IDENTIFICATION OF MASTER TRANSCRIPTIONAL REGULATORS OF NEUROENDOCRINE DIFFERENTIATION IN PROSTATE CANCER

Research PlanBackground: Neuroendocrine (NE) cells represent only a small fraction of the prostate epithelium in normal conditions, yet recent clinical studies have revealed that nearly 30% of patients with advanced prostate cancer and undergoing second-generation anti-androgen therapies (e.g., abiraterone and enzalutamide) relapse with tumors displaying a spectrum of NE features that are invariably associated with poor prognosis. The latest findings indicate that such treatment-emergent castration-resistant NE prostate cancer (CRPC-NE) develops by luminal cell transdifferentiation toward the NE lineage, yet the mechanisms underlying this process are poorly understood. Preliminary work carried out in the Shen laboratory has led to the selection of about 40 genes as putative key transcriptional regulators of NE differentiation, based on reverse-engineering of gene networks derived from RNA-seq analyses of mouse and human tumors with NE features.Objective: The key aim of this proposal is identification of master transcriptional regulators (MRs) of NE differentiation in prostate cancer.Specific Aims: (1) In vitro validation of master regulators of NE differentiation and (2) in vivo validation of the most promising master regulator of NE differentiation.Study Design: Aim 1. The list of gene candidates will be interrogated via a first round of in vitro validation in the human 2D androgen-responsive prostate cancer cell line LNCaP, which is known to be permissive for transdifferentiation to NE-like cells, followed by a second-round validation in mouse and human 3D organoids that display NE features. Such analyses will be based on CRISPR screens, mediated by either Cas9 nuclease for loss-of-function experiments or dCas9-VPR for gain-of-function assays. Aim 2. The most promising gene candidate will be investigated in vivo by gene targeting in an appropriate mouse model (e.g., Nkx3.1CreERT2/+Ptenflox/flox; Trp53flox/flox ). Finally, the expression of the functionally tested transcriptional regulator will be assessed in human CRPC genome-wide datasets and tissue microarrays, with or without treatment-induced NE features.PersonnelPrincipal Investigator (PI): Throughout my career path across three distinct research institutes in two different countries (Italy and the UK), I have studied fundamental principles in the fields of stem cell biology, epigenetics, and cell signaling. My career goal is to become an independent researcher investigating prostate stem cells and tissue biology, with the aim to improve survival and quality of life of prostate cancer patients.Mentor: Dr. Michael Shen is a leading expert in the field of prostate stem cells and cancer, with over 20 years of experience as a PI, as witnessed by his many contributions, including the identification of CARNs, the establishment of prostate 3D organoids, and most recently, the development of a clinically relevant mouse model of CRCP-NE. He has trained over 20 post-doctoral fellows.Training Plan: By pursuing this project under Dr. Shen’s supervision, I will learn how to apply high-throughput screens and analyses to different experimental models and how to integrate multiple datasets in order to tackle one of the key challenges faced by prostate cancer research. To do so, I will collaborate with experienced researchers across different fields at Columbia University Medical Center and in New York City.Impact: The identification of master regulators of NE transdifferentiation will open new avenues for predicting and controlling the emergence of therapy resistance in men with a high risk of metastatic prostate cancer.