The Androgen Receptor (AR) signaling axis is crucial for the onset and progression of prostate cancer (PCa). Androgen deprivation therapies (ADT) are commonly used as first-line treatments, but many patients eventually develop lethal castration-resistant prostate cancer (CRPC), which continues to grow without testosterone. Neuroendocrine markers can be detected in many tumors subjected to ADT, but are rarely detected in ADT-naïve tumors, supporting the idea that ADT triggers neuroendocrine differentiation (NED) in tumors (Patel et al., 2019). Long non-coding (lnc)RNAs play important regulatory roles in tumorigenesis and contribute to all hallmarks of cancer, including the onset of CRPC (Misawa et al., 2017). This project aims to identify and investigate lncRNA functions in the early response to ADT, specifically their role in regulating the AR signaling pathway and the onset of CRPC. To achieve this, we are using a dynamic in vitro cell system recapitulating progressive NED and CRPC phenotypes upon androgen deprivation (Terry et al., 2013). Two projects were set:1. Characterization of lncRNAs in the early response to ADT: We identified 964 lncRNAs induced by androgen deprivation, a subset of the most robust candidates was selected based on expression levels in cells and high-risk PCa, as well as co-expression with neurogenesis-associated protein-coding genes. This led to the identification of 15 novel lncRNAs named PROCAs, with PROCA11 being our top candidate. Three different isoforms of PROCA11 were characterized with distinct subcellular distribution, suggesting different fates and functions. PROCA11 expression is induced early after hormone depletion upon a release of AR from its promoter. Knockdown of AR results in increase of PROCA11 levels even in cells growing in presence of androgen, pinpointing the repressive activity of AR on the lncRNA transcription. Alterations in PROCA11 expression levels affect cell proliferation and apoptosis, perturbing the expression of genes involved in different signaling pathways. Hence, we propose that PROCA11 play a role in enabling PCa cells to circumvent ADT by regulating their proliferation and survival.2. Study of AR activity and epigenetic mechanisms during NED. Immunofluorescence and western blot experiments revealed nuclear localization of AR even in the absence of androgen. However, transcriptome analysis showed an extensive deregulation of all known androgen-responsive genes (ARGs), confirming the inhibition of the AR transactivation function. To investigate the relevance of AR nuclear persistence, we profiled AR and histone active and repressive modifications genome-wide. This experiment showed a global remodeling of the epigenetic landscape of cells undergoing NED with a decrease of the repressive mark (H3K27me3) and an increase of the active mark (H3K27ac) together with an overall loss of AR from chromatin, accompanied with the transcriptional activation of genes involved in neurogenesis and PROCA11. Studies in melanoma and PCa revealed various roles of lncRNAs in the direct control of AR activity due to RNA-protein interactions (Zhang et al., 2015, Schmidt et al., 2019, Hu et al., 2020). To test a hypothesis of differential AR binding to RNAs in PCa in response to ADT, we performed an immunoprecipitation of AR and RNA-sequencing in presence and absence of androgen. Preliminary analysis revealed AR association with numerous mature mRNAs and lncRNAs independent of its recruitment to chromatin. Remarkably, AR-bound RNAs were different in cells growing in presence or absence of androgen and 60% of them contained a consensus AR binding motif previously identified in melanoma (Schmidt et al., 2020). We propose that RNA play a role in maintenance of the nuclear residence and specificity of AR activity. Still, additional experiments are required to fully understand AR-RNA crosstalk and how AR activity is maintained at specific loci in the absence of androgen.