Identification of androgen-coregulated protein networks from the microsomes of human prostate cancer cells
© Wright et al 2003
Received: 3 September 2003
Accepted: 5 December 2003
Published: 23 December 2003
Androgens play a critical role in the development of prostate cancer-dysregulation of androgen-regulated growth pathways can led to hormone-refractory prostate cancer. A comprehensive understanding of androgen-regulated cellular processes has not been achieved to date. To this end, we have applied a large-scale proteomic approach to define cellular processes that are responsive to androgen treatment in LNCaP prostate cancer cells.
Using isotope-coded affinity tags and mass spectrometry we identified and quantified the relative abundance levels of 1,064 proteins and found that distinct cellular processes were coregulated by androgen while others were essentially unaffected. Subsequent pharmacological perturbation of the cellular process for energy generation confirmed that androgen starvation had a profound effect on this pathway.
Our results provide evidence for the role of androgenic hormones in coordinating the expression of critical components involved in distinct cellular processes and further establish a foundation for the comprehensive reconstruction of androgen-regulated protein networks and pathways in prostate cancer cells.
Androgens are critical for the growth of normal and diseased prostate epithelium . Androgenic ligands bind and activate the androgen receptor (AR) leading to the induction of prostate-specific genes associated with cell growth and the differentiated prostate epithelial phenotype . The biological impact that androgens have on prostate epithelial cell function is underscored by the fact that androgen deprivation therapy is an established therapeutic regime in the treatment of advanced prostate cancer (PCa) . However, PCa that emerges after androgen deprivation therapy routinely re-activates the androgen-response pathway during the development of hormone refractory PCa [4–9]. Large-scale transcript profiling studies have been initiated in order to identify androgen-regulated genes in human PCa cell lines with the intent that these targets will elucidate the pathways that result in hormone refractory PCa growth [10–13]. However, transcript-expression profiles do not always reflect changes that occur at the level of the protein . As proteins are the biological effectors of cell function, an assessment of their quantity and activation state provides a more informative description of a cellular phenotype. Therefore, we have initiated a large-scale quantitative proteomic analysis to systematically identify and quantify androgen-mediated protein abundance changes in the human LNCaP prostate cancer cell line using the isotope-coded affinity tags (ICAT) and mass spectrometry [15, 16]. This investigation represents a first-step towards mapping a protein network regulated by androgens in neoplastic prostate epithelium.
Results and discussion
Identification and quantification of microsomal proteins
Androgenic hormones have been reported to regulate the expression of a large number of genes in the prostate . One of the best-characterized androgen responsive targets is prostate specific antigen (PSA), a secreted serine protease that is commonly used to detect early stage PCa in men . The PSA polypeptide sequence is predicted to generate six cysteine-containing tryptic peptides, all of which were identified and quantified by the ICAT method (Figure 1b,c). PSA was easily detectable in the microsomes of AS cells (Figure 1d). We extended the orthogonal verification of protein abundance ratios determined by quantitative mass spectrometry by subjecting a small group of proteins to semi-quantitative western blot analysis (Figure 1f-l). A four-fold increase in AS cell PSA expression was determined by the ICAT method (Figure 1e and panel 2, page 59 in Additional data file 1); a finding that was confirmed by Western analysis. An approximately eight-fold increase in the expression of the known androgen-regulated transmembrane serine protease 2 (TMPRSS2) level in AS cells was measured by the ICAT method, and Western analysis supported this result (Figure 1f) [18, 19]. Androgens are known to activate the lipogenic pathway in PCa cells [20–22]. The ICAT method demonstrated that fatty acid synthase (FAS) and low-density lipoprotein receptor (LDLR) proteins were increased by approximately three- and approximately 1.8-fold, respectively in AS cells (Figure 1g,h). β-catenin, a component of cellular adherens junctions  that also functions as a co-activator of AR mediated transcription [24–26], was increased approximately 1.3-fold in AS cells (Figure 1i), while the heat shock chaperonin 60 protein (HSP60), was increased approximately 1.7-fold in AD cells, as measured by the ICAT method (Figure 1j). The abundance ratios for these proteins were calculated from the assessments of at least three independent peptide quantifications, providing an opportunity to estimate the amount of error in the quantitative measurement (panel 2, page 59 in Additional data file 1). We also confirmed protein quantifications that were derived from single ICAT peptide ratios. For example, the human sterile 20 serine/threonine protein kinase 3 (mst-3)  and the Type II transmembrane protein Fas ligand (FasL)  were quantified based upon single ICAT peptide ratios (panel 3, page 60 in Additional data file 1). The ICAT method measured an approximately 1.8-fold increase in mst-3 levels in AS cells, while FasL levels were relatively unchanged between AD and AS cells, which was verified by immunoblot analysis (Figure 1k,l, respectively). While it is preferable to assess multiple ICAT-labeled peptides corresponding to a particular protein for optimal quantitative accuracy, these validation experiments demonstrate the validity of protein quantifications determined by single ICAT measurements.
Assignment of proteins into cellular process categories
Identification of androgen-sensitive and androgen-insensitive cellular processes
Assignment of the 1,064 proteins to a specific cellular process provided a unique opportunity to determine whether certain cellular processes are under the control of androgens in LNCaP cells. For example, androgens are known to regulate lipid metabolism in PCa cells [20–22], and a group of proteins in the 'lipid metabolism' cellular process contained ICAT ratios (d0:d8) that were consistent with the androgen-mediated stimulation of this cellular process (panel 5, pages 239-242 in Additional data file 1). This led us to investigate the distribution among cellular process categories of proteins showing expression change (up or down) greater than a predetermined threshold. For example, among the 1,064 proteins detected, 350 (33%) showed an androgen-response change greater than or equal to 1.6-fold (panel 6, page 243 in Additional data file 1). If these 350 proteins were distributed randomly among the cellular process categories, it would be expected that about one third of the proteins in each category would show expression change ≥1.6. Alternatively, there may be categories in which androgen-responsive proteins are over-represented, thus identifying androgen-responsive cellular processes. Also, there may be categories in which androgen-responsive proteins are under-represented, thus identifying cellular processes likely to be androgen-insensitive.
Androgen-sensitive and androgen-insensitive cellular processes
ICAT-labeled protein ratios ≥1.6
ICAT-labeled proteins in cellular process
Observed compared with expected
Lipid, fat and sterol metabolism
Coregulated protein expression associated with energy and lipid metabolism
Chemical perturbations of energy metabolism in prostate cancer cells
In this article we have applied a large-scale quantitative mass spectrometry approach to reconstruct the androgen-responsive protein network in human prostate cancer cells. This study represents the largest dataset of identified and quantified proteins from human cells to date. Our primary goal was to construct an androgen-regulated protein abundance map from the microsomal fraction of prostate cancer cells. Future analyses that quantify androgen-mediated protein abundance changes from the nuclear and cytoplasmic fractions will provide a more global perspective of the androgen-responsive protein networks in prostate cancer cells. From the current analysis we were able to identify both androgen-sensitive and androgen-insensitive cellular processes. Androgen-responsive cellular processes displayed a coregulated protein expression pattern in response to androgen, suggesting that these cellular processes were coordinately controlled and share a common gene expression regulatory network (for example, common transcription factor binding sites in their promoters) . The androgen-responsive proteins of the 'energy generation' cellular process were specifically increased in AD cells, suggesting these cells were metabolically stressed. Pharmacological perturbation with drugs that specifically blocked mitochondrial respiration induced higher levels of cell death in AD cells when compared with AS cells, thus validating the utility of applying a large-scale quantitative proteomic approach to uncover biological differences between two cellular states. Systematic analysis of the other androgen-responsive cellular processes may increase our understanding of androgen and AR signaling in the development of androgen-refractory PCa. For example, proteins comprising the 'RNA splicing' and 'RNA processing/modification' cellular processes were specifically increased (panel 9, pages 262-269 in Additional data file 1, categories 37 and 38) in AD cells. Interestingly, steroid receptors can coordinate transcription and splicing through their association with specific nuclear receptor coregulatory proteins . AR may coordinate a similar activity in prostate epithelial cells, as AR can physically interact with FLH2 , a tissue-specific AR co-activator, which can bind the polypyrimidine tract binding protein-associated splicing factor . Future studies will be required to elucidate the relationship, if any, between androgen and RNA splicing/metabolism in prostate cancer cells, given that many cancers are associated with abnormalities in alternative splicing .
We also identified several generally androgen-insensitive cellular processes, including 'vesicular transport' and 'protein synthesis'. However, androgens may still influence these pathways by regulating the expression of a few critical modulators. These processes may also be regulated through alternative regulatory mechanisms that involve post-translational modifications (for example, phosphorylation and ubiquitination). Future analyses will be required to substantiate and test these hypotheses more directly. The androgen coregulated protein expression patterns described in this study are consistent with the coregulated gene expression patterns detected by large-scale cDNA microarray analyses . Coregulated gene and protein networks may represent a common theme for coordinating cellular processes in the cell. Targeting and blocking the activity of specific regulators through RNA interference or chemical inhibitors will help uncover the biological consequences of these specific coregulated cellular processes found in this analysis. Robust characterization of existing cellular processes and the creation of new cellular process categories may uncover the function of unknown coregulated genes and proteins. Overall, utilizing a large-scale quantitative proteomic approach via the ICAT reagent method may elucidate analogous growth factor and hormone responsive pathways in other model systems.
Materials and methods
Preparation of microsomal fractions, ICAT reagent labeling and mass spectrometric analysis
Microsomes were prepared and isolated from androgen-depleted and androgen-stimulated LNCaP cells (109). Cells were exposed to either phenol-red deficient RPMI 1640 (Invitrogen, Carlsbad, CA) media supplemented with 10% charcoal-stripped fetal bovine serum (androgen-depleted, AD cells) (Hyclone Laboratories Inc., Logan, UT) or androgen-depleted media supplemented with 10 nM R1881 (methyltrienolone, Perkin Elmer Life Sciences, Boston, MA) (androgen-stimulated, AS cells) for 72 h. A total of 5.0 mg of microsomal proteins, 2.5 mg from AD cells and 2.5 mg from AS cells, were ICAT reagent labeled (AD, d0-ICAT reagent, AS, d8-ICAT reagent) as previously described  with the following modifications: microsomes were reduced with 5 mM tributylphosphine (TBP) for 3 h, and TBP reduced extracts were ICAT reagent labeled for 3 h and digested with trypsin overnight at 37°C. The 5 mg trypsin digested ICAT-reagent labeled peptide mixture was subjected to cation exchange, and then 53 cation-exchanged and avidin-purified ICAT-reagent labeled fractions were analyzed by microcapillary liquid chromatography electrospray ionization tandem mass spectrometry (μLC-ESI-MS/MS) .
SEQUEST, INTERACT and XPRESS scoring criterion
Uninterpreted MS/MS spectra were searched against a database of approximately 73,600 human sequences generated as a subset of a non-redundant amino acid database downloaded from the National Cancer Institute's Advanced Biomedical Computing Center . Tandem mass spectra were analyzed using the SEQUEST database search software. The database search criteria included a static modification of cysteine residues of 503 Da (mass of cysteine plus light ICAT reagent tag) and a variable modification of 8 Da for cysteines (for the heavy ICAT reagent tag). Additionally, searches were performed with no enzyme-constraint on the peptides analyzed from the sequence database. SEQUEST output was analyzed using the INTERACT program where peptides containing the following values were considered to be correct identifications: Xcorr > 1.5, Δ correlation scores ≥ 0.1, Sp rank ≤ 8, singly tryptic, contain at least one cysteine residue. This generated a list that contained 5,450 MS/MS spectra. All peptide identifications were confirmed by manual inspection of the data. Comparing these criteria to those generated from the PeptideProphet software , 93% of the entries had a probability of 0.99 or higher and 97% of the entries in the list had a probability of 0.90 or higher. All d0/d8 peptide ratios were analyzed and validated/corrected using the XPRESS  quantification program. Quantitative ratios are generated by taking the ratios of the calculated heavy and light elution peak areas for ICAT-reagent labeled peptides. As we have calculated ratios only for those ions that we have acquired and confirmed the identification of by MS/MS, we can also quantify what are termed 'singleton' peaks. This is because we always start with a positive identification for the singleton elution peak so the presence of a peak for the counterpart ion is not required.
Immunoblotting, immunofluorescence and microscopy
Microsomal protein lysates derived from 72 h AD and AS cells (16 μg) were subjected to immunoblot analysis using 1 μg/ml mouse anti-PSA (Research Diagnostics Inc. RDI-PSA85abm/RDI-PSA85abm-1), 1:1,500 dilution of rabbit anti-TMPRSS2, 1:200 dilution of rabbit anti-LDLR (Research Diagnostics Inc.), 1 μg/ml mouse anti-MST-3 (BD Transduction Laboratories clone 47), 1 μg/ml mouse anti-FAS (BD Transduction Laboratories clone 23), 2 μg/ml mouse anti-β-catenin (BD Transduction Laboratories clone 14), 1 μg/ml mouse anti-Fas ligand (BD Transduction Laboratories clone 13) and 0.05 μg/ml mouse anti-Hsp-60 (Stressgen clone LK1). 72 h AD, AS and LNCaP cells grown in phenol-red deficient RPMI 1640 containing 10% fetal bovine serum (steady-state cells, SS cells) were labeled with the Mitotracker® Red CMXRos and Alexa Fluor 488 phalloidin and DAPI (4',6-diamidino-2-phenylindole, dihydrochloride) conjugates (Molecular Probes, Eugene, OR) according to the manufacturers instructions . Fluorescent images were acquired on an DeltaVision microscope (Applied Precision, Inc., Issaquah, WA) and image stacks were deconvolved using SoftWoRx 2.5 (API). All images were processed at the Scientific Imaging Lab, Fred Hutchinson Cancer Research Center. Light microscope photographs of cells were imaged with Fujifilm Super HQ 35 mm film (100 speed film) at 10X on an Olympus IX70 inverted microscope.
Cell death assay
LNCaP cells were seeded and exposed to three growth conditions for 72 hours prior to a 24 hour incubation with the respiratory chain inhibitors. These three growth conditions included normal growth medium (steady-state, SS; androgen-depleted, AD and androgen-stimulated, AS). 72 h AD, AS and SS cells were incubated with the respiratory chain inhibitors rotenone (10 μM), carbonyl cyanide m-chlorophenylhydrazone (CCCP) (50 μM), antimycin A (10 μg/ml), oligomycin (5 μg/ml), for 24 h. Untreated cells were incubated with vehicle (ethanol) for 24 h. SS cells were seeded at a density of 1 × 103 cells/cm2 while AD and AS cells were seeded at a density of 3 × 103 cells/cm2 to generate equivalent cell densities at the time of drug addition. Cell death was determined using the trypan-blue dye assay . At least 200 cells were counted in triplicate for all experiments. All drugs were purchased from Sigma.
Assignment of identified proteins into cellular process categories
All identified proteins that contained a Swiss-Prot annotation were searched at the Incyte Human Proteome Database  and assigned to one or several cellular process categories if applicable. All remaining proteins that contained a Genbank GenPept accession number were searched at the Incyte Human Proteome Database and assigned to a cellular process category if applicable. Non-annotated proteins were assigned to the cellular process category described as 'unknown process'.
Additional data file
We thank our colleague David K Han for helpful discussions and critical reading of the manuscript. We also thank Adrian Quintanilla for assistance with the scientific imaging, and Masazumi Matsumara for rabbit TMPRS22 antibodies. This work was supported by grants from the National Institutes of Health to R.A. (1R33CA-93302) and the Damon Runyon Cancer Research Foundation to P.S. N. A gift from Merck and Co. to support research at the Institute for Systems Biology is gratefully acknowledged. M.E.W. was supported in part by a NIH Postdoctoral Genome Training Fellowship Grant. M.E.W. is a United Negro College Fund/Merck postdoctoral research fellow.
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