Abstract A056: AR/FOXA1-mediated active gene repression as a mechanism of prostate carcinogenesis

Abstract

Abstract The identity of the prostate gland is defined by transcription factors (TF) like the androgen receptor (AR), FOXA1, and NKX3.1 that bind regulatory elements early in development to activate the lineage gene program. For instance, KLK3, is a canonical target gene activated by AR. While the mechanistic interactions between pioneer factors, chromatin remodelers, and epigenetic proteins required for gene activation is well understood, repressive activity of lineage-defining TFs remain largely uncharacterized. Here, we document and mechanistically describe the active, repressive transcriptional functions of AR and FOXA1 in the context of prostate tumorigenesis.We identified a shared repertoire of repressed gene targets of AR and FOXA1 by using transcriptomic profiles (RNA-seq) of PCa cells treated with dihydrotestosterone (DHT) or TF targeting siRNA. Chromatin binding of AR and/or FOXA1 (ChIPseq) at cis-regulatory elements (dCREs) within the gene’s topologically-associated domain was used as primary evidence for direct regulation. Here, the KLK3 and DEPTOR gene loci emerged as candidate-activated or repressed AR/FOXA1 targets in PCa cells. DEPTOR, a tumor suppressor gene, negatively regulates the mTORC1/2 complexes by inhibiting their kinase activity. In contrast to the passive model of repression, where sequestration of co-activators ensues in transcriptional down-regulation of genes, we found ligand-bound AR and FOXA1 bind CREs within DEPTORs’ TAD establishing looping interactions (H3K3me3 HiChIP-seq) with the DEPTOR gene promoter. Functional inactivation of AR or FOXA1 led to a 2-4 fold upregulation of DEPTOR in VCaP, LNCaP, and 22RV1 cells. Instead, DHT-stimulation of AR led to DEPTOR downregulation. To assess the rapidness of this effect, we used a SWI/SNF ATPase PROTAC, AU15330, which triggers instant compaction of CREs and dislodges AR/FOXA1 from the chromatin. Treatment of PCa cells with AU15330 for 4 hours led to an immediate and significant upregulation of DEPTOR, substantiating the direct transcriptional repression by the AR/FOXA1 complex. Consistently, AU-15330 treatment also abolished FOXA1 and AR binding and 3D loop formation into the DEPTOR promoter. Using an orthogonal genetic approach, CRISPR-KRAB-targeted heterochromatinization of the AR/FOXA1 repressive CREs led to increased production of the nascent DEPTOR transcript. Finally, monoclones devoid of the CRE had elevated DEPTOR levels. Altogether, our findings position DEPTOR as an actively repressed target of the AR/FOXA1 transcriptional complex, suggesting the “silencer” duties of driver TF oncogenes to play a critical role in prostate tumorigenesis. Furthermore, the repertoire of activated and repressed gene targets identified in this study serve as exciting loci to study differences in the co-factor machinery and/or other cis or trans-attributes of the encompassing TADs that regulate downstream transcriptional outcomes. We posit the active mode of gene repression plays a driver role in prostate carcinogenesis. Citation Format: Sanjana Eyunni, Abhijit Parolia, Eleanor Young, James George, Mustapha Jaber, Lanbo Xiao, Fengyun Su, Rui Wang, Xuhong Cao, Arul Chinnaiyan. AR/FOXA1-mediated active gene repression as a mechanism of prostate carcinogenesis [abstract]. In: Proceedings of the AACR Special Conference: Advances in Prostate Cancer Research; 2023 Mar 15-18; Denver, Colorado. Philadelphia (PA): AACR; Cancer Res 2023;83(11 Suppl):Abstract nr A056.