Metabolomic profiling reveals a role for androgen in activating amino acid metabolism and methylation in prostate cancer cells

Abstract

Prostate cancer is the second leading cause of cancer related death in American men. Development and progression of clinically localized prostate cancer is highly dependent on androgen signaling. Metastatic tumors are initially responsive to anti-androgen therapy, however become resistant to this regimen upon progression. Genomic and proteomic studies have implicated a role for androgen in regulating metabolic processes in prostate cancer. However, there have been no metabolomic profiling studies conducted thus far that have examined androgen-regulated biochemical processes in prostate cancer. Here, we have used unbiased metabolomic profiling coupled with enrichment-based bioprocess mapping to obtain insights into the biochemical alterations mediated by androgen in prostate cancer cell lines. Our findings indicate that androgen exposure results in elevation of amino acid metabolism and alteration of methylation potential in prostate cancer cells. Further, metabolic phenotyping studies confirm higher flux through pathways associated with amino acid metabolism in prostate cancer cells treated with androgen. These findings provide insight into the potential biochemical processes regulated by androgen signaling in prostate cancer. Clinically, if validated, these pathways could be exploited to develop therapeutic strategies that supplement current androgen ablative treatments while the observed androgen-regulated metabolic signatures could be employed as biomarkers that presage the development of castrate-resistant prostate cancer.

Figure 1. Metabolomic profiling of prostate cell lines.

Illustration of the various steps involved in metabolomic profiling of prostate cell lines. The major steps involved metabolite extraction and separation, mass spectrometry-based detection, spectral analysis, data normalization, delineation of class-specific metabolites and altered pathways and their functional characterization. The variation in sample extraction, separation and mass spectrometry were controlled using spiked standards and assessed using various quality control parameters (Figure S1).

Figure 2. Metabolome of prostate cancer cell lines.

A) Heat map representation of hierarchical clustering of 1,553 metabolites across 5 prostate cell lines. Sample classes are indicated by the colored bars [benign = green, androgen non-responsive PCa (ARI): yellow and androgen responsive PCa (ARD): red bar]. Columns represent individual cell lines and rows refer to distinct metabolites. Shades of yellow represent elevation of a metabolite and shades of blue represent decrease of a metabolite relative to the median metabolite levels (see color scale). B) Venn diagram representing the distribution of 1,553 metabolites measured across benign (RWPE), ARI (DU145 and PC3) and ARD (LNCaP and VCaP) cell lines using liquid chromatography coupled mass spectrometry. C) same as in (A), but for 72 named compounds D) Dendrogram representing hierarchical clustering of the prostate-related cell lines described in B, using compounds with significant differential expression.

Figure 3. Metabolic alterations in prostate cancer cell lines.

A) Heat map showing 29 named differential metabolites in prostate cancer cells relative to benign cell line (p<0.05, FDR = 20%), derived using a t-test coupled with permutations (n = 1,000) to determine the p-values for comparing the groups. The heat map was generated after log scaling and quantile normalization of the data. The color scheme is the same as in Fig. 2A . B) same as in (A) but for 52 named differential metabolites between ARI (yellow bar) and ARD (red bar) prostate cancer cells. C) Network view of the molecular concept analysis for the metabolomic profiles of our “altered in PCa cell line signature” (grey node). Each node represents a molecular concept or a set of biologically related genes. The node size is proportional to the number of genes in the concept. Each edge represents a statistically significant enrichment (FDR q-value<0.2). Enriched concepts describing “amino acid metabolism” are indicated by yellow bridges.

Figure 4. Metabolic alterations upon androgen-treatment of prostate cancer cells.

A) Dendrogram representing unsupervised hierarchical clustering of the androgen-treated and control cells using their total metabolic profiles. B) Heat map showing 48 named differential metabolites that are altered in VCaP prostate cancer cells 24 h after treatment with 10 nM synthetic androgen (R1881) compared to untreated controls, (p<0.05, FDR = 20%), derived using a t-test coupled with permutations (n = 1,000) to determine the p-values for comparing the groups. The heat map was generated after log scaling and quantile normalization of the data. The color scheme is the same as in Fig. 2A . C) same as in (B) but for metabolic alterations after 48 h of androgen-treatment D) Correlation plot for metabolites altered at 24 h and 48 h post-androgen treatment in VCaP prostate cancer cells E) Network view of the molecular concept analysis for the metabolomic profiles of our “Androgen-induced metabolomic signature” (grey node). Each node represents a molecular concept or a set of biologically related genes. The node size is proportional to the number of genes in the concept. Each edge represents a statistically significant enrichment (FDR q-value<0.2). Enriched concepts describing “amino acid metabolism” and “methylation potential” are indicated by yellow and red bridges respectively.

Figure 5. Measurement of metabolic flux in prostate cancer cells upon androgen-treatment.

Heat map showing extent of utilization 86 carbohydrate and nucleotide substrates by prostate cancer cells after 24 h of androgen-treatment. Essentially, 10 nM R1881 (treated for 24 h) or untreated VCaP cells were examined in replicates for their ability to utilize the various nutrient substrates (denoted as pathway activity on Y-axis) at different time points (plotted on X-axis) post-androgen-treatment. The ability to metabolize the nutrients was assessed by calculating the flux of NADH generated which was measured by reading the optical density (OD) at 590 nm. The heat map was generated after background subtraction, log scaling and quantile normalization of the data. Shades of yellow indicate increased activity while shades of blue indicate reduced activity (refer color scale). B) same as in (A), but for utilization of 29 amino acids. C) represents the box plot showing the median flux through the sugar and nucleotide (pink) as well as amino acid (brown) pathways in prostate cancer cells at different time points post-androgen treatment. For each boxplot, the median value is represented by the central, horizontal line; the upper (75%) and lower (25%) quartiles are represented by the upper and lower borders of the box. The upper and lower vertical lines extending from the box represent 1.5 times the inter-quartile range from the upper and lower quartiles.