Search

Understanding the regulation of the testicular endocrine function leading to testosterone production is a major objective as the alteration of endocrine function is associated with the development of many diseases such as infertility. In the last decades, it has been demonstrated that several endogenous molecules regulate the steroidogenic pathway. Among them, bile acids have recently emerged as local regulators of testicular physiology and particularly endocrine function. Bile acids act through the nuclear receptor FXRα (Farnesoid-X-receptor alpha; NR1H4) and the G-protein-coupled bile acid receptor (GPBAR-1; TGR5). While FXRα has been demonstrated to regulate testosterone synthesis within Leydig cells, no data are available regarding TGR5. Here, we investigated the potential role of TGR5 within Leydig cells using cell culture approaches combined with pharmacological exposure to the TGR5 agonist INT-777. The data show that activation of TGR5 results in a decrease in testosterone levels. TGR5 acts through the PKA pathway to regulate steroidogenesis. In addition, our data show that TGR5 activation leads to an increase in cholesterol ester levels. This suggests that altered lipid homeostasis may be a mechanism explaining the TGR5-induced decrease in testosterone levels. In conclusion, the present work highlights the impact of the TGR5 signaling pathway on testosterone production and reinforces the links between bile acid signaling pathways and the testicular endocrine function. The testicular bile acid pathways need to be further explored to increase our knowledge of pathologies associated with impaired testicular endocrine function, such as fertility disorders.

One of the most important but less understood step of epithelial tumourigenesis occurs when cells acquire the ability to leave their epithelial compartment. This phenomenon, described as basal epithelial cell extrusion (basal extrusion), represents the first step of tumour invasion. However, due to lack of adequate in vivo model, implication of emblematic signalling pathways such as Ras/Mitogen-Activated Protein Kinase (MAPK) and phosphoinositide 3 kinase (PI3K)/protein kinase B (AKT)/mammalian target of rapamycin (mTOR) signalling pathways, is scarcely described in this phenomenon. We have developed a unique model of basal extrusion in the Drosophila accessory gland. There, we demonstrate that both Ras/MAPK and PI3K/AKT/mTOR pathways are necessary for basal extrusion. Furthermore, as in prostate cancer, we show that these pathways are co-activated. This occurs through set up of Epidermal Growth Factor Receptor (EGFR) and Insulin Receptor (InR) dependent autocrine loops, a phenomenon that, considering human data, could be relevant for prostate cancer., The molecular mechanisms leading to basal extrusion are unclear. Here, the authors use the Drosophila accessory gland to model human prostate acini and show that Ras/MAPK and PI3K/AKT/mTOR pathways are co-activated in two autocrine loops by dEGF and dIGF, inducing basal extrusion and subsequent tumour formation.

International audience; The hypothalamic-pituitary axis exert a major control over endocrine and exocrine testicular functions. The hypothalamic-pituitary axis corresponds to a cascade with the Gonadotropin Releasing Hormone secreted by the hypothalamus, which stimulates the synthesis and the release of Luteinizing Hormone (LH) and Follicle Stimulating Hormone by the gonadotropic cells of the anterior pituitary. The LH signaling pathway controls the steroidogenic activity of the Leydig cells via the activation of the luteinizing hormone/choriogonadotropin receptor. In order to avoid a runaway system, sex steroids exert a negative feedback within hypothalamus and pituitary. Testicular steroidogenesis is locally controlled within Leydig cells. The present work reviews some local regulations of steroidogenesis within the Leydig cells focusing mainly on the roles of the Farnesoid-X-Receptor-alpha and its interactions with several orphan members of the nuclear receptor superfamily. Further studies are required to reinforce our knowledge of the regulation of testicular endocrine function, which is necessary to ensure a better understanding of fertility disorders and then proposed an adequate treatment of the diseases.

The facile synthesis of functionalized luminescent nanoparticles from LnL3 lanthanide complexes is described. The luminescence properties of the lanthanide chelates and of the corresponding nanohybrids are reported and compared. For a further application in bioimaging, the cytotoxicity of the nano-objects was investigated.

International audience; Primary bile acids are synthetized from cholesterol within the liver and then transformed by the bacteria in the intestine to secondary bile acids. In addition to their involvement in digestion and fat solubilization, bile acids also act as signaling molecules. Several receptors are sensors of bile acids. Among these receptors, this review focuses on the nuclear receptor FXRalpha and the G-protein-coupled receptor TGR5. This review briefly presents the potential links between bile acids and cancers that are discussed in more details in the other articles of this special issue of Molecular Aspects of Medicine focused on "Bile acids, roles in integrative physiology and pathophysiology".

The mammalian target of rapamycin (mTOR) plays essential roles in the regulation of growth-related processes such as protein synthesis, cell sizing and metabolism in both normal and pathological growing conditions. These functions of mTOR are thought to be largely a consequence of its cytoplasmic activity in regulating translation rate, but accumulating data highlight supplementary role(s) for this serine/threonine kinase within the nucleus. Indeed, the nuclear activities of mTOR are currently associated with the control of protein biosynthetic capacity through its ability to regulate the expression of gene products involved in the control of ribosomal biogenesis and proliferation. Using primary murine embryo fibroblasts (MEFs), we observed that cells with overactive mTOR signaling displayed higher abundance for the growth-associated Npm1 protein, in what represents a novel mechanism of Npm1 gene regulation. We show that Npm1 gene expression is dependent on mTOR as demonstrated by treatment of wild-type and Pten inactivated MEFs cultured with rapamycin or by transient transfections of small interfering RNA directed against mTOR. In accordance, the mTOR kinase localizes to the Npm1 promoter gene in vivo and it enhances the activity of a human NPM1-luciferase reporter gene providing an opportunity for direct control. Interestingly, rapamycin did not dislodge mTOR from the Npm1 promoter but rather strongly destabilized the Npm1 transcript by increasing its turnover. Using a prostate-specific Pten-deleted mouse model of cancer, Npm1 mRNA levels were found up-regulated and sensitive to rapamycin. Finally, we also showed that Npm1 is required to promote mTOR-dependent cell proliferation. We therefore proposed a model whereby mTOR is closely involved in the transcriptional and posttranscriptional regulation of Npm1 gene expression with implications in development and diseases including cancer.

International audience; Structural and functional studies have provided numerous insights over the past years on how members of the nuclear hormone receptor superfamily tightly regulate the expression of drug-metabolizing enzymes and transporters. Besides the role of the farnesoid X receptor (FXR) in the transcriptional control of bile acid transport and metabolism, this review provides an overview on how this metabolic sensor prevents the accumulation of toxic byproducts derived from endogenous metabolites, as well as of exogenous chemicals, in coordination with the pregnane X receptor (PXR) and the constitutive androstane receptor (CAR). Decrypting this network should provide cues to better understand how these metabolic nuclear receptors participate in physiologic and pathologic processes with potential validation as therapeutic targets in human disabilities and cancers.

International audience; Besides their well-known roles in digestion and fat solubilization, bile acids (BAs) have been described as signaling molecules activating the nuclear receptor Farnesoid-X-receptor (FXRα) or the G-protein-coupled bile acid receptor-1 (GPBAR-1 or TGR5). In previous reports, we showed that BAs decrease male fertility due to abnormalities of the germ cell lineage dependent on Tgr5 signaling pathways. In the presentstudy, we tested whether BA exposure could impact germ cell DNA integrity leading to potential implications for progeny. For that purpose, adult F0 male mice were fed a diet supplemented with cholic acid (CA) or the corresponding control diet during 3.5 months prior mating. F1 progeny from CA exposed founders showed higher perinatal lethality, impaired BA homeostasis and reduced postnatal growth, as well as altered glucose metabolism in later life. The majority of these phenotypic traits were maintained up to the F2 generation. In F0 sperm cells, differential DNA methylation associated with CA exposure may contribute to the initial programming of developmental and metabolic defects observed in F1 and F2 offspring. Tgr5 knock-out mice combined with in vitro strategies defined the critical role of paternal Tgr5 dependent pathways in the multigenerational impacts of ancestral CA exposure.

Radiotherapy is an essential component of glioma standard treatment. Glioblastomas (GBM), however, display an important radioresistance leading to tumor recurrence. To improve patient prognosis, there is a need to radiosensitize GBM cells and to circumvent the mechanisms of resistance caused by interactions between tumor cells and their microenvironment. STAT3 has been identified as a therapeutic target in glioma because of its involvement in mechanisms sustaining tumor escape to both standard treatment and immune control. Here, we studied the role of STAT3 activation on tyrosine 705 (Y705) and serine 727 (S727) in glioma radioresistance. This study explored STAT3 phosphorylation on Y705 (pSTAT3-Y705) and S727 (pSTAT3-S727) in glioma cell lines and in clinical samples. Radiosensitizing effect of STAT3 activation down-modulation by Go6976 was explored. In a panel of 15 human glioma cell lines, we found that the level of pSTAT3-S727 was correlated to intrinsic radioresistance. Moreover, treating GBM cells with Go6976 resulted in a highly significant radiosensitization associated to a concomitant pSTAT3-S727 down-modulation only in GBM cell lines that exhibited no or weak pSTAT3-Y705. We report the constitutive activation of STAT3-S727 in all GBM clinical samples. Targeting pSTAT3-S727 mainly in pSTAT3-Y705-negative GBM could be a relevant approach to improve radiation therapy.

We identified a ceftriaxone-resistant Neisseria gonorrhoeae isolate in a patient in Canada. This isolate carried the penA-60 allele, which differs substantially from its closest relative, mosaic penA XXVII (80% nucleotide identity). Epidemiologic and genomic data suggest spread from Asia. Antimicrobial susceptibility surveillance helps prevent spread of highly resistant N. gonorrhoeae strains.

International audience; Arboviruses are an expanding public health threat, with pregnant women facing unique complications from arbovirus infections. These infections, such as dengue and Crimean–Congo haemorrhagic fever, can be more severe in pregnant women than in the general population. Vertical transmission is reported for many arboviruses and can severely affect pregnancy outcome. Indeed, arboviruses—particularly flaviviruses and alphaviruses—are associated with increased risks of fetal loss and premature birth. Arboviruses can be teratogenic, as is the case for Zika virus and Venezuelan equine encephalitis virus. Finally, intrapartum transmission can result in severe neonatal infections, as is true for chikungunya virus. Although the global burden of arboviruses is well recognised, few studies have provided data on arbovirus infection specifically in the context of maternal and child health. Epidemiological and clinical studies are therefore needed to better assess the burden of arbovirus infections during pregnancy and to improve the prevention and clinical management of these viral infections. In this Review, we analyse the information available and identify gaps in knowledge that require further assessment.

International audience; Next to their involvement in digestion, bile acids have been defined as signaling molecules. They have been demonstrated to control many physiological functions among which lipid homeostasis, glucose and energy metabolisms. Bile acids are ligands of several receptors and multiple studies using transgenic mouse models defined the major roles of their respective nuclear and membrane receptors namely the Farnesoid-X-Receptor (FXRalpha) and the G-protein-coupled bile acid receptor 1(GPBAR1; TGR5). Here we review the reports highlighting the impacts of bile acids on testicular physiology and on male reproductive functions. The studies on mouse models open perspectives to better understand the deleterious effects of bile acids on testicular pathophysiologies and fertility disorders. Additional studies are needed to corroborate these correlations in humans.

Summary Spermatogenesis is the process by which spermatozoa are generated from spermatogonia. This cell population is heterogeneous, with self-renewing spermatogonial stem cells (SSCs) and progenitor spermatogonia that will continue on a path of differentiation. Only SSCs have the ability to regenerate and sustain spermatogenesis. This makes the testis a good model to investigate stem cell biology. The Farnesoid X Receptor alpha (FXRα) was recently shown to be expressed in the testis. However, its global impact on germ cell homeostasis has not yet been studied. Here, using a phenotyping approach in Fxrα−/− mice, we describe unexpected roles of FXRα on germ cell physiology independent of its effects on somatic cells. FXRα helps establish and maintain an undifferentiated germ cell pool and in turn influences male fertility. FXRα regulates the expression of several pluripotency factors. Among these, in vitro approaches show that FXRα controls the expression of the pluripotency marker Lin28 in the germ cells., Graphical Abstract, Highlights • FXRα regulated germ cell apoptotis independently of androgen homeostasis • FXRα controls germ cell differentiation • FXRα regulates the establishment and maintenance of undifferentiated germ cells • In germ cells, FXRα controls the expression of pluripotency markers such as Lin28, Martinot et al. report that FXRα controls the fate of undifferentiated spermatogonia.

International audience; Lysine Specific Demethylase 1 (LSD1) removes mono-and dimethyl groups from lysine 4 of histone H3 (H3K4) or H3K9, resulting in repressive or activating (respectively) transcriptional histone marks. The mechanisms that control the balance between these two antagonist activities are not understood. We here show that LSD1 and the orphan nuclear receptor estrogen-related receptor a (ERR alpha) display commonly activated genes. Transcriptional activation by LSD1 and ERR alpha involves H3K9 demethylation at the transcriptional start site (TSS). Strikingly, ERR alpha is sufficient to induce LSD1 to demethylate H3K9 in vitro. The relevance of this mechanism is highlighted by functional data. LSD1 and ERR alpha coregulate several target genes involved in cell migration, including the MMP1 matrix metallo-protease, also activated through H3K9 demethylation at the TSS. Depletion of LSD1 or ERR alpha reduces the cellular capacity to invade the extracellular matrix, a phenomenon that is rescued by MMP1 reexpression. Altogether our results identify a regulatory network involving a direct switch in the biochemical activities of a histone demethylase, leading to increased cell invasion.

Hantaviruspulmonary syndrome (HPS) is a rare illness in eastern Canada. We present three cases of HPS among military personnel in Quebec. The three cases shared a common exposure to mouse excreta while engaged in military training in Alberta, a western province of Canada.

Screening of bona fide ligands for nuclear receptors is a real tour de force as the identified molecules are supposed to be able to activate the targeted proteins in cell culture as well as in vivo. Indeed orphan nuclear receptors are putative pharmacologically targets for various diseases. It is thus necessary to have quick and reproductive systems that help in identifying new ligands, agonist or antagonist, before using them in vivo in animal models to check for secondary effects. Here, we describe the transient transfections (homologous and heterologous) used for the screening of ligands for liver X receptor α (LXRα, NR1H3) in HeLa cells.

The promotion and progression of prostate cancer (PCa) are associated with androgen receptor (AR) signalling. AR functions are modulated by a variety of co-factors amongst which we identified the nucleophosmin (NPM/B23), a member of the histone chaperone family. Here, we show that NPM is overexpressed in PCa compared to normal adjacent tissues. AR and NPM interact in vitro and in vivo, and NPM is critical for androgen-dependent transcriptional activation in LNCaP cells as an anti-NPM siRNA downregulates transcription of a transfected androgen response element (ARE)-containing reporter promoter as well as expression of the endogenous androgen responsive prostate-specific antigen (PSA) gene. By investigating the effect of NPM on AR, we have also observed that NPM enhances AR binding to an ARE in vitro in electrophoretic gel mobility-shift assay experiments. Chromatin immunoprecipitation studies further demonstrated that both AR and NPM associate with AREs of the PSA gene in vivo. Altogether, our data suggest that the molecular histone chaperone NPM could regulate AR functions by promoting assembly of AR-containing regulatory complexes and that high levels of NPM might alter AR functions in PCa.

The proto-oncoprotein Cbl is known to control several signaling processes. It is highly expressed in the testis, and because spermatogenesis is androgen dependent, we investigated the androgen dependency expression of Cbl through its testicular sublocalization and its expression levels in rats that were exposed to the antiandrogen flutamide or were hypophysectomized. We report the androgen dependency of Cbl as it localizes in pachytene spermatocytes during androgen-dependent stages, is down-regulated upon flutamide exposure, and is up-regulated with testosterone in hypophysectomized rats. Coculture experiments showed the key control exerted by the Sertoli cell on Cbl activity. As flutamide induces germ cell apoptosis, we investigate members of the Bcl-2 family upon flutamide exposure. We show that the proapoptotic Bcl-2 family member Bim mirrored Cbl expression through a posttranscriptional process. We also show that in Cbl knockout mouse testes, the imbalance between the high expression of Bim and Smac/Diablo and antiapoptotic factors such as cellular inhibitor of apoptosis 2 favors a survival process, which makes these mice unresponsive to androgen withdrawal and could explain their hypofertility.

A variety of transcription factors are post-translationally modified by SUMO, a 97-residue ubiquitin-like protein bound covalently to the targeted lysine. Here we describe SUMO modification of the Ets family member ERM at positions 89, 263, 293, and 350. To investigate how SUMO modification affects the function of ERM, Ets-responsive intercellular adhesion molecule 1 (ICAM-1) and E74 reporter plasmids were employed to demonstrate that SUMO modification causes inhibition of ERM-dependent transcription without affecting the subcellular localization, stability, or DNA-binding capacity of the protein. When the adenoviral protein Gam1 or the SUMO protease SENP1 was used to inhibit the SUMO modification pathway, ERM-dependent transcription was de-repressed. These results demonstrate that ERM is subject to SUMO modification and that this post-translational modification causes inhibition of transcription-enhancing activity.

The androgen receptor (AR) is a ligand-responsive transcription factor known to play a central role in the pathogenesis of prostate cancer. However, the regulation of AR gene expression in the normal and pathological prostate remains poorly understood. This study focuses on the effect of the phosphoinositide 3-kinase (PI 3-kinase)/Akt axis on AR expression in vas deferens epithelial cells (VDEC), a suitable model to study androgen regulation of gene expression, and LNCaP cells (derived from a metastasis at the left supraclavicular lymph node from a 50-year-old patient with a confirmed diagnosis of metastatic prostate carcinoma). Taken together, our data show for the first time that the PI 3-kinase/Akt pathway is required for basal and dihydrotestosterone-induced AR protein expression in both VDEC and LNCaP. Inhibition of the PI 3-kinase/Akt pathway reduced AR expression and the decline in AR protein level correlated with a decrease in AR mRNA in VDEC but not in LNCaP. Since PI 3-kinase/Akt axis is active in prostate cancer, cross-talk between PI 3-kinase/Akt and AR signalling pathways may have implications for endocrine therapy.

In studies comparing the performance of psychometric instruments and general practitioners in the identification of psychological disorders, authors usually treat the psychometric instrument as the gold standard. Some patients may have no psychiatric diagnosis and normal scores on self-report measures of distress, but still benefit from detection and treatment of their psychosocial problems. However, physicians may be spending valuable time identifying problems in patients who have no disability. The extent and implications of the discrepancy between clinician assessment and standard instruments requires further exploration. Adult patients of 40 family physicians completed the General Health Questionnaire (GHQ-28) before their visit. Immediately following the visit, physicians, who were blind to the patient's GHQ score, indicated whether they had detected any signs or symptoms of anxiety, depression, somatization, or other psychosocial problems. Of the 1,011 primary care patients that participated, 439 had normal GHQ-28 scores. Physicians detected psychological problems in 177 (38.3%) of the 439. In bivariate analyses, poorer general and mental health (as measured by SF-36) was associated with higher detection rates. The patient's belief that there was a psychological component of his or her problem (OR = 2.50), being in a marital relationship (OR = 1.87), and the physician's perception of the seriousness of the problem (OR = 1.84) were associated with detection. Detection was less frequent when the physician did not know the patient well (OR = 0.69), and when the physician was a woman (OR = 0.46). For the 28% of patients who themselves perceived a psychological element of their problem, physician detection was probably appropriate. However, it is unlikely that detection of the remaining patients was beneficial to the patients.

Radiotherapy is an essential component of glioma standard treatment. Glioblastomas (GBM), however, display an important radioresistance leading to tumor recurrence. To improve patient prognosis, there is a need to radiosensitize GBM cells and to circumvent the mechanisms of resistance caused by interactions between tumor cells and their microenvironment. STAT3 has been identified as a therapeutic target in glioma because of its involvement in mechanisms sustaining tumor escape to both standard treatment and immune control. Here, we studied the role of STAT3 activation on tyrosine 705 (Y705) and serine 727 (S727) in glioma radioresistance. This study explored STAT3 phosphorylation on Y705 (pSTAT3‐Y705) and S727 (pSTAT3‐S727) in glioma cell lines and in clinical samples. Radiosensitizing effect of STAT3 activation down‐modulation by Gö6976 was explored. In a panel of 15 human glioma cell lines, we found that the level of pSTAT3‐S727 was correlated to intrinsic radioresistance. Moreover, treating GBM cells with Gö6976 resulted in a highly significant radiosensitization associated to a concomitant pSTAT3‐S727 down‐modulation only in GBM cell lines that exhibited no or weak pSTAT3‐Y705. We report the constitutive activation of STAT3‐S727 in all GBM clinical samples. Targeting pSTAT3‐S727 mainly in pSTAT3‐Y705‐negative GBM could be a relevant approach to improve radiation therapy.

19 NO UV EL LE S M AG AZ IN E NOUVELLE C. Beaudoin, S. Maquaire, J. Allemand, C. Lours-Calet, G. Veyssiere, M. Manin, L. Morel : CNRS UMR 6247/ Clermont Universite/INSERM U931, Campus Universitaire des Cezeaux, 24, avenue des Landais, 63177 Aubiere Cedex, France. Laurent.MOREL@univ-bpclermont.fr L. Leotoing : Departement d’Hematologie, Inserm U567, Institut Cochin, 24, rue du Faubourg Saint-Jacques, 75014 Paris, France. NPM : un nouvel acteur dans le cancer de la prostate ?

Microsomal cytochrome P450c17 (17a-hydroxylase/17,20-Lyase) catalyzes two reactions in the delta5 and delta4 pathways leading to the production of C19 steroids. Transient expression of human, bovine, porcine, rat, and mouse P450c17 cDNAs showed that the protein has 17alpha-hydroxylase and 17,20-Lyase activities, converting pregnenolone and progesterone into delta5- and delta4-Cl9 steroids, respectively, although the rat and mouse proteins have a preferential pathway toward the delta4 steroids. The guinea pig (gp) P450c17 shares 46% to 70% amino acid identity with the corresponding proteins of other species, and further characterization indicated that the guinea pig enzyme only converts progesterone to androstenedione. In this study, we have tried to identify amino acid(s) in the gpP450c17 that governs such a steroid specificity. Among the various mutants that we have created, change of the arginine (R) residue at position 200 to an asparagine (N) (R200N) in the gpP450c17 protein increased reactivity toward pregnenolone compared with the wild-type enzyme. Pregnenolone was converted into 17alpha-hydroxypregnenolone and dehydroepiandrosterone. However, this gain occurred at the expense of the 17,20-lyase activity toward 17alpha-hydroxyprogesterone. The R200N mutation in the gpP450c17 protein introduced a potential N-linked glycosylation site (200Asn-X-Thr202); however, substitution of the Thr202 residue by an asparagine (R200N/T202N), which abolishes the site, did not change the preference of the gpP450c17 mutant for pregnenolone. Furthermore, introduction of a putative glycosylation site at amino acid 185 in the gpP450c17 enzyme did not alter substrate specificity. The properties of the amino acid were also investigated, and neither the charge nor the size of the sidechain elicited change in the substrate specificity of gpP450c17. Thus, our results demonstrate that the mutation of arginine to asparagine at position 200 changes the substrate specificity of the gpP450c17 enzyme.

In human placenta the cytochrome P450 side-chain cleavage (P450scc) and 3 beta-hydroxysteroid dehydrogenase type 1 (3 beta-HSD-1) convert cholesterol and pregnenolone producing progesterone, whereas 17 beta-hydroxysteroid dehydrogenase type 1 (17 beta-HSD-1) mediates the interconversion of estrone and estradiol. We have examined the effects of calcium on phorbol ester- and cAMP-induced P450scc, 3 beta-HSD-1 and 17 beta-HSD-1 mRNAs in human JEG-3 cells. A23187 increased in a dose-dependent fashion in the 1.3 kb 17 beta-HSD-1 mRNA whereas a weaker increase followed by a gradual depletion effect of A23187 was observed on 3 beta-HSD-1 mRNA. No significant effect of A23187 on P450scc mRNA was observed. Using 0.50 microM of A23187 the induction of 3 beta-HSD-1 and 17 beta-HSD-1 mRNAs was maximum within about 6 h whereas P450scc mRNA levels stayed unaffected throughout the time-course period. The action of A23187 was synergistic on cAMP-stimulated 17 beta-HSD-1 mRNA levels, while in a dose-dependent manner A23187 progressively depleted 3 beta-HSD-1 and P450scc mRNA abundance probably by activation of a calcium-/calmodulin-dependent phosphodiesterase. On the phorbol 12-myristate, 13-acetate (PMA)-stimulated 3 beta-HSD-1, 17beta-HSD-1 and P450scc mRNA levels only the lowest concentration of A23187 potentialized the PMA effect on the 17 beta-HSD-1 mRNA levels. Using thapsigargin (TG), a cell-permeable sesquiterpene lactone that releases calcium by inhibiting sarco/endoplasmic reticular calcium-ATPase, our data indicated the presence in JEG-3 cells of TG-sensitive and TG-insensitive calcium-ATPases regulating 3 beta-HSD-1 and 17 beta-HSD-1 mRNA levels. These results emphasized the complexity of calcium contribution with the protein kinase A and C pathways in the regulation of P450scc, 3 beta-HSD-1 and 17 beta-HSD-1 mRNA levels. In addition, the different sensitivity of these genes to calcium suggest they could be activated by different subclasses of PKCs.

In mammalian and fish species, P450c17 mediates both 17 alpha-hydroxylase and 17,20-lyase activities in the synthesis of steroid hormones. Previous results have shown that among the adrenal steroid hydroxylase enzymes involved in adrenal C19 steroid and glucocorticoid synthesis, regulation of cytochrome P450c17 is of primary importance because it is localized at the key branch between glucocorticoid and C19 steroid synthesis. A cDNA library from guinea pig adrenal was constructed, and the complete 17 alpha-hydroxylase cytochrome P450 cDNA was isolated. The guinea pig P450c17 cDNA includes the full-length coding region (1,524 nucleotide), the complete 3' untranslated region (169 nucleotide), and 39 bases of the 5' untranslated region. Our clone shares most of the features of the other P450c17 cDNAs; however, in addition, we identified a novel conserved region of 18 amino acids located in exon I between residues 80 and 97. This region presents the highest percentage of identity among the other P450c17 enzymes and is positioned one helixturn upstream of the important Ser106 on the corresponding human form. On Northern blot, the cDNA hybridizes with a major 1.8-kb mRNA and with two other related P450c17 mRNA of about 3 and 4 kb. P450c17 mRNA is equally distributed in male and female gonads and adrenals. Characterization of the enzymatic activity shows that 17 alpha-hydroxylase and 17,20-lyase are carried by a single protein, but in homogenates 17,20-lyase activity is barely detectable. Moreover, we demonstrate in vitro and in vivo that the guinea pig enzyme preferentially has very high levels of 17 alpha-hydroxylase and 17,20-lyase activities only toward delta 4 steroids. Second-messenger cyclic adenosine monophosphate and adrenocorticotropin specifically increased the abundance of P450c17 mRNA levels in guinea pig adrenal cells.

Although NHC-complexes are renowned for their catalytic properties, studies of their anti-cancer properties are scarce. In our search for new potent metal-based drugs, we envisioned that copper(I)-NHC could serve as a stable analogue of copper(I)-phenantroline. Herein, we report the results of the biological evaluation of the anticancer properties of [(SIMes)CuCl] 5. This complex showed impressive cytotoxicity against a panel of human tumor cell lines. Regarding IC50, up to 150 fold decrease is observed compared to cisplatin. Cellular effects of [(SIMes)CuCl] were evaluated at the cell cycle and the apoptotic levels. 5 causes cell cycle arrest at the G1 phase as demonstrated by the concomitant accumulation of protein p21 and the strong down-regulation of cyclin D1. Monitoring of poly-(ADP-ribose)-polymerase, implied in apoptosis response of cells, showed that [(SIMes)CuCl] induces apoptosis at low concentration. Finally, we showed that [(SIMes)CuCl] targets DNA by in vitro studies. When inducing aerobic DNA cleavage, [(SIMes)CuCl] behaves analogously to copper(I)-phenantroline. Thus, our experiments evidenced that employing NHC to deliver active copper(I) species in cellulo is a valuable strategy in anticancer research.

In the male, androgens promote growth and differentiation of sex reproductive organs through ligand activation of the androgen receptor (AR). Here, we show that androgens are not major actors of the cell cycle arrest associated with the differentiation process, and that the epidermal growth factor (EGF)-mediated signalling interferes with AR activities to regulate androgen response when epithelial cells are differentiated. Higher AR expression and enhanced androgen responsiveness correlate with reduction of phosphorylated ERK1/2 over differentiation. These modifications are associated with recruitment of cells in phase G(0)/G(1), up-regulation of p27(kip1), down-regulation of p21(Cip1) and p53 proteins, and accumulation of hypo-phosphorylated Rb. Exposure to EGF reduces AR expression levels and blocks androgen-dependent transcription in differentiated cells. It also restores p53 and p21(Cip1) levels, Rb hyper-phosphorylation, ERK1/2 activation and promotes cell cycle re-entry as p27(kip1) protein levels are decreased. Treatment with a MEK inhibitor reverses the EGF-mediated AR down-regulation in differentiated cells, thus suggesting the existence of an inverse correlation between EGF and androgen signalling in non-tumoural epithelia. Interestingly, when androgen signalling is set in differentiated cells, dihydrotestosterone exerts an inhibitory effect on ERK activity but paradoxically does not modify EGFR (ErbB1) phosphorylation, indicating that androgens are able to disrupt the EGFR-ERK cascade. Overall, our data demonstrate the existence of a balance between AR and mitogen-activated protein kinase activities that favours either the maintenance of differentiated conditions or the enhancement of cell proliferation capacities.

Nucleophosmin NPM1 is a molecular chaperone involved in many aspect of cellular physiology, eg. ribosomal biogenesis and cell cycle regulation. NPM1 is overexpressed in numerous types of solid tumors, including prostate cancer but the underlying molecular mechanisms are largely unknown. Using both cell lines and transgenic mouse models, we show that NPM1 expression is significantly increased in cells where the PI3K-AKT-mTOR pathway is activated through PTEN deletion. This overexpression is reversed when cells are treated with the pharmacological inhibitor of mTOR rapamycin. In accordance, transfection of small interfering RNA (siRNA) directed against mTOR leads to mTOR and NPM1 downregulation in these cells. In vivo, we found that NPM1 is overexpressed in PTEN knock-out murine prostate, and a one week treatment of these mice with rapamycin leads to NPM1 downregulation. Chromatin Immunoprecipitation (ChIP) assays show that mTOR is localized on NPM1 proximal promoter, close to a binding site for the transcription factor Ying-Yang 1 (YY1). mTOR binding on the chromatin seems to be constitutive, since rapamycin treatment did not alter its localization. mTOR could therefore regulates NPM1 expression by phosphorylating others factors involved in NPM1 gene expression, as previously described (Cunningham et al., 2007). We have previously shown that NPM1 overexpression enhance the migration and invasion abilities of prostate cancer cells (Loubeau et al., 2014). Moreover, NPM1 overexpression in mouse prostate epithelium leads to p27 protein downregulation and cyclin E upregulation, two molecular features of aggressive human prostate cancer. We thus propose that NPM1 could be a downstream effector of PI3K-AKT-mTOR pathway, supporting prostate cancer progression by altering expression of a set of genes involved in cellular homeostasis and proliferation. Citation Format: Rafik Boudra, Gaelle Loubeau, Corinne Lours-Calet, Cyrille Dejoussineau, Laurent Morel, Claude Beaudoin. NPM1: A new downstream effector of PI3K-AKT-mTOR pathway in prostate cancer? [abstract]. In: Proceedings of the AACR Special Conference: Targeting the PI3K-mTOR Network in Cancer; Sep 14-17, 2014; Philadelphia, PA. Philadelphia (PA): AACR; Mol Cancer Ther 2015;14(7 Suppl):Abstract nr B04.

Androgens are known to modulate many cellular processes such as cell growth and survival by binding to the androgen receptor (AR) and activating the transcription of target genes. Recent data suggested that AR can also mediate non-transcriptional actions outside the nucleus in addition to its ligand-inducible transcription factor function. Here, we describe a transcription-independent activation of the phosphatidylinositol 3-OH kinase (PI3-K) signaling pathway by androgens. Using non-transformed androgen-sensitive epithelial cells, we show that androgens enhance the PI3-K activity by promoting accumulation of phosphoinositide-3-P phospholipids in vitro. This activation is found in conjunction with an increased time-dependent phosphorylation of the downstream kinase AKT/protein kinase B on both Ser(473) and Thr(308) residues. Hormone-stimulated phosphorylation of AKT requires AR since incubation with the anti-androgen bicalutamide completely abolishes the androgen-stimulated AKT phosphorylation. Accordingly, we show that androgens increase AKT phosphorylation level in prostatic carcinoma PC3 cells only once they have been transfected with AR. Downstream, androgens enhance phosphorylation of transcription factor FKHR (Forkhead in rhabdomyosarcoma)-L1 and proapoptotic Bad protein and promote cell survival as they can counteract an apoptotic process. We also report that non-genomic effects of androgens are based on direct interaction between AR and the p85alpha regulatory subunit of class I(A) PI3-K. Together, these novel findings point out an important and physiologically relevant link between androgens and the PI3-K/AKT signaling pathway in governing cell survival.

The ets genes encode eukaryotic transcription factors that are involved in tumorigenesis and developmental processes. The signature of the Ets family is the ETS-domain, which binds to sites containing a central 5,-GGAA/T-3, motif. They can be sub-classified primarily because of the high amino acid conservation in their ETS-domains and, in addition, in the conservation of other domains generally characterized as transactivating. This is the case for the PEA3 group, which is currently made up of three members, PEA3/E1AF, ER81/ETV1 and ERM, which are more than 95% identical in the ETS-domain and more than 85% in the transactivation acidic domain. The members of the PEA3 group are activated through both the Ras-dependent and other kinase pathways, a function which emphasizes their involvement in several oncogenic mechanisms. The expression pattern of the three PEA3 group genes during mouse embryogenesis suggests that they are differentially regulated, probably to serve important functions such as tissue interaction. Although the target genes of these transcription factors are multiple, their most frequently studied role concerns their involvement in the metastatic process. In fact, PEA3 group members are over-expressed in metastatic human breast cancer cells and mouse mammary tumors, a feature which suggests a function of these transcription factors in mammary oncogenesis. Moreover, when they are ectopically over-expressed in non-metastatic breast cancer cells, these latter become metastatic with the activation of transcription of matrix metalloproteinases or adhesion molecules, such as ICAM-1.

The chaperone nucleophosmin (NPM1) is over-expressed in the epithelial compartment of prostate tumours compared to adjacent healthy epithelium and may represent one of the key actors that support the neoplastic phenotype of prostate adenocarcinoma cells. Yet, the mechanisms that underlie NPM1 mediated phenotype remain elusive in the prostate. To better understand NPM1 functions in prostate cancer cells, we sought to characterize its impact on prostate cancer cells behaviour and decipher the mechanisms by which it may act. Here we show that NPM1 favors prostate tumour cell migration, invasion and colony forming. Furthermore, knockdown of NPM1 leads to a decrease in the growth of LNCaP-derived tumours grafted in Nude mice in vivo. Such oncogenic-like properties are found in conjunction with a positive regulation of NPM1 on the ERK1/2 (Extracellular signal-Regulated Kinases 1/2) kinase phosphorylation in response to EGF (Epidermal Growth Factor) stimulus, which is critical for prostate cancer progression following the setting of an autonomous production of the growth factor. NPM1 could then be a target to switch off specifically ERK1/2 pathway activation in order to decrease or inhibit cancer cell growth and migration.

The final step of the transduction pathway is the activation of gene transcription, which is driven by kinase cascades leading to changes in the activity of many transcription factors. Among these latter, PEA3/E1AF, ER81/ETV1, and ERM, members of the well conserved PEA3 group from the Ets family are involved in these processes. We show here that protein kinase A (PKA) increases the transcriptional activity of human ERM and human ETV1, through a Ser residue situated at the edge of the ETS DNA-binding domain. PKA phosphorylation does not directly affect the ERM transactivation domains but does affect DNA binding activity. Unphosphorylated wild-type ERM bound DNA avidly, whereas after PKA phosphorylation it did so very weakly. Interestingly, S367A mutation significantly reduced the ERM-mediated transcription in the presence of the kinase, and the DNA binding of this mutant, although similar to that of unphosphorylated wild-type protein, was insensitive to PKA treatment. Mutations, which may mimic a phosphorylated serine, converted ERM from an efficient DNA-binding protein to a poor DNA binding one, with inefficiency of PKA phosphorylation. The present data clearly demonstrate a close correlation between the capacity of PKA to increase the transactivation of ERM and the drastic down-regulation of the binding of the ETS domain to the targeted DNA. What we thus demonstrate here is a relatively rare transcription activation mechanism through a decrease in DNA binding, probably by the shift of a non-active form of an Ets protein to a PKA-phosphorylated active one, which should be in a conformation permitting a transactivation domain to be active.

Background and objectives The training of caring physicians represents an important goal of medical education. Little is known however, on whether medical faculty constitute good role models for teaching humanistic skills to medical students. In this study, we examined to what extent medical students at innovative and traditional schools perceived their teachers as humanistic physicians and teachers. We also explored whether pre-clinical and clinical students shared the same perceptions. Methods A mail survey was conducted in Canada of all second-year students and senior clerks at one innovative medical school (problem-based learning (PBL), patient-centred, community-oriented) and three traditional medical schools. Students were asked to what extent they agreed or disagreed that the majority of their teachers behaved as humanistic physicians and teachers; 10 statements were used. Overall, 65% of the 1039 students returned the questionnaire. Results Over 25% of second-year students and 40% of senior clerks did not agree that their teachers behaved as humanistic caregivers with patients or were good role models in teaching the doctor–patient relationship. More than half of second-year students and senior clerks did not agree that their teachers valued human contact with them or were supportive of students who had difficulties. There were few differences in the way medical students at innovative and traditional schools perceived their teachers’ humanistic qualities. At the pre-clinical level however, there were more students from the innovative school than from the traditional schools (around 60% vs. 40%, P

Cytochrome P450c17 is a single enzyme that catalyzes two successive reactions within the delta 5 and delta 4 pathways. The proteins expressed with human, bovine, and rat cDNAs convert both pregnenolone and progesterone into delta 5-delta 4-C19 steroids, although the rat cDNA prefers the delta 4 pathway. Our results showed that the guinea pig adrenal possesses the enzymatic machinery to produce C19 steroids and suggest that the lyase activity plays a major role in regulating these syntheses. To obtain more information on the structure-function relationship we isolated a full-length cDNA clone encoding guinea pig P450c17. Northern blots of total RNA extracted from the testis, ovary, and adrenals of the guinea pig show that the P450c17 cDNA hybridized with a predicted 1.8-kb mRNA and with two other mRNAs of 3 and 4 kb. No signal other than the 1.8-kb mRNA was observed in the human adrenocortical NCI-H295 cells. Activation of the cAMP-dependent protein kinase A pathway increased the levels of the three mRNAs. Transfection of vectors expressing guinea pig P450c17 cDNA into nonsteroidogenic cells confers 17 alpha-hydroxylase and 17,20-lyase activities, showing that a single protein in the guinea pig supports both activities. However, the analysis of the enzymatic properties showed that the guinea pig P450c17 recombinant, in contrast to the human, supports hydroxylase and lyase activities only with delta 4 substrates. These results were further confirmed with isolated guinea pig adrenocortical cells. Our data demonstrate, first, that guinea pig P450c17 cDNA hybridizes with three different transcripts and second, that the expressed protein has characteristics associated exclusively with the guinea pig enzyme.