22 research outputs found
The antiproliferative role of the liver X receptors in breast and colorectal cancer
The liver X receptors (LXR
α
and LXR
β
) are members of the nuclear receptor superfamily of
ligand activated transcription factors and have
functions as regulators of lipid and glucose
metabolism, as well as inflammatory response. In recent years, several reports have
demonstrated an important role of LXRs
in the control of cell proliferation.
In
Paper I
we demonstrate that LXR activation with synthetic agonist GW3965 leads to a
strong antiproliferative effect in four different
human breast cancer cell
lines. We show that
LXR activation induces an arrest at the G
1
/S check point of the cell cycle with a
hypophosphorylation of retinoblasto
ma protein and a downregulation of cell cycle modulators
such as Skp2, cyclin A2 and cy
clin D1. We further show that
the antiproliferative function of
LXRs is independent of lipid biosynthesis.
In
Paper II
we follow up the results in Paper I to
elucidate more mechanisms of LXR
activation in human breast cancer cell lines. Us
ing microarray analysis, we find both cell line
specific and common LXR target
genes. The common responsive genes that were upregulated
upon LXR activation are annotated
to known metabolic functions of LXR, while the common
downregulated genes mostly include those
with function in cell cycle regulation and
proliferation. Comparing the common downregulat
ed gene set, with br
east cancer tumour
samples and patient data we find
that patients with tumours expr
essing lower levels of these
LXR target genes had better survival compared
to patients with a higher expression of these
genes. In addition, we identify the E2F family
of transcription factor
s as mediators of the
antiproliferative effect of LXR activation.
In
Paper III
we demonstrate that activa
tion of LXRs with
GW3965 decreases proliferation in
human colorectal cell lines with a cell cycle arrest in the G
1
to S phase transition. We
demonstrate a decreased expression of cell
cycle promoters such
as Skp2, CDK1, CDK2,
CDK4, cyclin E, cyclin B1 and c-myc, as
well as hypophosphorylation of retinoblastoma
protein. Moreover, we show that LXR deficient
mice have an increased proliferation in the
colonic crypt compared to wild
type mice. Also, activation of
LXRs with GW3965 reduces
proliferation in the colonic
crypt of wild type mice.
In
Paper IV
we demonstrate that activation of LXRs
dampens the inflammatory response by
downregulating pro-inflammatory
mediators in two different
mouse models of colitis. In
addition, LXR deficient mice have
a faster and more severe di
sease progression. We further
demonstrate that expression of LXR regulated
genes is suppressed in colon samples from
patients with either Crohn’s disease or ulcer
ative colitis compared
to healthy controls.
Inflammatory bowel disease (IBD), including Crohn’s disease and ulcerative colitis, is
associated to increased risk of developing colore
ctal cancer. The data in Paper IV suggests the
potential for LXR mediated inhib
ition of inflammation during IBD,
thus reducing the risk for
developing colorectal cancer
Insights From Liver-Humanized Mice on Cholesterol Lipoprotein Metabolism and LXR-Agonist Pharmacodynamics in Humans
Background and Aims Genetically modified mice have been used extensively to study human disease. However, the data gained are not always translatable to humans because of major species differences. Liver-humanized mice (LHM) are considered a promising model to study human hepatic and systemic metabolism. Therefore, we aimed to further explore their lipoprotein metabolism and to characterize key hepatic species-related, physiological differences. Approach and Results Fah(-/-), Rag2(-/-), and Il2rg(-/-) knockout mice on the nonobese diabetic (FRGN) background were repopulated with primary human hepatocytes from different donors. Cholesterol lipoprotein profiles of LHM showed a human-like pattern, characterized by a high ratio of low-density lipoprotein to high-density lipoprotein, and dependency on the human donor. This pattern was determined by a higher level of apolipoprotein B100 in circulation, as a result of lower hepatic mRNA editing and low-density lipoprotein receptor expression, and higher levels of circulating proprotein convertase subtilisin/kexin type 9. As a consequence, LHM lipoproteins bind to human aortic proteoglycans in a pattern similar to human lipoproteins. Unexpectedly, cholesteryl ester transfer protein was not required to determine the human-like cholesterol lipoprotein profile. Moreover, LHM treated with GW3965 mimicked the negative lipid outcomes of the first human trial of liver X receptor stimulation (i.e., a dramatic increase of cholesterol and triglycerides in circulation). Innovatively, LHM allowed the characterization of these effects at a molecular level. Conclusions LHM represent an interesting translatable model of human hepatic and lipoprotein metabolism. Because several metabolic parameters displayed donor dependency, LHM may also be used in studies for personalized medicine.Peer reviewe
Liver × receptor ligands disrupt breast cancer cell proliferation through an E2F-mediated mechanism
Knockdown of SF-1 and RNF31 Affects Components of Steroidogenesis, TGFβ, and Wnt/β-catenin Signaling in Adrenocortical Carcinoma Cells
The orphan nuclear receptor Steroidogenic Factor-1 (SF-1, NR5A1) is a critical regulator of development and homeostasis of the adrenal cortex and gonads. We recently showed that a complex containing E3 ubiquitin ligase RNF31 and the known SF-1 corepressor DAX-1 (NR0B1) interacts with SF-1 on target promoters and represses transcription of steroidogenic acute regulatory protein (StAR) and aromatase (CYP19) genes. To further evaluate the role of SF-1 in the adrenal cortex and the involvement of RNF31 in SF-1-dependent pathways, we performed genome-wide gene-expression analysis of adrenocortical NCI-H295R cells where SF-1 or RNF31 had been knocked down using RNA interference. We find RNF31 to be deeply connected to cholesterol metabolism and steroid hormone synthesis, strengthening its role as an SF-1 coregulator. We also find intriguing evidence of negative crosstalk between SF-1 and both transforming growth factor (TGF) β and Wnt/β-catenin signaling. This crosstalk could be of importance for adrenogonadal development, maintenance of adrenocortical progenitor cells and the development of adrenocortical carcinoma. Finally, the SF-1 gene profile can be used to distinguish malignant from benign adrenocortical tumors, a finding that implicates SF-1 in the development of malignant adrenocortical carcinoma
Genes changed by both siSF-1 and siRNF31 treatment.
<p>Values given in log<sub>2</sub> (fold change).</p
Genes changed by both siSF-1 and cAMP treatment.
<p>Values given in log<sub>2</sub> (fold change).</p
Analysis of differentially expressed genes in siRNF31 ± cAMP-treated H295R cells.
<p>(<b>A</b>) qPCR showing approximately 60% efficiency of RNF31 RNAi-treatment on mRNA level. (<b>B</b>) Western blot showing efficient knockdown of RNF31 protein and upregulation of RNF31 target StAR in RNF31 RNAi-treated H295R cells. (<b>C</b>) Venn diagram showing overlap of differentially expressed genes in siSF-1 and siRNF31 microarrays. (<b>D</b>) Venn diagram overlap among the differentially expressed genes in all cAMP-treated samples.</p
Hypothesis of SF-1 mechanism acting both <i>in cis</i> and <i>in trans</i>.
<p>(<b>A</b>) Classical SF-1 action on steroidogenic enzyme gene promoters. SF-1 binds promoters <i>in cis</i> and recruit coactivators and the general transcription machinery to activate transcription. Raised intracellular cAMP levels due to ACTH (in the adrenal) activation of MC2R activates the CREB/CREM transcription factors that work synergistically to further increase transcription rates. Input from Wnt/β-catenin signalling through direct binding of β-catenin to SF-1 can also increase transcription. (<b>B</b>) Possible mechanisms of SF-1 dependent repression of Wnt/β-catenin signaling. 1. SF-1 binds <i>in cis</i> to promoters but due to post-translational modifications and/or specific corepressor recruitment represses instead of activates target gene transcription. 2. SF-1 binds <i>in trans</i> to transcription factor complex and directs corepressors to the site to repress transcription. This could also be mediated by post-translational modifications like SUMOylation. A third option is that transcription factors or corepressors whose expression is activated by SF-1 acts as repressors of the TGFβ and Wnt/β-catenin signalling making SF-1 an indirect regulator.</p
Confirmation of steroidogenic target genes differentially expressed in microarrays and promoter assays of StAR, DAX-1 and SULT2A1 proximal promoters after cAMP treatment and flow-cytometry assay of siSF-1 treated cells.
<p>(<b>A</b>) qPCR showing mRNA levels of six steroidogenic target genes. (<b>B</b>) Luciferase reporter assays of StAR, DAX-1 and SULT2A1 promoters after 16 h of ± cAMP treatment show that the down regulation of DAX-1 and SULT2A1 mRNA levels after cAMP-treatment cannot be recreated with the proximal 1 kb of the promoters alone. (<b>C</b>) Expression targets of DAX-1 differentially expressed in the Control vs. siRNF31 microarray as identified by Pathway Studio. Error bars show standard deviation. Statistical analysis done with Student's t-test (*p<0.05; **p<0.01; ***p<0.001).</p
Putative SF-1 targets in the Wnt/β-catenin-signaling pathway.
<p>(<b>A</b>) qPCR data of three genes in the Wnt/β-catenin pathway that are changed after siSF-1 knockdown. (<b>B</b>) Change in β-catenin responsive reporter plasmid TopFlash activity with increasing amounts of SF-1. Values shown are fold change compared to reporter plasmid luciferase activity without transfected β-catenin. (<b>C</b>) Schematic Wnt/β-catenin pathway with genes changed by siSF-1 marked in red (upregulation). Error bars show standard deviation. Statistical analysis done with Student's t-test (* p<0.05; **p<0.01;***p<0.001).</p