EXPERIMENTAL IDENTIFICATION AND FUNCTIONAL VALIDATION OF TRANSCRIPTION FACTORS AND ASSOCIATED PROTEINS DRIVING NEUROENDOCRINE PROSTATE CANCER (NEPC)

Abstract

Background:
Neuroendocrine prostate cancer (NEPC) is an aggressive, therapy-resistant subtype that frequently arises from prostate adenocarcinoma following prolonged androgen receptor (AR) pathway inhibition. The transition toward NEPC involves extensive transcriptional and epigenetic reprogramming; however, the key transcription factors (TFs) and their co-regulatory protein partners driving this lineage plasticity remain insufficiently characterized.

Objectives:
This study aimed to (i) identify transcription factors significantly associated with NEPC differentiation, (ii) characterize their interacting protein partners, and (iii) validate their functional relevance in vitro and in vivo. A secondary objective was to map the regulatory networks controlling neuroendocrine lineage commitment.

Methods:
We employed NEPC cell lines (NCI-H660, DU145-NE, LASCPC-01), adenocarcinoma models (LNCaP, VCaP), and human tumor samples. Transcriptomic (RNA-Seq), epigenomic (ATAC-Seq, ChIP-Seq), and proteomic (Co-IP/MS) analyses were integrated with computational network modeling using ARACNe, MARINa, and SCENIC. Candidate TFs were validated using gene knockdown/knockout, overexpression, phenotypic assays, rescue experiments, and xenograft models. Statistical significance was assessed using FDR-corrected analyses.

Results:
RNA-Seq and ATAC-Seq identified a distinct NEPC transcriptional state marked by upregulation of ASCL1, BRN2, SOX2, FOXA2, and PEG10, accompanied by broad enhancer reactivation at neuronal regulatory regions. Proteomic profiling revealed TF interactions with key chromatin remodelers, including BRD4, CHD7, SMARCA4, and KDM1A. Network modeling identified ASCL1, BRN2, SOX2, and PEG10 as master regulators linking chromatin remodeling to NE transcriptional programs. Functional assays showed that TF knockdown reduced NE markers (CHGA, SYP, INSM1), suppressed proliferation, and increased apoptosis, while TF overexpression induced NE-like phenotypes in adenocarcinoma cells. Xenograft studies confirmed reduced tumor growth and diminished NEPC histology following TF silencing.

Conclusion:
This study identifies and validates a core transcriptional-epigenetic network involving ASCL1, BRN2, SOX2, and PEG10 as key drivers of neuroendocrine differentiation and NEPC progression. By defining essential TF–chromatin remodeler interactions, the findings establish potential therapeutic targets for mitigating lineage plasticity and improving outcomes in advanced prostate cancer.