Phenotypic and transcriptomic characterization of bicalutamide and enzalutamide resistance in castration-resistant prostate cancer cells

Natkin, R.; Paloneva, M. I.; Raittinen, P. V.; Syvala, H.; Blauer, M.; Tammela, T. L.; Ilmonen, P.; Nykter, M.; Siltari, A.; Murtola, T. J.

doi:10.1101/2025.05.30.656961

bioRxiv · 10.1101/2025.05.30.656961

Phenotypic and transcriptomic characterization of bicalutamide and enzalutamide resistance in castration-resistant prostate cancer cells

Natkin, R.·Paloneva, M. I.·Raittinen, P. V.·Syvala, H.·Blauer, M.·Tammela, T. L.·Ilmonen, P.·Nykter, M.·Siltari, A.·Murtola, T. J.

View original source ↗Export metadata ↓

Abstract

The cornerstone treatment for aggressive prostate cancer (PCa) is androgen deprivation therapy (ADT). Eventually PCa cells develop castration resistance, i.e. resistance to ADT. Castration resistant PCa initially responds to androgen signaling inhibitors such as bicalutamide and enzalutamide, but the cells become resistant to these drugs as well overtime. We used long-term cell cultures to create testosterone-dependent, testosterone-independent (CT), bicalutamide-resistant (BR), enzalutamide-resistant (ER), and sequential resistant BR-ER VCaP cell lines to investigate transcriptomic and proteomic changes during the development of drug resistance. Phenotypical changes were evaluated based on cellular morphology and sensitivity to docetaxel. We observed marked changes in transcriptome during the development of drug resistance and differences between ER and sequential BRER. Androgen response and fatty acid metabolism were upregulated in both ER and BRER. MYC target hallmarks were positively enriched only in BRER. Negative regulation of cell death was upregulated in ER while in BRER cell cycle related pathways were upregulated. Direct comparison of ER and BRER revealed androgen signaling signature being higher in ER than in BRER. BRER was more resistant to docetaxel than ER. Clinical significance of our results was confirmed using Finnish patient data and public patient data. In castration resistant patients response to enzalutamide treatment were decreased in patients first treated with bicalutamide comparing patients treated only with enzalutamide. In conclusion, we showed that transcriptomic and phenotypic changes occurring during formation of drug resistances and docetaxel sensitivity depend on the sequence of treatments. Treatment responses were also different in sequentially treated patients. In the future, this may inform treatment sequencing to retain the PCa cells more sensitivity to subsequent treatment lines.

Explore related subjects

cancer biology Preprint

Keep this discovery

Explore connections, maps & timelines

BibTeX RIS

Natkin, R., Paloneva, M. I., Raittinen, P. V., Syvala, H., Blauer, M., Tammela, T. L., Ilmonen, P., Nykter, M., Siltari, A., Murtola, T. J.. 2025-06-02. Phenotypic and transcriptomic characterization of bicalutamide and enzalutamide resistance in castration-resistant prostate cancer cells. https://doi.org/10.1101/2025.05.30.656961

Cite the original work for its findings. Save a collection to share your selection of sources.

KEEP EXPLORING

Related preprints

bioRxiv2026

The Phospholipase A2 Activity of Peroxiredoxin 6 Protects Hepatocarcinoma Cells Against Ferroptosis and Mitophagy Impairment

Peroxiredoxin 6 (PRDX6) is a moonlighting protein that plays a key role in regulating redox homeostasis and lipid metabolism through its unique dual peroxidase and calcium-independent phospholipase A2 (iPLA2) activities. Here, we generated a stable HepG2 cell line harboring a D140A mutation to selectively ablate iPLA2 activity, and we compared its phenotype with that of wild-type (WT) and PRDX6-knockout (KO) cells. D140A exhibited low ROS leves, similar to WT cells, confirming that oxidative stress is due to peroxidase deficiency. In contrast, D140A cells displayed higher lipid peroxidation than WT cells, though significantly lower than that of KO cells. Furthermore, the D140A mutant mirrored the KO phenotype regarding intracellular ferrous iron accumulation, impaired lipid droplet biogenesis, and the downregulation of the key ferroptosis regulator GPX4. Mechanistically, integrated proteomic, lipidomic, and enzymatic analyses revealed that a deficiency in iPLA2 activity of PRDX6 disrupted the release of key lipid species necessary for cell structure and signaling. Consequently, organelles involved in lipid synthesis and trafficking, such as peroxisomes, endoplasmic reticulum, mitochondria and lipid droplet, were affected. Specifically, we found that mitochondria exhibited a reduction in cristae and a diminished capacity for mitophagy and autophagy, which ultimately makes cells much more susceptible to erastin-induced ferroptotic cell death. Taken together, our findings demonstrate that PRDX6 iPLA2 activity uniquely coordinates organelle lipid homeostasis and mitophagy to protect cancer cells from ferroptotic cell death. Consequently, the use of iPLA2 inhibitors such as MJ33, which decreased PRDX6 iPLA2 activity to a level comparable to the mutant D140A, could represent a promising therapeutic strategy for inducing ferroptotic cell death in hepatocellular carcinoma.

cancer biology↗

bioRxiv2026

Serum extracellular vesicle proteomics reveals treatment-associated biomarkers in pancreatic ductal adenocarcinoma

Background Longitudinal biomarkers of treatment response in pancreatic ductal adenocarcinoma (PDAC) are needed. Extracellular vesicle (EV)-enriched serum proteomics may provide a minimally invasive approach for monitoring systemic and tumor-associated responses to therapy. Methods Paired pre-treatment and post-treatment serum samples were analyzed from 51 patients with pancreatic cancer, including 25 treated with FOLFIRINOX and 20 treated with gemcitabine-based therapy. EVs were enriched using the Mag-Net method and profiled by data independent acquisition mass spectrometry. After batch correction and filtering, 941 proteins were retained for differential and pathway analyses. Paired linear models and gene set enrichment analyses were used to assess treatment-associated changes. Baseline protein abundance and longitudinal changes were evaluated in relation to overall survival (OS) using Cox models. Results A total of 2,089 EV-associated proteins were identified, with an average of 1,186 proteins quantified per sample. Across the cohort, 26 proteins differed between post- and pretreatment samples. Proteins that increased after treatment were primarily related to extracellular matrix organization and cell adhesion, whereas proteins that decreased after treatment were associated with actin cytoskeletal organization, coagulation, and hemostasis. Treatment-associated changes were more pronounced in the FOLFIRINOX group, in which 32 proteins were differentially expressed. FOLFIRINOX was characterized by coordinated reductions in platelet- and coagulation-associated proteins, including PF4, PF4V1, PPBP, GP5, ITGA2B, and ITGB3, accompanied by enrichment of platelet activation, coagulation, and hemostasis pathways among down-regulated processes. No individual proteins met the same significance threshold in the gemcitabine group. A composite seven-protein platelet score decreased significantly after FOLFIRINOX but not for gemcitabine, and the between-regimen difference in score change was significant. Higher pretreatment ITGB1 abundance was associated with longer OS after multiple-testing correction (HR, 0.112; BH-FDR = 0.0063). In multivariable analysis, greater treatment-associated platelet-score depletion was independently associated with shorter OS (HR per unit increase in {Delta}score, 0.56; P = 0.030). Conclusions Longitudinal serum EV-enriched proteomics identified treatment-associated biological changes in PDAC, including a coordinated platelet-related response predominantly observed after FOLFIRINOX. Baseline ITGB1 abundance and longitudinal platelet-score changes represent potentially complementary prognostic and pharmacodynamic markers. These findings are exploratory and require validation in larger, independent cohorts incorporating platelet counts, coagulation measurements, and targeted proteomic assays.

cancer biology↗

bioRxiv2026

BAF complexes cooperate with lineage-restricted transcription factors to establish cell-state-specific regulatory programs

BRG1/BRM-associated factor (BAF) complexes are essential regulators of chromatin organization and cell identity. Genes encoding BAF subunits are among the most frequently mutated in human cancer. Yet, how disruption of a single subunit gives rise to distinct tumor states remains poorly understood. Atypical teratoid rhabdoid tumors (ATRTs) provide a compelling model, as they are uniformly driven by bi-allelic loss of the BAF subunit SMARCB1, yet segregate into distinct molecular subtypes. Using integrated multi-omics profiling of patient-derived ATRT organoids and primary tumor tissues, we show that transcriptional and chromatin accessibility changes upon SMARCB1 reconstitution are directed by subtype- and lineage-specific transcription factor (TF) repertoires. Genome-wide binding and transcriptomic analyses reveal that SMARCB1 reconstitution enables the lineage-specific TF RFX4 to engage regulatory elements and activate neuronal differentiation programs, partially reprogramming ATRT-MYC toward an ATRT-SHH-like identity. Together, these findings demonstrate that SMARCB1 loss disrupts TF-dependent chromatin priming, revealing that the lineage-specific regulatory output of BAF complexes is constrained by the underlying TF repertoire.

cancer biology↗