Chromatin-directed proteomics-identified network of endogenous androgen receptor in prostate cancer cells. Chromatin-directed proteomics-identified network of endogenous androgen receptor in prostate cancer cells

Treatment of prostate cancer confronts resistance to androgen receptor (AR)-targeted therapies. AR-associated coregulators and chromatin proteins hold a great potential for novel therapy targets. Here, we employed a powerful chromatin-directed proteomics approach termed ChIP-SICAP to uncover the composition of chromatin protein network, the chromatome, around endogenous AR in castration resistant prostate cancer (CRPC) cells. In addition to several expected AR coregulators, the chromatome contained many nuclear proteins not previously associated with the AR. In the context of androgen signaling in CRPC cells, we further investigated the role of a known AR-associated protein, a chromatin remodeler SMARCA4 and that of SIM2, a transcription factor without a previous association with AR. To understand their role in chromatin accessibility and AR target gene expression, we integrated data from ChIP-seq, RNA-seq, ATAC-seq and functional experiments. Despite the wide co-occurrence of SMARCA4 and AR on chromatin, depletion of SMARCA4 influenced chromatin accessibility and expression of a restricted set of AR target genes, especially those involved in cell morphogenetic changes in epithelial-mesenchymal transition. The depletion also inhibited the CRPC cell growth, validating its functional role in CRPC cells. Although silencing of SIM2 reduced chromatin accessibility similarly, it affected the expression of a much larger group of androgen-regulated genes, including those involved in cellular responses to external stimuli and steroid hormone stimulus. The silencing also reduced proliferation of CRPC cells and tumor size in chick embryo chorioallantoic membrane assay, further emphasizing the importance of SIM2 in CRPC cells and pointing to the functional relevance of this potential prostate cancer biomarker in CRPC cells. Overall, the chromatome of AR identified in this work is an important resource for the field focusing on this important drug target. Overall design: Examination of AR, and SMARCA4 binding by ChIP-seq from VCaP prostate cancer cells, in biological duplicates. Input sample from VCaP cells was used as control. AR binding by ChIP-seq from VCaP prostate cancer cells treated with non-targeting siRNA, SIM2 targeting siRNA, or FOXA1 targeting siRNA, in biological duplicates. Chromatin accessibility examined by ATAC-seq from VCaP cells treated with non-targeting siRNA, SMARCA4 targeting siRNA, or SIM2 targeting siRNA, in biological duplicates. Gene expression profiling by RNA-seq from VCaP cells treated with non-targeting siRNA, SMARCA4 targeting siRNA, or SIM2 targeting siRNA, in biological triplicates. All sequencing was done with Illumina NextSeq 500.

前列腺癌治疗面临雄激素受体（androgen receptor, AR）靶向治疗耐药的挑战。AR相关共调控因子与染色质蛋白具备成为新型治疗靶点的巨大潜力。本研究采用一种名为ChIP-SICAP的高效染色质导向蛋白质组学方法，旨在解析去势抵抗性前列腺癌（castration resistant prostate cancer, CRPC）细胞中内源性AR周围的染色质蛋白网络——即染色质组（chromatome）的组成。除多种已报道的AR共调控因子外，该染色质组还包含诸多此前未被发现与AR相关的核蛋白。
针对CRPC细胞中的雄激素信号通路，我们进一步研究了已知AR相关蛋白——染色质重塑因子SMARCA4，以及此前未被发现与AR存在关联的转录因子SIM2的功能。为阐明二者在染色质开放状态调控与AR靶基因表达中的作用，我们整合了ChIP-seq、RNA-seq、ATAC-seq及功能实验的数据。尽管SMARCA4与AR在染色质上广泛共结合，但敲低SMARCA4仅会影响有限一组AR靶基因的染色质开放状态与表达水平，其中尤以参与上皮间质转化过程中细胞形态发生变化的基因为主。敲低SMARCA4还可抑制CRPC细胞的增殖，验证了其在CRPC细胞中的功能作用。
虽然敲低SIM2同样能够降低染色质开放程度，但其影响的雄激素调控基因数量要多得多，涵盖了参与细胞对外界刺激及类固醇激素刺激应答的相关基因。在鸡胚绒毛尿囊膜实验中，敲低SIM2同样可降低CRPC细胞的增殖能力并缩小肿瘤体积，进一步证实了SIM2在CRPC细胞中的重要性，同时表明这一潜在前列腺癌生物标志物在CRPC细胞中具备功能相关性。
综上，本研究鉴定得到的AR染色质组，可为以该重要药物靶点为核心的研究领域提供宝贵的研究资源。
整体实验设计：采用ChIP-seq检测VCaP前列腺癌细胞中AR与SMARCA4的结合情况，设置生物学重复2次；以VCaP细胞的Input样本作为对照。采用ChIP-seq检测经非靶向小干扰RNA（small interfering RNA, siRNA）、SIM2靶向siRNA或叉头框A1（forkhead box A1, FOXA1）靶向siRNA处理的VCaP前列腺癌细胞中的AR结合情况，设置生物学重复2次。采用ATAC-seq检测经非靶向siRNA、SMARCA4靶向siRNA或SIM2靶向siRNA处理的VCaP细胞的染色质开放状态，设置生物学重复2次。采用RNA-seq对经非靶向siRNA、SMARCA4靶向siRNA或SIM2靶向siRNA处理的VCaP细胞进行基因表达谱分析，设置生物学重复3次。所有测序实验均使用Illumina NextSeq 500平台完成。