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

Pommier AJ, Dufour J, Alves G, Viennois E, De Boussac H, Trousson A, Volle DH, Caira F, Val P, Arnaud P, Lobaccaro JM, Baron S - PLoS Genet. (2013)

Bottom Line: LXR (Liver X Receptors) act as "sensor" proteins that regulate cholesterol uptake, storage, and efflux.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.

View Article: PubMed Central - PubMed

Affiliation: Clermont Université, Université Blaise Pascal, Génétique Reproduction et Développement, BP 10448 Clermont-Ferrand, France.

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.

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Related in: MedlinePlus

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 [45] (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  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 [21] and [20] (n.s.; non-significant). * p<0.05, ** p<0.01 in Student's t test. Error bars represent the ± mean SEM.
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pgen-1003483-g006: 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 [45] (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 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 [21] and [20] (n.s.; non-significant). * p<0.05, ** p<0.01 in Student's t test. Error bars represent the ± mean SEM.

Mentions: Our data showed that control of cholesterol homeostasis by LXR is crucial to restrain epithelial cell proliferation in the prostate. In order to determine key molecular events resulting from elevation of cholesterol in the prostate, we designed microarray experiments. We compared prostatic gene expression of WT and LXR mutant mice in normal and high dietary cholesterol conditions (Figure 4A). The list of up- and down-regulated genes has been established on the basis of signal intensity, Log ratio and p-value (Figure S3). The highest number of deregulated genes was observed when WT and LXR knockout mice were exposed to high circulating cholesterol levels, again emphasizing the central role of cholesterol in the establishment of the phenotype (Figure 4A). In order to determine gene expression signature of the PIN phenotype in LXR mutant mice fed a high cholesterol diet and to identify relevant molecular events, we have restricted the gene list using Venn analysis. We selected common deregulated genes associated with the PIN phenotype and eliminated those that were sensitive to diet and/or LXR ablation alone. Therefore, we focused on the genes involved in the establishment of the PIN phenotype by selecting genes that were deregulated in both arrays 3 (lxr-/- normal vs. lxr-/- high chol.) and 4 (+/+ high chol. vs. lxr-/- high chol.) and by subtracting genes that were deregulated in both arrays 1 (+/+ normal vs. +/+ high chol.) and 2 (lxr-/- normal vs. +/+ normal). This resulted in a list of 463 genes (Dataset S1), 253 up and 210 down (Figure 4B). Ingenuity Pathway Analysis (IPA) was used to investigate potential biological processes that underlay the PIN phenotype of LXR mutant mice (Figure S4). The second most significantly enriched gene-category was ‘cancer’, which was associated with a large list of 146 genes (Dataset S2). More than 50% of these 146 genes were also deregulated in a mouse model of prostate cancer resulting from PTEN deletion in prostatic epithelium [18] (data not shown). This strongly suggested that the PIN lesions observed in LXR knockout mice in the high cholesterol condition were genuine pre-cancerous alterations. Interestingly, this analysis showed down-regulation of two well described prostatic tumor suppressor genes Nkx3.1 and Msmb (Dataset S2, highlighted in red), which was further confirmed by qPCR analysis (Figure 5A, Figure S5). These two genes were specifically found in gene categories such as tumor development, cell proliferation and prostate organogenesis (Dataset S3, highlighted in red). Nkx3.1 and Msmb promoters have recently been demonstrated to be targets of the histone methyl transferase EZH2 that represses gene expression through H3K27 trimethylation. qPCR and western blot analyses showed that Ezh2 was specifically overexpressed in LXR knockout prostates when animals were fed a high cholesterol diet (Figure 5A, 5B). Immunohistochemistry further confirmed overaccumulation of EZH2 in proliferative PCNA+ cells in LXR knockout prostates, when animals fed a high cholesterol condition (Figure 5C). This suggested that the effect of cholesterol on the development of PIN was dependent on down-regulation of Nkx3.1 and Msmb, resulting from EZH2-mediated modification of their promoter chromatin. Indeed, ChIP analyses confirmed that nucleosomes at both Nkx3.1 and Msmb promoters were significantly trimethylated on H3K27 in the prostates of LXR -mice fed a high cholesterol diet (Figure 6A, 6B). Interestingly, Msmb expression was increased by a high cholesterol diet in WT mice. This was independent of Ezh2, whose expression was unaltered (Figure 5A). Such observation indicates that other mechanisms are involved in the regulation of this tumor suppressor gene expression and that it is highly sensitive to metabolic changes in prostate tissue. To further confirm the potential link between LXR and EZH2 expression, we performed a retrospective study of publicly available DNA microarray data of human PCa cohorts, using Oncomine. These analyses showed that LXRβ expression was significantly down-regulated in prostate carcinomas compared to normal tissue and that this down-regulation was associated with increased EZH2 expression (Figure 6C). Interestingly, careful analysis of normal prostate gland as well as metastasis heat maps revealed that levels of LXRβ, EZH2 and MSMB were tightly coordinated between each other (Figure S8). The expression pattern of NKX3.1 present no significant modification. Therefore, the connection between LXR, cholesterol homeostasis, EZH2 and MSMB expression that we uncovered in mouse could also be relevant in human PCa.


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

Pommier AJ, Dufour J, Alves G, Viennois E, De Boussac H, Trousson A, Volle DH, Caira F, Val P, Arnaud P, Lobaccaro JM, Baron S - PLoS Genet. (2013)

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 [45] (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  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 [21] and [20] (n.s.; non-significant). * p<0.05, ** p<0.01 in Student's t test. Error bars represent the ± mean SEM.
© Copyright Policy
Related In: Results  -  Collection

License
Show All Figures
getmorefigures.php?uid=PMC3649972&req=5

pgen-1003483-g006: 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 [45] (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 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 [21] and [20] (n.s.; non-significant). * p<0.05, ** p<0.01 in Student's t test. Error bars represent the ± mean SEM.
Mentions: Our data showed that control of cholesterol homeostasis by LXR is crucial to restrain epithelial cell proliferation in the prostate. In order to determine key molecular events resulting from elevation of cholesterol in the prostate, we designed microarray experiments. We compared prostatic gene expression of WT and LXR mutant mice in normal and high dietary cholesterol conditions (Figure 4A). The list of up- and down-regulated genes has been established on the basis of signal intensity, Log ratio and p-value (Figure S3). The highest number of deregulated genes was observed when WT and LXR knockout mice were exposed to high circulating cholesterol levels, again emphasizing the central role of cholesterol in the establishment of the phenotype (Figure 4A). In order to determine gene expression signature of the PIN phenotype in LXR mutant mice fed a high cholesterol diet and to identify relevant molecular events, we have restricted the gene list using Venn analysis. We selected common deregulated genes associated with the PIN phenotype and eliminated those that were sensitive to diet and/or LXR ablation alone. Therefore, we focused on the genes involved in the establishment of the PIN phenotype by selecting genes that were deregulated in both arrays 3 (lxr-/- normal vs. lxr-/- high chol.) and 4 (+/+ high chol. vs. lxr-/- high chol.) and by subtracting genes that were deregulated in both arrays 1 (+/+ normal vs. +/+ high chol.) and 2 (lxr-/- normal vs. +/+ normal). This resulted in a list of 463 genes (Dataset S1), 253 up and 210 down (Figure 4B). Ingenuity Pathway Analysis (IPA) was used to investigate potential biological processes that underlay the PIN phenotype of LXR mutant mice (Figure S4). The second most significantly enriched gene-category was ‘cancer’, which was associated with a large list of 146 genes (Dataset S2). More than 50% of these 146 genes were also deregulated in a mouse model of prostate cancer resulting from PTEN deletion in prostatic epithelium [18] (data not shown). This strongly suggested that the PIN lesions observed in LXR knockout mice in the high cholesterol condition were genuine pre-cancerous alterations. Interestingly, this analysis showed down-regulation of two well described prostatic tumor suppressor genes Nkx3.1 and Msmb (Dataset S2, highlighted in red), which was further confirmed by qPCR analysis (Figure 5A, Figure S5). These two genes were specifically found in gene categories such as tumor development, cell proliferation and prostate organogenesis (Dataset S3, highlighted in red). Nkx3.1 and Msmb promoters have recently been demonstrated to be targets of the histone methyl transferase EZH2 that represses gene expression through H3K27 trimethylation. qPCR and western blot analyses showed that Ezh2 was specifically overexpressed in LXR knockout prostates when animals were fed a high cholesterol diet (Figure 5A, 5B). Immunohistochemistry further confirmed overaccumulation of EZH2 in proliferative PCNA+ cells in LXR knockout prostates, when animals fed a high cholesterol condition (Figure 5C). This suggested that the effect of cholesterol on the development of PIN was dependent on down-regulation of Nkx3.1 and Msmb, resulting from EZH2-mediated modification of their promoter chromatin. Indeed, ChIP analyses confirmed that nucleosomes at both Nkx3.1 and Msmb promoters were significantly trimethylated on H3K27 in the prostates of LXR -mice fed a high cholesterol diet (Figure 6A, 6B). Interestingly, Msmb expression was increased by a high cholesterol diet in WT mice. This was independent of Ezh2, whose expression was unaltered (Figure 5A). Such observation indicates that other mechanisms are involved in the regulation of this tumor suppressor gene expression and that it is highly sensitive to metabolic changes in prostate tissue. To further confirm the potential link between LXR and EZH2 expression, we performed a retrospective study of publicly available DNA microarray data of human PCa cohorts, using Oncomine. These analyses showed that LXRβ expression was significantly down-regulated in prostate carcinomas compared to normal tissue and that this down-regulation was associated with increased EZH2 expression (Figure 6C). Interestingly, careful analysis of normal prostate gland as well as metastasis heat maps revealed that levels of LXRβ, EZH2 and MSMB were tightly coordinated between each other (Figure S8). The expression pattern of NKX3.1 present no significant modification. Therefore, the connection between LXR, cholesterol homeostasis, EZH2 and MSMB expression that we uncovered in mouse could also be relevant in human PCa.

Bottom Line: LXR (Liver X Receptors) act as "sensor" proteins that regulate cholesterol uptake, storage, and efflux.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.

View Article: PubMed Central - PubMed

Affiliation: Clermont Université, Université Blaise Pascal, Génétique Reproduction et Développement, BP 10448 Clermont-Ferrand, France.

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.

Show MeSH
Related in: MedlinePlus