Blood-based proteomic profiling reveals novel biomarkers of neuroendocrine prostate cancer

Neuroendocrine prostate cancer (NEPC) is an aggressive, AR-indifferent subtype of metastatic prostate cancer, with a median survival of 7-15 months. Trans-differentiation from prostate adenocarcinoma to NEPC occurs through a mechanism termed lineage plasticity. Diagnosing NEPC remains a major clinical challenge due to the lack of robust biomarkers and pronounced spatial and temporal heterogeneity. There is an urgent need for diagnostic biomarkers of NEPC before clinical manifestation. To characterize the proteomic landscape of NEPC, we first performed a bottom-up proteomic analysis using LC-MS/MS in NEPC (H660) and adenocarcinoma (VCaP) cell lines. An average of 10,230 proteins were identified in both cell lines, with over 1,300 proteins significantly enriched in NEPC compared to VCaP. NCAM1, a clinical marker of NEPC, was among the significantly enriched proteins ( adjusted p =0.004; Log2FC=6.5), supporting the biological significance of our findings. Pathway analysis revealed enrichment in E2F, EMT, and p53 signaling pathways, consistent with established mechanisms of lineage plasticity. Next, we conducted a prospective pilot study using plasma from patients with pathologically confirmed NEPC or concurrent RB1/TP53 loss (n=21) and from patients with RB1/TP53wt prostate adenocarcinoma without evidence of histological transformation (n=20). To enhance the detection of low-abundance proteins, we applied sequential enrichment and elution (SEER) followed by Proteograph XT digestion and high-resolution mass spectrometry. Putative proteoforms were identified using data-independent acquisition-neural network (DIA-NN) processing. A total of 6,619 proteins were identified in both cohorts, 93 of which were significantly enriched in the NEPC samples, including NCAM1, validating the approach in plasma. In addition, potential new markers were detected such as methionine adenosyltransferase 2A (MAT2A) ( adjusted p =2.59E-09; Log2FC=2.8), a critical metabolic driver of androgen-independent cellular state. Neuronal-specific enolase (NSE/ENO2), a glycolytic enzyme associated with poor prognosis and tumor dedifferentiation in prostate cancer, was exclusively expressed in H660 cells and NEPC patient samples. Pathway analysis revealed co-enrichment of neuroendocrine and mitotic germ cell-associated programs, reflecting extensive phenotypic reprogramming that enhances metastatic capacity during lineage plasticity. These findings demonstrate the utility of blood proteomic profiling for the discovery of prognostic and predictive NEPC biomarkers. These markers may help elucidate molecular mechanisms underlying lineage plasticity. Furthermore, high-resolution plasma proteomics offers a promising approach to overcome the limitations of tissue biopsy, which is often restricted by high intra-tumoral heterogeneity and the lack of specificity of current pathological markers.