Next-generation sequencing of advanced prostate cancer treated with androgen-deprivation therapy

Abstract

Background: Androgen-deprivation therapy (ADT) is standard treatment for locally advanced or metastatic prostate cancer (PCa). Many patients develop castration resistance (castration-resistant PCa [CRPC]) after approximately 2-3 yr, with a poor prognosis. The molecular mechanisms underlying CRPC progression are unclear.

Objective: To undertake quantitative tumour transcriptome profiling prior to and following ADT to identify functionally important androgen-regulated pathways or genes that may be reactivated in CRPC.

Design, setting, and participants: RNA sequencing (RNA-seq) was performed on tumour-rich, targeted prostatic biopsies from seven patients with locally advanced or metastatic PCa before and approximately 22 wk after ADT initiation. Differentially regulated genes were identified in treatment pairs and further investigated by quantitative reverse transcription-polymerase chain reaction (qRT-PCR) on cell lines and immunohistochemistry on a separate CRPC patient cohort. Functional assays were used to determine the effect of pathway modulation on cell phenotypes.

Outcome measurements and statistical analysis: We searched for gene expression changes affecting key cell signalling pathways that may be targeted as proof of principle in a CRPC in vitro cell line model.

Results and limitations: We identified ADT-regulated signalling pathways, including the Wnt/β-catenin signalling pathway, and observed overexpression of β-catenin in a subset of CRPC by immunohistochemistry. We validated 6 of 12 (50%) pathway members by qRT-PCR on LNCaP/LNCaP-AI cell RNAs, of which 4 (67%) demonstrated expression changes consistent with RNA-seq data. We show that the tankyrase inhibitor XAV939 (which promotes β-catenin degradation) reduced androgen-independent LNCaP-AI cell line growth compared with androgen-responsive LNCaP cells via an accumulation of cell proportions in the G0/G1 phase and reduction in the S and G2/M phases. Our biopsy protocol did not account for tumour heterogeneity, and pathway inhibition was limited to pharmacologic approaches.

Conclusions: RNA-seq of paired PCa samples revealed ADT-regulated signalling pathways. Proof-of-principle inhibition of the Wnt/β-catenin signalling pathway specifically delays androgen-independent PCa cell cycle progression and proliferation and warrants further investigation as a potential target for therapy for CRPC.

Keywords: Androgen-deprivation therapy; Castration resistant; Prostate cancer; Wnt; β-catenin.

Fig. 1

Differential expression of genes after androgen-deprivation therapy. (A) Correlation between KLK3 messenger RNA transcript expression levels (x-axis) normalised by trimmed means of M value in normalised counts per million and serum prostate-specific antigen levels (nanograms per millilitre; y-axis). (B) Overlap between genes identified as twofold differentially expressed and previously reported microarray experiments , . (C) Hierarchical clustering of Kyoto Encyclopaedia of Genes and Genomics (KEGG) pathways enriched in upregulated gene sets. Dendrograms show clustering of KEGG terms based on the number of differentially expressed genes two pathways have in common as a proportion of the total number of differentially regulated genes in the two pathways combined. This shows, for example, that the set of cardiac-related pathways cluster with pathways connected to adhesion and matrix interaction. These categories are driven by differential expression of the integrin genes, which are known to be involved in cell–cell and cell–matrix adhesion . FDR = false discovery rate; PSA = prostate-specific antigen.

Fig. 2

Differential expression of genes encoding components of the Wnt/β-catenin–signalling pathway in individual patients. Fold change (logarithmic base 2) after androgen-deprivation therapy of genes in both up- and downregulated gene sets, annotated as being part of the Kyoto Encyclopaedia of Genes and Genomes term Wnt signalling pathway.

Fig. 3

Inhibition of the Wnt/β-catenin signalling pathway limits growth and delays cell cycle progression of LNCaP-AI cells. (A) WST-1 proliferation assays were performed using either LNCaP cells grown in full medium or LNCaP-AI cells grown in DCC. Cells were treated with 10 μm XAV939 in 0.1% dimethyl sulfoxide (DMSO) or vehicle. Values were normalised to the growth of LNCaP cells in full medium without any treatment. Data from at least three independent experiments were used to obtain the mean relative growth plus or minus standard deviation (SD) for each cell type and treatment condition. (B) Cell cycle analysis was performed using either LNCaP cells grown in full medium or LNCaP-AI cells grown in DCC. Cells were treated with 10 μm XAV939 in 0.1% DMSO or vehicle. Plots shown are representative of at least three independent experiments, from which (C) percentages of cells in each phase of the cell cycle were calculated for each cell type and treatment condition to obtain mean percentage plus or minus SD. BrdU = BrdU fluorescence intensity.