analysis clustered the whole samples by paired of initial and recurrent tumors. However only 44% of CGH Array alterations were shared between initial and recurrent tumors (amplifications: 55%; deletions: 30%). The new alterations detected at relapse were amplifications in 25% and deletions in 23% of tumors. Two regions corresponding to 171 genes were lost at relapse (p = 0.03): 19q13.33 and 19q13.41. Using DAVID genome, 3/171 genes (related to neutrophil chemotactic factors) were identified: FPR1, FPR2, FPR3. Moreover, 24 genes were lost (including MPDZ) and 2 genes were gained in 20% of recurrent tumors. Totally, 29 genes were analyzed by NGS and 4 genes showed pathogenic mutations shared by initial and recurrent tumors: FPR2, REL, TYRP1 and MPDZ. Only MPDZ showed, at relapse, an increasing rate of mutated variants and a new mutation affecting the splicing site. These alterations were independent from classical prognostic factors (age, sexe, karnofsky performans status, MMS and MGMT status) and from patient survivals. To explore MPDZ expression, we used TCGA initial dataset and observed that a lower RNA expression of MPDZ was associated with IDHwt ( p< 0.001) and grade IV ( p< 0.001) gliomas, reinforcing the potential pejorative impact of MPDZ loss. Conclusions: Our results suggest that MPDZ is frequently altered at initial diagnosis with increased alterations in recurrent IDHwt GB after RTCT, suggesting that MPDZ impairment could contribute to the resistance/relapse mechanisms. Further investigations are needed to validate these results.

International audience; Glioblastomas are the most common primary brain tumors, highly vascularized, infiltrating, and resistant to current therapies. This cancer leads to a fatal outcome in less than 18 months. The aggressive behavior of glioblastomas, including resistance to current treatments and tumor recurrence, has been attributed to glioma stemlike/progenitor cells. The transcription factor EGR1 (early growth response 1), a member of a zinc finger transcription factor family, has been described as tumor suppressor in gliomas when ectopically overexpressed. Although EGR1 expression in human glioblastomas has been associated with patient survival, its precise location in tumor territories as well as its contribution to glioblastoma progression remain elusive. In the present study, we show that EGR1-expressing cells are more frequent in high grade gliomas where the nuclear expression of EGR1 is restricted to proliferating/progenitor cells. We show in primary cultures of glioma stemlike cells that EGR1 contributes to stemness marker expression and proliferation by orchestrating a PDGFA-dependent growth-stimulatory loop. In addition, we demonstrate that EGR1 acts as a positive regulator of several important genes, including SHH, GLI1, GLI2, and PDGFA, previously linked to the maintenance and proliferation of glioma stemlike cells.

International audience; BACKGROUND: Cellular self-renewal capacity in glioblastomas is heterogeneous, with only stem-like cells having this property. These cells generate a specific tumor phenotype, but no link with tumor location or molecular characteristics has ever been made. METHODS: Two cells lines, established from cell-dissociated glioblastomas and A2B5+ magnetic cell sorting, were used to decipher the mechanisms of cell migration in glioblastomas. GBM6 was derived from a glioblastoma close to the subventricular zone, whereas GBM9 was derived from a cortical glioblastoma and contained a high number of CD133(+) cells. RESULTS: Orthotopic injections in both the subventricular zone and the cortex of nude mice showed that GBM6 and GBM9 cells had a differential pattern of migration that mirrored that of adult and fetal normal neural stem cells, respectively. GBM6 demonstrated higher tumorigenicity than GBM9, and whichever cell line was injected, subventricular zone-implanted tumors were larger than cortical ones. In vitro, GBM6 and GBM9 displayed high autorenewal and proliferation rates, and their expression profiles and genomic status showed that they had distinctive molecular signatures: GBM6 was classified as a mesenchymal glioblastoma and GBM9 as a proneural glioblastoma. CONCLUSIONS: Altogether, our findings suggest that tumor location in addition to molecular signature influence tumor growth and migration pattern.

International audience; Glioblastomas in adults are highly heterogeneous tumors that can develop throughout the brain. To date no predictive-location marker has been identified. We previously derived two glioblastoma cell lines from cortical and periventricular locations and demonstrated distinct transcriptomic profiles. Based on these preliminary results, the aim of this study was to correlate glioblastoma locations with the expression of ten selected genes (VEGFC, FLT4, MET, HGF, CHI3L1, PROM1, NOTCH1, DLL3, PDGFRA, BCAN). Fifty nine patients with newly diagnosed glioblastomas were retrospectively included. Tumors were classified into cortical and periventricular locations, which were subsequently segregated according to cerebral lobes involved: cortical fronto-parietal (C-FP), cortical temporal (C-T), periventricular fronto-parietal (PV-FP), periventricular temporal (PV-T), and periventricular occipital (PV-O). Gene expression levels were determined using RT-qPCR. Compared to cortical glioblastomas, periventricular glioblastomas were characterized by a higher expression of two mesenchymal genes, VEGFC (p = 0.001) and HGF (p = 0.001). Among cortical locations, gene expressions were homogeneous. In contrast, periventricular locations exhibited distinct expression profiles. PV-T tumors were associated with higher expression of two proneural and cancer stem cell genes, NOTCH1 (p = 0.028) and PROM1 (p = 0.033) while PV-FP tumors were characterized by high expression of a mesenchymal gene, CHI3L1 (p = 0.006). Protein expression of NOTCH1 was correlated with RNA expression levels. PV-O glioblastomas were associated with lower expression of VEGFC (p = 0.032) than other periventricular locations, whereas MET overexpression remained exceptional. These data suggest a differential gene expression profile according to initial glioblastoma location.

We conducted a population-based study on glioblastomas in the Canton of Zurich, Switzerland (population, 1.16 million) to determine the frequency of major genetic alterations and their effect on patient survival. Between 1980 and 1994, 715 glioblastomas were diagnosed. The incidence rate per 100,000 population/year, adjusted to the World Standard Population, was 3.32 in males and 2.24 in females. Observed survival rates were 42.4% at 6 months, 17.7% at 1 year, and 3.3% at 2 years. For all of the age groups, younger patients survived significantly longer, ranging from a median of 8.8 months (80 years). Loss of heterozygosity (LOH) 10q was the most frequent genetic alteration (69%), followed by EGFR amplification (34%), TP53 mutations (31%), p16INK4a deletion (31%), and PTEN mutations (24%). LOH 10q occurred in association with any of the other genetic alterations and was predictive of shorter survival. Primary (de novo) glioblastomas prevailed (95%), whereas secondary glioblastomas that progressed from low-grade or anaplastic gliomas were rare (5%). Secondary glioblastomas were characterized by frequent LOH 10q (63%) and TP53 mutations (65%). Of the TP53 mutations in secondary glioblastomas, 57% were in hotspot codons 248 and 273, whereas in primary glioblastomas, mutations were more equally distributed. G:C→A:T mutations at CpG sites were more frequent in secondary than primary glioblastomas (56% versus 30%; P = 0.0208). This suggests that the acquisition of TP53 mutations in these glioblastoma subtypes occurs through different mechanisms.

The tumor suppressor gene PTEN on chromosome 10q23.3 regulates the Akt signaling pathway and modulates cell growth and apoptosis. The PTEN gene is mutated in 20-40% of glioblastomas. In this study, we assessed whether loss of PTEN expression is also caused epigenetically. Methylation-specific PCR revealed that CpG islands of the PTEN promoter were methylated in 27 of 77 (35%) glioblastomas and in 4 of 11 (36%) glioblastoma cell lines. Only two glioblastomas showed loss of PTEN immunoreactivity in the entire biopsy; both had a missense PTEN mutation and LOH at the PTEN locus, but lacked PTEN methylation. In biopsy specimens with focal loss of PTEN expression, DNA samples extracted from microdissected foci showed PTEN methylation only in areas with loss of PTEN expression. These results suggest that PTEN methylation occurs frequently in glioblastomas and may be associated with focal loss of PTEN expression. However, the correlation between PTEN methylation, PTEN mutations, LOH at the PTEN locus, and loss of PTEN protein expression was inconsistent. Possible reasons for discrepancies between gene status and protein expression include differences in the biological effect of specific PTEN mutations and the possibility that the processed PTEN pseudogene on 9p21 is expressed in glioblastomas and co-reacts with the PTEN antibody.

International audience; End-binding 1 protein (EB1) is a key player in the regulation of microtubule (MT) dynamics. Here, we investigated the role of EB1 in glioblastoma (GBM) tumor progression and its potential predictive role for response to Vinca-alkaloid chemotherapy. Immunohistological analysis of the 109 human GBM cases revealed that EB1 overexpression correlated with poor outcome including progression-free survival and overall survival. Downregulation of EB1 by shRNA inhibited cell migration and proliferation in vitro. Conversely, EB1 overexpression promoted them and accelerated tumor growth in orthotopically-transplanted nude mice. Furthermore, EB1 was largely overexpressed in stem-like GBM6 that display in vivo a higher tumorigenicity with a more infiltrative pattern of migration than stem-like GBM9. GBM6 showed strong and EB1-dependent migratory potential. The predictive role of EB1 in the response of GBM cells to chemotherapy was investigated. Vinflunine and vincristine increased survival of EB1-overexpressing U87 bearing mice and were more effective to inhibit cell migration and proliferation in EB1-overexpressing clones than in controls. Vinca inhibited the increase of MT growth rate and growth length induced by EB1 overexpression. Altogether, our results show that EB1 expression level has a prognostic value in GBM, and that Vinca-alkaloid chemotherapy could improve the treatment of GBM patients with EB1-overexpressing tumor.

Glioblastoma is the most frequent primary brain tumor in adults. Because of molecular and cellular heterogeneity, high proliferation rate and significant invasive ability, prognosis of patients is poor. Recent therapeutic advances increased median overall survival but tumor recurrence remains inevitable. In this context, we used a high throughput screening approach to bring out novel compounds with anti-proliferative and anti-migratory properties for glioblastoma treatment. Screening of the Prestwick chemical library® of 1120 molecules identified proscillaridin A, a cardiac glycoside inhibitor of the Na(+)/K(+) ATPase pump, with most significant effects on glioblastoma cell lines. In vitro effects of proscillaridin A were evaluated on GBM6 and GBM9 stem-like cell lines and on U87-MG and U251-MG cell lines. We showed that proscillaridin A displayed cytotoxic properties, triggered cell death, induced G2/M phase blockade in all the glioblastoma cell lines and impaired GBM stem self-renewal capacity even at low concentrations. Heterotopic and orthotopic xenotransplantations were used to confirm in vivo anticancer effects of proscillaridin A that both controls xenograft growth and improves mice survival. Altogether, results suggest that proscillaridin A is a promising candidate as cancer therapies in glioblastoma. This sustains previous reports showing that cardiac glycosides act as anticancer drugs in other cancers.

Fibroblast growth factor (FGF) signalling has been implicated in patterning, proliferation and cell differentiation in many organs, including the developing pancreas. Here we show that the FGF receptors (FGFRs) 1 and 2, together with the ligands FGF1, FGF2, FGF4, FGF5, FGF7 and FGF10, are expressed in adult mouse beta-cells, indicating that FGF signalling may have a role in differentiated beta-cells. When we perturbed signalling by expressing dominant-negative forms of the receptors, FGFR1c and FGFR2b, in the pancreas, we found that that mice with attenuated FGFR1c signalling, but not those with reduced FGFR2b signalling, develop diabetes with age and exhibit a decreased number of beta-cells, impaired expression of glucose transporter 2 and increased proinsulin content in beta-cells owing to impaired expression of prohormone convertases 1/3 and 2. These defects are all characteristic of patients with type-2 diabetes. Mutations in the homeobox gene Ipf1/Pdx1 are linked to diabetes in both mouse and human. We also show that Ipf1/Pdx1 is required for the expression of FGFR1 signalling components in beta-cells, indicating that Ipf1/Pdx1 acts upstream of FGFR1 signalling in beta-cells to maintain proper glucose sensing, insulin processing and glucose homeostasis.

A differential pancreatic behavior observed between male and female mice in diabetes and pancreatitis led us to study the gene and protein expressions of endocrine and exocrine pancreatic proteins in normal mice. We compared the levels of expression of six pancreatic genes and of four of the corresponding proteins in male and female mice OF1. Amylase gene expression was found to be significantly higher in females than in males, whereas trypsinogen and lipase gene expression were significantly lower. For chymotrypsinogen, reg, and insulin the differences were not significant. This sexual dimorphism did not exist in rat pancreas, where no gender difference was observed. After characterization of mice enzymes by sodium dodecylsulfate-polyacrylamide gel electrophoresis (SDS-PAGE) and antibodies directed to the closely related human pancreatic enzymes, we have compared the levels of these proteins in mice pancreatic homogenates. No significant difference was observed between males and females at the level of protein expression. These data suggest a hormonal sexual difference in the regulation of pancreatic protein synthesis at the pre- and posttranscriptional levels in normal mice, which may play a role in the development of mice pancreatic diseases.

Peptide 23, the rat homolog of the human pancreatitis-associated protein (PAP)/hepatocarcinoma-intestine-pancreas (HIP) protein, has been identified in primary culture of rat pituitary cells. Its secretion was shown to be stimulated by GH-releasing factor and inhibited by somatostatin in a similar fashion to GH. This observation led the researchers to speculate that peptide 23 does have a physiological hormonal role. We tested this hypothesis by screening by RT-PCR reactions the expression of the PAP/HIP gene in several human pituitary adenomas, especially GH-producing adenomas. Our results show a weak expression of the PAP/HIP gene in the pituitary gland and in most of the tumors, but independent of their origin. The significant homology of the PAP/HIP gene to the Reg gene family prompted us to study in the same pituitary adenomas the presence of the related Reg genes. Reg expression was never observed in the adenomas tested or in the pituitary gland. In contrast, the RegL transcript was observed in pitu...

Identification of new drugs and predicting drug response are major challenges in oncology, especially for brain tumors, because total surgical resection is difficult and radiation therapy or chemotherapy is often ineffective. With the aim of developing a culture system close to in vivo conditions for testing new drugs, we characterized an ex vivo three-dimensional culture system based on a hyaluronic acid-rich hydrogel and compared it with classical two-dimensional culture conditions. U87-MG glioblastoma cells and seven primary cell cultures of human glioblastomas were subjected to radiation therapy and chemotherapy drugs. It appears that 3D hydrogel preserves the original cancer growth behavior and enables assessment of the sensitivity of malignant gliomas to radiation and drugs with regard to inter-tumoral heterogeneity of therapeutic response. It could be used for preclinical assessment of new therapies.

Human tracheal gland (HTG) serous cells are now believed to play a major role in the physiopathology of cystic fibrosis. Because of the persistent inflammation and the specific infection by Pseudomonas aeruginosa in the lung, we looked for the action of the lipopolysaccharide (LPS) of this bacteria on human tracheal gland cells in culture by studying the secretion of the secretory leukocyte proteinase inhibitor (SLPI) which is a specific serous secretory marker of these cells. Treatment with Pseudomonas aeruginosa LPS resulted in a significant dose-dependent increase in the basal production of SLPI (+250 ± 25%) whilst the SLPI transcript mRNA levels remained unchanged. This LPS-induced increase in secretion was inhibited by glucocorticoides. Furthermore, LPS treatment of HTG cells induces a loss of responsiveness to carbachol and isoproterenol but not to adenosine triphosphate. These findings indicate that HTG cells treated by Pseudomonas aeruginosa LPS have the same behavior as those previously observed with CF-HTG cells. Exploration by using reverse transcriptase polymerase chain reaction amplification showed that LPS downregulated cystic fibrosis transmembrane conductance regulator (CFTR) mRNA expression in HTG cells indicative of a link between CFTR function and consequent CF-like alteration in protein secretory process.

Purpose: To study the role of the adrenomedullin system [adrenomedullin and its receptors (AMR), CLR, RAMP2, and RAMP3] in prostate cancer androgen-independent growth. Experimental Design: Androgen-dependent and -independent prostate cancer models were used to investigate the role and mechanisms of adrenomedullin in prostate cancer hormone-independent growth and tumor-associated angiogenesis and lymphangiogenesis. Results: Adrenomedullin and AMR were immunohistochemically localized in the carcinomatous epithelial compartment of prostate cancer specimens of high grade (Gleason score >7), suggesting a role of the adrenomedullin system in prostate cancer growth. We used the androgen-independent Du145 cells, for which we demonstrate that adrenomedullin stimulated cell proliferation in vitro through the cAMP/CRAF/MEK/ERK pathway. The proliferation of Du145 and PC3 cells is decreased by anti-adrenomedullin antibody (αAM), supporting the fact that adrenomedullin may function as a potent autocrine/paracrine growth factor for prostate cancer androgen-independent cells. In vivo, αAM therapy inhibits the growth of Du145 androgen-independent xenografts and interestingly of LNCaP androgen-dependent xenografts only in castrated animals, suggesting strongly that adrenomedullin might play an important role in tumor regrowth following androgen ablation. Histologic examination of αAM-treated tumors showed evidence of disruption of tumor vascularity, with depletion of vascular as well as lymphatic endothelial cells and pericytes, and increased lymphatic endothelial cell apoptosis. Importantly, αAM potently blocks tumor-associated lymphangiogenesis, but does not affect established vasculature and lymphatic vessels in normal adult mice. Conclusions: We conclude that expression of adrenomedullin upon androgen ablation in prostate cancer plays an important role in hormone-independent tumor growth and in neovascularization by supplying/amplifying signals essential for pathologic neoangiogenesis and lymphangiogenesis. Clin Cancer Res; 19(22); 6138–50. ©2013 AACR.

Background Pilocytic astrocytomas occur predominantly in childhood. In contrast to the posterior fossa location, hypothalamo-chiasmatic pilocytic astrocytomas display a worse prognosis often leading to multiple surgical procedures and/or several lines of chemotherapy and radiotherapy to achieve long-term control. Hypothalamo-chiasmatic pilocytic astrocytomas and cerebellar pilocytic astrocytomas have a distinctive gene signature and several differential expressed genes (ICAM1, CRK, CD36, and IQGAP1) are targets for available drugs: fluvastatin and/or celecoxib. Results Quantification by RT-Q-PCR of the expression of these genes was performed in a series of 51 pilocytic astrocytomas and 10 glioblastomas: they were all significantly overexpressed in hypothalamo-chiasmatic pilocytic astrocytomas relative to cerebellar pilocytic astrocytomas, and CRK and ICAM1 were significantly overexpressed in pilocytic astrocytomas versus glioblastomas. We used two commercially available glioblastoma cell lines and three pilocytic astrocytoma explant cultures to investigate the effect of celecoxib/fluvastatin alone or in combination. Glioblastoma cell lines were sensitive to both drugs and a combination of 100 μM celecoxib and 240 μM fluvastatin was the most synergistic. This synergistic combination was used on the explant cultures and led to massive cell death of pilocytic astrocytoma cells. As a proof of concept, a patient with a refractory multifocal pilocytic astrocytoma was successfully treated with the fluvastatin/celecoxib combination used for 18 months. It was well tolerated and led to a partial tumor response. Conclusion This study reports evidence for new targets and synergistic effect of celecoxib/fluvastatin combination in pilocytic astrocytoma. Because it is non-toxic, this new strategy offers hope for the treatment of patients with refractory pilocytic astrocytoma.

Glioblastomas (GBMs) are the most aggressive primary brain tumors in adult and remain a therapeutic challenge. Targeting key apoptosis regulators with the ultimate aim to restore apoptosis in tumor cells could be an interesting therapeutic strategy. The inhibitors of apoptosis proteins (IAPs) are regulators of cell death and represent attractive targets, especially because they can be antagonized by SMAC mimetics. In this study, we first investigated the expression of cIAP1, cIAP2, XIAP and ML-IAP in human GBM samples and in four different cell lines. We showed that all GBM samples and GBM cell lines expressed all these IAPs, although the expression of each IAP varied from one case to another. We then showed that high level of ML-IAP predicted worse progression-free survival and overall survival in both univariate and multivariate analyses in two independent cohorts of 58 and 43 primary human GBMs. We then used GDC-0152, a SMAC mimetic that antagonizes these IAPs and confirmed that GDC-0152 treatment in vitro decreased IAPs in all the cell lines studied. It affected cell line viability and triggered apoptosis, although the effect was higher in U87MG and GL261 than in GBM6 and GBM9 cell lines. In vivo, GDC-0152 effect on U87MG orthotopic xenografts was dose dependent; it postponed tumor formation and slowed down tumor growth, significantly improving survival of GBM-bearing mice. This study revealed for the first time that ML-IAP protein expression correlates with GBM patient survival and that its antagonist GDC-0152 improves outcome in xenografted mouse.

International audience; L'étude présentée porte sur une formation de licence en intervention socio-sportive. Les compétences et l'identité professionnelles se développent dans des ateliers d'intervention. Elaborés en partenariat avec des institutions, ces expériences permettent aux étudiants une plongée dans une complexité proche du réel. L'analyse de ces moments permet à l'étudiant de s'approprier et d'objectiver sa progression dans la formation, dans l'objectif de faciliter son entrée dans le métier. Les interventions sont systématiquement filmées. Les étudiants visionnent ces séquences, en sélectionnent des « moments » qui leur font sens… Ils procèdent à une analyse, une caractérisation (« simplexe ») de leur activité et un échange avec le collectif en formation. Des extensions sont possibles vers la formation des enseignants.

Glioblastomas, like other cancers, harbor small cell populations with the capability of sustaining tumor formation. These cells are referred to as cancer stem cells. We isolated cells expressing the surface marker A2B5 from three human glioblastomas (GBM) and showed that after grafting into nude mice, they generated dense and highly infiltrative tumors. Then, we extensively studied A2B5(+) cells isolated from 11 human GBM. These cells display neurosphere-like, self-renewal, asymmetrical cell division properties and have multipotency capability. Stereotactic xenografts of dissociated A2B5(+)-derived secondary spheres revealed that as few as 1000 cells produced a tumor. Moreover, flow cytometry characterization of A2B5(+)-derived spheres revealed three distinct populations of cells: A2B5(+)/CD133(+), A2B5(+)/CD133(-) and A2B5(-)/CD133(-), with striking proportion differences among GBM. Both A2B5(+)/CD133(+) and A2B5(+)/CD133(-) cell fractions displayed a high proliferative index, the potential to generate spheres and produced tumors in nude mice. Finally, we generated two green fluorescent protein-cell lines that display--after serum induction--distinct proliferative and migratory properties, and differ in their CD133 level of expression. Taken together, our results suggest that transformed A2B5(+) cells are crucial for the initiation and maintenance of GBM, although CD133 expression is more involved in determining the tumor's behavior.

2030 Background: Initial GB topography occurred throughout the brain without established specific molecular profile. Our previous study suggested that cortical and subventricular GB exhibit distinc...

Impaired glucose-stimulated insulin secretion (GSIS) and perturbed proinsulin processing are hallmarks of beta cell dysfunction in type 2 diabetes. Signals that can preserve and/or enhance beta cell function are therefore of great therapeutic interest. Here we show that bone morphogenetic protein 4 (Bmp4) and its high-affinity receptor, Bmpr1a, are expressed in beta cells. Mice with attenuated BMPR1A signaling in beta cells show decreased expression of key genes involved in insulin gene expression, proinsulin processing, glucose sensing, secretion stimulus coupling, incretin signaling, and insulin exocytosis and develop diabetes due to impaired insulin secretion. We also show that transgenic expression of Bmp4 in beta cells enhances GSIS and glucose clearance and that systemic administration of BMP4 protein to adult mice significantly stimulates GSIS and ameliorates glucose tolerance in a mouse model of glucose intolerance. Thus, BMP4-BMPR1A signaling in beta cells plays a key role in GSIS.

The AP-HM tissue bank is the largest one in France regarding some collections, including brain tumors. This collection was used to better characterize some gliomas. In particular because some benign gliomas such as pilocytic astrocytomas (WHO grade I) can be misclassified as malignant ones such as glioblastomas (grade IV) the first aim of our study was to find accurate diagnostic markers. This was done mainly by suppressive substractive hybridization (SSH). This study also provides a restrictive list of genes selectively involved in angiogenesis and invasion, which were highly expressed in GBM. Results were confirmed by real time quantitative RT-PCR in a large cohort of patients. In addition in order to find accurate markers which can predict GBM overall survival (OS) we selected three cohorts of GBM patients with distinctive OS (short survival6 months, long survival18 months and intermediate). Quantification of a series of markers involved in angiogenesis and invasion was done as well as cDNA array analysis.

We conducted a population-based study on glioblastomas in the Canton of Zurich, Switzerland (population, 1.16 million) to determine the frequency of major genetic alterations and their effect on patient survival. Between 1980 and 1994, 715 glioblastomas were diagnosed. The incidence rate per 100,000 population/year, adjusted to the World Standard Population, was 3.32 in males and 2.24 in females. Observed survival rates were 42.4% at 6 months, 17.7% at 1 year, and 3.3% at 2 years. For all of the age groups, younger patients survived significantly longer, ranging from a median of 8.8 months (50 years) to 1.6 months (80 years). Loss of heterozygosity (LOH) 10q was the most frequent genetic alteration (69%), followed by EGFR amplification (34%), TP53 mutations (31%), p16(INK4a) deletion (31%), and PTEN mutations (24%). LOH 10q occurred in association with any of the other genetic alterations and was predictive of shorter survival. Primary (de novo) glioblastomas prevailed (95%), whereas secondary glioblastomas that progressed from low-grade or anaplastic gliomas were rare (5%). Secondary glioblastomas were characterized by frequent LOH 10q (63%) and TP53 mutations (65%). Of the TP53 mutations in secondary glioblastomas, 57% were in hotspot codons 248 and 273, whereas in primary glioblastomas, mutations were more equally distributed. G:C--A:T mutations at CpG sites were more frequent in secondary than primary glioblastomas (56% versus 30%; P = 0.0208). This suggests that the acquisition of TP53 mutations in these glioblastoma subtypes occurs through different mechanisms.

Medulloblastoma is a malignant, invasive embryonal tumour of the cerebellum which manifests preferentially in children. A subset of cases is associated with colon cancer and APC germline mutations (Turcot syndrome), and APC and beta-catenin point mutations occur in up to 10% of sporadic cases, indicating the involvement of the Wnt pathway in the development of medulloblastoma. In 39 sporadic cerebellar medulloblastomas screeened for alterations in the AXIN1 gene, another component of the Wnt pathway, we found missense AXIN1 mutations in two tumours, CCC--TCC at codon 255 (exon 1, Pro--Ser) and TCT--TGT at codon 263 (exon 1, Ser--Cys). Furthermore, the A allele at the G/A polymorphism at nucleotide 16 in intron 4 was significantly over-represented in medulloblastomas (39 cases; G 0.76 vs-A 0.24) compared to healthy individuals (86 cases; G 0.91 vs A 0.09; P=0.0027). RT-PCR revealed large deletions in the AXIN1 gene in 5/12 (42%) medulloblastomas, consistent with a previous report. However, we observed such deletions at a similar frequency also in normal brain tissue (6/12, 50%). Since there are multiple complementary, inverted sequences present in the AXIN1 gene, these large deletions may represent RT-PCR errors due to stem-loop secondary structures.

In the adult pancreas, the � -cells sense glucose and control blood glucose levels by secreting active insulin. To address the role of IPF- 1 in the adult � -cell, we previously generated transgenic mice in which the IPF-1 gene has been disrupted specifically in the � -cells using the CRE-lox system. This model revealed that IPF-1 was required to maintain correct hormone expression in the � -cell and to maintain high levels of expression of Glut2, the key component in the glucose-sensing machinery (5). More recent work has shown that in these mice, the expression of the proinsulin pro- cessing enzymes PC1/3 and PC2 are also affected, leading to par- tially processed insulin being stored and subsequently secreted from the � -cells. All together, these results show that IPF-1 is nec- essary to maintain glucose homeostasis by controlling several aspects of � -cell identity, because aberrant IPF-1 expression leads to � -cell dysfunction and consequently type 2 diabetes. We believe that these approaches will generate data that will expand our understanding of the intrinsic regulatory molecules that act upstream and downstream of IPF-1 dur- ing pancreatic development and in the adult � -cell. We hope this knowledge will contribute to the development of improved diabetes therapies.

The reg gene has previously been shown to be associated with regeneration of pancreatic islets. Strategies for influencing the replication and the growth of the β-cell mass may be important for prevention and/or treatment of type I diabetes. In this study, we have examined the level of reg gene expression at various degrees of diabetogenesis in the pancreas of the NOD mouse (male, female, and cyclophosphamide-treated male) using both human reg cDNA as the probe and dot blot analysis. The expression of the reg gene was found to be significantly increased in female mice compared with male mice, and in both cases, the expression level was not influenced by age. Nondiabetic female mice have a significantly higher expression of the gene than diabetic female mice, and there was a positive correlation between the age of diabetes onset and the reg mRNA level. In addition, overexpression of the reg gene was found in male mice treated by cyclophosphamide, an agent known to be a potent inducer of diabetes in male NOD mice. None of these results were found in the diabetes-resistant control OF1 mice, in which pancreatic reg gene expression did not differ between female and male mice treated or untreated with cyclophosphamide. All of these data suggest that there is a strong correlation between reg gene expression in the pancreas of the NOD mouse and the likelihood of developing diabetes.

Resume Lors de la prise de controle d’une classe en EPS, des « zones de rencontre » se construisent selon des modeles de rites d’interaction (Goffman, 1974). Elles renvoient a un espace de rencontre entre l’enseignant et ses eleves qui participe de la construction de l’identite professionnelle. La methodologie comporte un questionnaire (13 questions ouvertes, 20 sujets enquetes) permettant de modeliser des profils. L’analyse de contenu fait apparaitre trois conduites typiques d’enseignants : (le « protecteur », le « transmetteur » et l’« acteur ») s’appuyant sur trois logiques : sociale, corporelle et professionnelle. Les conduites ainsi identifiees devoilent une dialectique entre la construction du sujet professionnel et celle des rituels de rencontre avec les eleves, dialectique presentant une dimension adaptatrice et creative. Les « zones de rencontre » caracterisent les profils d’enseignants dans l’exercice de leur metier.