Role of ETS factors in specifying prostate luminal cell identity and androgen receptor dependence

摘要 ETS家族蛋白质改变（主要是ERG易位）存在于>50%的人类前列腺癌中 在西方男人。我们是第一个开发出一个强大的ERG驱动的前列腺癌GEM模型的人，揭示了 增强管腔分化和扩大雄激素受体（AR）顺式组在肿瘤中，表明 通过染色质编程的ERG致癌性的新机制（Chen，2013）。我们和其他人 随后确定ERG激活前列腺类器官中的管腔分化程序，人 前列腺细胞系，并通过计算分析前列腺癌基因组，在临床样品中（Blee等， 2018; Kron等人，2017年; Li等人，2020年b）。其他ETS功能获得性改变，例如ETV 4易位（Li et 例如，2020 a）和ETS功能丧失改变，例如ERF阻遏物突变/缺失（Bose等，（2017） 显示这种表型，FOXA 1（也在前列腺癌中扩增或突变）也是如此，但AR收缩 顺反子（亚当斯等人，2019年）。已经证明腔分化是多个细胞的主要特征， 致癌ETS蛋白（和FOXA 1），我们在下一个资金周期的主要目标是了解ERG如何 激活这种分化程序以及这种程序如何导致致癌表型。我们将奉行 三条平行调查线首先，从我们使用纯化的全长蛋白质的生化研究中， 我们发现ERG（和其他ETS因子）协同增强AR DNA结合， 通过直接蛋白质-蛋白质相互作用的变构效应;在生物学上，这扩大了AR顺式组， 包括新的AR结合位点（Wasperson等人，2020年）。目标1将扩大这一分析，以评估 FOXA 1对AR/ERG相互作用的影响。第二，我们已经建立了基因定义的前列腺类器官模型， 在精确定义的时间过程内概括ERG的腔分化效应。使用这个系统， 我们鉴定了使基底上皮主调节因子p63转录沉默的表观遗传变化， 解释了基底细胞的减少。目标2将使用谱系追踪，单细胞分析和CRISPR 筛选，以进一步阐明ERG如何扩大管腔细胞的数量，并启动致癌 转型第三，我们发现另一种致癌ETS蛋白（ETV 4）也驱动管腔分化， 并且单独足以引发前列腺上皮内瘤形成（PIN）。值得注意的是，这些管腔上皮细胞 细胞获得对AR的高度依赖性以存活（与正常的腔上皮细胞相反）， 因此显示对雄激素剥夺疗法（ADT）的敏感性增强。Aim 3将探索 通过检查AR顺式组的变化， 与系统性雄激素相比， 通过手术去势剥夺（损害前列腺基质和上皮中的AR功能）。我们将 用ADT前后ETS阳性患者样品的单细胞分析补充这些实验。

ABSTRACT ETS family protein alterations (primarily ERG translocations) are present in >50% of human prostate cancers in Western men. We were the first to develop a robust GEM model of ERG-driven prostate cancer, revealing enhanced luminal differentiation and an expanded androgen receptor (AR) cistrome in tumors, suggesting a novel mechanism for ERG oncogenicity via chromatin programming (Chen, 2013). We and others subsequently established that ERG activates a luminal differentiation program in prostate organoids, human prostate cell lines and, by computational analysis of prostate cancer genomes, in clinical samples (Blee et al., 2018; Kron et al., 2017; Li et al., 2020b). Other ETS gain-of-function alterations e.g. ETV4 translocations (Li et al., 2020a) and ETS loss-of-function alterations e.g. ERF repressor mutations/deletions (Bose et al., 2017) also show this phenotype, as does FOXA1 (also amplified or mutated in prostate cancer) but with a contracted AR cistrome (Adams et al., 2019). Having demonstrated that luminal differentiation is a primary feature of multiple oncogenic ETS proteins (and FOXA1), our major goal during the next funding cycle is to understand how ERG activates this differentiation program and how this program results in an oncogenic phenotype. We will pursue three parallel lines of investigation. First, from our biochemical studies using purified full-length proteins and various DNA templates, we find that that ERG (and other ETS factors) cooperatively enhance AR DNA binding through allosteric effects via direct protein-protein interaction; biologically, this broadens the AR cistrome to include novel AR binding sites (Wasmuth et al., 2020). Aim 1 will expand this analysis to assess the role of FOXA1 on AR/ERG interactions. Second, we have built genetically defined prostate organoid models that recapitulate the luminal differentiation effect of ERG within a precisely defined time course. Using this system, we identified epigenetic changes that silence transcription of the basal epithelial master regulator p63, likely explaining the reduction in basal cells. Aim 2 will use lineage tracing, single cell analysis, and CRISPR screening to further elucidate how ERG expands the number of luminal cells and initiates oncogenic transformation. Third, we showed that another oncogenic ETS protein (ETV4) also drives luminal differentiation and is sufficient, alone, to initiate prostatic intraepithelial neoplasia (PIN). Remarkably, these luminal epithelial cells acquire exquisite dependence on AR for survival (in contrast to normal luminal epithelial cells) and consequently display enhanced sensitivity to androgen deprivation therapy (ADT). Aim 3 will explore mechanisms underlying this shift to cell-intrinsic AR dependence by examining changes in the AR cistrome and transcriptome following luminal-specific AR ablation (by genetic deletion) versus systemic androgen deprivation through surgical castration (impairs AR function in prostate stroma and epithelium). We will complement these experiments with single cell analysis of ETS-positive patient samples before and after ADT.