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

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 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

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

NPM1 does not act directly on EGF expression but upstream of the MEKK effector to activate the MAPK pathway in prostate cancer cells.

<p>(a) ERK1/2 activation is rescued by caMEKK1. shScr and shNPM1 LNCaP cells are transfected with a constitutively activated construct of MEKK1 (caMEKK1) and ERK1/2 phosphorylation level was analysed using western blotting. (b) ERK1/2 pathway activation restores EGF promoter activity in LNCaP cells downregulated for NPM1. LNCaP shSCR and shNPM1 cells were transiently co-transfected with phEGF-luc and caMEKK1. EGF promoter activity was evaluated by measuring the luciferase activity 24 hours after. Results of the assay were standardized against control reporter activity CMV-Luc and expressed as fold-induction over control cells (shScr). Data are representative of at least three independent experiments and are expressed as the mean ± SD.</p

LXRs control expression of genes involved in cholesterol homeostasis and fatty acid synthesis in MPECs.

<p>(A) qPCR analysis of <i>Abca1</i>, <i>Abcg1</i> and <i>Idol</i> levels in WT (+/+) and <i>Lxrαβ−/−</i> (lxr<i>−</i>/<i>−</i>) MPECs after DMSO (vehicle) or T0901317 stimulation (B) Effect of 9-<i>cis</i> retinoic acid and/or T0901317 stimulation on <i>Abca1</i> accumulation levels in WT (+/+) and <i>Lxrαβ−/−</i> (lxr<i>−/−</i>) MPECs (C) qPCR analysis of <i>Srebp1</i>, <i>Acc</i>, and <i>Fas</i> levels in WT (+/+) and <i>Lxrαβ−/−</i> (lxr<i>−/−</i>) MPECs after DMSO (vehicle) or T0901317 stimulation. (qPCR analysis results from 4 independant experiments and was normalized using <i>36b4</i> gene). *p<0.05, **p<0.01, ***p<0.001 in Student’s <i>t</i> test. Error bars represent mean ± SEM. (D) Western blot analysis was performed on WT (+/+) or <i>Lxr</i>αβ<i>−/−</i> MPECs using Srebp1c, Abca1 and β-Actin antibodies. (E) Oil-Red O staining (ORO) and Normarski/Dapi of WT (+/+) and <i>Lxrαβ−/−</i> MPECs, or WT MEFs, treated for 48h with DMSO (vehicle) or T0901317 (1 µM). Head arrows indicate lipid droplets. Scale bars 100 µm.</p

Analysis of stromal and epithelial markers in WT or <i>Lxrαβ−/−</i> MPECs.

<p>(A) WT and <i>Lxrαβ−/−</i> MPECs and WT MEFs were immunostained using anti-E-cadherin (E-Cad), anti-Cytokeratin8 (CK8) and anti-αTubulin (Tub) antibodies. Scale bar 100 µm. (B) mRNA relative levels of <i>Tenascin, Snail, Keratin 8</i> and <i>E-cadh</i> were measured in WT MEFs, WT (+/+) and <i>Lxrαβ−/−</i> MPECs by qPCR. *p<0.05, **p<0.01, ***p<0.001 in Student’s <i>t</i> test. Error bars represent mean ± SEM. (qPCR analysis results from 3 independant experiments and was normalized using <i>36b4</i> gene) (C) Western blot analysis was performed on WT MEFs, WT (+/+) and <i>Lxrαβ−/−</i> MPECs using CK8, Psca antibodies. βActin was used as a loading control.</p

LXRs are necessary to warrant proper transduction pathway activities.

<p>(A) Western blot analysis was performed on WT (+/+) or <i>Lxr</i>αβ<i>−/−</i> MPECs, treated with DMSO (vehicle), T0901317 or 22(R)-hydroxycholesterol (22(R)-OHC), using antibodies against PTEN, pAKT S473, pAKT T308, AKT, pGSK3β, GSK3β, pBAD, BAD, pp42/44, p42/44 and α-Tubulin as a loading control. (B) Quantification of pAKT S473 / AKT and pp42/44 / p42/44 ratios of WT (+/+) or <i>Lxr</i>αβ<i>−/−</i> MPECs, treated with DMSO (vehicle) or T0901317 (N = 3). (C) WT (+/+) and <i>Lxrαβ−/−</i> MPECs were treated with DMSO (vehicle), T0901317 (1 µM) or IGF-1 (50 ng/ml) as a control and immunofluorescence was observed by microscopy using pAKT S473 antibody (nuclear staining is nonspecific). Scale bars 100 µm. (D) MTT assays were performed on WT (+/+) or <i>Lxr</i>αβ<i>−/−</i> MPECs, treated with DMSO (vehicle), LY-294002 (5, 10, 30 µM) or PD-98059 (10, 25, 50 µM) inhibitors. *p<0.05, **p<0.01, ***p<0.001 in Student’s <i>t</i> test <i>vs.</i> DMSO treatment, # p<0.01, µ p<0.001 in Student’s <i>t</i> test <i>vs.</i> WT (+/+) MPECs. Error bars represent mean ± SEM. N = 3. Treatment efficiency was checked by western blot using antibodies against, pAKT S473, AKT, pp42/44 and p42/44.</p