Integrated Expression Profiling and ChIP-seq Analyses of the Growth Inhibition Response Program of the Androgen Receptor

Abstract

BackgroundThe androgen receptor (AR) plays important roles in the development of male phenotype and in different human diseases including prostate cancers. The AR can act either as a promoter or a tumor suppressor depending on cell types. The AR proliferative response program has been well studied, but its prohibitive response program has not yet been thoroughly studied.Methodology/Principal FindingsPrevious studies found that PC3 cells expressing the wild-type AR inhibit growth and suppress invasion. We applied expression profiling to identify the response program of PC3 cells expressing the AR (PC3-AR) under different growth conditions (i.e. with or without androgens and at different concentration of androgens) and then applied the newly developed ChIP-seq technology to identify the AR binding regions in the PC3 cancer genome. A surprising finding was that the comparison of MOCK-transfected PC3 cells with AR-transfected cells identified 3,452 differentially expressed genes (two fold cutoff) even without the addition of androgens (i.e. in ethanol control), suggesting that a ligand independent activation or extremely low-level androgen activation of the AR. ChIP-Seq analysis revealed 6,629 AR binding regions in the cancer genome of PC3 cells with an FDR (false discovery rate) cut off of 0.05. About 22.4% (638 of 2,849) can be mapped to within 2 kb of the transcription start site (TSS). Three novel AR binding motifs were identified in the AR binding regions of PC3-AR cells, and two of them share a core consensus sequence CGAGCTCTTC, which together mapped to 27.3% of AR binding regions (1,808/6,629). In contrast, only about 2.9% (190/6,629) of AR binding sites contains the canonical AR matrix M00481, M00447 and M00962 (from the Transfac database), which is derived mostly from AR proliferative responsive genes in androgen dependent cells. In addition, we identified four top ranking co-occupancy transcription factors in the AR binding regions, which include TEF1 (Transcriptional enhancer factor), GATA (GATA transcription factors), OCT (octamer transcription factors) and PU1 (PU.1 transcription factor).Conclusions/SignificanceOur data provide a valuable data set in understanding the molecular basis for growth inhibition response program of the AR in prostate cancer cells, which can be exploited for developing novel prostate cancer therapeutic strategies.

Interaction between angiotensin II and relaxin 2 in the progress of growth and spread of prostate cancer cells

“…For example Niu et al (25) noted that restoring AR in PC3 cells could suppress their ability to invade in vitro and in vivo. whereas Lin et al (26) hypothesize that PC3 cells possess cellular machinery that turns the AR in PC3-AR cells into a growth suppressor. In another study, an equally interesting observation is that RLN2 is negatively regulated by androgens in prostate cancer (in vitro and in vivo) through the AR, implying that RLN2 levels increase in the both the absence of androgens and their ablation (27).…”

Integration of Regulatory Networks by NKX3-1 Promotes Androgen-Dependent Prostate Cancer Survival

“…Expression of AR in the AR-null prostate cancer cell line PC3 under different doses of androgen stimulation has been shown to result in differential gene expression, with approximately 5.7% of these genes involved in cell survival/apoptosis pathways (43). Such phenotypic effects observed upon androgen signaling generally occur through regulation of critical cell survival pathways, such as the insulin-like growth factor 1 (IGF-1), epidermal growth factor (EGF), and mitogen-activated protein kinase (MAPK) signaling pathways, as well as cell death pathways, such as the transforming growth factor ␤1 (TGF-␤1), p53, or death receptor-mediated, caspase-dependent apoptotic pathway (21,77).…”

Androgen Receptor Signaling in Prostate Cancer: New Twists for an Old Pathway

“…Recently by combining chromatin immunoprecipitation with tiled oligonucleotide microarrays (ChIP-on-chip) or with massively parallel sequencing (ChIP-Seq), the genome-wide AR binding sites (termed "AR cistrome") have been identified from a variety of PCa cell lines in multiple independent studies [46][47][48][49][50]. It was found that contrary to our expectation, most of the experimentally defined AR binding sites contain noncanonical AREs that are shown to be functional.…”

“…Another noteworthy discovery is that co-occupancy of AR with other transcription factors, such as FOXA1, TEF1, PU1, GATA2 and OCT1 [46,49], was frequently found in AR-binding sites, suggesting that AR may be working in collaboration with other transcription factors to control the expression of androgen-responsive genes. In support of this view, FOXA1, the DNA-binding transcription factor that contains chromatin-remodeling activity, has been found to serve as a pioneer factor binding to the FKH motif that is often concurrent with the AREs (> 60% concurrent rate in PCa cells at the basal state [51]), opening the compacted chromatin, and allowing subsequent association of AR at the AREs within these enhancers to cooperatively regulate target gene expression.…”