Wnt5a augments intracellular free cholesterol levels and promotes castration resistance in prostate cancer

Abstract

Background: Prostate cancer (PCa) is a leading cause of cancer-related mortality in men globally. While androgen deprivation therapy (ADT) can extend the asymptomatic phase and overall survival of patients with metastatic PCa, prolonged ADT often leads to the development of castration-resistant prostate cancer (CRPC) within 18-24 months. The mechanisms underlying CRPC remain incompletely understood, presenting a significant challenge in clinical prostate cancer treatment.

Methods: In this study, we investigated the role of Wnt5a, a member of the Wnt family, in CRPC. Tumor tissues from CRPC patients were analyzed to assess the expression levels of Wnt5a. Prostate cancer cells were used to examine the impact of Wnt5a on androgen-dependent and -independent growth, as well as sensitivity to bicalutamide. RNA-seq analysis, qRT-PCR, intracellular cholesterol content and the activation of the androgen receptor (AR) signaling pathway were evaluated to elucidate the mechanistic role of Wnt5a in CRPC progression. Drug target Mendelian randomization analysis was performed to investigate the effect of PCSK9 inhibitor on prostate cancer.

Results: Our study revealed a significant overexpression of Wnt5a in tissues from CRPC tumors. Wnt5a was found to enhance both androgen-dependent and -independent growth in prostate cancer cells while reducing their sensitivity to bicalutamide. Mechanistically, Wnt5a was shown to upregulate intracellular free cholesterol content and activate the AR signaling pathway, contributing to hormone therapy resistance in CRPC. PCSK9 inhibitor significantly reduced the risk of PCa.

Conclusions: The findings of this study highlight a novel molecular mechanism underlying endocrine therapy resistance in CRPC mediated by Wnt5a. Targeting Wnt5a or reducing cholesterol level would be a promising therapeutic strategy for the treatment of CRPC, providing new insights into potential avenues for combating this challenging form of prostate cancer.

Keywords: Androgen receptor signaling; Castration resistance; Cholesterol; Prostate cancer.

Fig. 1

Wnt5a is associated with castration resistance in prostate cancer and promotes androgen-dependent and -independent growth of PCa cells. (A) Comparison of mRNA expression of Wnt5a in normal, paraneoplastic, primary and metastatic prostate cancer tissues in the GSE6919 dataset from the GEO Database. (B) Expression of Wnt5a in patients with different Gleason score levels or (C) tissue type were analyzed using GSE70768 dataset from the GEO database. (D) Expression levels of Wnt5a in tumor tissues formed by LNCaP prostate cancer cells in mice with or without surgical castration in GSE33316 dataset from the GEO database. (E) Wnt5a expression of LNCaP cells cultured in regular medium and hormone-deprived medium were compared using GSE8702 dataset from the GEO database. (F) Protein and mRNA expression levels of Wnt5a in LNCaP cells with or without Wnt5a overexpression were tested by Western blot and RT-qPCR respectively. (G) Protein and mRNA expression of Wnt5a in 22RV1 cells with Wnt5a knockdown (Wnt5a-KD) or not (Control) were tested by Western blot and RT-qPCR respectively. (H) The proliferation capacity of LNCaP cells with Wnt5a overexpression or 22RV1 cells with Wnt5a knockdown were assessed by colony formation assay and (I) MTS assay. (J) Androgen-independent growth of LNCaP cells with Wnt5a overexpression or 22RV1 cells with Wnt5a knockdown were detected by colony formation assay and (K) MTS assay. *P < 0.05; **P < 0.01; ns, not significant

Fig. 2

Wnt5a promotes bicalutamide resistance of prostate cancer cells. (A, B) Comparison of the sensitivity of bicalutamide-resistant LNCaP cell line (LNCaP-BicR) and LNCaP parental cell line (LNCaP-Parental) to bicalutamide by MTS assay. (C) Protein and mRNA levels of Wnt5a in LNCaP-BicR cells and LNCaP-Parental cells were detected by Western blot analysis and RT-qPCR. (D) The inhibitory effect of bicalutamide on LNCaP cells with or without Wnt5a overexpression was tested by colony formation assay and (E, F) MTS assay. (G, H) The inhibitory effect of bicalutamide on 22RV1 cells with or without Wnt5a knockdown was tested by MTS assay. *P < 0.05; **P < 0.01

Fig. 3

Wnt5a affects steroid hormone anabolic pathway in PCa cells. (A) Volcano plot showing differentially expressed genes between LNCaP-Wnt5a and LNCaP-Control cells under hormone-deprived medium (HDM) culturing conditions. (B) Heat map displays the expression levels of the top 100 genes with the most significant differences. (C) Differential genes were enriched for the pathways with significant differences after KEGG analysis

Fig. 4

Wnt5a regulates expression of enzymes involved in steroid hormone biosynthesis. (A) Diagram of metabolites and enzymes involved in androgen biosynthesis, with enzymes differentially expressed in Wnt5a-overexpressed LNCaP cells marked in red. (B) Differential genes enriched in steroid hormone anabolic pathways and their expression ploidy changes in data of RNA-seq analysis (LNCaP-Wnt5a vs. LNCaP-Control). (C) RT-qPCR was employed to investigate the impact of Wnt5a on the expression levels of enzymes related to steroid hormone anabolic pathways in LNCaP cells and (D) 22RV1 cells. *P < 0.05; **P < 0.01; ns, not significant

Fig. 5

Detection of steroid hormones in LNCaP-Wnt5a and LNCaP-Control cells under HDM culturing condition. (A) Score scatter plot of PCA model for group LNP_Wnt (LNCaP cells with Wnt5a overexpression) vs. LNP_Ctrl (LNCaP control cells). (B) Score scatter plot of OPLS-DA model for group LNP_Wnt vs. LNP_Ctrl. (C) Correlation analysis of the four detected hormones in the indicated LNCaP cells. (D) Volcano plot, (E) Z-score plot, and (F) Boxplot analysis for group LNP_Wnt vs. LNP_Ctrl

Fig. 6

Wnt5a upregulates free cholesterol levels and activates AR signaling. (A) Enzymatic assays of total and free cholesterol in LNCaP and (B) 22RV1 cells with different Wnt5a expression levels. (C) Filipin staining was used to detect the content of free cholesterol in the indicated cells. (D) RT-qPCR was performed to detect the effect of Wnt5a on the expression levels of AR target genes in LNCaP and (E) 22RV1 cells. Data are presented as mean ± SD. *P < 0.05; **P < 0.01; ns, not significant

Fig. 7

PCSK9 inhibitor significantly reduced the risk of PCa. (A) The effect of PCSK9 inhibitor on the risk of coronary heart disease and PCa. OR, odds ratio; CI, confidence interval; PCSK9, proprotein convertase subtilisin/kexin 9; CHD, coronary heart disease; PC, prostate cancer. (B) Sensitivity analysis of PCSK9 on coronary heart disease (CHD) and (C) PCa