Anterior gradient 2 and 3 – two prototype androgen‐responsive genes transcriptionally upregulated by androgens and by oestrogens in prostate cancer cells

Abstract

Androgens and oestrogens have been implicated in prostatic carcinogenesis and tumour progression. Although the actions of androgens have been studied extensively, the mechanisms underlying oestrogen signalling in prostate cancer are not fully understood. In the present study, we analyzed the effect of androgens and oestrogens on the expression of anterior gradient 2 (AGR2) and anterior gradient 3 (AGR3), comprising two highlyrelated genes encoding secretory proteins that are expressed in prostate cancer and one of which (AGR2) has been associated with tumour metastasis. Quantitative reverse-transcriptase PCR and western blot analysis showed androgen induction of AGR2 and AGR3 in three androgen receptor positive cell lines, starting at concentrations of 0.1 nM. Both AGR genes were also transcriptionally activated by ! 5 nM oestradiol but not by isotype selective or nonselective oestrogen receptor agonists in DUCaP cells that harbour a high-level of wild-type androgen receptor. A functional androgen receptor but not oestrogen receptor turned out to be required for both androgen and oestrogen regulation. This pattern of androgen and oestrogen regulation was confirmed in VCaP cells and was also observed for FKBP5, a well-characterized androgen-regulated gene. Genome-wide chromatin-immunoprecipitation studies coupled with deep sequencing identified androgen receptor binding sites localized in the distal promoter and intron regions of the AGR2 and AGR3 genes, respectively. The androgen responsiveness of these enhancers was verified by luciferase reporter gene assays and site-directed mutagenesis analysis. Androgen treatment also induced p300 and RNA Pol II recruitment to androgen receptor enhancers of AGR2 and initiated local chromatin remodelling and the formation of RNA Pol II-containing androgen receptor transcription complexes.

Putative Prostate Cancer Risk SNP in an Androgen Receptor‐Binding Site of the Melanophilin Gene Illustrates Enrichment of Risk SNPs in Androgen Receptor Target Sites

“…ChIP-seq was performed as described (Bu, et al, 2013b). AR antibodies used for ChIP were from Cell Signaling (Danvers, MA, 3202) and Upstate (PG-21, UB 06-680).…”

“…The renilla luciferase vector PGL4.73 was used to normalize for transfection efficiency and the empty PGL3P vector served as a baseline control (Contr) in PC-3 cells. As a positive control (+Contr) a known androgen regulated reporter vector containing the AGR3 gene enhancer, which harbours a consensus ARE3 motif (Bu, et al, 2013b) and its mutated counterpart (+Contr mutated ) were employed in DUCaP cells. The androgen induced changes in luciferase activity were measured after 48 h of treatment with 1 nM R1881 using a dual luciferase assays.…”

Mechanistic rationale for MCL1 inhibition during androgen deprivation therapy

“…2H), we hypothesized that this regulation is not directly mediated by AR but rather indirectly by another AR-stimulated transcription factor. Indeed, in a genome-wide chromatin-immunoprecipitation experiment coupled with deep sequencing (ChIP-seq) in DuCaP cells [19] we were not able to identify AR binding sites neighboring or within the MCL1 gene, in contrast to other known AR targets and, surprisingly, other BCL2 family members (Fig. 3A and S2A).…”

miR-22 and miR-29a Are Members of the Androgen Receptor Cistrome Modulating LAMC1 and Mcl-1 in Prostate Cancer

“…ChIP AR-precipitated DNA fragments were then detected by deep sequencing. Peaks were identified by macs (v1.4) and annotated to the whole human genome sequence as described in more detail elsewhere (27).…”