Figure 5. Stemness-high PCa are associated with aggressive molecular subtypes and a 12-gene PCa-stem signature prognosticates poor patient survival.

A, Schematic illustrating the profiling strategy used to assess associations between Stemness and molecular features in PCa. Patients were ranked by Stemness score and stratified into Stemness-high (top 33%) and Stemness-low (bottom 33%) groups for downstream analyses. Genome-wide functional analyses were performed using transcriptomic, genomic, and clinical data.

B-C, Volcano plots showing differentially expressed genes (DEGs) between Stemness-high and Stemness-low groups in Pri-PCa (B) and mCRPC (C) cohorts. Red and blue indicate upregulated and downregulated DEGs, respectively. Genes with FDR > 0.05 are shown in gray. Sample sizes (n) of each group are indicated. See Supplementary Table S3 for complete DEG lists.

D Venn diagram showing the 12-gene “PCa-Stem signature” derived from overlapping upregulated DEGs (FC ≥ 2, FDR < 0.05) in both Pri-PCa and mCRPC.

E-F, GSEA showing low Stemness is associated with immune signaling and inflammatory response (E) while high Stemness in both cohorts is enriched for embryonic stem cell traits, aggressiveness, DNA repair, MYC activation, and mTORC1 signaling (F). All enrichments are significant (P < 0.05, FDR < 0.05).

G-H, PAM50 subtyping showing a higher frequency of LumB subtype in Stemness-high versus Stemness-low samples in Pri-PCa (G) and mCRPC (H). (****, P < 0.0001, χ² test).

I-L, GSEA showing enrichment of the aggressive PCS1 gene expression signature in Stemness-high groups and the less aggressive PCS3 signature in Stemness-low groups in Pri-PCa (I, K) and mCRPC (J, L). The Prostate Cancer Subtype (PCS) classification system (45,46) stratifies tumors into three molecular subtypes based on transcriptomic features: PCS1 (most aggressive), PCS2 (intermediate), and PCS3 (least aggressive). See Supplementary Table S2 for PCS gene signature.

M-R, GSEA showing AR signature enrichment in Pri-PCa Stemness-high (M), NEPC signature enrichment in mCRPC Stemness-high (N), and shared enrichment of cell cycle (O, P) and lineage plasticity (Q, R) programs in both cohorts. See Supplementary Table S5 for gene sets.

S-V, Kaplan-Meier analyses showing that the 12-gene PCa-Stem signature predicts worse progression-free survival (PFS) in TCGA Pri-PCa (S) and worse overall survival (OS) in SU2C mCRPC (U). Multivariable Cox models adjusting for clinicopathologic variables confirm the PCa-Stem-High subgroup as independently prognostic for PFS (T) and OS (V).