ABSTRACT Background The Hippo pathway effector YAP1 has been implicated in prostate cancer progression, migration, and castration resistance, yet how pharmacological YAP1 inhibition reshapes the prostate cancer proteome remains uncharacterized. Methods We used label‐free quantitative proteomics together with functional assays to characterize the proteomic and phenotypic effects of CA3, a selective YAP1 inhibitor, in androgen‐independent PC3 and androgen‐sensitive LNCaP prostate cancer cells, and validated key findings with wound‐healing, western blot, and immunodetection assays for focal adhesion kinase (FAK), vimentin, occludin, and junctional adhesion molecule A (JAM‐A). Results CA3 reduced cell viability and YAP1 protein levels in both cell lines in a dose‐ and time‐dependent manner ( p < 0.05). Nonlinear regression analysis yielded 24 h IC50 values of 1.59 µM (95% CI, 1.38–1.83) for PC3 and 0.63 µM (95% CI, 0.53–0.77) for LNCaP, with corresponding 48 h values of 0.59 µM (95% CI, 0.50–0.69) and 0.49 µM (95% CI, 0.39–0.62). CA3 showed markedly lower potency in the non‐tumorigenic PNT1A prostate epithelial cell line (IC50 18.87 µM at 24 h; 4.47 µM at 48 h), corresponding to an approximately 7‐ to 30‐fold selectivity window favoring the cancer cell lines. Label‐free quantitative proteomics identified 365 and 153 differentially expressed proteins (DEPs) in PC3 and LNCaP cells, respectively. Functional enrichment analysis revealed cell line‐dependent alterations in pathways related to RNA metabolism and translation, alongside migration‐ and adhesion‐associated processes, including focal adhesion, cytoskeletal organization, and guidance signaling. Comparison of these proteins with the Gene Ontology cell migration gene set (GO:0016477) identified EGFR, SRC, TGFB1, IL6, and SCARB1 as hub proteins in PC3 cells, and RAC1, RHOC, NF1, FSCN1, and ASPM in LNCaP cells. Functional validation confirmed that CA3 reduced wound closure in both cell lines, decreased FAK protein levels, selectively reduced vimentin in PC3 cells, increased occludin in both cell lines, and increased JAM‐A specifically in PC3 cells (all p < 0.05). Conclusions To our knowledge, this is the first study to characterize the CA3‐induced proteome in prostate cancer cells and link these changes to migration‐associated protein networks, in cell lines with differing androgen dependence.
ABSTRACT Background Benign prostatic hyperplasia (BPH) and lower urinary tract symptoms are strongly associated with chronic prostate inflammation and stromal remodeling, yet the stromal cell programs that emerge during inflammatory, nodular remodeling remain incompletely defined. A Toxoplasma gondii infection model produces persistent inflammation and nodular formations, providing an experimentally tractable system to map inflammation‐linked stromal states. Methods We performed single‐cell RNA sequencing of prostates from control and chronically infected male CBA/J mice harvested at 28 days post‐infection (DPI). Stromal subset reprogramming was assessed by cluster‐resolved pseudobulk differential expression and Ingenuity Pathway Analysis. Stromal expansion and Cd52 + fibroblast subset changes were validated by whole‐section immunofluorescence at 28 and 60 DPI using a CellposeSAM‐based tissue cytometry pipeline. To assess human relevance, CD52 + fibroblasts were evaluated in FFPE human prostate sections by comparing inflamed versus non‐inflamed stromal regions. Results Whole‐prostate single‐cell profiling resolved stromal, epithelial, immune, and seminal vesicle compartments. Infection increased immune‐cell abundance and increased stromal representation among non‐immune cells (53.1% in controls to 69.0% in infected prostates). Stromal reclustering identified eight subsets, including a Cd52 + /Ccl5 + immune‐reactive fibroblast population that showed the strongest selective expansion during infection. Whole‐section imaging supported overall stromal expansion over time, and Cd52 + fibroblasts were higher during infection at 60 DPI (270% increase, p = 0.0507), whereas Cxcl13 + fibroblasts remained rare and stable across conditions. Pathway analysis indicated immune‐like stromal reprogramming during infection, with antigen‐presentation programs enriched in larger fibroblast subsets and interferon/JAK–STAT signaling most consistently concentrated in the Cd52 + /Ccl5 + subset. In human prostate tissue, CD52 + VIM + CD45 − fibroblasts were significantly enriched in inflamed regions compared with paired non‐inflamed regions within the same specimen (paired Wilcoxon p = 0.0026). Conclusions Chronic, pathogen‐driven inflammation produces selective, program‐specific stromal remodeling rather than uniform fibroblast activation. An interferon‐reactive Cd52 + fibroblast state expands during infection in mice and marks inflamed stromal microenvironments in human prostate, supporting discrete immune‐reactive fibroblast programs as a prominent feature of inflammation‐associated prostate remodeling.
ABSTRACT Background Management after suspicious multiparametric MRI (mpMRI; PI‐RADS ≥ 3) and an initial biopsy negative for clinically significant prostate cancer (csPCa) remains uncertain. We described the frequency of subsequent csPCa detection and explored baseline factors. Methods This post hoc longitudinal analysis of MULTIPROS trial data included 153 men with PI‐RADS ≥ 3 lesions and no csPCa detected on baseline transrectal systematic biopsy, with or without targeted biopsy. Participants contributed follow‐up from the date of the initial mpMRI until csPCa detection or their last available clinical follow‐up, with records reviewed through May 2025. Follow‐up was not protocol‐mandated, and repeat biopsy was guided by clinical findings. The primary outcome was time to histological csPCa detection. Kaplan–Meier and multivariable logistic regression analyses were exploratory. Results Over a median observed time to csPCa detection or censoring of 64 months, csPCa was detected in 37/153 men (24.2%). Of these, 28/37 (75.7%) were detected within 6 months, and the median time to detection was 3 months. This early clustering was compatible with possible baseline sampling limitations, although timing alone could not establish this mechanism. Overall, 84/153 men underwent repeat biopsy and 69/153 did not; 69/116 men without detected csPCa lacked repeat histological confirmation. The between‐group Kaplan–Meier comparison was not significant ( p = 0.210) and was difficult to interpret because repeat‐biopsy rates differed. Transition‐zone location, anterior fibromuscular stroma location, and atypical small acinar proliferation were associated with detection. The hypothesis‐generating model had moderate apparent discrimination (AUC 0.756) but low sensitivity at a 0.50 threshold (24.3%). Conclusions Most detections occurred early, a pattern compatible with possible baseline sampling limitations, although the biological timing of disease onset cannot be established from these data. Selective follow‐up limits the interpretation of the detection proportion and between‐group comparison. The identified associations require external validation, and the model should not be used clinically.
ABSTRACT Background Whether younger age independently predicts biochemical recurrence (BCR) after radical prostatectomy (RP) remains controversial because previous studies have included heterogeneous pathological stages and treatment populations. We aimed to determine whether age independently predicts BCR following RP in patients with organ‐confined, node‐negative, margin‐negative (pT2N0R0) prostate cancer. Methods We performed a retrospective multicenter cohort study of 4223 patients with pT2N0R0 prostate cancer treated with RP. Patients were stratified into ≤ 55 years ( n = 875; including 223 aged < 50 years) and > 55 years ( n = 3348). BCR was defined as a confirmed prostate‐specific antigen level ≥ 0.2 ng/mL. Kaplan–Meier analyses, multivariable Cox regression, restricted cubic spline modeling, subgroup analyses, and sensitivity analyses were performed. Results Younger patients had lower PSA levels, lower Grade Group, and lower crude BCR rates than older patients (3.8% vs. 5.9%; p = 0.018). However, younger age was not independently associated with BCR after adjustment for clinicopathological variables (hazard ratio 0.52, 95% confidence interval 0.26–1.04; p = 0.075). Higher PSA and Grade Group 3–5 independently predicted recurrence (all p < 0.05). Age analyzed continuously, alternative age thresholds, restricted cubic spline modeling, and subgroup analyses consistently demonstrated no significant association between age and BCR. Limitations include the retrospective design, relatively short follow‐up, and use of BCR as a surrogate endpoint. Conclusions Among men with pT2N0R0 prostate cancer treated with RP, age was not a statistically significant independent predictor of BCR, although a clinically meaningful effect cannot be excluded. Postoperative risk stratification should rely on established pathological factors rather than age alone.
ABSTRACT Background Chronic exposure to androgen receptor (AR)‐targeted therapies can drive lineage plasticity, leading to treatment resistance and neuroendocrine prostate cancer (NEPC), characterized by loss of AR expression and acquisition of neuroendocrine (NE) markers including synaptophysin (SYP). We evaluated concordance between circulating tumor cell (CTC) AR and SYP expression patterns and evidence of NE differentiation defined by clinical, pathologic, and genomic features. Methods CTCs from patients with metastatic castration‐resistant prostate cancer (mCRPC) were analyzed using the RareCyte platform with quantitative immunofluorescence imaging for SYP and AR. In patients with ≥ 5 CTCs, associations of CTC phenotype with PSA at collection and overall survival (OS) were assessed using the Wilcoxon rank‐sum test, Fisher's exact test, and Kaplan–Meier analysis. Patients were retrospectively classified as AR‐positive adenocarcinoma or NE‐differentiated based on clinical, pathologic, and genomic data. Results The AR/SYP CTC assay was analytically validated using cell‐line spike‐in models, achieving > 90% accuracy for both biomarkers. Of 52 patients with mCRPC, 27 had ≥ 5 evaluable CTCs and were included in the primary CTC phenotype analyses. The NE‐differentiated cohort had a higher fraction of SYP‐positive CTCs (median 28.6% vs 0.0%, p = 0.0084), whereas the AR‐positive adenocarcinoma cohort had a higher fraction of AR‐positive CTCs (median 96.7% vs 0.0%, p = 0.0004). Classification based on high AR or SYP expression (≥ 80% of CTCs positive) significantly distinguished cohorts and was associated with tissue histology ( p = 0.0048). PSA at CTC collection was lower in the NE‐differentiated cohort (median 3.83 vs 486.89 ng/mL; 95% CI: −1016 to −190.9). OS did not significantly differ between groups. Conclusion SYP and AR CTC expression showed strong concordance with tissue histology and clinical features of NE differentiation and AR‐positive adenocarcinoma. These findings support the feasibility of CTC immunophenotyping to characterize lineage‐associated phenotypes including SYP‐expressing NE differentiation in mCRPC. Prospective validation in larger cohorts with matched tissue biopsies is needed to define the clinical utility of this approach.
