To validate our findings, we analyzed four publicly available RNA sequencing datasets from the Gene Expression Omnibus (GEO) database: GSE163539 (LNCaP cells treated with DHT), GSE128749 (LNCaP cells treated with R1881), GSE150807 (LNCaP cells treated with enzalutamide), and GSE163240 (enzalutamide-resistant LNCaP cells). Differential expression analysis of ELOVL2 was performed across all datasets using standardized bioinformatics pipelines.

For clinical validation of our findings, we collected a cohort of nine prostate tissue specimens from patients with localized PCa who underwent either prostate biopsy or radical prostatectomy at the Fifth Affiliated Hospital of Southern Medical University. Among these specimens, two cases were histologically confirmed as enzalutamide-resistant PCa. Comprehensive clinical and pathological characteristics of these patients are detailed in Supplementary Table S1. All participants provided written informed consent prior to sample collection, and the study protocol was approved by the Institutional Review Board of the Fifth Affiliated Hospital of Southern Medical University (Approval No. 2025-MNWK-K-001), ensuring compliance with international ethical standards for human subject research.

IHC analysis was performed following our previously established protocol (Lv et al., 2019). Briefly, paraffin-embedded tissue blocks were sectioned at 10 μm thickness and mounted on slides. After deparaffinization and rehydration through a graded ethanol series, endogenous peroxidase activity was quenched by incubating sections in 0.3% hydrogen peroxide for 15 min at room temperature. Tissue sections were then incubated with primary rabbit anti-ELOVL2 antibody (1:400 dilution; 20308-1-AP,proteintech) overnight at 4°C. Following primary antibody incubation, slides were treated with horseradish peroxidase-conjugated anti-rabbit secondary antibodies (Dako, K4003) for 1 h at room temperature. Immunoreactivity was visualized using liquid 3,3′-diaminobenzidine tetrahydrochloride (DAB) substrate (Dako, K3468), followed by counterstaining with Mayer’s hematoxylin. Whole slide images were acquired using a panoramic MIDI scanner (3Dhistech, Budapest, Hungary) at ×20 magnification. Quantitative analysis of immunohistochemical staining was performed using ImageJ software (NIH, Bethesda, MD, United States of America) to determine the percentage of DAB-positive areas relative to total tissue area.

The human benign prostatic hyperplastic cell line BPH-1 and PCa cell lines (PC-3, DU145, 22Rv1, C4-2, and LNCaP) were obtained from the Stem Cell Bank of the Chinese Academy of Sciences. All cell lines were cultured in RPMI-1640 medium supplemented with 10% fetal bovine serum (FBS; Hyclone), 25 U/mL penicillin, and 25 μg/mL streptomycin. Cells were maintained at 37°C in a humidified atmosphere containing 5% CO₂.

To establish enzalutamide-resistant PCa cell lines, LNCaP and C4-2 cells were first adapted to androgen-deprived conditions by culturing in phenol red-free RPMI-1640 medium supplemented with 10% charcoal-stripped FBS for 1 week. Enzalutamide resistance was induced by stepwise exposure to increasing concentrations of enzalutamide (5 μM, 10 μM, 20 μM, and finally 30 µM), with each concentration maintained for 7 days. Following the establishment of resistance, the cells were maintained in medium containing 10 µM enzalutamide. The resulting enzalutamide-resistant cell lines, designated as LNCaP-enzR and C4-2-enzR, were used for subsequent experiments.

To investigate the functional role of ELOVL2, gene silencing was achieved using both transient siRNA transfection and stable lentiviral transduction approaches. For transient knockdown, Lipofectamine 3,000 (Invitrogen, Carlsbad, CA, United States of America,L3000008) was used to transfect cells with ELOVL2-specific small interfering RNAs (siRNAs) or negative control (NC) siRNA, following the manufacturer’s protocol. Three independent siRNAs targeting ELOVL2, along with their corresponding sequences (provided in Supplementary Table S2), were designed and synthesized by RiboBio Co. (Guangzhou, China).

For stable ELOVL2 knockdown, lentiviral particles containing short hairpin RNA (shRNA) targeting ELOVL2 were generated using the pLKO.1 vector system (GeneChem Bio-Medical Biotechnology, Shanghai, China). Lentiviral transduction was performed according to the manufacturer’s instructions, followed by puromycin selection (2 μg/mL) to establish stable shELOVL2 cell lines. Knockdown efficiency was validated at both mRNA and protein levels using quantitative real-time PCR and Western blot analysis, respectively.

Total RNA was extracted from PCa cells using the Total RNA Extraction Kit (Seven, China), following the manufacturer’s instructions. cDNA synthesis was performed using the PrimeScript RT Reagent Kit (TaKaRa, Japan,RR047A), and qRT-PCR was carried out using the TB-Green PCR Master Mix Kit (TaKaRa, Japan,RR430B). Glyceraldehyde 3-phosphate dehydrogenase (GAPDH) was used as an internal control, and relative gene expression levels were calculated using the 2^−ΔΔCt method. Primer sequences for target genes are provided in Supplementary Table S3.

PCa cells were lysed using RIPA buffer supplemented with Proteinase inhibitors, protein phosphatase inhibitors, PMSF. Protein concentrations were quantified using the BCA assay, and equal amounts of protein (30 µg) were separated by 10% SDS-PAGE and transferred onto polyvinylidene fluoride (PVDF) membranes.

Membranes were blocked with 5% skimmed milk in TBST for 1 h at room temperature and then incubated overnight at 4°C with the following primary antibodies: anti-Tubulin (Cell Signaling Technology, United States of America, 2128S), anti-ELOVL2 (Abcam, United States of America,ab176327), anti-AR (Cell Signaling Technology, United States of America), and anti-Ubiquitin Enzymes (Abcam, United States of America,ab134953). After washing, membranes were incubated with horseradish peroxidase (HRP)-conjugated secondary antibodies for 1 h at room temperature. Protein bands were visualized using an enhanced chemiluminescence (ECL) kit (Pierce Biotechnology, United States of America, 32109) and quantified using ImageJ software.

Cell viability was assessed using the Cell Counting Kit-8 (CCK-8) assay. Briefly, PCa cells were seeded at a density of 2,000 cells per well in 96-well plates and allowed to adhere overnight. After treatment, 10% CCK-8 reagent was added to each well, and plates were incubated at 37°C for 2 h. Absorbance was measured at 450 nm using a microplate reader.

For enzalutamide sensitivity assays, 1 × 10⁴ cells were seeded per well in 96-well plates and transfected with siRNA or plasmids as indicated. After 24 h, cells were treated with enzalutamide at final concentrations of 0, 0.05, 0.1, 0.5, and 2 µM (with a final ethanol concentration of 0.1%). Following 24 h of treatment, 10 µL of CCK-8 solution was added to each well, and plates were incubated for 90 min. Absorbance was measured at 450 nm, and the half-maximal inhibitory concentration (IC₅₀) was calculated.

PCa cells were seeded at a density of 300 cells per well in 6-well plates and cultured for 2 weeks to allow colony formation. Colonies were fixed with 4% paraformaldehyde for 15 min and stained with 0.5% Giemsa solution for 30 min. Colonies containing >50 cells were counted under a microscope. Each experimental condition was performed in triplicate, and the assay was repeated three times independently.

To assess the degradation rate of AR protein, PCa cells were treated with cycloheximide (CHX, 10 μg/mL, HY-12320), a protein synthesis inhibitor (Astapova et al., 2021). Cells in good growth condition were divided into two groups: control and ELOVL2 knockdown. Each group was seeded in 6-well plates at 50% confluence, with three replicate wells per group. When cells reached 70%–80% confluence, CHX was added to block new protein synthesis. Cells were harvested at 0, 4, and 8 h post-treatment, and total protein was extracted. AR protein levels were analyzed by Western blot, and band intensities were quantified using ImageJ software. The half-life (t₁/₂) of AR protein was calculated based on the degradation kinetics.

To determine whether the ubiquitin-proteasome system mediates AR protein degradation, co-immunoprecipitation (Co-IP) was performed using protein extracts from PCa cells. Cells were cultured to a density equivalent to one 15 cm² dish or three 10 cm² dishes, washed with PBS, trypsinized, and collected. After resuspension and centrifugation to remove residual medium and trypsin, cells were lysed in ice-cold lysis buffer supplemented with 1% protease inhibitor, phosphatase inhibitor, and PMSF. The lysate was incubated on ice for 30 min and centrifuged to collect the supernatant. The supernatant was divided into two groups: Input (100 μL) and IP (400 μL). The Input group was used to determine protein concentration, mixed with loading buffer, and stored at −80°C for subsequent analysis. The IP group was incubated with AR primary antibody (2–5 μg) at 4°C overnight. Protein A/G beads (40–50 μL) were then added to the IP group and incubated at 4°C overnight. Beads were washed 2–3 times with PBST:PMSF (100:1) and PBS:PMSF (100:1) to remove non-specific binding. Bound proteins were eluted using 1× loading buffer (diluted with wash buffer) and heat-denatured at 95°C for 5 min before storage at −80°C. Finally, AR protein and its ubiquitinated forms were detected by Western blot. The ubiquitination levels of AR were quantified to assess the role of the ubiquitin-proteasome pathway in AR degradation.

To investigate the involvement of the ubiquitin-proteasome system in AR protein degradation, ELOVL2-knockdown enzalutamide-resistant PCa cells and control cells were treated with MG132 (MCE, HY-13259), a proteasome inhibitor. Cells were seeded in 6-well plates at 70% confluence and cultured until reaching 90% confluence. The experimental group was treated with complete medium containing 10 μg/mL CHX and 10 μM MG132, while the control group was treated with complete medium containing 10 μg/mL CHX alone. Cells were incubated at 37°C for 8 h to allow proteasome inhibition and protein degradation analysis. After treatment, cells were washed with PBS, harvested, and lysed in RIPA buffer supplemented with 1% PMSF, protease inhibitors, and phosphatase inhibitors. The lysates were incubated on ice for 30 min and centrifuged at 12,000 rpm, 4°C for 30 min. The supernatants were collected and mixed with 25% volume of 4× loading buffer, followed by storage at −80°C for subsequent analysis. Target protein expression, particularly AR, was detected by Western blot. Band intensities were quantified using ImageJ software, and statistical analysis was performed to assess the effects of MG132 on AR protein stability in ELOVL2-knockdown and control cells.

Data are presented as mean ± standard error of the mean (SEM) unless otherwise stated. Statistical significance was determined using unpaired two-tailed Student’s t-tests or one-way ANOVA, as appropriate. Significance levels were defined as  *p < 0.05,  **p < 0.01,  ***p < 0.001, and  ****p < 0.0001. All statistical analyses were performed using SPSS software (version 22.0; IBM Corporation, Armonk, NY, United States of America).

To identify genes potentially involved in enzalutamide resistance, we conducted a comprehensive analysis of multiple enzalutamide-resistant PCa models. Four independent transcriptomic datasets were analyzed to compare gene expression profiles between enzalutamide-resistant (ENZ-R) and enzalutamide-sensitive samples. Through cross-comparison of differentially expressed genes, we identified a consensus signature of four genes (AKAP12, ELOVL2, SLC2A3, and WWTR1) that were significantly upregulated in ENZ-R samples (>1.5-fold change, P < 0.05; Figure 1A).

To validate these findings, we quantified the mRNA levels of these genes in the TCGA dataset and a panel of PCa cell lines (LNCaP, C4-2, PC3, and DU145). ELOVL2 exhibited the most pronounced upregulation in both PCa tissues (Figure 1B) and PCa cell lines compared to the benign prostatic hyperplasia (BPH) cell line BPH-1(Figure 1C; Supplementary Figure S1). Western blot analysis further confirmed elevated ELOVL2 protein expression in three PCa cell lines (C4-2, LNCaP, and DU145) relative to BPH-1 cells(Figure 1D). IHC analysis of archived paraffin-embedded PCa specimens (n = 9) revealed significantly higher ELOVL2 expression in tumor tissues, particularly in enzalutamide-resistant cases, whereas adjacent non-tumorous tissues showed minimal expression (Supplementary Figure S2).

These multi-level analyses demonstrate that ELOVL2 is consistently overexpressed in enzalutamide-resistant PCa and suggest its potential role as a critical mediator of therapeutic resistance. These findings highlight ELOVL2 as a promising biomarker and therapeutic target for overcoming enzalutamide resistance in advanced PCa.

LNCaP and C4-2 cell lines are widely used to model the biological behavior of primary tumor cells from CRPC patients and tumor cells following ADT, given their clinical relevance and origins (Guo et al., 2024; Mourkioti et al., 2023). To investigate the mechanisms of enzalutamide resistance, we established enzalutamide-resistant sublines derived from LNCaP and C4-2 cells, designated as LNCaP-enzR and C4-2-enzR, respectively (Figure 2A).

Notably, both LNCaP-enzR and C4-2-enzR cells exhibited distinct morphological changes compared to their parental counterparts (Figure 2B). LNCaP-enzR cells displayed a slightly shrunken phenotype with elongated synaptic extensions, while C4-2-enzR cells transitioned from a spindle-shaped morphology to a polygonal form with prominent dendrite-like structures. Functional characterization confirmed increased enzalutamide resistance in these sublines, as evidenced by a significantly higher half-maximal inhibitory concentration (IC₅₀) compared to parental cells (Figure 2C). Furthermore, to assess the reversibility of the ENZ-R phenotype, we cultured ENZ-R cells in enzalutamide-free medium for 7 days and re-exposed them to the drug. No significant cell death was observed upon re-challenge, and repeated IC₅₀ measurements remained consistent with the initial resistance profile (Figure 2C; Supplementary Figure S3). These data indicate that the ENZ-R phenotype is stable even after short-term drug withdrawal, underscoring the robustness of our model system.

Furthermore, qPCR analysis demonstrated significant upregulation of ELOVL2 mRNA in both LNCaP-enzR and C4-2-enzR cells compared to their parental counterparts (Figure 2D). This transcriptional induction was consistently reflected at the protein level, as Western blot analysis revealed markedly elevated expression of both ELOVL2 and AR in the resistant cells (Figure 2E; Supplementary Figure S4). The concordant upregulation of ELOVL2 at both mRNA and protein levels, along with increased AR expression, suggests that prolonged enzalutamide treatment induces coordinated upregulation of ELOVL2 and AR in resistant cells. Importantly, the positive correlation between ELOVL2 and AR levels in enzalutamide-resistant PCa cells reveals a potential functional interplay between ELOVL2-mediated fatty acid metabolism and AR signaling in driving therapeutic resistance.

To further investigate the functional role of ELOVL2 in prostate cancer (PCa), we employed siRNA-mediated knockdown to deplete ELOVL2 expression in enzalutamide-resistant LNCaP-enzR and C4-2-enzR cells. Among the three siRNAs tested, SI3 exhibited the most significant knockdown efficiency, as confirmed by qRT-PCR and Western blotting at the mRNA and protein levels, respectively (Figure 3A). Subsequently, we generated stable shELOVL2 cell lines using lentiviral delivery of SI3. Further validation by qRT-PCR and Western blotting demonstrated a marked reduction in ELOVL2 expression in both cell lines (Figure 3B; Supplementary Figure S5).

Functional assays revealed that ELOVL2 knockdown markedly inhibited cell proliferation, as evidenced by CCK-8 assays and colony formation experiments (Figures 3C,D; Supplementary Figure S6). To determine whether ELOVL2 contributes to enzalutamide resistance, we assessed the sensitivity of ELOVL2-depleted cells to enzalutamide. Notably, ELOVL2 silencing significantly reduced IC₅₀ of enzalutamide in both LNCaP-enzR (IC₅₀: 21.03 µM vs. 51.15 µM,  ****<0.0001) and C4-2-enzR (IC₅₀: 32.78 µM vs. 80.72 µM,  ****<0.0001) cells (Figure 3E). Furthermore, we silenced ELOVL2 in both parental cell lines (LNCaP and C4-2) and measured their IC₅₀ values. The results showed no significant differences in IC₅₀ changes upon ELOVL2 silencing in these cells (Supplementary Figure S7), further highlighting the specific role of ELOVL2 in mediating enzalutamide resistance within the CRPC context.

These findings collectively demonstrate that ELOVL2 promotes the growth of enzalutamide-resistant PCa cells and plays a critical role in maintaining therapeutic resistance. Targeting ELOVL2 may therefore represent a promising strategy to overcome enzalutamide resistance in advanced PCa.

AR is the primary target of enzalutamide, and AR-associated mechanisms are closely linked to therapeutic resistance in PCa. To investigate whether ELOVL2 influences AR signaling, we examined the expression of AR and its downstream coactivators (Klk3 and NKX3-1) in enzalutamide-resistant PCa cells. While ELOVL2 knockdown did not significantly alter AR mRNA levels, it markedly reduced the expression of Klk3 and NKX3-1, which are downstream targets of AR signaling (Figure 4A). Western blot analysis further revealed that ELOVL2 depletion led to a significant decrease in AR protein levels in enzalutamide-resistant cells (Figure 4B; Supplementary Figure S8), suggesting that ELOVL2 regulates AR at the post-translational level.

To explore this mechanism, we treated ELOVL2-depleted cells with cycloheximide, a protein synthesis inhibitor, and observed accelerated AR protein degradation compared to control cells (Figure 4C). This finding indicates that ELOVL2 plays a critical role in maintaining AR protein stability. To further elucidate the pathway involved, we treated ELOVL2-knockdown C4-2-enzR and LNCaP-enzR cells with the proteasome inhibitor MG132. Western blot analysis demonstrated that MG132 treatment significantly attenuated AR protein degradation, confirming the involvement of the ubiquitin-proteasome system (Figure 4D).

To directly assess the role of ELOVL2 in AR ubiquitination, we performed co-immunoprecipitation assays. Results showed that ELOVL2 knockdown significantly increased the ubiquitination levels of AR protein (Figure 4E), providing direct evidence that ELOVL2 regulates AR stability through the ubiquitin-proteasome pathway.

Collectively, these findings demonstrate that ELOVL2 maybe upregulates AR protein expression by inhibiting the ubiquitin-proteasome pathway, thereby enhancing AR signaling and contributing to enzalutamide resistance in PCa cells.