Defining the epigenetic landscape in human prostate cancer

 描述(由申请人提供)：雄激素受体(AR)是前列腺癌发生、发展和耐药的核心。AR是一种核转录因子，与DNA结合并调节基因活性。全基因组范围的AR-DNA结合位点的集合被称为AR序列。AR及其共同调节因子之间复杂的相互作用决定了转录调控的目标基因。使用新开发的AR染色质免疫沉淀和高通量测序(CHIP-SEQ)技术，我们明确地表明，AR程序是高度动态的，至少在一定程度上是由可用的协同调节因子的存在决定的。尤其是， 先锋组织因子FOXA1和前列腺系特异性因子HOXB13似乎共同定位于肿瘤组织特异性AR部位。本提案的总体目标是表征肿瘤发生过程中AR重新编程的潜在机制，并首次表征从局限性前列腺癌到转移性、耐药疾病进展过程中人体组织中的AR程序。在第一个目标中，CHIP-SEQ将在AR表达的正常前列腺上皮(LHSAR)和前列腺癌(LNCaP和VCaP)的细胞系模型中进行。当每个TF通过shRNA和基因组编辑被击倒或通过慢病毒转导过表达时，AR、FOXA1和HOXB13的信号通路的变化将被测量。还将对每个细胞系的状况进行RNA-SEQ，以确定受表观遗传重新编程影响的基因。在第二和第三个目标中，AR的表观遗传学图景将通过CHIP-SEQ在人类标本中系统地绘制：局部组织、未经处理的转移组织、耐去势转移组织和耐苯那鲁胺疾病。完成这项提案中概述的新颖实验将提供一个无与伦比的视角，了解主要转录因子是如何推动前列腺癌进展的。具体地说，我们将发现：(I)在前列腺癌的发生和发展过程中，AR是如何被重新编程的，哪些协同调节因子促进了这一过程；(Ii)在对苯扎鲁胺产生耐药性的过程中，AR是如何被重新编程的，如果是这样的话，哪些协同调节因子正在促进这一过程；以及(Iii)其他推动前列腺癌进展的非AR主调节因子。拟议的实验还将使我们能够识别受AR重新编程影响的目标基因。该项目将产生一份随着疾病进展到耐阉割转移状态的表观遗传图集，这种转移状态通常是致命的。遗传基因座 组成这个数据集的靶基因将刺激新的靶点进行治疗干预。

 DESCRIPTION (provided by applicant): The androgen receptor (AR) is central to prostate cancer development, progression, and drug resistance. The AR is a nuclear transcription factor (TF) that binds to DNA and regulates gene activity. The set of genome-wide AR-DNA binding sites is termed the AR cistrome. A complex interplay between AR and its co-regulators determines the genes targeted for transcriptional regulation. Using newly developed techniques for AR chromatin immunoprecipitation followed by high-throughput sequencing (ChIP-seq) in human prostate specimens, we unambiguously show that the AR program is highly dynamic and is determined, at least in part, by the presence of available co-regulators. In particular, the pioneer TF FOXA1 and the prostate lineage- specific TF HOXB13 appear to co-localize at tumor tissue-specific AR sites. The overall objectives of the present proposal are to characterize the mechanisms underlying AR reprogramming during tumorigenesis and - for the first time - to characterize the AR program in human tissue during progression from localized prostate cancer to metastatic, drug-resistant disease. In the first aim, ChIP-seq will be performed in cell line models for AR-expressing normal prostate epithelium (LHSAR) and prostate cancer (LNCaP and VCaP). Changes in the cistromes of AR, FOXA1 and HOXB13 will be measured as each TF is knocked down via shRNA and genome editing or overexpressed via lentiviral transduction. RNA-seq will also be performed for each cell line condition to determine the genes affected by epigenetic reprogramming. In the second and third aims, the epigenetic landscape of the AR will be systematically charted via ChIP-seq in human specimens: localized tissue, untreated metastatic tissue, castration-resistant metastases and enzalutamide-resistant disease. Completion of the novel experiments outlined in this proposal will provide an unparalleled look at how master transcription factors drive prostate cancer progression. Specifically, we will discover: (i) how the AR is reprogrammed during prostate cancer development and progression, and which co-regulators are facilitating this process, (ii) how the AR is reprogrammed during the acquisition of resistance to enzalutamide and, if so, which co-regulators are facilitating this process, and (iii) other non-AR master regulators that are driving prostate cancer progression. The proposed experiments will also enable us to identify the target genes that are affected by AR reprogramming. The project will result in an atlas of the epigenetic landscape as the disease progresses to the castration-resistant metastatic state, which is uniformly fatal. The genetic loci and target genes comprising this dataset will stimulate new targets for therapeutic intervention.