Rôle de la Nucléophosmine (NPM1) dans la physiopathologie prostatique

Nucleophosmin (NPM1/B23) is a small molecular chaperone involved in a large array of cellular processes, including the regulation of gene expression and the control of the cell cycle. Several studies have reported the overexpression of NPM1 in solid tumors from various histological origin, including prostate cancer, and its proliferative impact on several human cancer cell line is being well described. The first part of our work aimed at assessing the NPM1 oncogenic properties in the prostate gland in vivo. To do so, we generated a new transgenic mouse model that overexpresses NPM1 specifically in the prostatic epithelium. These mice harbor prostatic hyperplasia associated with an increase of the ki67 proliferative index. Our molecular investigations revealed that NPM1 could be an inhibitor of the quiescent state of epithelial cells through a dysregulation of key cell-cycle controlers such as Cyclin E or p27kip1. Although these mice do not develop neoplastic lesions, our data suggest that NPM1 overexpression accelerate prostate cancer progression when associated with other oncogenic alterations. The second part of the work aimed at understanding the mechanisms underlying NPM1 overexpression in prostate tumors. The serine/threonine Kinase mTOR was recently shown to bind to the proximal promoter of NPM1 in the mouse liver. In order to characterize a fonctionnal link between NPM1 and mTOR, we took advantage of murine embryonic fibroblast (MEF) deleted for PTEN, since these cells display a constitutive mTOR activity. In such cells, NPM1 protein and mRNA levels are increased compared to wild type MEF. We also demonstrated that mTOR controls NPM1 expression i) through its binding to NPM1 promoter, thus stimulating NPM1 gene expression and ii) by stabilizing NPM1 mRNA. We have confirmed the functional link between NPM1 and mTOR in vivo in a mouse model deleted for PTEN specifically in the prostatic epithelium. Finally, we have shown that NPM1 expression is necessary for the proliferation of PTEN knock-out MEF. These data set NPM1 as a new downstream effector of the PI3K/AKT/mTOR pathway, and suggest that it could be a new potential therapeutic target in PTEN negative human prostate cancer.La Nucléophosmine (NPM1/B23) est une petite chaperonne moléculaire impliquée dans de nombreux processus cellulaires, tels que la régulation de l’expression génique ou le contrôle du cycle cellulaire. De nombreuses études rapportent une surexpression de NPM1 dans divers types de tumeurs solides incluant les cancers de la prostate, et son rôle proH prolifératif dans des lignées cellulaires tumorales d’origines variées est bien établi. La première partie de notre travail s’est attaché à évaluer le potentiel oncogénique de NPM1 dans l’épithélium prostatique in vivo. Pour cela, nous avons généré un modèle de souris transgéniques qui surexpriment NPM1 spécifiquement dans l’épithélium de la prostate. Ces animaux présentent une hyperplasie prostatique associée à une augmentation de l’index prolifératif de l’épithélium. Nos expériences révèlent que NPM1 pourrait lever la quiescence des cellules épithéliales différenciées en dérégulant l’expression de gènes clés de la régulation du cycle cellulaire, comme la Cycline E ou p27kip1. Bien que ces souris ne développent pas de lésions néoplasiques, ces données suggèrent que NPM1 participe à la carcinogenèse prostatique en association avec d’autres lésions oncogéniques. La seconde partie du travail visait à comprendre la nature des mécanismes qui supportent la surexpression de NPM1 dans les tumeurs prostatiques. Des données récentes de la littérature indiquent un enrichissement de la protéine kinase mTOR au niveau du promoteur proximal de NPM1 dans des foies de souris. Pour déterminer s’il existe un lien fonctionnel entre mTOR et NPM1, nous avons tiré parti d’un modèle de fibroblastes embryonnaires de souris invalidés pour le suppresseur de tumeur PTEN dont l’inactivation mène à une hyperactivité de mTOR. Dans ce contexte, les taux d’ARNm et de protéines NPM1 sont augmentés par rapport aux cellules sauvages. Nos résultats montrent également que mTOR contrôle l’expression de NPM1 i) en se fixant sur son promoteur et en stimulant l’expression du gène et ii) en stabilisant l’ARNm de NPM1. Nous avons confirmé le lien entre NPM1 et mTOR in vivo grâce à notre modèle de souris invalidées pour PTEN dans l’épithélium prostatique. Enfin, nous avons montré que l’expression de NPM1 est nécessaire pour transduire les effets prolifératifs de la voie PI3K/AKT/mTOR. Ces données placent donc NPM1 comme nouvel effecteur en aval de cette voie de signalisation, faisant de cette protéine une potentielle cible thérapeutique dans les tumeurs présentant une perte de PTEN

Sepsis promotes splenic production of a protective platelet pool with high CD40 ligand expression.

Platelets have a wide range of functions including critical roles in hemostasis, thrombosis, and immunity. We hypothesized that during acute inflammation, such as in life-threatening sepsis, there are fundamental changes in the sites of platelet production and phenotypes of resultant platelets. Here, we showed during sepsis that the spleen was a major site of megakaryopoiesis and platelet production. Sepsis provoked an adrenergic-dependent mobilization of megakaryocyte-erythrocyte progenitors (MEPs) from the bone marrow to the spleen, where IL-3 induced their differentiation into megakaryocytes (MKs). In the spleen, immune-skewed MKs produced a CD40 ligandhi platelet population with potent immunomodulatory functions. Transfusions of post-sepsis platelets enriched from splenic production enhanced immune responses and reduced overall mortality in sepsis-challenged animals. These findings identify a spleen-derived protective platelet population that may be broadly immunomodulatory in acute inflammatory states such as sepsis.NHS Blood and Transplant European Union's Horizon 2020 (ERC-2014-CoG-648765) MRC-AMED (MR/V005421/1

Role of Nucleophosmin (NPM1) in prostate physiopathology

La Nucléophosmine (NPM1/B23) est une petite chaperonne moléculaire impliquée dans de nombreux processus cellulaires, tels que la régulation de l’expression génique ou le contrôle du cycle cellulaire. De nombreuses études rapportent une surexpression de NPM1 dans divers types de tumeurs solides incluant les cancers de la prostate, et son rôle proH prolifératif dans des lignées cellulaires tumorales d’origines variées est bien établi. La première partie de notre travail s’est attaché à évaluer le potentiel oncogénique de NPM1 dans l’épithélium prostatique in vivo. Pour cela, nous avons généré un modèle de souris transgéniques qui surexpriment NPM1 spécifiquement dans l’épithélium de la prostate. Ces animaux présentent une hyperplasie prostatique associée à une augmentation de l’index prolifératif de l’épithélium. Nos expériences révèlent que NPM1 pourrait lever la quiescence des cellules épithéliales différenciées en dérégulant l’expression de gènes clés de la régulation du cycle cellulaire, comme la Cycline E ou p27kip1. Bien que ces souris ne développent pas de lésions néoplasiques, ces données suggèrent que NPM1 participe à la carcinogenèse prostatique en association avec d’autres lésions oncogéniques. La seconde partie du travail visait à comprendre la nature des mécanismes qui supportent la surexpression de NPM1 dans les tumeurs prostatiques. Des données récentes de la littérature indiquent un enrichissement de la protéine kinase mTOR au niveau du promoteur proximal de NPM1 dans des foies de souris. Pour déterminer s’il existe un lien fonctionnel entre mTOR et NPM1, nous avons tiré parti d’un modèle de fibroblastes embryonnaires de souris invalidés pour le suppresseur de tumeur PTEN dont l’inactivation mène à une hyperactivité de mTOR. Dans ce contexte, les taux d’ARNm et de protéines NPM1 sont augmentés par rapport aux cellules sauvages. Nos résultats montrent également que mTOR contrôle l’expression de NPM1 i) en se fixant sur son promoteur et en stimulant l’expression du gène et ii) en stabilisant l’ARNm de NPM1. Nous avons confirmé le lien entre NPM1 et mTOR in vivo grâce à notre modèle de souris invalidées pour PTEN dans l’épithélium prostatique. Enfin, nous avons montré que l’expression de NPM1 est nécessaire pour transduire les effets prolifératifs de la voie PI3K/AKT/mTOR. Ces données placent donc NPM1 comme nouvel effecteur en aval de cette voie de signalisation, faisant de cette protéine une potentielle cible thérapeutique dans les tumeurs présentant une perte de PTEN.Nucleophosmin (NPM1/B23) is a small molecular chaperone involved in a large array of cellular processes, including the regulation of gene expression and the control of the cell cycle. Several studies have reported the overexpression of NPM1 in solid tumors from various histological origin, including prostate cancer, and its proliferative impact on several human cancer cell line is being well described. The first part of our work aimed at assessing the NPM1 oncogenic properties in the prostate gland in vivo. To do so, we generated a new transgenic mouse model that overexpresses NPM1 specifically in the prostatic epithelium. These mice harbor prostatic hyperplasia associated with an increase of the ki67 proliferative index. Our molecular investigations revealed that NPM1 could be an inhibitor of the quiescent state of epithelial cells through a dysregulation of key cell-cycle controlers such as Cyclin E or p27kip1. Although these mice do not develop neoplastic lesions, our data suggest that NPM1 overexpression accelerate prostate cancer progression when associated with other oncogenic alterations. The second part of the work aimed at understanding the mechanisms underlying NPM1 overexpression in prostate tumors. The serine/threonine Kinase mTOR was recently shown to bind to the proximal promoter of NPM1 in the mouse liver. In order to characterize a fonctionnal link between NPM1 and mTOR, we took advantage of murine embryonic fibroblast (MEF) deleted for PTEN, since these cells display a constitutive mTOR activity. In such cells, NPM1 protein and mRNA levels are increased compared to wild type MEF. We also demonstrated that mTOR controls NPM1 expression i) through its binding to NPM1 promoter, thus stimulating NPM1 gene expression and ii) by stabilizing NPM1 mRNA. We have confirmed the functional link between NPM1 and mTOR in vivo in a mouse model deleted for PTEN specifically in the prostatic epithelium. Finally, we have shown that NPM1 expression is necessary for the proliferation of PTEN knock-out MEF. These data set NPM1 as a new downstream effector of the PI3K/AKT/mTOR pathway, and suggest that it could be a new potential therapeutic target in PTEN negative human prostate cancer

NPM1 Silencing Reduces Tumour Growth and MAPK Signalling in Prostate Cancer Cells

<div><p>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 <i>in vivo</i>. 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.</p></div

mTOR transcriptionally and post-transcriptionally regulates Npm1 gene expression to contribute to enhanced proliferation in cells with Pten inactivation

International audienc

Regulation of LNCaP cell clonogenic capacities and proliferation rate by NPM1.

<p>(<b>a</b>) NPM1 knockdown does not alter LNCaP cells morphology. LNCaP cells were stably transfected with control shRNA (shScr) or specific NPM1 shRNA (shNPM1). NPM1 mRNA and protein levels were analysed by RT-qPCR and western blotting respectively. Morphology of cells was observed by inverted microscopy and photographed. (<b>b</b>) NPM1 knockdown inhibits LNCaP cells clonogenicity. Cells were seeded at low confluence over one week, then fixed with methanol and stained with 5% Giemsa blue before microscopic observation. Pictures are representative of three independent experiments with consistent results. The graph represents the number of cell clones (>50 cells) in the shNPM1 condition, calculated as the mean ± SD of the number of clones counted per field, on 5 random fields, using the ImageJ free software and expressed relatively to the number of clones counted in the control condition. (<b>c, d</b>) NPM1 controls proliferation of prostate cancer cells. Five thousand cells were seeded per well in a 96-wells plate and cultured for 48 hours. (<b>c</b>) Cells were then incubated with a BrdU labeling solution for 2.5 hours and BrdU incorporation was measured by densitometric analysis at 655 nm. (<b>d</b>) Proliferation was also analysed by RT-qPCR assay by evaluating PCNA relative mRNA level accumulation normalized using β-actin mRNA level. All data are representative of at least three independent triplicate experiments and BrdU incorporation as mean of triplicate experiments of 96 points each. Data are expressed as the mean ± SD.</p

NPM1 knockdown decreases EGF expression.

<p>(<b>a</b>) NPM1 controls EGF expression. Relative EGF mRNA levels compared to β-actin were analyzed by RT-qPCR in LNCaP cells expressing control (shScr) or NPM1 specific shRNA (shNPM1). (<b>b</b>) NPM1 control EGF promoter activity. shScr and shNPM1 LNCaP cells were transfected with the phEGF-luciferase reporter plasmid. EGF promoter activity was evaluated by measuring the luciferase activity 24 hours later. Results of the assay were standardized using the CMV promoter as control and expressed as fold-induction over control cells (shScr). (<b>c</b>) NPM1 controls activation of the EGF/EGFR pathway downstream effectors. Proteins, extracted from shScr and shNPM1 LNCaP cells cultured in RPMI 1640 10%FBS, were electrophoresed by SDS-PAGE. Transferred membranes were immunoblotted with indicated antibodies. Histograms show the band quantification reported to the β-actin level. Blots are representative of three independent experiments with consistent results. Data are representative of at least three independent experiments and are expressed as the mean ± SD.</p

NPM1 knockdown in prostate cancer cells reduces proliferation and migration capacities by inhibiting the EGF/EGFR pathway activity.

<p>(a) NPM1 down-regulation inhibits EGF induced ERK1/2 pathway activity. shScr and shNPM1 LNCaP cells were treated with 100 nM EGF and phosphorylation of the EGF/EGFR pathway effectors was analysed using Western Blotting. Histograms show the band quantification reported to the β-actin level. The blot is representative of three independent experiments with consistent results. (b) Despite EGF treatment, migration capacities of LNCaP decreased for NPM1 were not restored. Wound closure was analysed 72 hours after continuous treatment with 100 nM EGF. Cells were observed under inverted microscope and photographed. Histograms show wound area quantification using ImageJ. (c) EGF treatment does not rescue proliferation of NPM1 knockdown LNCaP cells. BrdU incorporation assay was performed in shScr and shNPM1 LNCaP 24 hours after 100 nM EGF treatment. Densitometry was measured at 655 nm. The data are representative of at least three independent experiments and are expressed as the mean ± SD.</p

ERRα induces H3K9 demethylation by LSD1 to promote cell invasion

International audienceLysine 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

NPM1 knockdown impacts migration, invasion and growth of prostate cancer cells.

<p>(a) NPM1 controls migration capacities of LNCaP cells. Cells were seeded at confluence in order to create a wound 24 hrs later. Wound recolonization was observed after 72 hours of culture by inverted microscopy and photographed. Histograms show wound area quantification using the ImageJ and pictures are representative of three independent experiments with consistent results. (<b>b</b>) NPM1 knockdown inhibits the invasive potential of prostate cancer cells. shScr and shNPM1 LNCaP cells were seeded at confluency in RPMI 1640 serum free medium on matrigel coated microporous membrane. 48 hrs later, migrated cells present on the opposite side of the membrane were fixed and stained with 5% Giemsa blue and observed at microscope (200× magnification). The graph represents the number of cells in the shNPM1 condition, calculated as the mean ± SD of the number of cells counted per field, on 5 random fields, using the ImageJ free software and expressed relatively to the number of cells counted in the control condition. (<b>c</b>) NPM1 impacts three-dimensional growth of prostate cancer cells. Control or NPM1 knocked down cells were seeded at low confluency on agarose/RPMI 1640 10% FBS for 2 weeks. Number and size of the emerging clones were then observed under inverted microscope (x100) and photographed. The graph represents the number of cell clones (>50 cells) in the shNPM1 condition, calculated as the mean ± SD of the number of clones counted per field, on 5 random fields, using the ImageJ free software and expressed relatively to the number of clones counted in the control condition. (<b>d, e, f,g</b>) NPM1 knock-down abrogates tumourigenicity of LNCaP cells when injected in nude mice. shScr (n = 14) and shNPM1 (n = 14) LNCaP cells were subcutaneously grafted on nude mice and tumour volume was measured every 2 days following engraftment (<b>d</b>). Graph in (<b>e</b>) is the quantitation of shScr and shNPM1-derived tumours volume at day 24 post-injection. NPM1 relative expression level was evaluated by western blotting in tumours when mice were sacrified (f) and tumour weight was measured (g). The data are representative of at least three independent experiments and are expressed as the mean ± SD.</p