Profiling of Androgen Receptor in prostate cancer cells in response to  androgen deprivation and the onset of the neuroendocrine phenotype
Androgen deprivation therapy (ADT), also called chemical castration, is one of the prevailing treatments of local and metastatic prostate cancer enabling inhibition of the Androgen Receptor (AR) pathway and tumor regression. However, the response to therapy varies among patients with some showing symptomatic relief and prolonged survival, while others relapsing in less than 5 years or failing to respond (reviewed in Harris et al, 2009). These clinical trajectories are in part due to emergence of mechanisms allowing maintenance of the AR activity, often observed in CRPC tumors, and progressive cell reprograming enabling androgen-independent growth. In some case CRPC tumors progressively evolve in highly agressive AR-negative neuroendocrine prostate cancer (NEPC). In this project we aimed to profile Andrigen Receptor in the dynamic in vitro system recapitulating the CRPC and NEPC onset. Hormone-sensitive LNCaP cells were grown in  DHT-supplemented medium. In this condition cells exhibit an epithelial-like morphology and actively proliferate under the control of AR.  Androgen deprivation results in growth arrest and progressive emergence of the neuroendocrine phenotype characterized by AR signalling inactivation, expression of neuroendocrine genes and change in morphology  (Terry et al, 2013). Overall design: The experiment was performed in duplicates following the protocol from Henikoff lab (Skene et al, 2018) with modifications described in (Jarroux et al, 2021) in prostate cancer adenocarcinoma cells LNCaP growing in the presence of androgen (DHT) or in hormone-depleted media for 1 month (NE1m). Antibodies: against AR (Abcam, ab108341) and IgG (Thermo Fisher, 31235). DNA fragments were purified with NEB Monarch PCR & DNA purification kit following the protocol enrichment for short DNA fragments and was quantified by Qubit HS DNA kit. CUT & RUN libraries were prepared using the TruSeq ChIP-Sample Prep Kit-Set A and PCR Box (Illumina) from 5 ng of DNA with 10 PCR cycles for amplification following the manufacture's procedures. Concentration and quality were assessed by Qubit with dsDNA HS Assay Kit (Thermo Fisher Scientific) and High Sensitivity DNA Analysis Chip Kit (Agilent Technologies). Libraries were sequenced on a NovaSeq – S1 device in a pair-end 100 bp read length mode.