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Genome-wide analysis of AR binding and transcriptomic analysis of mesenchymal subsets during prostate development

ABSTRACT: Prostate organogenesis is regulated by interactions between mesenchymal and epithelial cells, and via androgen signalling though androgen receptors (AR) expressed in mesenchymal cells. However, there is little knowledge of AR target genes and we have used high resolution genomic methods to examine AR function in subsets of mesenchyme during prostate development. Using ChIP-seq, we defined genomic AR binding sites (ARBS) in microdissected mesenchymal tissues of neonatal male and female rats during prostate growth. By comparing female tissue subsets (ventral mesenchymal pad and urethral smooth muscle) with male tissues (ventral prostate and dorsolateral prostate), we were able to identify sexually dimorphic ARBS. Females had a distinct AR binding profile from males with enrichment of ARBS proximal to gene transcriptional start sites as well as binding to non-classical AR binding sequence motifs. Using both tissue RNA-sequencing and single-cell RNA-sequencing of the same mesenchymal subsets, we identified 128 differentially expressed transcripts between males and females. Comparison of these to ChIP-seq ARBS allowed us to define sexually dimorphic AR target genes. Selected candidates were validated as sexually dimorphic at both the mRNA and protein level in both rat and human developing prostate tissues by qPCR and western blot. Response to testosterone stimulation was examined using ex vivo rat tissue organ cultures and qPCR. In single cell RNAseq data we observed subpopulations of mesenchymal cells in males and females as well as subpopulations common to both. The distribution of AR and target genes did not follow the subpopulation distribution, suggesting that AR action is not restricted by subset identity.Our study is the first to apply multiple transcriptomic approaches to prostate mesenchyme, and we have identified unique AR targets. We have identified sexually dimorphic, androgen responsive mesenchymal target genes that may underlie sexually dimorphic development of the prostate. We suggest that the analysis of transcription factor binding and transcriptomes in cell subsets will be informative when applied to tumour cells and stroma – since these show similar cellular heterogeneity and subset specific activity.

ORGANISM(S): Rattus norvegicus

PROVIDER: GSE116397 | GEO | 2019/06/30

REPOSITORIES: GEO

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Project description:Androgen receptor (AR) orchestrates an intricate transcriptional regulatory network that governs prostate cancer initiation, development and progression. To understand this network in detail, we generated genome-wide maps of AR occupancy by ChIP-seq in LNCaP cells. We found NKX3-1, an androgen-dependent homeobox protein well-characterized for its role in prostate development and differentiation, being recruited to AR binding sites (ARBS) in response to androgen signaling. We identified 6,359 NKX3-1 binding sites, most of which overlapped with AR. In addition to its novel collaborative transcriptional role at well-known prostate cancer model genes, our binding and knockdown studies further suggested that NKX3-1 potentially regulates AR in a feed-forward manner. Integrative analysis of Oncomine molecular concepts showed that these androgen-regulated AR and NKX3-1 associated genes are significantly overexpressed in prostate carcinoma as well as advanced and recurrent prostate tumors. From our transcriptomic profiling and Gene Ontology analysis, we observed that AR and NKX3-1 co-regulate genes involved in "protein trafficking" processes, which are mandatory events in the integration of oncogenic signaling pathways leading to prostate cancer development and progression. Interestingly, we found that AR and NKX3-1 co-regulate several members of the RAB GTPase family of secretory/trafficking proteins via the involvement of FoxA1 in a ternary complex and we believe that these AR/NKX3-1/FoxA1 co-regulated RAB genes could serve as expression signatures in prostate carcinogenesis. More specifically, through functional analyses, we showed that NKX3-1, together with AR and FoxA1, could promote prostate cancer cell survival through activation of RAB3B expression. Collectively, our study has provided important insights into the hierarchical transcriptional regulatory network established between AR and NKX3-1 and sought to elucidate the important genetic-molecular-phenotypic paradigm in androgen-dependent prostate cancer. Genome-wide binding analyses of AR, NKX3-1 and FoxA1 in LNCaP with or without DHT (5alpha-dihydrotestosterone) stimulation using ChIP-Seq.

Project description:Androgen receptor (AR) orchestrates an intricate transcriptional regulatory network that governs prostate cancer initiation, development and progression. To understand this network in detail, we generated genome-wide maps of AR occupancy by ChIP-seq in LNCaP cells. We found NKX3-1, an androgen-dependent homeobox protein well-characterized for its role in prostate development and differentiation, being recruited to AR binding sites (ARBS) in response to androgen signaling. We identified 6,359 NKX3-1 binding sites, most of which overlapped with AR. In addition to its novel collaborative transcriptional role at well-known prostate cancer model genes, our binding and knockdown studies further suggested that NKX3-1 potentially regulates AR in a feed-forward manner. Integrative analysis of Oncomine molecular concepts showed that these androgen-regulated AR and NKX3-1 associated genes are significantly overexpressed in prostate carcinoma as well as advanced and recurrent prostate tumors. From our transcriptomic profiling and Gene Ontology analysis, we observed that AR and NKX3-1 co-regulate genes involved in ‘protein trafficking’ processes, which are mandatory events in the integration of oncogenic signaling pathways leading to prostate cancer development and progression. Interestingly, we found that AR and NKX3-1 co-regulate several members of the RAB GTPase family of secretory/trafficking proteins via the involvement of FoxA1 in a ternary complex and we believe that these AR/NKX3-1/FoxA1 co-regulated RAB genes could serve as expression signatures in prostate carcinogenesis. More specifically, through functional analyses, we showed that NKX3-1, together with AR and FoxA1, could promote prostate cancer cell survival through activation of RAB3B expression. Collectively, our study has provided important insights into the hierarchical transcriptional regulatory network established between AR and NKX3-1 and sought to elucidate the important genetic-molecular-phenotypic paradigm in androgen-dependent prostate cancer. Gene expression profiling of LNCaP in response to ETOH (vehicle) or DHT (5α-dihydrotestosterone) stimulation across different treatment time-points using microarray.

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Genome-wide analysis of AR binding and transcriptomic analysis of mesenchymal subsets during prostate development

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