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

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. 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. 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. 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.