Synergy of the antibiotic colistin with echinocandin antifungals in Candida species.

International audienceOBJECTIVES: Candida albicans is the most prevalent fungal pathogen of humans, causing a wide range of infections from harmless superficial to severe systemic infections. Improvement of the antifungal arsenal is needed since existing antifungals can be associated with limited efficacy, toxicity and antifungal resistance. Here we aimed to identify compounds that act synergistically with echinocandin antifungals and that could contribute to a faster reduction of the fungal burden. METHODS: A total of 38 758 compounds were tested for their ability to act synergistically with aminocandin, a β-1,3-glucan synthase inhibitor of the echinocandin family of antifungals. The synergy between echinocandins and an identified hit was studied with chemogenomic screens and testing of individual Saccharomyces cerevisiae and C. albicans mutant strains. RESULTS: We found that colistin, an antibiotic that targets membranes in Gram-negative bacteria, is synergistic with drugs of the echinocandin family against all Candida species tested. The combination of colistin and aminocandin led to faster and increased permeabilization of C. albicans cells than either colistin or aminocandin alone. Echinocandin susceptibility was a prerequisite to be able to observe the synergy. A large-scale screen for genes involved in natural resistance of yeast cells to low doses of the drugs, alone or in combination, identified efficient sphingolipid and chitin biosynthesis as necessary to protect S. cerevisiae and C. albicans cells against the antifungal combination. CONCLUSIONS: These results suggest that echinocandin-mediated weakening of the cell wall facilitates colistin targeting of fungal membranes, which in turn reinforces the antifungal activity of echinocandins

Identifying the Deleterious Effect of Rare LHX4 Allelic Variants, a Challenging Issue

International audienceLHX4 is a LIM homeodomain transcription factor involved in the early steps of pituitary ontogenesis. To date, 8 heterozygous LHX4 mutations have been reported as responsible of combined pituitary hormone deficiency (CPHD) in Humans. We identified 4 new LHX4 heterozygous allelic variants in patients with congenital hypopituitarism: W204X, delK242, N271S and Q346R. Our objective was to determine the role of LHX4 variants in patients' phenotypes. Heterologous HEK293T cells were transfected with plasmids encoding for wild-type or mutant LHX4. Protein expression was analysed by Western Blot, and DNA binding by electro-mobility shift assay experiments. Target promoters of LHX4 were cotransfected with wild type or mutant LHX4 to test the transactivating abilities of each variant. Our results show that the W204X mutation was associated with early GH and TSH deficiencies and later onset ACTH deficiency. It led to a truncated protein unable to bind to alpha-Gsu promoter binding consensus sequence. W204X was not able to activate target promoters in vitro. Cotransfection experiments did not favour a dominant negative effect. In contrast, all other mutants were able to bind the promoters and led to an activation similar as that observed with wild type LHX4, suggesting that they were likely polymorphisms. To conclude, our study underlines the need for functional in vitro studies to ascertain the role of rare allelic variants of LHX4 in disease phenotypes. It supports the causative role of the W204X mutation in CPHD and adds up childhood onset ACTH deficiency to the clinical spectrum of the various phenotypes related to LHX4 mutations

Anti-proliferative and anti-secretory effects of everolimus on human pancreatic neuroendocrine tumors primary cultures: is there any benefit from combination with somatostatin analogs?

Therapeutic management of gastroenteropancreatic neuroendocrine tumors (GEP-NETs) is challenging. The mammalian target of rapamycin (mTOR) inhibitor everolimus recently obtained approval from the Food and Drug Administration for the treatment of patients with advanced pancreatic neuroendocrine tumors (pNETs). Despite its promising antitumor efficacy observed in cell lines, clinical benefit for patients is unsatisfactory. The limited therapeutic potential of everolimus in cancer cells has been attributed to Akt activation due to feedback loops relief following mTOR inhibition. Combined inhibition of Akt might then improve everolimus antitumoral effect. In this regard, the somatostatin analog (SSA) octreotide has been shown to repress the PI3K/Akt pathway in some tumor cell lines. Moreover, SSAs are well tolerated and routinely used to reduce symptoms caused by peptide release in patients carrying functional GEP-NETs. We have recently established and characterized primary cultures of human pNETs and demonstrated the anti-proliferative effects of both octreotide and pasireotide. In this study, we aim at determining the antitumor efficacy of everolimus alone or in combination with the SSAs octreotide and pasireotide in primary cultures of pNETs. Everolimus reduced both Chromogranin A secretion and cell viability and upregulated Akt activity in single treatment. Its anti-proliferative and anti-secretory efficacy was not improved combined with the SSAs. Both SSAs did not overcome everolimus-induced Akt upregulation. Furthermore, caspase-dependent apoptosis induced by SSAs was lost in combined treatments. These molecular events provide the first evidence supporting the lack of marked benefit in patients co-treated with everolimus and SSA

Caractérisation et fonctionnalité des sous-types des récepteurs de la somatostatine dans les adénomes hypophysaires humains

Les adénomes hypophysaires par leur croissance tumorale et l'hypersécrétion hormonale sont responsables d'une morbi-mortalité importante. Les analogues de la somatostatine utilisés actuellement contrôlent l'hypersécrétion hormonale des 50 % des adénomes somatotropes et présentent une efficacité partielle dans les adénomes gonadotropes et lactotropes. Ils lient de façon préférentielle, parmi les 5 soustypes de récepteurs somatostatinergiques (sst1-5), le sst2. Ce travail met en évidence un profil d'expression de l'ARNm des ssts spécifique à chaque phénotype tumoral : sst2+sst5 pour les adénomes somatotropes, sst2+sst3 pour les adénomes gonadotropes et sst1+sst5 pour les adénomes lactotropes. L'étude de leur fonctionnalité, avec les agonistes préférentiels de sous-type, montre que sst2 est le principal médiateur de l'inhibition de la sécrétion de GH dans les adénomes somatotropes en culture cellulaire. Néanmoins, dans les adénomes partiellement sensibles à l'octréotide, ou sst2 est sous exprimé, l'agoniste sst5 inhibe la GH de façon équivalente à l'agoniste sst2. Cette sauvegarde fonctionnelle via sst5, est illustrée par un agoniste bispécifique, sst2 et sst5 (BIM-23244), qui réalise un effet d'inhibition de GH meilleur que l'octréotide dans ces tumeurs. Nous proposons une explication à cette coopération fonctionnelle sst2-sst5 dans l'inhibition hormonale via le mécanisme de dimérisation des récepteurs. Nous avons montré l'existence d'une autre coopération fonctionnelle dans l'inhibition de GH (et PRL) en utilisant un ligand bivalent somatostatinergique sst2 et dopaminergique D2R (BIM-23A387). Le gain en EC50 d'inhibition de GH et PRL (100 fois inférieures à l'octréotide) peut-être expliqué par les conséquences de l'hétérodimérisation sst2-D2R, favorisée par l'agoniste bivalent sst2-D2R. Ce ligand " dopastatinergique " nous parait un bon candidat en tant que nouvel agent thérapeutique dans l'acromégalie. L'activation concomitante de récepteurs reliés aux voies de transduction inhibitrices potentiellement additives, par les ligands bivalents ou bispécifiques, est une nouvelle approche de développement des drogues efficaces dans le contrôle de l'hypersécrétion hormonale et peut-être de la prolifération des adénomes hypophysairesThe pituitary adenomas, by their tumoral growth and hormonal hypersecretion, are responsible for a significant increase in morbidity and mortality. The current somatostatin analogs control hormonal hypersecretion in 50 % of the somatotroph adenomas and present a partial efficiency in the gonadotroph and lactotroph adenomas. They bind preferentially, among the 5 subtypes of somatostatine receptors (sst1-5), the sst2 subtype. This work highlights specific profiles of mRNA expression of the ssts for each tumoral phenotype: sst2+sst5 for the somatotroph adenomas, sst2+sst3 for the gonadotroph adenomas and sst1+sst5 for the lactotroph adenomas. The study of their functionality, with preferential subtype agonists, showed that sst2 is the principal mediator of GH secretion suppression of the somatotroph adenomas in cell culture. Nevertheless, in the adenomas partially responders to octreotide, in which sst2 is underexpressed, the sst5 agonist inhibited the GH release in a similar manner to the sst2 agonist. Such a functional rescue, via sst5, is enhanced by a bispecific agonist, sst2 and sst5 (BIM-23244), which realized a better GH inhibitory effect than octreotide in these tumours. We propose an explanation to this functional cooperation of sst2 and sst5 in hormonal suppression by the mechanism of receptors dimerization. We showed the existence of another functional cooperation in GH (and PRL) inhibition by using a bivalent ligand directed to sst2 and to D2R dopamine receptors (BIM-23A387). Its lower EC50 for GH and PRL suppressions (100 times lower than those of octreotide) may be explained by the consequences of sst2-D2R heterodimerization, favored by the bivalent sst2-D2R ligand. This "dopastatinergic" ligand appears as a good candidate drug in the treatment of acromegaly. Concomitant activation of receptors related to potentially additive inhibiting pathways of transduction by the bivalent or bispecific ligands represent a new approach in the development of effective drugs for the control of hormonal hypersecretion and possibly of the proliferation of the pituitary adenomasAIX-MARSEILLE2-BU Méd/Odontol. (130552103) / SudocPARIS-BIUP (751062107) / SudocSudocFranceF

Relevance of coexpression of somatostatin and dopamine D2 receptors in pituitary adenomas.

International audienceDopamine and somatostatin are both involved in the negative control of normal pituitary cells. Dopamine subtype 2 receptor (D2DR) and somatostatin receptor (sst) agonists, mainly directed to sst2, are used in the treatment of pituitary adenomas. Nevertheless, a majority of corticotroph and gonadotroph adenomas and a third of somatotroph adenomas are still not sufficiently controlled by these treatments. D2DR and sst1, 2, 3 and 5 are present in most pituitary adenomas. These receptors may interact by heterodimerization as shown for sst1-sst5, sst5-D2DR, sst2-sst3 and sst2-D2DR suggesting possible additive effects. D2DR and sst2 agonist cotreatment showed limited additivity on GH secretion in acromegaly. Moreover, new chimeric compounds with sst2, D2DR and sst5 affinity have shown an increased control of secretion and/or proliferation of different types of pituitary adenomas in cell culture. Together with the multi-sst ligand drugs recently developed, these dopamine-somatostatin ligands represent a new opportunity in the combinatory treatment of pituitary adenomas