ELOVL2 mediated stabilization of AR contributes to enzalutamide resistance in prostate cancer

Abstract

Introduction: To investigate the molecular mechanisms underlying enzalutamide resistance in castration-resistant prostate cancer (CRPC) and explore potential therapeutic strategies to overcome resistance.

Methods: We conducted comprehensive bioinformatic analysis using LNCaP/enzalutamide-resistant cells to identify key pathways associated with resistance. Functional validation was performed through targeted inhibition of the elongation of very-long chain fatty acid protein 2 (ELOVL2), followed by assays to assess cancer cell proliferation and enzalutamide sensitivity. Mechanistic studies were conducted to evaluate the impact of ELOVL2 on the ubiquitin-proteasome system and AR signaling pathways.

Results: Bioinformatic analysis revealed that activation of fatty acid metabolism, particularly through upregulation of ELOVL2, plays a critical role in driving enzalutamide resistance in PCa. Functional studies demonstrated that targeted inhibition of ELOVL2 significantly suppressed cancer cell proliferation and restored enzalutamide sensitivity in resistant cells. Mechanistically, ELOVL2 facilitates enzalutamide resistance by impairing the ubiquitin-proteasome system, leading to the subsequent activation of AR signaling pathways.

Discussion: Our findings demonstrate that ELOVL2 drives enzalutamide resistance in CRPC by stabilizing AR through inhibition of ubiquitin-proteasome-mediated degradation. Targeting ELOVL2 represents a promising therapeutic strategy to overcome resistance in CRPC, with potential to improve clinical outcomes for patients.

Keywords: CRPC; ELOVL2; androgen receptor; enzalutamide resistance; prostate cancer.

FIGURE 1

Identification of ELOVL2 as a Key Regulator in Enzalutamide Resistance. (A) Bioinformatics analysis of GEO datasets was performed to identify differentially expressed genes associated with enzalutamide resistance in PCa. (B) Comparative analysis of ELOVL2 expression levels between prostate tumor tissues and adjacent normal tissues, demonstrating significant upregulation in malignant samples. (C) qRT-PCR analysis revealing differential expression of ELOVL2 in benign prostatic hyperplasia (BPH) tissues versus PCa cell lines (LNCaP, C4-2, PC3, and DU145),Data are presented as mean ± SD;  **P < 0.01.  ***P < 0.001. ****P < 0.0001. (D) Western blot analysis revealing protein expression level of ELOVL2 in benign prostatic hyperplasia (BPH) tissues versus PCa cell lines (LNCaP, C4-2, PC3, and DU145). (E) Representative immunohistochemical images showing elevated ELOVL2 expression (brown-yellow staining) in PCa tissues compared to non-tumorous tissues.

FIGURE 2

Characterization of Enzalutamide-Resistant PCa Cell Lines. (A) Schematic illustration of the experimental workflow for generating AR-positive enzalutamide-resistant PCa cell lines (LNCaP-enzR and C4-2-enzR). (B) Representative images showing morphological changes in LNCaP and C4-2 cells following the development of enzalutamide resistance. LNCaP-enzR cells exhibited a shrunken phenotype with elongated synapses, while C4-2-enzR cells transitioned from a spindle-shaped to a polygonal morphology with dendritic extensions. (C) Dose-response curves and half-maximal inhibitory concentration (IC₅₀) values for enzalutamide in parental and resistant cell lines. IC₅₀ values were significantly higher in resistant cells (C4-2-enzR: 75.46 µM, 95% CI: 72.02–78.99,  ****P < 0.0001; LNCaP-enzR: 45.13 µM, 95% CI: 41.45–48.75,  ****P < 0.01) compared to their parental counterparts (C4-2: 30.70 µM, 95% CI: 28.31–33.12; LNCaP: 18.96 µM, 95% CI: 15.85–21.61). (D) qRT-PCR analysis demonstrating upregulated expression of ELOVL2 in LNCaP-enzR and C4-2-enzR cells compared to their parental cell lines, Data are presented as mean ± SD,  *P < 0.05,  ***P < 0.001. (E) Western blot analysis demonstrating upregulated expression of ELOVL2 and AR in LNCaP-enzR and C4-2-enzR cells compared to their parental cell lines.

FIGURE 3

ELOVL2 inhibition suppresses growth and restores enzalutamide sensitivity in resistant PCa cells. (A) Validation of ELOVL2 knockdown efficiency using three independent small interfering RNAs (siRNAs) by qRT-PCR and Western blot analysis. siRNA#3 showed the most potent knockdown effect and was selected for subsequent experiments. (B) Stable ELOVL2 knockdown using lentiviral shRNA (derived from siRNA#3 sequence). Knockdown efficiency was confirmed by qRT-PCR and Western blot. (C) Cell proliferation assessed by CCK-8 assay in C4-2-enzR and LNCaP-enzR cells following ELOVL2 knockdown. Data represent mean ± SD (n = 5), ***p < 0.001. (D) Colony formation assay demonstrating the proliferative capacity of C4-2-enzR and LNCaP-enzR cells after ELOVL2 inhibition. Colonies were counted after 14 days (mean ± SD, n = 3), *p < 0.05, ***p < 0.001. (E) Dose-response curves and calculated half-maximal inhibitory concentration (IC₅₀) values for enzalutamide in ELOVL2-depleted cells. ELOVL2 knockdown significantly reduced IC₅₀ values in both C4-2-enzR (32.78 μM, 95% CI: 27.95–37.54; ***p < 0.0001) and LNCaP-enzR (21.03 μM, 95% CI: 19.62–22.36; ***p < 0.0001) cells compared to control (C4-2-NC: 80.72 μM, 95% CI: 75.26–86.48; LNCaP-NC: 51.15 μM, 95% CI: 47.92–54.33).

FIGURE 4

ELOVL2 Regulates AR Protein Stability via the Ubiquitin-Proteasome Pathway. (A) qRT-PCR analysis of AR mRNA and its downstream target genes (Klk3 and NKX3-1) in ELOVL2-depleted PCa cell lines. ELOVL2 knockdown did not significantly alter AR mRNA levels but reduced the expression of Klk3 and NKX3-1. (B) Western blot analysis showing decreased AR protein levels in ELOVL2-knockdown C4-2-enzR and LNCaP-enzR cells compared to controls. (C) AR protein stability assay using cycloheximide treatment. AR protein degradation was significantly accelerated in ELOVL2-depleted cells, C4-2-enzR-shELOVL2 t_1/2 = 5.04 h, LNCaP-enzR-shELOVL2 t_1/2 = 7.38 h,ns no significant difference, ***P < 0.001, ****P < 0.0001). (D) Western blot analysis of AR protein levels in enzR cells treated with or without the proteasome inhibitor MG132 (10 μM, 8 h). “-” and “+” indicate untreated control and MG132 treatment, respectively. (E) CO-IP and Western blot analysis demonstrating increased ubiquitination of AR protein in ELOVL2-knockdown enzR cells, indicating enhanced proteasomal degradation.

FIGURE 5

A schematic illustration of the molecular mechanism by which ELOVL2-mediated stabilization of AR contributes to Enzalutamide resistance in PCa.