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Pegvisomant (PEG), an antagonist of growth hormone (GH)-receptor (GHR), normalizes insulin-like growth factor 1 (IGF1) oversecretion in most acromegalic patients unresponsive to somatostatin analogs (SSAs) and/or uncontrolled by transsphenoidal surgery. The residual GH-secreting tumor is therefore exposed to the action of circulating PEG. However, the biological effect of PEG at the pituitary level remains unknown. To assess the impact of PEG in vitro on the hormonal secretion (GH and prolactin (PRL)), proliferation and cellular viability of eight human GH-secreting tumors in primary cultures and of the rat somatolactotroph cell line GH4C1. We found that the mRNA expression levels of GHR were characterized in 31 human GH-secreting adenomas (0.086 copy/copy β-Gus) and the GHR was identified by immunocytochemistry staining. In 5/8 adenomas, a dose-dependent inhibition of GH secretion was observed under PEG with a maximum of 38.2±17% at 1μg/mL (PP

Contexte Les agonistes somatostatinergiques et dopaminergiques inhibient la secretion de GH (Growth Hormone) des adenomes somatotropes (AdGH) par leur action sur SSTR2 et D2DR. En depit de resultats precliniques encourageants dans l’acromegalie, le developpement du BIM-23A760, une dopastatine SSTR2/D2DR, fut interrompu en raison d’un metabolite interferant derive du compose source et competitif pour la valence D2DR. Objectif notre avons teste le BIM-065, une nouvelle Dopastatine, sur la secretion de GH des AdGH. Materiel et methodes Les tumeurs de 17 patients acromegales operes au CHU de Marseille furent mises en culture primaire et soumises a des concentrations croissantes (0,1 pM a 0,1 μM) de BIM-065, BIM-23A760, octreotide (OCT) et cabergoline (CAB). Nous avons determine la secretion de GH dans le surnageant et teste parallelement le metabolite D2DR (=BIM-133) du BIM-065 en coincubation. Resultats Tous les AdGH exprimaient l’ARNm de D2DR et SSTR2. L’inhibition de la secretion de GH etait plus importante avec le BIM-065 qu’avec le BIM-23A760 (Emax = 57 ± 5,6 % vs 41,1 ± 12,5 %, p Discussion En demontrant une superiorite sur l’inhibition de la secretion de GH par rapport au BIM-23A760 ou a l’association OCT + CAB et sans interference de son metabolite, le BIM-065 constitue un nouvelle molecule prometteuse dans l’arsenal therapeutique du traitement de l’acromegalie.

International audience; Treatment for recurrent and aggressive meningiomas remains an unmet medical need in neuro-oncology, and chemotherapy exhibits limited clinical activity, if any. Merlin expression, encoded by the NF2 gene, is lost in a majority of meningiomas, and merlin is a negative regulator of mTORC1. The sst2 somatostatin receptor, targeted by octreotide, is highly expressed in meningiomas. To investigate new therapeutic strategies, we evaluated the activity of everolimus (mTOR inhibitor), BKM-120 and BEZ-235 (new Pi3K/Akt/mTOR inhibitors), octreotide and a combined treatment (octreotide plus everolimus), on cell proliferation, signaling pathways, and cell cycle proteins, respectively. The in vitro study was conducted on human meningioma primary cells extracted from fresh tumors, allowing the assessment of somatostatin analogs at the concentration levels used in patients. The results were correlated to WHO grades. Further, everolimus decreased cell viability of human meningiomas, but concomitantly, induced Akt activation, reducing the antiproliferative effect of the drug. The new Pi3K inhibitors were not more active than everolimus alone, limiting their clinical relevance. In contrast, a clear cooperative inhibitory effect of octreotide and everolimus was observed on cell proliferation in all tested meningiomas, including WHO grades II-III. Octreotide not only reversed everolimus-induced Akt phosphorylation but also displayed additive and complementary effects with everolimus on downstream proteins involved in translation (4EB-P1), and controlling cell cycle (p27Kip1 and cyclin D1). We have demonstrated a co-operative action between everolimus and octreotide on cell proliferation in human meningiomas, including aggressive ones, establishing the basis for a clinical trial.

OBJECTIVEMeningiomas express somatostatin receptor subtype 2 (SST2), which is targeted by the somatostatin analog octreotide. However, to date, using somatostatin analog therapy for the treatment of these tumors in clinical practice has been debated. This study aims to clarify the in vitro effects of octreotide on meningiomas for precise clinical applications.METHODSThe effects of octreotide were analyzed in a large series of 80 meningiomas, including 31 World Health Organization (WHO) Grade II and 4 WHO Grade III tumors, using fresh primary cell cultures to study the impact on cell viability, apoptosis, and signal transduction pathways.RESULTSSST2 mRNA was detected in 100% of the tested meningiomas at levels similar to those observed in other SST2-expressing tumors, neuroendocrine tumors, or pituitary adenomas. Octreotide significantly decreased cell proliferation in 88% of meningiomas but did not induce cell death. On average, cell proliferation was more inhibited in the meningioma group expressing a high level of SST2 than in the low-SST2 group. Moreover, octreotide response was positively correlated to the level of merlin protein and inversely correlated to the level of phosphorylated p70-S6 kinase, a downstream effector of the PI3K/Akt/mammalian target of rapamycin (mTOR) pathway. Octreotide inhibited Akt phosphorylation and activated tyrosine phosphatase without impacting the extracellular regulated kinase (ERK) pathway.CONCLUSIONSOctreotide acts exclusively as an antiproliferative agent and does not promote apoptosis in meningioma in vitro. Therefore, in vivo, octreotide is likely to limit tumor growth rather than induce tumor shrinkage. A meta-analysis of the literature reveals an interest in octreotide for the treatment of WHO Grade I tumors, particularly those in the skull base for which the 6-month progression-free survival level reached 92%. Moreover, somatostatin analogs, which are well-tolerated drugs, could be of interest for use as co-targeting therapies for aggressive meningiomas.

International audience; Pasireotide is a somatostatin analog (SSA) that targets somatostatin receptor subtype 1 (SST1), SST2, SST3, and SST5 with a high affinity. Pasireotide has a better antisecretory effect in acromegaly, Cushing's disease, and neuroendocrine tumors than octreotide. In this study, we compared the effects of pasireotide to those of octreotide in vitro on meningioma primary cell cultures, both alone and in combination with the mTOR inhibitor everolimus. Significant mRNA expression levels of SST1, SST2, and SST5 were observed in 40.5%, 100%, and 35% of meningioma samples, respectively. Pasireotide had a significantly stronger inhibitory effect on cell proliferation than octreotide. The effect of pasireotide, but not of octreotide, was significantly stronger in the group expressing the highest level of SST1 mRNA. Combined treatment with pasireotide and everolimus induced a higher reduction in cell viability than that with octreotide plus everolimus. Moreover, pasireotide decreased Akt phosphorylation and reversed everolimus-induced Akt hyperphosphorylation to a higher degree than octreotide. Using 4E-BP1 siRNA (si4E-BP), we demonstrated that 4E-BP1 protein silencing significantly reversed the response to everolimus, both alone and in combination with SSAs. Moreover, si4E-BP completely reversed the inhibition of cyclin D1 expression level and the increase in p27kip1 induced by SSAs, both alone and in combination with everolimus. Our results strongly support the need for further studies on the combination of pasireotide and everolimus in medical therapy for meningiomas.

Fagot and Paleressompoulle [Fagot and Paleressompoulle (2009) Behav Res Methods 41: 396–404] described a new automated learning device for monkeys (ALDM) to test the cognitive functions of nonhuman primates within their social groups. However, the impact of the ALDM procedure on animal well‐being needs to be investigated. The present study assessed the consequences of ALDM testing on the behavioral repertoire of Guinea baboons (Papio papio) and their stress levels as inferred from measurements of saliva cortisol. Accessibility to ALDM test computers reduced the number of resting periods as well as the number of stereotypies. Lower cortisol levels were also found during ALDM testing. These findings and others demonstrate that ALDM testing hasapositiveimpact on animal well‐ being and can be considered as a means for behavioral enrichment in captive primates. Am. J. Primatol. 76:56–64, 2014. © 2013 Wiley Periodicals, Inc.

Objective As prolactinomas fail to respond to dopamine agonist (DA) in 10–20% of cases, we hypothesized that somatostatin subtype 2 receptor (sst2) overexpression in DA-resistant prolactinomas may enhance suppression of prolactine (PRL) using chimeric agonist (dopastatin) that simultaneously binds sst2 and the dopamine subtype 2 receptor (D2DR). Design and methods PRL suppression by octreotide, sst5 agonist, sst2-D2DR agonist (BIM-23A760 dopastatin) and cabergoline was assessed in primary cultures of seven DA-resistant prolactinomas overexpressing sst2. Results sst2 was effectively overexpressed via adenoviral expression in prolactinomas (38.1 ± 7.4 vs. 0.1 ± 0.1 copy/copy β-Gus) and induced octreotide sst2-mediated PRL suppression that remained lower than that induced by DA. BIM-23A760 inhibited PRL similarly to cabergoline both in the control and sst2-expressing cells. Antagonist experiments confirmed predominant dopaminergic effect in dopastatin activity. Conclusion sst2 was successfully overexpressed in prolactinomas. However BIM-23A760 was unable to enhance PRL suppression underlining a predominant dopaminergic contribution in its action.

International audience; Nonfunctioning pituitary adenomas (NFPA; gonadotroph derived), even not inducing hormonal hypersecretion, cause significant morbidity by compression neighboring structures. No effective and specific medical methods are available so far for treating these tumors. The pituitary homeobox 2 (PITX2) gene is a member of the bicoid-like homeobox transcription factor family, which is involved in the Wnt/Dvl/β-catenin pathway. PITX2 is overexpressed in NFPA. PITX2 mutations are known to be responsible for Axenfield Rieger syndrome, a genetic disorder in which pituitary abnormalities have been detected. The R91P mutant identified in Axenfeld Rieger syndrome is a dominant-negative factor, which is able to block the expression of several pituitary genes activated by PITX2. To better understand the role of Pitx2 on gonadotroph tumorigenesis and to explore new approach for inhibiting tumoral growth, the R91P mutant was transferred via a lentiviral vector in tumoral gonadotroph cells of two kinds: the αT3-1 cell line and human adenoma cells. R91P mutant and small interfering RNA directed against Pitx2 both decreased the viability of αT3-1 cells via an apoptotic mechanism involving the activation of executioner caspase. Similar effects of the R91P mutant were observed on human gonadotroph cells in primary culture. Therefore, Pitx2 overexpression may play an antiapoptotic role during NFPA tumorigenesis.

Context: The ghrelin receptor GHS-R1a is highly expressed in human somatotroph adenomas and exhibits unusually high basal signaling activity. In humans, the suppression of this constitutive activity by mutation induces a short stature. Objective: Using a GHS-R1a inverse agonist, modified substance P (MSP), we explored the role of GHS-R1a constitutive activity in GH hypersecretion from somatotroph adenomas and as a putative therapeutic target. Design: The effects of MSP were assessed on GH secretion from 19 human somatotroph tumors in vitro. Moreover, these effects were compared with those of octreotide (somatostatin receptor subtype 2 [sst2] agonist) and with the combination of both drugs. Expression and localization of GHS-R1a and sst2 were studied. Results: For all tumors, MSP inhibited GH secretion in a dose-dependent manner from 13 to 64%. Moreover, MSP enhanced octreotide-induced GH inhibition. For five tumors, the effects of combined MSP plus octreotide treatment were significantly higher than the sum of effects of each drug alone. MSP increased the membrane localization of GHS-R1a and of microdomains colocalizing sst2-GHS-R1a, highlighting the cooperation between the two drugs. Conclusions: The GHS-R1a inverse agonist could open new therapeutic options for acromegalic patients, particularly patients partially sensitive to octreotide whose GH secretion is not completely controlled by the sst2 agonist.

Introduction Le pegvisomant (PEG), un antagoniste du recepteur de la GH (GH-R) normalise, dans certains cas d’acromegalie non controles par le traitement chirurgical et/ou les analogues de la somatostatine, l’IGF-1 plasmatique. Lorsqu’il persiste, le reliquat adenomateux est expose a l’action du pegvisomant circulant. L’impact de ce dernier au niveau hypophysaire demeure neanmoins toujours inconnu. Objectif Evaluer l’impact du PEG in vitro sur la secretion hormonale (GH et prolactine [PRL]), la proliferation, et la viabilite cellulaire de huit cultures primaires d’adenomes somatotropes humains (dont 4 GH/PRL) et sur la lignee somatolactotrope de rat GH4C1. Resultats L’expression de l’ARNm du GH-R est caracterisee dans 33 adenomes somatotropes (0,125 ± 0,125 copy/copy b-Gus), semblable a celle de l’hypophyse saine. Le GH-R est localise a la membrane en immunocytochimie. Dans 6/8 adenomes, une inhibition dose dependante de la secretion de GH est observee sous PEG avec un maximal de 33,8 ± 19,1 % a 1 mg/mL (p Conclusion Le pegvisomant inhibe la secretion de GH et de PRL des adenomes somato(lacto) tropes humains, phenomene non decrit jusqu’a present. Les mecanismes intimes mis en jeu sont en cours d’investigations.

Germline aryl hydrocarbon receptor interacting protein (AIP) gene mutations confer a predisposition to pituitary adenoma (PA), predominantly GH-secreting (GH-PA). As recent data suggest a role for AIP in the pathogenesis of sporadic GH-PA and their response to somatostatin analogues (SSA), the expression of AIP and its partner, aryl hydrocarbon receptor (AHR), was determined by semiquantitative immunohistochemistry scoring in 62 sporadic GH-PA (37 treated with SSA preoperatively). The influence ofGspstatus was studied in a subset of tumours (n=39, 14Gsp+) and six GH-PA were available for primary cultures. AIP and AHR were detected in most cases, with a positive correlation between AIP and cytoplasmic AHR (P=0.012). Low AIP expression was significantly more frequent in untreated vs SSA-treated tumours (44.0 vs 20.5%,P=0.016). AHR expression or localisation did not differ between the two groups. Similarly,in vitrooctreotide induced a median twofold increase in AIP expression (range 1.2–13.9,P=0.027) in GH-PA. In SSA-treated tumours, the AIP score was significantly higher in the presence of preoperative IGF1 decrease or tumour shrinkage (P=0.008 andP=0.014 respectively). In untreated tumours, low AIP expression was significantly associated with invasiveness (P=0.028) and suprasellar extension (P=0.019). The only effect ofGspstatus was a significantly lower nuclear AHR score inGsp+vsGsp−tumours (P=0.025), irrespective of SSA. In conclusion, AIP is involved in the aggressiveness of sporadic GH-PA, regardless ofGspstatus, and AIP up-regulation in SSA-treated tumours is associated with a better preoperative response, with no clear role for AHR.

International audience; Integrins are essential protagonists of the complex multi-step process of angiogenesis that has now become a major target for the development of anticancer therapies. We recently reported and characterized that MVL-PLA2, a novel phospholipase A2 from Macrovipera lebetina venom, exhibited anti-integrin activity. In this study, we show that MVL-PLA2 also displays potent anti-angiogenic properties. This phospholipase A2 inhibited adhesion and migration of human microvascular-endothelial cells (HMEC-1) in a dose-dependent manner without being cytotoxic. Using Matrigel and chick chorioallantoic membrane assays, we demonstrated that MVL-PLA2, as well as its catalytically inactivated form, significantly inhibited angiogenesis both in vitro and in vivo. We have also found that the actin cytoskeleton and the distribution of alphav beta3 integrin, a critical regulator of angiogenesis and a major component of focal adhesions, were disturbed after MVL-PLA2 treatment. In order to further investigate the mechanism of action of this protein on endothelial cells, we analyzed the dynamic instability behavior of microtubules in living endothelial cells. Interestingly, we showed that MVL-PLA2 significantly increased microtubule dynamicity in HMEC-1 cells by 40%. We propose that the enhancement of microtubule dynamics may explain the alterations in the formation of focal adhesions, leading to inhibition of cell adhesion and migration.

International audience; Crosstalk between integrins is involved in the regulation of various cell functions including cell migration. Here we identify the interplay between the integrins alpha v beta 5/beta 6 and alpha 2 beta 1 during cell migration toward type I collagen. Human colon cancer cell lines HT29-D4 and SW480 were used as cell models. To improve our Understanding of the consequences of alpha v beta 5/beta 6 function on alpha 2 beta 1, we decreased the expression of alpha v integrins by either siRNA or lysosomal targeting strategies, or inhibited their function using, as antagonists, blocking antibodies or disintegrins. In all cases, we observed a greatly enhanced alpha 2 beta 1 integrin-dependent cell migration associated with focal adhesion rearrangements and increased outside-in signaling as demonstrated by elevated phosphorylation of focal adhesion kinase and MAPKinase (ERK1 and ERK2). The alpha v beta 5/beta 6-dependent limitation of alpha 2 beta 1 function could be overridden by TS2/16, an activating anti-beta 1 antibody. Interestingly, compared to control cells, the pharmacological inhibition of PI3Kinase or the siRNA-mediated knockdown of AKT had little effect on the high alpha 2 beta 1-mediated cell migration observed in the absence of alpha v integrins or following activation of alpha 2 beta 1 integrins by the TS2/16. These results suggest that integrins alpha v beta 5/beta 6 repress alpha 2 beta 1 possibly by interfering with their activation process and thereby modify the cell signaling regulation of alpha 2 beta 1-mediated migration. (C) 2009 Elsevier Inc. All rights reserved.

Crosstalk between integrins is involved in the regulation of various cell functions including cell migration. Here we identify the interplay between the integrins alpha v beta 5/beta 6 and alpha 2 beta 1 during cell migration toward type I collagen. Human colon cancer cell lines HT29-D4 and SW480 were used as cell models. To improve our Understanding of the consequences of alpha v beta 5/beta 6 function on alpha 2 beta 1, we decreased the expression of alpha v integrins by either siRNA or lysosomal targeting strategies, or inhibited their function using, as antagonists, blocking antibodies or disintegrins. In all cases, we observed a greatly enhanced alpha 2 beta 1 integrin-dependent cell migration associated with focal adhesion rearrangements and increased outside-in signaling as demonstrated by elevated phosphorylation of focal adhesion kinase and MAPKinase (ERK1 and ERK2). The alpha v beta 5/beta 6-dependent limitation of alpha 2 beta 1 function could be overridden by TS2/16, an activating anti-beta 1 antibody. Interestingly, compared to control cells, the pharmacological inhibition of PI3Kinase or the siRNA-mediated knockdown of AKT had little effect on the high alpha 2 beta 1-mediated cell migration observed in the absence of alpha v integrins or following activation of alpha 2 beta 1 integrins by the TS2/16. These results suggest that integrins alpha v beta 5/beta 6 repress alpha 2 beta 1 possibly by interfering with their activation process and thereby modify the cell signaling regulation of alpha 2 beta 1-mediated migration. (C) 2009 Elsevier Inc. All rights reserved.

Here, we report the purification and characterization of an acidic Asp49 phospholipase A2, named MVL-PLA2, with a molecular mass of 13,626.64 Da. The complete MVL-PLA2 cDNA was cloned from Macrovipera lebetina transmediterranea venom gland cDNA library. MVL-PLA2 possesses 122 amino acid residues, including 14 cysteines, and belongs to group II snake venom phospholipase A2 enzymes. MVL-PLA2 was not cytotoxic up to 2 muM and completely abolished cell adhesion and migration of various human tumor cells. Chemical modification with p-bromophenacyl bromide abolished the enzymatic activity of MVL-PLA2 without affecting its anti-tumor effect, suggesting the presence of 'pharmacological sites' distinct from the catalytic site in snake venom phospholipase A2. We demonstrated for the first time that the anti-tumor effect of MVL-PLA2 was mediated by alpha5beta1 and alphav-containing integrins. This finding may serve as starting point for structure-function relationship studies leading to design a new generation of specific anti-cancer drugs.

Disclosure A. Barlier: the research prgramm was partially supported by Novartis. All other authors have declared no conflicts of interest. Meningiomas are the most frequent brain tumors after 35 years-old. When total surgical removal is not possible, radiotherapy and radiosurgery are able to control tumoral growth in some cases. But no chemotherapy has ever been demonstrated to be really efficient. Then, in cases of recurrence (particularly in WHO grade 2 and 3 meningiomas), no therapeutic exist at present, leading to fatal issue. Pi3Kinase-Akt-mTor pathway is activated in meningiomas, due to Merlin protein (encoded by NF2 gene) inactivation. SST2 somatostatin receptors are strongly expressed in meningiomas. These receptors are targeted by somatostatin agonists (octreotide and a new “pan-sst” agonist, pasireotide) which antiproliferative effect is known in other types of tumors. The aim of our study is to test in vitro effects of m-Tor inhibitor (Everolimus) and/or somatostatin agonists Octreotide or pasireotide, on human meningiomas in primary culture. By real time PCR, we found that sst2 was expressed in all meningiomas. The expression level of Merlin was highly variable between tumors and was inversely correlated with mTor pathway activation (p