Enzalutamide promotes an early plasticity-associated transcriptional state without terminal neuroendocrine differentiation in prostate cancer cells.

INTRODUCTION: Neuroendocrine prostate cancer (NEPC) is an aggressive treatment-associated lineage state emerging with potent androgen receptor (AR) pathway inhibition. Although treatment-emergent NEPC is increasingly recognized, the transcriptional consequences of sustained AR suppression remain incompletely defined. It remains unclear to what extent AR-targeted therapies reshape cellular identity and engage neuroendocrine-associated transcriptional programs without full lineage-defining differentiation. MATERIALS AND METHODS: Bulk RNA sequencing was performed in AR-dependent LNCaP and castration-resistant C4-2 prostate cancer cells under untreated conditions, androgen deprivation, first-generation AR antagonism with bicalutamide, and second-generation AR pathway inhibition with enzalutamide, using NCI-H660 as a NEPC reference. Transcriptomic organization and pathway-level changes were assessed using principal component analysis, comparative analyses, module score analysis of lineage-associated programs, and Gene Ontology-based functional enrichment. RESULTS: In LNCaP cells, AR suppression induced a progressive transcriptomic shift toward the NEPC reference cell line H660, with this trend being most pronounced in enzalutamide-treated cells. Canonical AR target genes (KLK3, TMPRSS2, NKX3-1, and FKBP5) were suppressed, whereas neuroendocrine-associated genes (NCAM1, ENO2, PEG10, and DLL3) showed partial induction. Canonical NEPC markers and lineage-defining transcription factors, including CHGA, INSM1, and SOX2, were not activated. Module score analysis demonstrated selective activation of lineage plasticity-associated programs corresponding to an intermediate Phase 2 state without engagement of canonical neuroendocrine differentiation programs. Gene-wise analyses further showed preferential induction of developmental and plasticity-associated transcriptional programs by enzalutamide compared with bicalutamide. In contrast, C4-2 cells exhibited context-dependent and incomplete neuroendocrine-associated transcriptional changes, consistent with modulation of a pre-existing permissive transcriptional state rather than stable neuroendocrine differentiation. CONCLUSIONS: Potent AR pathway inhibition does not induce terminal neuroendocrine differentiation in vitro but promotes an adaptive plastic intermediate state characterized by partial activation of neuroendocrine-associated transcriptional programs. These findings suggest that enzalutamide preferentially enhances early lineage plasticity rather than bona fide NEPC differentiation and provide a framework for distinguishing adaptive transcriptional reprogramming from true neuroendocrine lineage conversion in treatment-emergent prostate cancer.