Liver x receptors protect from development of prostatic intra-epithelial neoplasia in mice

Abstract

LXR (Liver X Receptors) act as "sensor" proteins that regulate cholesterol uptake, storage, and efflux. LXR signaling is known to influence proliferation of different cell types including human prostatic carcinoma (PCa) cell lines. This study shows that deletion of LXR in mouse fed a high-cholesterol diet recapitulates initial steps of PCa development. Elevation of circulating cholesterol in Lxrαβ-/- double knockout mice results in aberrant cholesterol ester accumulation and prostatic intra-epithelial neoplasia. This phenotype is linked to increased expression of the histone methyl transferase EZH2 (Enhancer of Zeste Homolog 2), which results in the down-regulation of the tumor suppressors Msmb and Nkx3.1 through increased methylation of lysine 27 of histone H3 (H3K27) on their promoter regions. Altogether, our data provide a novel link between LXR, cholesterol homeostasis, and epigenetic control of tumor suppressor gene expression.

Figure 1. High-cholesterol diet induces proliferation in LXR mutant mouse prostate.

(A) Histological sections of dorsal prostate lobes of 5 month-old WT (a,b,c,d) and LXR null mice (e,f,g,h) fed normal or high cholesterol diet were analyzed after H&E staining (Left) or Ki67 IHC (Right). Arrowheads point Ki67-positive cells. Higher magnification of the prostatic epithelium of LXR null mice fed a high cholesterol diet revealed abnormal features (i). Arrowheads indicate atypical cells with enlarged nuclei and prominent nucleoli which represent typical signs of PIN. Ep: Epithelium, St: Stroma (Scale bars = 50 µm). (B) IHC for Ki67 was quantified by counting the percentage of prostatic acini with proliferative cells and the average Ki67+ cell number in proliferative acini (N = 6 per group). (C) qPCR analysis of CyclinD1 and CyclinD2 expression (N = 9/13 per group). * p<0.05, ** p<0.01, *** p<0.001 in Student's t test. Error bars represent the ± mean SEM.

Figure 2. LXR null mice exhibit aberrant epithelial cell renewal.

(A) Proliferative cells in LXR knockout prostates under high cholesterol condition were identified by H&E staining and double-IHC with antibodies directed against PCNA and specific markers for luminal epithelial cells (CK18) (a,b,c), basal cells (p63) (d,e,f) and smooth muscle (SMA - smooth muscle actin) (g,h,i). Ep: Epithelium, St: Stroma (Scale bar = 10 µm). (B) PCNA immunodetection (proliferation), TUNEL staining (apoptotic nuclei) and BrdU immunodetection (cumulative proliferation) were performed on dorsal prostates of LXR null mice under high cholesterol condition (Scale bar = 10 µm). Arrowheads point to regions of interest.

Figure 3. Prostates of LXR mutant mice accumulate cholesterol esters through inappropriate LXR target genes regulation.

(A) Plasma concentrations of cholesterol were determined (N = 9/13 per group) after 5 weeks dietary conditional exposure in each genotype. (B) Neutral lipids accumulation was observed after Oil-Red-O staining (ORO) (Scale bars = 50 µm). (C) Cholesterol esters, free cholesterol and triglycerides were quantified by thin layer chromatography (N = 3 per group). (D) Srebp1c, Fas and Scd2, (E) Abca1, Ldlr and Idol transcript levels were determined by qPCR (N = 9/13 per group). (F) Total protein lysates of WT and LXR null mice under normal or high cholesterol diet were analyzed by western blotting with antibodies against ABCA1, LDLR and ACTIN as a loading control (left panel), quantification of ABCA1 and LDLR protein accumulation levels (right panel). * p<0.05, *** p<0.001 in Student's t test. Error bars represent the ± mean SEM.

Figure 4. Identification of genes associated with the occurence of PIN lesions.

(A) Experimental design of gene expression profiling studies. (B) Venn diagram analysis was used to isolate genes associated with PIN development in LXR null mice under high cholesterol diet: genes deregulated in both arrays 3 and 4 were selected and genes deregulated in arrays 1 and 2 were further subtracted from this list. This method leads to the extraction of 463 genes (253 up- and 210 down-regulated).

Figure 5. Disruption of cholesterol homeostasis induces the repression of Nkx3.1 and Msmb tumor suppressor genes and upregulation of the Ezh2 histone methyltransferase gene.

(A) Nkx3.1, Msmb and Ezh2 expression levels were analyzed by qPCR (N = 9/13 per group). (B) Western blot analysis of EZH2 accumulation in total protein lysates from dorsal prostate of WT and LXR null mice under normal or high cholesterol diet. (C) Immunofluorescence analyses were carried out on LXR null mice under normal or high cholesterol diet using antibodies directed against PCNA and EZH2 (Scale bar = 5 µm). * p<0.05, *** p<0.001 in Student's t test. Error bars represent the ± mean SEM.

Figure 6. Upregulation of Ezh2 leads to increased enrichment of the H3K27me3 histone mark on Nkx3.1 and Msmb promoter regions.

(A) Location of loci I, II and III amplified by qPCR on H3K27me3 mark profiles and Ezh2 occupancy sites on Nkx3.1 and Msmb promoters as identified by ChIP-seq in ES cells (http://www.broadinstitute.org/scientific-community/science/programs/epigenomics/chip-seq-data). (B) ChIP analyses using antibodies raised against trimethylated H3K27 vs. negative control IgG (N = 3/6 per group). Histograms show relative enrichment values of Loci I, II and III (bound/input) on chromatin obtained from WT and LXR null mice under normal or high cholesterol diet. (C) Oncomine boxed plot analysis (http://www.oncomine.org) of LXRα, LXRβ and EZH2 expression levels between healthy prostate glands and human PCa in datasets referenced in and (n.s.; non-significant). * p<0.05, ** p<0.01 in Student's t test. Error bars represent the ± mean SEM.