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

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 prostatectomy-treated 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. Overall design: Examination of AR binding with 9 cell line samples + control and 2 xenograft samples + control. ChIPseq with AR antibody in 3 different LNCaP derivative cell lines overexpressing AR upon treatment with different androgen concentrations and 2 different tumor xenografts expressing different AR levels.

雄激素受体（Androgen receptor, AR）在绝大多数去势抵抗性前列腺癌（castration-resistant prostate cancers, CRPC）中呈过表达状态。本研究旨在探究AR过表达对该受体染色质结合的影响，并筛选出可能在CRPC发生进程中发挥关键作用的AR靶基因。
我们构建了两株LNCaP前列腺癌细胞系的亚系：其中一株的AR表达量较对照细胞高2~3倍，另一株则高4~5倍。采用染色质免疫共沉淀（chromatin immunoprecipitation, ChIP）与深度测序（deep-sequencing, seq）技术，我们鉴定得到了AR结合位点（AR-binding sites, ARBSs）。
研究结果显示，当细胞经低浓度雄激素刺激时，AR结合位点的数量与AR结合强度均与AR表达水平呈正相关。在AR过表达的细胞中，受体结合染色质所需的配体浓度较对照细胞低100倍。我们在两株前列腺癌异种移植瘤模型中验证了AR水平与染色质结合的相关性：其中一株存在AR基因扩增且AR高表达，另一株则呈低表达状态。
通过整合ChIP测序与表达谱分析，我们筛选出了在前列腺癌中上调的AR靶基因。其中，ZWINT、SKP2（S期激酶相关蛋白2（p45）, S-phase kinase-associated protein 2 (p45)）以及FEN1（瓣状结构特异性核酸内切酶1, flap structure-specific endonuclease 1）的表达在CRPC中被证实上调，而SNAI2的表达在前列腺癌与CRPC中均呈下调趋势。此外，FEN1蛋白的表达水平与接受前列腺切除术治疗的患者不良预后显著相关。最后，通过小干扰RNA（small interfering RNA, siRNA）敲低FEN1的表达可抑制LNCaP细胞的增殖。
本研究数据表明，AR过表达可增强受体与染色质的结合能力，由此解释了CRPC细胞中AR信号通路如何被重新激活。
总体实验设计：共纳入9株细胞系样本+对照，以及2株异种移植瘤样本+对照。实验采用AR抗体进行ChIP测序，分别检测3株不同AR过表达水平的LNCaP衍生细胞系在不同浓度雄激素处理下的AR结合情况，以及2株AR表达水平不同的肿瘤异种移植瘤的AR结合特征。