Figure 1.
Outline of the strategy employed in this study (see text for details).
Androgen-stimulated LNCaP cells were profiled using both quantitative iTRAQ MALDI- TOF/TOF and semi-quantitative MudPIT ESI- LTQ proteomics platforms. Data from each of these platforms were normalized independently and combined to generate a list of androgen-regulated proteins. This list was interrogated for biological associations using Molecular Concepts Mapping (MCM). The concept describing aminoacyl tRNA synthetases was selected for further examination; selected proteins were validated using immunoblot and immunofluorescence staining. Transcriptomic profiling and chromatin immunoprecipitation showed that androgen drives expression of aminoacyl-tRNA synthases at the transcript level. This was further confirmed using cancer tissue gene expression data and immunoblot analysis on prostate tissue samples, which demonstrated the existence of the elevated aminoacyl-tRNA synthase niche during prostate cancer progression.
Figure 2.
Total and androgen-regulated proteins identified from two mass-spectrometric platforms.
A) Venn diagram showing the number of total and the sub-sets of androgen-regulated proteins identified from iTRAQ MALDI TOF- MS/MS analysis. A minimum threshold of 1.25 SD from the global mean in both androgen-treated samples and not less than 1.5 SD in one of the androgen treated samples was set. B) Venn diagram showing the numbers of total and androgen-regulated LNCaP proteins identified in MudPIT (ESI ion trap- MS/MS) analysis. Proteins shown in brackets were not considered for differential analysis because of their lower spectral counts. C) Venn diagram showing the total number of proteins identified from two mass-spectrometric platforms and their overlap. D) Venn diagram showing the total number of androgen up-regulated proteins identified from two mass-spectrometric platforms and their overlap. E. Venn diagram showing the total number of androgen down-regulated proteins identified from two mass-spectrometric platforms and their overlap.
Figure 3.
Androgen-induced expression of LNCaP proteins.
A) Expression fold change of androgen-regulated proteins in mass-spectrometric quantitation compared to immunoblot assessment. Shown are the immunoblot bands and their intensities for androgen up-regulated proteins and their calculated intensity ratios as well as iTRAQ ratios and MudPIT spectral counts for the corresponding proteins. Beta-tubulin is used as a sample loading control. B) Immunofluorescence staining of AR (Red) and FASN (Green) following vehicle (ethanol) and 1 nM R1881 treatments for 48 h on LNCaP cells. Treatment was given after 48 h of androgen deprivation.
Figure 4.
Molecular concepts of androgen up-regulated proteins from two mass spectrometry platforms.
Network view of molecular concepts or sets of biologically related genes enriched in androgen up-regulated protein set obtained through Molecular Concepts Mapping (MCM) analysis. Each node represents a biological concept; the node size is proportional to the number of genes in each concept. Each edge represents a statistically significant enrichment. P-value of each concept and the MCM number of the concept are given in brackets. The most enriched concept is indicated by a thick edge. Red colored edges indicate enrichment of known prostate cancer-specific and androgen-regulated concepts. Interconnected aminoacyl tRNA synthetase concepts are indicated in blue edges at the bottom of the network.
Figure 5.
Androgen dependent up-regulation of aminoacyl tRNA synthetases in LNCaP, and their expression in prostate cancer.
A) Heat map showing the elevated expression of aminoacyl-tRNA synthetases identified from iTRAQ experiment. Only proteins in bold face are designated as androgen up-regulated in the iTRAQ data based on the threshold cut-off used. B) Heat map showing the concordant over-expression of transcripts measured using oligonucleotide microarray (Affymetrix U133 Plus 2.0) for aminoacyl-tRNA synthetases shown in A. C) Immunoblot analysis of aminoacyl t-RNA synthetases GARS, KARS, and WARS in response to androgen treatment in LNCaP cells. Beta-tubulin is used as loading control. D) Immunoblot analysis of KARS and GARS in localized prostate cancer (PCa), and metastatic prostate cancer (Met) compared to normal (benign) prostate tissues. Beta-actin is used as loading control. E) Promoter-occupancy of AR on aminoacyl tRNA genes. Chromatin immunoprecipitation (ChIP) -PCR analysis shows the androgen-dependent enrichment of AR-binding to the target promoters of GARS and KARS. Enrichment ratio was calculated based on the amount of target amplification against the input DNA, with primers for GAPDH promoter used as control. The primer sequences spanning the gene promoters are given in Table S5. F) Meta-analysis of the aminoacyl t-RNA synthetase genes (of proteins shown in A) across three prostate cancer gene expression profiling studies. Oncomine heat map view showing the over-expression of a subset of aminoacyl-tRNA synthetase genes in localized prostate cancer compared to their benign controls. Gene symbols of proteins that pass through the cut-off value and were assigned as androgen up-regulated in iTRAQ data are indicated in bold face. P-value represents the significance of expression in two of the three studies. Red, white, and blue (not present in the figure) indicates relative over, unchanged, and under expression, respectively.