Overexpression of androgen receptor enhances the binding of the receptor to the chromatin in prostate cancer

Abstract

Androgen receptor (AR) is overexpressed in the majority of castration-resistant prostate cancers (CRPCs). Our goal was to study the effect of AR overexpression on the chromatin binding of the receptor and to identify AR target genes that may be important in the emergence of CRPC. We have established two sublines of LNCaP prostate cancer (PC) cell line, one overexpressing AR 2-3-fold and the other 4-5-fold compared with the control cells. We used chromatin immunoprecipitation (ChIP) and deep-sequencing (seq) to identify AR-binding sites (ARBSs). We found that the number of ARBSs and the AR-binding strength were positively associated with the level of AR when cells were stimulated with low concentrations of androgens. In cells overexpressing AR, the chromatin binding of the receptor took place in 100-fold lower concentration of the ligand than in control cells. We confirmed the association of AR level and chromatin binding in two PC xenografts, one containing AR gene amplification with high AR expression, and the other with low expression. By combining the ChIP-seq and expression profiling, we identified AR target genes that are upregulated in PC. Of them, the expression of ZWINT, SKP2 (S-phase kinase-associated protein 2 (p45)) and FEN1 (flap structure-specific endonuclease 1) was demonstrated to be increased in CRPC, while the expression of SNAI2 was decreased in both PC and CRPC. FEN1 protein expression was also associated with poor prognosis in prostatectomytreated patients. Finally, the knock-down of FEN1 with small interfering RNA inhibited the growth of LNCaP cells. Our data demonstrate that the overexpression of AR sensitizes the receptor binding to chromatin, thus, explaining how AR signaling pathway is reactivated in CRPC cells.

Androgen-regulated miR-32 targets BTG2 and is overexpressed in castration-resistant prostate cancer

“…Our data link the PI3K and AR pathways, which are both important to PC progression. Finally, the data here confirm our previous findings (Urbanucci et al, 2011) indicating that chromatin binding of AR takes place in lower DHT concentrations in the cells overexpressing the receptor. Thus, the overexpression seems to make cancer cells hypersensitive to the low levels of residual androgens in patients even after castration.…”

“…We were also able to confirm the presence of ARBSs in close proximity to both of the miRNA genomic loci by ChIP-qPCR. The same ARBSs are also shown by ChIP-seq analysis of two xenografted tumors originating from CRPC patients (Urbanucci et al, 2011). This finding confirms that such ARBSs are not only LNCaP specific but can also be found in vivo.…”

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

“…Higher expression levels of AR in DUCaP and VCaP cells was proposed to be responsible for a higher number of genomic binding sites. This is in line with increasing numbers of ARBSs in LNCaP cells manipulated to express higher levels of AR (Urbanucci, et al, 2012). …”

The ETS Domain Transcription Factor ELK1 Directs a Critical Component of Growth Signaling by the Androgen Receptor in Prostate Cancer Cells

“…Recent reports have demonstrated enrichment of possible ELK1 binding sites in relation to chromatin sites of AR binding (52)(53)(54). The observation that ELK1, fully or in part, supported a substantial proportion (ϳ27%) of all gene activation (direct or indirect) by androgen in PC cells suggests that only a few ARtethering proteins may be adequate to direct AR signaling toward supporting growth.…”