Purpose: The high prevalence of osteoblastic bone metastases in prostate cancer involves the production of osteoblast-stimulating factors by prostate cancer cells. Prostate-specific antigen (PSA) is a serine protease uniquely produced by prostate cancer cells and is an important serologic marker for prostate cancer. In this study, we examined the role of PSA in the induction of osteoblast differentiation.

Experimental Design: Human cDNA containing a coding region for PSA was transfected into human osteosarcoma SaOS-2 cells. SaOS-2 cells were also treated with exogenously added PSA. We evaluated changes in global gene expression using cDNA arrays and Northern blot analysis resulting from expression of PSA in human osteosarcoma SaOS-2 cells.

Results: SaOS-2 cells expressing PSA had markedly up-regulated expression of genes associated with osteoblast differentiation including runx-2 and osteocalcin compared with the controls. Consistent with these results, the stable clones expressing PSA showed increased mineralization and increased activity of alkaline phosphatase in vitro compared with controls, suggesting that these cells undergo osteoblast differentiation. We also found that osteoprotegerin expression was down-regulated and that the receptor activator of NF-κB ligand expression was up-regulated in cells expressing PSA compared with controls.

Conclusions: Modulation of the expression of osteogenic genes and alteration of the balance between osteoprotegerin–receptor activator of NF-κB ligand by PSA suggests that PSA produced by metastatic prostate cancer cells may participate in bone remodeling in favor of the development of osteoblastic metastases in the heterogeneous mixture of osteolytic and osteoblastic lesions. These findings provide a molecular basis for understanding the high prevalence of osteoblastic bone metastases in prostate cancer.

Bone is the frequent site of many types of cancer metastasis including prostate cancer. Advanced prostate cancer is frequently accompanied by the development of unique bone metastases characterized as osteoblastic (bone forming), which results in significant complications including bone pain, fractures, and spinal cord compression, even hemiparesis, leading to morbidity with no curable treatment (13). Although osteoblastic lesions are the most dominant bone metastases associated with prostate cancer, osteoclastic lesions (bone resorption) also infrequently occur in prostate bone metastases (4, 5). In contrast, cancers from other tissues that metastasize to bone are frequently associated with osteoclast formation. Osteoblastic characterization associated with prostate bone metastases suggests that factors derived from prostate cancer cells which influence bone remodeling may be unique from other types of cancer cells.

Several osteogenic factors produced by prostate cancer cells have been identified including bone morphogenetic proteins (BMP; refs. 6, 7), endothelin-1 (8), insulin-like growth factors (IGF; ref. 9), parathyroid hormone–related peptide (10), transforming growth factor-β (TGF-β; ref. 11), and prostate-specific antigen (PSA; refs. 11, 12). Among them, only PSA is uniquely produced and secreted abundantly by prostate cancer cells, although recently, trace amounts of PSA were found to be produced by breast cancer cells. PSA is a widely used serologic marker for prostate cancer. The physiologic functions of PSA are largely unknown. PSA is a serine protease which could cleave the parathyroid hormone–related peptide, resulting in abolishment of the ability of parathyroid hormone–related peptide to stimulate cyclic AMP production leading to a decrease in bone resorption (13). PSA could enhance IGF function by cleaving IGF-binding protein and activating latent TGF-β, leading to an increase in bone formation (14). These studies suggest that PSA may be involved in prostate cancer bone metastases associated with the osteoblastic phenotype.

In the present study, we examined the role of PSA in the differentiation of human osteosarcoma SaOS-2 cells by ectopically expressing a cDNA encoding human PSA. Using cDNA arrays and Northern blot analysis, we evaluated the changes in global gene expression resulting from the expression of PSA in human osteosarcoma SaOS-2 cells. We found a number of osteogenic genes induced by PSA to be implicated in bone remodeling, and unveiled a molecular basis for prostate cancer bone metastases associated with osteoblastic phenotype.

Cell culture. Human SaOS-2 osteosarcoma cells were obtained from American Type Culture Collection (Manassas, VA) and maintained in RPMI 1640 supplemented with 10% fetal bovine serum. The cells were grown at 37°C in 5% CO2 and 95% air.

Cloning of human PSA cDNA and construction of PSA expression vectors. Human PSA cDNA was cloned by RT-PCR from the LNCaP human prostate cancer cells. Total RNAs were isolated by TRIzol kit (Life Technologies, Inc., Gaithersburg, MD) and cDNAs were reverse-transcribed by using first-strand cDNA synthesis reagents (Pharmacia, Piscataway, NJ) according to the manufacturer's instructions. A full open reading frame of the human PSA cDNA was isolated by PCR using primers forward (5′-GATGACTCCAGCCACGACCT) and reverse (5′-CACAGACACCCCATCCTATC) based on the human PSA cDNA sequence. Briefly, 5 μg of total RNA was reverse-transcribed and 2 μL of the reverse transcription reaction mix was amplified. The PCR was done for 30 seconds at 94°C, 1 minute at 55°C, and 2 minutes at 72°C for 35 cycles. The PCR products were cloned into pCR II vector (Invitrogen, San Diego, CA) and subcloned into pCDNA3.1+ (Invitrogen). The sense and antisense PSA constructs were selected and confirmed by DNA sequencing using DNasequenaseII in accordance with the manufacturer's instructions (Amersham, Arlington Heights, IL).

Northern blot. Total RNA was extracted from cells with TRIzol reagent (Life Technologies, Rockville, MD). Twenty micrograms of each sample were electrophoresed in 1.2% denaturing agarose gels and transferred to a nylon membrane (MSI, Westborough, MA). A 1.2-kb BamHI fragment of the PSA cDNA was labeled with [α-32P]dCTP (3,000 Ci/mmol, ICN, Costa Mesa, CA) using the Ready-To-Go DNA Labeling Beads (Amersham Pharmacia Biotech, Piscataway, NJ). Hybridization was carried out for 3 hours at 65°C in Rapid-hyb buffer (Amersham). Membranes were washed for 15 minutes at 65°C in 2× SSC, 0.1% SDS (twice), 0.5× SSC, 0.1% SDS, and 0.1× SSC, 0.1% SDS. Radioactivity in the membranes was analyzed with a Molecular Imager FX System (Bio-Rad, Hercules, CA).

PSA protein analysis. PSA secretion was quantitated by ELISA with the use of anti-PSA as primary antibody as described by the manufacturer's protocol (Beckman Coulter, Fullerton, CA). An equal number of cells were plated in phenol red–free RPMI containing 10% fetal bovine serum. Cells were allowed to grow for 24 hours and 50 μL of supernatant was assayed for PSA.

Production of cDNA microarrays. The 6K Cancer-Specific arrays used in this experiment were produced at the RPCI Microarray and Genomics Core Facility. A total of 6,116 cDNA clones (Research Genetics, Huntsville, AL) were selected based on their association with oncogenesis. Each clone was amplified from 100 ng plasmid DNA by performing PCR amplification of the insert using M13 universal primers for the plasmids represented in the clone set (5′-TGAGCGGATAACAATTTCACACAG-3′, 5′-GTTTTCCCAGTCACGACGTTG-3′). Each PCR product (75 μL) was purified by ethanol precipitation, resuspended in 25% DMSO and adjusted to 200 ng/μL. The PCR amplicons were spotted in duplicate on type A glass slides (Schott North American, Inc., Elmsfor, NY) using a MicroGrid II TAS arrayer and MicroSpot 2500 split pins (Genomic Solutions, Inc., Ann Arbor, MI).

Preparation and hybridization of fluorescent-labeled cDNA. To screen the samples for gene expression, cDNA was synthesized and indirectly labeled using the Atlas Powerscript Fluorescent Labeling Kit (BD BioSciences, San Jose, CA). Total RNA isolated from the transfected cells was labeled with Cy5 or Cy3. For each reverse transcription reaction, 2.5 μg total RNA was mixed with 2 μL random hexamer primers (Invitrogen) in a total volume of 10 μL, heated to 70°C for 5 minutes and cooled to 42°C. An equal volume of reaction mix was added to this sample (4 μL 5× first-strand buffer, 2 μL 10× deoxynucleotide triphosphate mix, 2 μL DTT, 1 μL of deionized H2O, and 1 μL Powerscript reverse transcriptase) according to the manufacturer's instructions. After 1 hour of incubation at 42°C, the reverse transcriptase was inactivated by incubating at 70°C for 5 minutes. The mixture was cooled to 37°C and incubated for 15 minutes with 0.2 μL RNase H (10 units/μL). The resultant amino-modified cDNA was purified, precipitated, and fluorescently labeled as described by the manufacturer's instructions. Uncoupled dye is removed by washing twice using a Qiaquick PCR Purification Kit (Qiagen, Chatsworth, CA). The probe is eluted in 60 μL elution buffer and dried down to completion in a SpeedVac.

Prior to hybridization, the two separate probes were resuspended in 10 μL dH2O, combined and mixed with 2 μL of human Cot-1 DNA (20 μg/μL, Invitrogen) and 2 μL of polyadenylic acid (20 μg/μL, Sigma, St. Louis, MO). The probe mixture was denatured at 95°C for 5 minutes, placed on ice for 1 minute and prepared for hybridization with the addition of 110 μL of preheated (65°C) SlideHyb no. 3 buffer (Ambion). After a 5-minute incubation at 65°C, the probe solution was placed on the array in an assembled GeneTAC hybridization station module (Genomic Solutions, Inc.). The slides were incubated overnight at 55°C for 16 to 18 hours with occasional pulsation of the hybridization solution. After hybridization, the slides were automatically washed in the GeneTAC station with reducing concentrations of SSC and SDS. The final wash was 30 seconds in 0.1× SSC, followed by a 5-second 100% ethanol dip. Two hybridizations for each RNA sample were done, switching the dyes in the second hybridization to account for possible dye bias.

Microarray image and data analysis. The hybridized slides were scanned using a GenePix 4200A scanner (Molecular Devices Corp., Sunnyvale, CA) to generate high-resolution (10 μm) images for both the Cy3 and Cy5 channels. Image analysis was done using the ImaGene program (version 6.0.1, BioDiscovery, Inc., El Segundo, CA). Each cDNA spot is defined by a circular region. For each spot, the size of which is programmed to adjust to match the size of the spot. The local background for a spot is determined by ignoring a two- to three-pixel buffer region around the spot and then measuring the signal intensity in a two- to three-pixel–wide area outside the buffer region. Raw signal intensity values for each spot and its background region are segmented using a proprietary segmentation algorithm, which excludes pixels that are not representative of the majority of pixels in that region. The background-corrected signal for each cDNA spot was obtained by subtracting the mean local background from the mean signal of all the pixels in the region. The output of the image analysis is two raw fluorescence data files, one for each channel, and is further processed by the RPCI-developed Perl program. Spots that are not significantly above background or have a poor coefficient of variance are excluded. For each spot, a ratio is calculated from the background subtracted mean signal of the two channels. The ratios are then normalized on the log scale across the entire slide using a linear normalization algorithm. For each slide, the expression ratios are displayed as the log2 mean of all replicate spots. The results from the two slides that make up the dye flip are then averaged on the log scale and becomes the final expression ratio of that clone.

Gene annotation and classification. The accession numbers of the cDNAs on the microarray were first mapped to the UniGene database (build 184) at the National Center for Biotechnology Information to extract full information of the genes represented by these cDNA sequences. The UniGene cluster IDs of these genes were then used to query the SOURCE database at Stanford University (http://source.stanford.edu/cgi-bin/source/sourceSearch) to extract the Gene Ontology terms associated with these genes. The genes were classified into 11 categories based on their Gene Ontology annotations. These analyses were carried out using custom Perl scripts. Due to space limitations, we could only list the 10 most modulated genes in each category. These genes are ranked by the absolute values of their folds of changes. The full list can be found at our web site (http://falcon.roswellpark.org/publication/Gao/).

von Kossa staining. SaOS-2 cells, vector controls, and PSA-expressing clones were seeded at 105 cells/plate in 60 mm plates in medium supplemented with 1.4 mmol/L CaCl2, 10 nmol/L dexamethasone, and 50 μg/mL ascorbic acid. β-Glycerophosphate (10 mmol/L) was added to the plates on day 8 and incubation continued for 3 more days. At the end of 11 days, cells were fixed in neutral buffered formalin and stained in situ using the standard von Kossa technique. Briefly, cells were fixed overnight and subjected to dehydration using ethanol gradients of 70%, 90%, and 100%. The plates were rinsed in distilled water and stained with 5% silver nitrate solution by placing in front of a UV light source until calcium deposits turned black. Plates were then rinsed in distilled water with three changes and treated with 5% sodium thiosulfate for 5 minutes, rinsed in distilled water and allowed to dry. Plates were stored in the dark for analysis.

Treatment of SaOS-2 cells with PSA. Enzymatically active PSA was obtained from Calbiochem (La Jolla, CA). SaOS-2 cells were grown in RPMI supplemented with 10% fetal bovine serum and 100 units/mL penicillin and 100 μg/mL streptomycin. Cells were plated in 24-well plates at a density of 5 × 104 cells/well and treated after 24 hours with 0 to 1,000 ng/mL PSA in differentiation medium containing 1.4 mmol/L CaCl2, 10 nmol/L dexamethasone, 50 μg/mL ascorbic acid, and 10 mmol/L β-glycerophosphate. The cells were incubated at 37°C for 4 days with PSA being replenished every 36 hours.

Alkaline phosphatase activity assay. After PSA treatment, alkaline phosphatase activity was assayed in cell lysates by determining the release of p-nitrophenol from p-nitrophenyl phosphate using Sigma Fast p-nitrophenyl phosphate tablet sets according to the manufacturer's instructions. Cells were lysed in 20 mmol/L Tris-HCl (pH 7.4), 0.1 mmol/L ZnCl2, 1 mmol/L MgCl2, and 0.5% Triton X-100 and the release of p-nitrophenol was determined by measuring the absorbance at 405 nm over a period of 4 minutes. Total protein content was determined using Coomassie blue protein assay reagent (Pierce, Rockford, IL) and alkaline phosphatase activity was normalized to milligrams of protein. The experiments were repeated at least thrice.

Generation of PSA expressing osteosarcoma SaOS-2 cells. Human osteosarcoma SaOS-2 cells were transfected with cDNA encoding a full-length PSA protein under a cytomegalovirus promoter. Clones were selected in G418 and PSA mRNA expression was assessed by Northern blots and PSA protein secreted in the medium was assessed by ELISA. Several PSA-producing clones and vector controls were selected and PSA expression was shown in Fig. 1. Northern blot analysis revealed that PSA-producing clones express PSA mRNA; parental SaOS-2 and vector control lack PSA expression (Fig. 1). These clones also express ∼500 ng/mL of secreted PSA in the medium (Fig. 1).

Fig. 1.

PSA expression in SaOS-2, vector (V), and PSA cDNA-transfected clones (C12, C14, and C21). Top, Northern blot analysis using PSA cDNA labeled probe. Glyceraldehyde-3-phosphate dehydrogenase (GAPDH) was a RNA loading control. The levels of PSA protein secretion were measured by ELISA and expressed as ng/mL of medium.

Fig. 1.

PSA expression in SaOS-2, vector (V), and PSA cDNA-transfected clones (C12, C14, and C21). Top, Northern blot analysis using PSA cDNA labeled probe. Glyceraldehyde-3-phosphate dehydrogenase (GAPDH) was a RNA loading control. The levels of PSA protein secretion were measured by ELISA and expressed as ng/mL of medium.

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Identification of PSA-regulated genes in osteosarcoma SaOS-2 cells using cDNA microarrays. To investigate whether PSA regulates genes involved in bone remodeling, we investigated genome-wide changes in gene expression in PSA-producing and PSA-nonproducing vector control osteosarcoma SaOS-2 cells. To control for biological and experimental variation, the same cell numbers of PSA-producing and vector control cells were seeded. Total RNA was extracted when the cells reached ∼90% confluence and were used for microarray analysis using the 6K Cancer-Specific arrays. Differentially expressed genes between PSA-producing and vector control SaOS-2 cells were identified. A treatment to control signal ratio of ≥2 or ≤0.5 was chosen as the criteria for induction or repression, respectively. We identified total of 1,000 genes that are differentially regulated by PSA expression in SaOS-2 cells. Among them, the expression of ∼454 genes are increased by PSA that were considered significant and ∼400 genes are repressed by PSA. The top 50 genes in each of these two groups are shown in Tables 1 and 2, respectively. Complete lists of the significant up-regulated and down-regulated genes are included in the Supplementary Data. The PSA regulated genes were then grouped into distinct known biological functions including signal transduction, cell death, cell motility, metabolism, cell growth and/or maintenance, and transporter functions. Functional annotation of transcripts was done by using the Gene Ontology database and literature review (Table 3).

Table 1.

Top 50 up-regulated genes

Accession no.Unigene IDSymbolDescriptionFold of change
R55809 Hs.284122 WIF1 WNT inhibitory factor 1 31.12 
R53942 Hs.246506 SLC25A4 Solute carrier family 25, member 4 28.43 
AA858175 Hs.535845 RUNX2 Runt-related transcription factor 2 25.80 
AA156964 Hs.471200 NRP2 Neuropilin 2 19.22 
AA425628 Hs.77578 USP9X Ubiquitin specific protease 9, X-linked (fat facets-like, Drosophila17.23 
H06516 Hs.212838 A2M α-2-macroglobulin 16.64 
T51538 Hs.368592 SORL1 Sortilin-related receptor, L(DLR class) A repeats-containing 14.85 
AA677306 Hs.158560 TAF1 TAF1 RNA polymerase II, TATA box binding protein (TBP)-associated factor, 250 kDa 13.32 
AI016456 Hs.333497 CYP2D7P1 Cytochrome P450, family 2, subfamily D, polypeptide 7 pseudogene 1 13.28 
AA442092 Hs.476018 CTNNB1 Catenin (cadherin-associated protein), β 1, 88 kDa 13.15 
AA055979 Hs.524484 ITGA7 Integrin, α 7 13.06 
AA490459 Hs.417948 TCN2 Transcobalamin II; macrocytic anemia 12.68 
AA464532 Hs.164226 THBS1 Thrombospondin 1 12.53 
AA705237 Hs.370480 ABCB7 ATP-binding cassette, subfamily B (MDR/TAP), member 7 12.20 
AA676805 Hs.336994 MTSS1 Metastasis suppressor 1 11.36 
AA404486 Hs.522767 SLC25A5 Solute carrier family 25, member 5 10.28 
AA487231 Hs.148641 CTSH Cathepsin H 10.14 
H96738 Hs.116471 CDH11 Cadherin 11, type 2, OB-cadherin (osteoblast) 9.82 
AA446600 Hs.136905 UREB1 HECT, UBA and WWE domain containing 1 9.70 
AA227885 Hs.80395 MAL Mal, T cell differentiation protein 8.97 
N29914 Hs.82002 EDNRB Endothelin receptor type B 8.93 
AA810225 Hs.552573 GPR30 G protein–coupled receptor 30 8.39 
AA452149 Hs.173135 DYRK2 Dual-specificity tyrosine-(Y)-phosphorylation regulated kinase 2 8.34 
AA443093 Hs.533683 FGFR2 Fibroblast growth factor receptor 2 (bacteria-expressed kinase, keratinocyte growth factor receptor, craniofacial dysostosis 1, Crouzon syndrome, Pfeiffer syndrome, Jackson-Weiss syndrome) 8.29 
AA682819 Hs.470171 ACVR2 Activin A receptor, type II 8.26 
AA970865 Hs.21145 ODAG GATA zinc finger domain containing 1 7.48 
AA775616  SPP1 Secreted phosphoprotein 1 (osteopontin, bone sialoprotein 1) 7.45 
N69689 Hs.310645 RAB1A RAB1A, member RAS oncogene family 7.40 
R85213 Hs.22543 UBE3A Ubiquitin protein ligase E3A (human papilloma virus E6-associated protein, Angelman syndrome) 7.28 
AA706987 Hs.514806 GALNT1 UDP-N-acetyl-α-d-galactosamine: polypeptide N-acetylgalactosaminyltransferase 1 (GalNAc-T1) 7.28 
R41839 Hs.154073 SLC35B1 Solute carrier family 35, member B1 7.27 
N71628 Hs.437905 SPIB Spi-B transcription factor (Spi-1/PU.1 related) 7.26 
AI129421 Hs.83077 IL18 Interleukin 18 (IFN-γ-inducing factor) 7.25 
AA101617 Hs.220971 FOSL2 FOS-like antigen 2 7.18 
AA180742 Hs.75318 TUBA1 Tubulin, α 1 (testis specific) 7.15 
AA446108 Hs.76753 ENG Endoglin (Osler-Rendu-Weber syndrome 1) 7.11 
T60048 Hs.516105 ACTG2 Actin, γ 2, smooth muscle, enteric 7.06 
AA454585 Hs.73965 SFRS2 Splicing factor, arginine/serine-rich 2 6.96 
AA418813 Hs.309090 SFRS7 Splicing factor, arginine/serine-rich 7, 35 kDa 6.93 
AA626845 Hs.380774 DDX3X DEAD (Asp-Glu-Ala-Asp) box polypeptide 3, X-linked 6.82 
T66839 Hs.325846 FLJ11806 Nuclear protein UKp68 6.78 
T50498 Hs.433795 SHC1 SHC (Src homology 2 domain containing) transforming protein 1 6.73 
AA496691 Hs.76111 DAG1 Dystroglycan 1 (dystrophin-associated glycoprotein 1) 6.72 
AA598670 Hs.533273 UBE1 Ubiquitin-activating enzyme E1 (A1S9T and BN75 temperature sensitivity complementing) 6.63 
AA281784 Hs.518451 PIK3CD Phosphoinositide-3-kinase, catalytic, δ polypeptide 6.37 
H20138 Hs.503222 RAB6A RAB6A, member RAS oncogene family 6.35 
AA912448 Hs.46523 ELK3 ELK3, ETS-domain protein (SRF accessory protein 2) 6.33 
N68166 Hs.185172 GNB2 Guanine nucleotide binding protein (G protein), β polypeptide 2 6.22 
AI392759 Hs.515840 DNMT3A DNA (cytosine-5-)-methyltransferase-3α 6.21 
AA281635 Hs.411311 IL24 Interleukin 24 6.16 
Accession no.Unigene IDSymbolDescriptionFold of change
R55809 Hs.284122 WIF1 WNT inhibitory factor 1 31.12 
R53942 Hs.246506 SLC25A4 Solute carrier family 25, member 4 28.43 
AA858175 Hs.535845 RUNX2 Runt-related transcription factor 2 25.80 
AA156964 Hs.471200 NRP2 Neuropilin 2 19.22 
AA425628 Hs.77578 USP9X Ubiquitin specific protease 9, X-linked (fat facets-like, Drosophila17.23 
H06516 Hs.212838 A2M α-2-macroglobulin 16.64 
T51538 Hs.368592 SORL1 Sortilin-related receptor, L(DLR class) A repeats-containing 14.85 
AA677306 Hs.158560 TAF1 TAF1 RNA polymerase II, TATA box binding protein (TBP)-associated factor, 250 kDa 13.32 
AI016456 Hs.333497 CYP2D7P1 Cytochrome P450, family 2, subfamily D, polypeptide 7 pseudogene 1 13.28 
AA442092 Hs.476018 CTNNB1 Catenin (cadherin-associated protein), β 1, 88 kDa 13.15 
AA055979 Hs.524484 ITGA7 Integrin, α 7 13.06 
AA490459 Hs.417948 TCN2 Transcobalamin II; macrocytic anemia 12.68 
AA464532 Hs.164226 THBS1 Thrombospondin 1 12.53 
AA705237 Hs.370480 ABCB7 ATP-binding cassette, subfamily B (MDR/TAP), member 7 12.20 
AA676805 Hs.336994 MTSS1 Metastasis suppressor 1 11.36 
AA404486 Hs.522767 SLC25A5 Solute carrier family 25, member 5 10.28 
AA487231 Hs.148641 CTSH Cathepsin H 10.14 
H96738 Hs.116471 CDH11 Cadherin 11, type 2, OB-cadherin (osteoblast) 9.82 
AA446600 Hs.136905 UREB1 HECT, UBA and WWE domain containing 1 9.70 
AA227885 Hs.80395 MAL Mal, T cell differentiation protein 8.97 
N29914 Hs.82002 EDNRB Endothelin receptor type B 8.93 
AA810225 Hs.552573 GPR30 G protein–coupled receptor 30 8.39 
AA452149 Hs.173135 DYRK2 Dual-specificity tyrosine-(Y)-phosphorylation regulated kinase 2 8.34 
AA443093 Hs.533683 FGFR2 Fibroblast growth factor receptor 2 (bacteria-expressed kinase, keratinocyte growth factor receptor, craniofacial dysostosis 1, Crouzon syndrome, Pfeiffer syndrome, Jackson-Weiss syndrome) 8.29 
AA682819 Hs.470171 ACVR2 Activin A receptor, type II 8.26 
AA970865 Hs.21145 ODAG GATA zinc finger domain containing 1 7.48 
AA775616  SPP1 Secreted phosphoprotein 1 (osteopontin, bone sialoprotein 1) 7.45 
N69689 Hs.310645 RAB1A RAB1A, member RAS oncogene family 7.40 
R85213 Hs.22543 UBE3A Ubiquitin protein ligase E3A (human papilloma virus E6-associated protein, Angelman syndrome) 7.28 
AA706987 Hs.514806 GALNT1 UDP-N-acetyl-α-d-galactosamine: polypeptide N-acetylgalactosaminyltransferase 1 (GalNAc-T1) 7.28 
R41839 Hs.154073 SLC35B1 Solute carrier family 35, member B1 7.27 
N71628 Hs.437905 SPIB Spi-B transcription factor (Spi-1/PU.1 related) 7.26 
AI129421 Hs.83077 IL18 Interleukin 18 (IFN-γ-inducing factor) 7.25 
AA101617 Hs.220971 FOSL2 FOS-like antigen 2 7.18 
AA180742 Hs.75318 TUBA1 Tubulin, α 1 (testis specific) 7.15 
AA446108 Hs.76753 ENG Endoglin (Osler-Rendu-Weber syndrome 1) 7.11 
T60048 Hs.516105 ACTG2 Actin, γ 2, smooth muscle, enteric 7.06 
AA454585 Hs.73965 SFRS2 Splicing factor, arginine/serine-rich 2 6.96 
AA418813 Hs.309090 SFRS7 Splicing factor, arginine/serine-rich 7, 35 kDa 6.93 
AA626845 Hs.380774 DDX3X DEAD (Asp-Glu-Ala-Asp) box polypeptide 3, X-linked 6.82 
T66839 Hs.325846 FLJ11806 Nuclear protein UKp68 6.78 
T50498 Hs.433795 SHC1 SHC (Src homology 2 domain containing) transforming protein 1 6.73 
AA496691 Hs.76111 DAG1 Dystroglycan 1 (dystrophin-associated glycoprotein 1) 6.72 
AA598670 Hs.533273 UBE1 Ubiquitin-activating enzyme E1 (A1S9T and BN75 temperature sensitivity complementing) 6.63 
AA281784 Hs.518451 PIK3CD Phosphoinositide-3-kinase, catalytic, δ polypeptide 6.37 
H20138 Hs.503222 RAB6A RAB6A, member RAS oncogene family 6.35 
AA912448 Hs.46523 ELK3 ELK3, ETS-domain protein (SRF accessory protein 2) 6.33 
N68166 Hs.185172 GNB2 Guanine nucleotide binding protein (G protein), β polypeptide 2 6.22 
AI392759 Hs.515840 DNMT3A DNA (cytosine-5-)-methyltransferase-3α 6.21 
AA281635 Hs.411311 IL24 Interleukin 24 6.16 
Table 2.

Top 50 down-regulated genes

Accession no.Unigene IDSymbolDescriptionFold of change
AA194983 Hs.81791 TNFRSF11B Tumor necrosis factor receptor superfamily, member 11b (osteoprotegerin) −95.28 
AA486444 Hs.505687 PYM Within bgcn homologue (Drosophila−30.49 
AA598794 Hs.410037 CTGF Connective tissue growth factor −27.45 
N35316 Hs.153952 NT5E 5′-nucleotidase, ecto (CD73) −27.19 
AA629591 Hs.35052 TEGT Testis enhanced gene transcript (BAX inhibitor 1) −26.86 
AA917374 Hs.104839 TIMP2 Tissue inhibitor of metalloproteinase 2 −25.98 
AI262978 Hs.435974 MTHFD1 Methylenetetrahydrofolate dehydrogenase (NADP+ dependent) 1, methenyltetrahydrofolate cyclohydrolase, formyltetrahydrofolate synthetase −20.70 
H13623 Hs.26139 EPS8 Epidermal growth factor receptor pathway substrate 8 −20.60 
H23235 Hs.74615 PDGFRA Platelet-derived growth factor receptor, α-polypeptide −20.51 
H88540 Hs.187199 MALAT1 Metastasis associated lung adenocarcinoma transcript 1 (non-coding RNA) −16.50 
AA136710 Hs.268849 GLO1 Glyoxalase I −16.36 
AA495936 Hs.389700 MGST1 Microsomal glutathione S-transferase 1 −15.68 
AA487429 Hs.352018 TAP1 Transporter 1, ATP-binding cassette, subfamily B (MDR/TAP) −14.88 
R09561 Hs.527653 DAF Decay accelerating factor for complement (CD55, Cromer blood group system) −13.88 
AA598817 Hs.30743 PRAME Preferentially expressed antigen in melanoma −13.75 
T62048 Hs.458355 C1S Complement component 1, s subcomponent −13.35 
AA670438 Hs.518731 UCHL1 Ubiquitin carboxyl-terminal esterase L1 (ubiquitin thiolesterase) −13.12 
AA130042 Hs.438970 TBL1XR1 Transducin (β)-like 1X-linked receptor 1 −12.99 
N66644 Hs.132513 HSD17B12 Hydroxysteroid (17-β) dehydrogenase 12 −12.60 
AA464246 Hs.534125 HLA-C MHC, class I, C −12.07 
H11003 Hs.511899 EDN1 Endothelin 1 −12.02 
AA937895 Hs.495605 CD99 CD99 antigen −11.79 
AA043133 Hs.75231 SLC16A1 Solute carrier family 16 (monocarboxylic acid transporters), member 1 −11.70 
AA181300 Hs.180062 PSMB8 Proteasome (prosome, macropain) subunit, β type, 8 (large multifunctional protease 7) −11.59 
N21573 Hs.514685 TWSG1 Twisted gastrulation homologue 1 (Drosophila−11.10 
H99676 Hs.474053 COL6A1 Collagen, type VI, α1 −10.72 
H15574 Hs.127826 EPOR Erythropoietin receptor −10.53 
AA447561 Hs.90691 NPM3 Nucleophosmin/nucleoplasmin, 3 −10.53 
AA630771 Hs.413812 RAC1 Ras-related C3 botulinum toxin substrate 1 (rho family, small GTP binding protein Rac1) −10.48 
W73874 Hs.418123 CTSL Cathepsin L −10.42 
AA878257 Hs.173894 CSF1 Colony-stimulating factor 1 (macrophage) −10.21 
R76394 Hs.25338 PRSS23 Protease, serine, 23 −10.13 
AA865464 Hs.521903 LY6E Lymphocyte antigen 6 complex, locus E −10.11 
AA504482 Hs.185597 SPG7 Spastic paraplegia 7, paraplegin (pure and complicated autosomal recessive) −9.82 
AA056232 Hs.75652 GSTM5 Glutathione S-transferase M5 −9.80 
AA486570 Hs.348387 GSTM4 Glutathione S-transferase M4 −9.62 
N52911 Hs.436687 SET SET translocation (myeloid leukemia-associated) −9.46 
AI356709 Hs.332197 D2S448 Melanoma associated gene −9.45 
AI084613 Hs.56186 EGFL3 EGF-like-domain, multiple 3 −9.36 
AA424956 Hs.151777 EIF2S1 Eukaryotic translation initiation factor 2, subunit 1 α, 35 kDa −9.34 
R25377 Hs.484813 DEK DEK oncogene (DNA binding) −9.22 
AA453749 Hs.506748 HDGF Hepatoma-derived growth factor (high-mobility group protein 1-like) −8.98 
AA487486 Hs.523852 CCND1 Cyclin D1 (PRAD1: parathyroid adenomatosis 1) −8.93 
AA485353 Hs.514535 LGALS3BP Lectin, galactoside-binding, soluble, 3 binding protein −8.83 
AA504348 Hs.156346 TOP2A Topoisomerase (DNA) II α 170 kDa −8.80 
AA479090 Hs.295137 AMFR Autocrine motility factor receptor −8.69 
AA464849 Hs.434367 TXNRD1 Thioredoxin reductase 1 −8.57 
AA676466 Hs.160786 ASS Argininosuccinate synthetase −8.54 
AA410636 Hs.445403 IARS Isoleucine-tRNA synthetase −8.51 
R38933 Hs.491582 PLAT Plasminogen activator, tissue −8.32 
Accession no.Unigene IDSymbolDescriptionFold of change
AA194983 Hs.81791 TNFRSF11B Tumor necrosis factor receptor superfamily, member 11b (osteoprotegerin) −95.28 
AA486444 Hs.505687 PYM Within bgcn homologue (Drosophila−30.49 
AA598794 Hs.410037 CTGF Connective tissue growth factor −27.45 
N35316 Hs.153952 NT5E 5′-nucleotidase, ecto (CD73) −27.19 
AA629591 Hs.35052 TEGT Testis enhanced gene transcript (BAX inhibitor 1) −26.86 
AA917374 Hs.104839 TIMP2 Tissue inhibitor of metalloproteinase 2 −25.98 
AI262978 Hs.435974 MTHFD1 Methylenetetrahydrofolate dehydrogenase (NADP+ dependent) 1, methenyltetrahydrofolate cyclohydrolase, formyltetrahydrofolate synthetase −20.70 
H13623 Hs.26139 EPS8 Epidermal growth factor receptor pathway substrate 8 −20.60 
H23235 Hs.74615 PDGFRA Platelet-derived growth factor receptor, α-polypeptide −20.51 
H88540 Hs.187199 MALAT1 Metastasis associated lung adenocarcinoma transcript 1 (non-coding RNA) −16.50 
AA136710 Hs.268849 GLO1 Glyoxalase I −16.36 
AA495936 Hs.389700 MGST1 Microsomal glutathione S-transferase 1 −15.68 
AA487429 Hs.352018 TAP1 Transporter 1, ATP-binding cassette, subfamily B (MDR/TAP) −14.88 
R09561 Hs.527653 DAF Decay accelerating factor for complement (CD55, Cromer blood group system) −13.88 
AA598817 Hs.30743 PRAME Preferentially expressed antigen in melanoma −13.75 
T62048 Hs.458355 C1S Complement component 1, s subcomponent −13.35 
AA670438 Hs.518731 UCHL1 Ubiquitin carboxyl-terminal esterase L1 (ubiquitin thiolesterase) −13.12 
AA130042 Hs.438970 TBL1XR1 Transducin (β)-like 1X-linked receptor 1 −12.99 
N66644 Hs.132513 HSD17B12 Hydroxysteroid (17-β) dehydrogenase 12 −12.60 
AA464246 Hs.534125 HLA-C MHC, class I, C −12.07 
H11003 Hs.511899 EDN1 Endothelin 1 −12.02 
AA937895 Hs.495605 CD99 CD99 antigen −11.79 
AA043133 Hs.75231 SLC16A1 Solute carrier family 16 (monocarboxylic acid transporters), member 1 −11.70 
AA181300 Hs.180062 PSMB8 Proteasome (prosome, macropain) subunit, β type, 8 (large multifunctional protease 7) −11.59 
N21573 Hs.514685 TWSG1 Twisted gastrulation homologue 1 (Drosophila−11.10 
H99676 Hs.474053 COL6A1 Collagen, type VI, α1 −10.72 
H15574 Hs.127826 EPOR Erythropoietin receptor −10.53 
AA447561 Hs.90691 NPM3 Nucleophosmin/nucleoplasmin, 3 −10.53 
AA630771 Hs.413812 RAC1 Ras-related C3 botulinum toxin substrate 1 (rho family, small GTP binding protein Rac1) −10.48 
W73874 Hs.418123 CTSL Cathepsin L −10.42 
AA878257 Hs.173894 CSF1 Colony-stimulating factor 1 (macrophage) −10.21 
R76394 Hs.25338 PRSS23 Protease, serine, 23 −10.13 
AA865464 Hs.521903 LY6E Lymphocyte antigen 6 complex, locus E −10.11 
AA504482 Hs.185597 SPG7 Spastic paraplegia 7, paraplegin (pure and complicated autosomal recessive) −9.82 
AA056232 Hs.75652 GSTM5 Glutathione S-transferase M5 −9.80 
AA486570 Hs.348387 GSTM4 Glutathione S-transferase M4 −9.62 
N52911 Hs.436687 SET SET translocation (myeloid leukemia-associated) −9.46 
AI356709 Hs.332197 D2S448 Melanoma associated gene −9.45 
AI084613 Hs.56186 EGFL3 EGF-like-domain, multiple 3 −9.36 
AA424956 Hs.151777 EIF2S1 Eukaryotic translation initiation factor 2, subunit 1 α, 35 kDa −9.34 
R25377 Hs.484813 DEK DEK oncogene (DNA binding) −9.22 
AA453749 Hs.506748 HDGF Hepatoma-derived growth factor (high-mobility group protein 1-like) −8.98 
AA487486 Hs.523852 CCND1 Cyclin D1 (PRAD1: parathyroid adenomatosis 1) −8.93 
AA485353 Hs.514535 LGALS3BP Lectin, galactoside-binding, soluble, 3 binding protein −8.83 
AA504348 Hs.156346 TOP2A Topoisomerase (DNA) II α 170 kDa −8.80 
AA479090 Hs.295137 AMFR Autocrine motility factor receptor −8.69 
AA464849 Hs.434367 TXNRD1 Thioredoxin reductase 1 −8.57 
AA676466 Hs.160786 ASS Argininosuccinate synthetase −8.54 
AA410636 Hs.445403 IARS Isoleucine-tRNA synthetase −8.51 
R38933 Hs.491582 PLAT Plasminogen activator, tissue −8.32 
Table 3.

PSA-regulated genes grouped into function classes

Accession no.Unigene IDSymbolDescriptionFold of change
Metabolism (566 genes)     
    R53942 Hs.246506 SLC25A4 Solute carrier family 25 (mitochondrial carrier; adenine nucleotide translocator), member 4 28.43 
    AA598794 Hs.410037 CTGF Connective tissue growth factor −27.45 
    N35316 Hs.153952 NT5E 5′-nucleotidase, ecto (CD73) −27.19 
    AA858175 Hs.535845 RUNX2 Runt-related transcription factor 2 25.80 
    AI262978 Hs.435974 MTHFD1 Methylenetetrahydrofolate dehydrogenase (NADP+ dependent) 1, methenyltetrahydrofolate cyclohydrolase, formyltetrahydrofolate synthetase −20.70 
    H23235 Hs.74615 PDGFRA Platelet-derived growth factor receptor, α polypeptide −20.51 
    AA156964 Hs.471200 NRP2 Neuropilin 2 19.22 
    AA425628 Hs.77578 USP9X Ubiquitin specific protease 9, X-linked (fat facets-like, Drosophila17.23 
    AA136710 Hs.268849 GLO1 Glyoxalase I −16.36 
    T51538 Hs.368592 SORL1 Sortilin-related receptor, L(DLR class) A repeats-containing 14.85 
Catalytic activity (399 genes)     
    R55809 Hs.284122 WIF1 WNT inhibitory factor 1 31.12 
    N35316 Hs.153952 NT5E 5′-nucleotidase, ecto (CD73) −27.19 
    AI262978 Hs.435974 MTHFD1 Methylenetetrahydrofolate dehydrogenase (NADP+ dependent) 1, methenyltetrahydrofolate cyclohydrolase, formyltetrahydrofolate synthetase −20.70 
    H23235 Hs.74615 PDGFRA Platelet-derived growth factor receptor, α polypeptide −20.51 
    AA156964 Hs.471200 NRP2 Neuropilin 2 19.22 
    AA425628 Hs.77578 USP9X Ubiquitin specific protease 9, X-linked (fat facets-like, Drosophila17.23 
    AA136710 Hs.268849 GLO1 Glyoxalase I −16.36 
    AA495936 Hs.389700 MGST1 Microsomal glutathione S-transferase 1 −15.68 
    AA487429 Hs.352018 TAP1 Transporter 1, ATP-binding cassette, subfamily B (MDR/TAP) −14.88 
    T62048 Hs.458355 C1S Complement component 1, s subcomponent −13.35 
Cell growth and/or maintenance (307 genes)     
    R53942 Hs.246506 SLC25A4 Solute carrier family 25 (mitochondrial carrier; adenine nucleotide translocator), member 4 28.43 
    AA598794 Hs.410037 CTGF Connective tissue growth factor −27.45 
    H13623 Hs.26139 EPS8 Epidermal growth factor receptor pathway substrate 8 −20.60 
    H23235 Hs.74615 PDGFRA Platelet-derived growth factor receptor, α polypeptide −20.51 
    H06516 Hs.212838 A2M α-2-macroglobulin 16.64 
    AA487429 Hs.352018 TAP1 Transporter 1, ATP-binding cassette, subfamily B (MDR/TAP) −14.88 
    T51538 Hs.368592 SORL1 Sortilin-related receptor, L(DLR class) A repeats-containing 14.85 
    AA677306 Hs.158560 TAF1 TAF1 RNA polymerase II, TATA box binding protein (TBP)-associated factor, 250 kDa 13.32 
    AA055979 Hs.524484 ITGA7 Integrin, α 7 13.06 
    AA490459 Hs.417948 TCN2 Transcobalamin II; macrocytic anemia 12.68 
Cell communication (277 genes)     
    AA194983 Hs.81791 TNFRSF11B Tumor necrosis factor receptor superfamily, member 11b (osteoprotegerin) −95.28 
    R55809 Hs.284122 WIF1 WNT inhibitory factor 1 31.12 
    AA598794 Hs.410037 CTGF Connective tissue growth factor −27.45 
    H13623 Hs.26139 EPS8 Epidermal growth factor receptor pathway substrate 8 −20.60 
    H23235 Hs.74615 PDGFRA Platelet-derived growth factor receptor, α polypeptide −20.51 
    AA156964 Hs.471200 NRP2 Neuropilin 2 19.22 
    AA442092 Hs.476018 CTNNB1 Catenin (cadherin-associated protein), β 1, 88 kDa 13.15 
    AA055979 Hs.524484 ITGA7 Integrin, α 7 13.06 
    AA464532 Hs.164226 THBS1 Thrombospondin 1 12.53 
    H11003 Hs.511899 EDN1 Endothelin 1 −12.02 
Signal transduction (141 genes)     
    AA194983 Hs.81791 TNFRSF11B Tumor necrosis factor receptor superfamily, member 11b (osteoprotegerin) −95.28 
    H13623 Hs.26139 EPS8 Epidermal growth factor receptor pathway substrate 8 −20.60 
    H23235 Hs.74615 PDGFRA Platelet-derived growth factor receptor, α polypeptide −20.51 
    AA156964 Hs.471200 NRP2 Neuropilin 2 19.22 
    T51538 Hs.368592 SORL1 Sortilin-related receptor, L(DLR class) A repeats-containing 14.85 
    AA055979 Hs.524484 ITGA7 Integrin, α 7 13.06 
    AA464246 Hs.534125 HLA-C MHC, class I, C −12.07 
    H11003 Hs.511899 EDN1 Endothelin 1 −12.02 
    AA676805 Hs.336994 MTSS1 Metastasis suppressor 1 11.37 
    H15574 Hs.127826 EPOR Erythropoietin receptor −10.53 
Transcriptional regulation (117 genes)     
    AA858175 Hs.535845 RUNX2 Runt-related transcription factor 2 25.80 
    R25377 Hs.484813 DEK DEK oncogene (DNA binding) −9.22 
    AA970865 Hs.21145 ODAG GATA zinc finger domain containing 1 7.48 
    N71628 Hs.437905 SPIB Spi-B transcription factor (Spi-1/PU.1 related) 7.26 
    AA101617 Hs.220971 FOSL2 FOS-like antigen 2 7.18 
    AA664389 Hs.507916 TGFB1I4 TSC22 domain family 1 −6.68 
    AI364369 Hs.493096 PBX1 Pre–B cell leukemia transcription factor 1 −6.58 
    R42479 Hs.517296 ETS2 V-ets erythroblastosis virus E26 oncogene homologue 2 (avian) −6.34 
    AA912448 Hs.46523 ELK3 ELK3, ETS-domain protein (SRF accessory protein 2) 6.33 
    AA495962 Hs.412293 NCOA1 Nuclear receptor coactivator 1 6.08 
Cell death (70 genes)     
    AA194983 Hs.81791 TNFRSF11B Tumor necrosis factor receptor superfamily, member 11b (osteoprotegerin) −95.28 
    AA629591 Hs.35052 TEGT Testis enhanced gene transcript (BAX inhibitor 1) −26.86 
    AA227885 Hs.80395 MAL Mal, T cell differentiation protein 8.97 
    AI129421 Hs.83077 IL18 Interleukin 18 (IFN γ-inducing factor) 7.25 
    AA281635 Hs.411311 IL24 Interleukin 24 6.16 
    AA598601 Hs.450230 IGFBP3 Insulin-like growth factor binding protein 3 −5.96 
    N62514 Hs.283454 BNIP2 BCL2/adenovirus E1B 19 kDa interacting protein 2 −5.83 
    AA418744 Hs.55220 BAG2 BCL2-associated athanogene 2 −5.77 
    N69521 Hs.397465 HIPK2 Homeodomain interacting protein kinase 2 −5.62 
    N70463 Hs.255935 BTG1 B cell translocation gene 1, antiproliferative 5.32 
Transporter (54 genes)     
    R53942 Hs.246506 SLC25A4 Solute carrier family 25 (mitochondrial carrier; adenine nucleotide translocator), member 4 28.43 
    AA156964 Hs.471200 NRP2 Neuropilin 2 19.22 
    H06516 Hs.212838 A2M α-2-macroglobulin 16.64 
    AA487429 Hs.352018 TAP1 Transporter 1, ATP-binding cassette, subfamily B (MDR/TAP) −14.88 
    T51538 Hs.368592 SORL1 Sortilin-related receptor, L(DLR class) A repeats-containing 14.85 
    AA490459 Hs.417948 TCN2 Transcobalamin II; macrocytic anemia 12.68 
    AA705237 Hs.370480 ABCB7 ATP-binding cassette, subfamily B (MDR/TAP), member 7 12.20 
    AA043133 Hs.75231 SLC16A1 Solute carrier family 16 (monocarboxylic acid transporters), member 1 −11.70 
    AA404486 Hs.522767 SLC25A5 Solute carrier family 25 (mitochondrial carrier; adenine nucleotide translocator), member 5 10.28 
    AA227885 Hs.80395 MAL Mal, T cell differentiation protein 8.97 
Cell motility (18 genes)     
    AA156964 Hs.471200 NRP2 Neuropilin 2 19.22 
    AA496691 Hs.76111 DAG1 Dystroglycan 1 (dystrophin-associated glycoprotein 1) 6.72 
    AA056693 Hs.405156 PPAP2B Phosphatidic acid phosphatase type 2B −6.70 
    H20759 Hs.224008 FEZ1 Fasciculation and elongation protein ζ 1 (zygin I) −6.17 
    N94616 Hs.213861 LAMA4 Laminin, α 4 −5.6 
    N70463 Hs.255935 BTG1 B cell translocation gene 1, antiproliferative 5.32 
    R59598 Hs.371720 SYK Spleen tyrosine kinase 3.23 
    AA398400 Hs.465929 CNN1 Calponin 1, basic, smooth muscle −3.21 
    AA496565 Hs.476209 PLXNB1 Plexin B1 3.12 
    AA676840 Hs.133135 UTRN Utrophin (homologous to dystrophin) −2.98 
Translational regulation (15 genes)     
    AA424956 Hs.151777 EIF2S1 Eukaryotic translation initiation factor 2, subunit 1 α, 35 kDa −9.34 
    AA878570 Hs.534314 EIF5A Eukaryotic translation initiation factor 5A 4.84 
    AA669443 Hs.433702 EIF5 Eukaryotic translation initiation factor 5 4.49 
    AA460838 Hs.355348 GTF2H3 General transcription factor IIH, polypeptide 3, 34 kDa −3.85 
    AA459999 Hs.534582 MTIF3 Mitochondrial translational initiation factor 3 −3.84 
    AA053129 Hs.396644 PAIP2 Poly(A) binding protein interacting protein 2 3.60 
    R43766 Hs.515070 EEF2 Eukaryotic translation elongation factor 2 3.34 
    AA486233 Hs.528780 GSPT1 G1 to S phase transition 1 3.30 
    R15111 Hs.530727 CUGBP1 CUG triplet repeat, RNA binding protein 1 2.92 
    R51607 Hs.150580 SUI1 Putative translation initiation factor −2.64 
Other/unknown (146 genes)     
    AA486444 Hs.505687 PYM Within bgcn homologue (Drosophila−30.49 
    AA917374 Hs.104839 TIMP2 Tissue inhibitor of metalloproteinase 2 −25.98 
    H88540 Hs.187199 MALAT1 Metastasis associated lung adenocarcinoma transcript 1 (noncoding RNA) −16.50 
    R09561 Hs.527653 DAF Decay accelerating factor for complement (CD55, Cromer blood group system) −13.88 
    AA598817 Hs.30743 PRAME Preferentially expressed antigen in melanoma −13.75 
    N21573 Hs.514685 TWSG1 Twisted gastrulation homologue 1 (Drosophila−11.10 
    AI084613 Hs.56186 EGFL3 EGF-like domain, multiple 3 −9.36 
    AA400234 Hs.523789 TncRNA Trophoblast-derived noncoding RNA −7.81 
    AA464417 Hs.374650 IFITM3 IFN induced transmembrane protein 3 (1-8U) −7.65 
    AA424824 Hs.304192 DSTN Destrin (actin depolymerizing factor) −7.40 
Accession no.Unigene IDSymbolDescriptionFold of change
Metabolism (566 genes)     
    R53942 Hs.246506 SLC25A4 Solute carrier family 25 (mitochondrial carrier; adenine nucleotide translocator), member 4 28.43 
    AA598794 Hs.410037 CTGF Connective tissue growth factor −27.45 
    N35316 Hs.153952 NT5E 5′-nucleotidase, ecto (CD73) −27.19 
    AA858175 Hs.535845 RUNX2 Runt-related transcription factor 2 25.80 
    AI262978 Hs.435974 MTHFD1 Methylenetetrahydrofolate dehydrogenase (NADP+ dependent) 1, methenyltetrahydrofolate cyclohydrolase, formyltetrahydrofolate synthetase −20.70 
    H23235 Hs.74615 PDGFRA Platelet-derived growth factor receptor, α polypeptide −20.51 
    AA156964 Hs.471200 NRP2 Neuropilin 2 19.22 
    AA425628 Hs.77578 USP9X Ubiquitin specific protease 9, X-linked (fat facets-like, Drosophila17.23 
    AA136710 Hs.268849 GLO1 Glyoxalase I −16.36 
    T51538 Hs.368592 SORL1 Sortilin-related receptor, L(DLR class) A repeats-containing 14.85 
Catalytic activity (399 genes)     
    R55809 Hs.284122 WIF1 WNT inhibitory factor 1 31.12 
    N35316 Hs.153952 NT5E 5′-nucleotidase, ecto (CD73) −27.19 
    AI262978 Hs.435974 MTHFD1 Methylenetetrahydrofolate dehydrogenase (NADP+ dependent) 1, methenyltetrahydrofolate cyclohydrolase, formyltetrahydrofolate synthetase −20.70 
    H23235 Hs.74615 PDGFRA Platelet-derived growth factor receptor, α polypeptide −20.51 
    AA156964 Hs.471200 NRP2 Neuropilin 2 19.22 
    AA425628 Hs.77578 USP9X Ubiquitin specific protease 9, X-linked (fat facets-like, Drosophila17.23 
    AA136710 Hs.268849 GLO1 Glyoxalase I −16.36 
    AA495936 Hs.389700 MGST1 Microsomal glutathione S-transferase 1 −15.68 
    AA487429 Hs.352018 TAP1 Transporter 1, ATP-binding cassette, subfamily B (MDR/TAP) −14.88 
    T62048 Hs.458355 C1S Complement component 1, s subcomponent −13.35 
Cell growth and/or maintenance (307 genes)     
    R53942 Hs.246506 SLC25A4 Solute carrier family 25 (mitochondrial carrier; adenine nucleotide translocator), member 4 28.43 
    AA598794 Hs.410037 CTGF Connective tissue growth factor −27.45 
    H13623 Hs.26139 EPS8 Epidermal growth factor receptor pathway substrate 8 −20.60 
    H23235 Hs.74615 PDGFRA Platelet-derived growth factor receptor, α polypeptide −20.51 
    H06516 Hs.212838 A2M α-2-macroglobulin 16.64 
    AA487429 Hs.352018 TAP1 Transporter 1, ATP-binding cassette, subfamily B (MDR/TAP) −14.88 
    T51538 Hs.368592 SORL1 Sortilin-related receptor, L(DLR class) A repeats-containing 14.85 
    AA677306 Hs.158560 TAF1 TAF1 RNA polymerase II, TATA box binding protein (TBP)-associated factor, 250 kDa 13.32 
    AA055979 Hs.524484 ITGA7 Integrin, α 7 13.06 
    AA490459 Hs.417948 TCN2 Transcobalamin II; macrocytic anemia 12.68 
Cell communication (277 genes)     
    AA194983 Hs.81791 TNFRSF11B Tumor necrosis factor receptor superfamily, member 11b (osteoprotegerin) −95.28 
    R55809 Hs.284122 WIF1 WNT inhibitory factor 1 31.12 
    AA598794 Hs.410037 CTGF Connective tissue growth factor −27.45 
    H13623 Hs.26139 EPS8 Epidermal growth factor receptor pathway substrate 8 −20.60 
    H23235 Hs.74615 PDGFRA Platelet-derived growth factor receptor, α polypeptide −20.51 
    AA156964 Hs.471200 NRP2 Neuropilin 2 19.22 
    AA442092 Hs.476018 CTNNB1 Catenin (cadherin-associated protein), β 1, 88 kDa 13.15 
    AA055979 Hs.524484 ITGA7 Integrin, α 7 13.06 
    AA464532 Hs.164226 THBS1 Thrombospondin 1 12.53 
    H11003 Hs.511899 EDN1 Endothelin 1 −12.02 
Signal transduction (141 genes)     
    AA194983 Hs.81791 TNFRSF11B Tumor necrosis factor receptor superfamily, member 11b (osteoprotegerin) −95.28 
    H13623 Hs.26139 EPS8 Epidermal growth factor receptor pathway substrate 8 −20.60 
    H23235 Hs.74615 PDGFRA Platelet-derived growth factor receptor, α polypeptide −20.51 
    AA156964 Hs.471200 NRP2 Neuropilin 2 19.22 
    T51538 Hs.368592 SORL1 Sortilin-related receptor, L(DLR class) A repeats-containing 14.85 
    AA055979 Hs.524484 ITGA7 Integrin, α 7 13.06 
    AA464246 Hs.534125 HLA-C MHC, class I, C −12.07 
    H11003 Hs.511899 EDN1 Endothelin 1 −12.02 
    AA676805 Hs.336994 MTSS1 Metastasis suppressor 1 11.37 
    H15574 Hs.127826 EPOR Erythropoietin receptor −10.53 
Transcriptional regulation (117 genes)     
    AA858175 Hs.535845 RUNX2 Runt-related transcription factor 2 25.80 
    R25377 Hs.484813 DEK DEK oncogene (DNA binding) −9.22 
    AA970865 Hs.21145 ODAG GATA zinc finger domain containing 1 7.48 
    N71628 Hs.437905 SPIB Spi-B transcription factor (Spi-1/PU.1 related) 7.26 
    AA101617 Hs.220971 FOSL2 FOS-like antigen 2 7.18 
    AA664389 Hs.507916 TGFB1I4 TSC22 domain family 1 −6.68 
    AI364369 Hs.493096 PBX1 Pre–B cell leukemia transcription factor 1 −6.58 
    R42479 Hs.517296 ETS2 V-ets erythroblastosis virus E26 oncogene homologue 2 (avian) −6.34 
    AA912448 Hs.46523 ELK3 ELK3, ETS-domain protein (SRF accessory protein 2) 6.33 
    AA495962 Hs.412293 NCOA1 Nuclear receptor coactivator 1 6.08 
Cell death (70 genes)     
    AA194983 Hs.81791 TNFRSF11B Tumor necrosis factor receptor superfamily, member 11b (osteoprotegerin) −95.28 
    AA629591 Hs.35052 TEGT Testis enhanced gene transcript (BAX inhibitor 1) −26.86 
    AA227885 Hs.80395 MAL Mal, T cell differentiation protein 8.97 
    AI129421 Hs.83077 IL18 Interleukin 18 (IFN γ-inducing factor) 7.25 
    AA281635 Hs.411311 IL24 Interleukin 24 6.16 
    AA598601 Hs.450230 IGFBP3 Insulin-like growth factor binding protein 3 −5.96 
    N62514 Hs.283454 BNIP2 BCL2/adenovirus E1B 19 kDa interacting protein 2 −5.83 
    AA418744 Hs.55220 BAG2 BCL2-associated athanogene 2 −5.77 
    N69521 Hs.397465 HIPK2 Homeodomain interacting protein kinase 2 −5.62 
    N70463 Hs.255935 BTG1 B cell translocation gene 1, antiproliferative 5.32 
Transporter (54 genes)     
    R53942 Hs.246506 SLC25A4 Solute carrier family 25 (mitochondrial carrier; adenine nucleotide translocator), member 4 28.43 
    AA156964 Hs.471200 NRP2 Neuropilin 2 19.22 
    H06516 Hs.212838 A2M α-2-macroglobulin 16.64 
    AA487429 Hs.352018 TAP1 Transporter 1, ATP-binding cassette, subfamily B (MDR/TAP) −14.88 
    T51538 Hs.368592 SORL1 Sortilin-related receptor, L(DLR class) A repeats-containing 14.85 
    AA490459 Hs.417948 TCN2 Transcobalamin II; macrocytic anemia 12.68 
    AA705237 Hs.370480 ABCB7 ATP-binding cassette, subfamily B (MDR/TAP), member 7 12.20 
    AA043133 Hs.75231 SLC16A1 Solute carrier family 16 (monocarboxylic acid transporters), member 1 −11.70 
    AA404486 Hs.522767 SLC25A5 Solute carrier family 25 (mitochondrial carrier; adenine nucleotide translocator), member 5 10.28 
    AA227885 Hs.80395 MAL Mal, T cell differentiation protein 8.97 
Cell motility (18 genes)     
    AA156964 Hs.471200 NRP2 Neuropilin 2 19.22 
    AA496691 Hs.76111 DAG1 Dystroglycan 1 (dystrophin-associated glycoprotein 1) 6.72 
    AA056693 Hs.405156 PPAP2B Phosphatidic acid phosphatase type 2B −6.70 
    H20759 Hs.224008 FEZ1 Fasciculation and elongation protein ζ 1 (zygin I) −6.17 
    N94616 Hs.213861 LAMA4 Laminin, α 4 −5.6 
    N70463 Hs.255935 BTG1 B cell translocation gene 1, antiproliferative 5.32 
    R59598 Hs.371720 SYK Spleen tyrosine kinase 3.23 
    AA398400 Hs.465929 CNN1 Calponin 1, basic, smooth muscle −3.21 
    AA496565 Hs.476209 PLXNB1 Plexin B1 3.12 
    AA676840 Hs.133135 UTRN Utrophin (homologous to dystrophin) −2.98 
Translational regulation (15 genes)     
    AA424956 Hs.151777 EIF2S1 Eukaryotic translation initiation factor 2, subunit 1 α, 35 kDa −9.34 
    AA878570 Hs.534314 EIF5A Eukaryotic translation initiation factor 5A 4.84 
    AA669443 Hs.433702 EIF5 Eukaryotic translation initiation factor 5 4.49 
    AA460838 Hs.355348 GTF2H3 General transcription factor IIH, polypeptide 3, 34 kDa −3.85 
    AA459999 Hs.534582 MTIF3 Mitochondrial translational initiation factor 3 −3.84 
    AA053129 Hs.396644 PAIP2 Poly(A) binding protein interacting protein 2 3.60 
    R43766 Hs.515070 EEF2 Eukaryotic translation elongation factor 2 3.34 
    AA486233 Hs.528780 GSPT1 G1 to S phase transition 1 3.30 
    R15111 Hs.530727 CUGBP1 CUG triplet repeat, RNA binding protein 1 2.92 
    R51607 Hs.150580 SUI1 Putative translation initiation factor −2.64 
Other/unknown (146 genes)     
    AA486444 Hs.505687 PYM Within bgcn homologue (Drosophila−30.49 
    AA917374 Hs.104839 TIMP2 Tissue inhibitor of metalloproteinase 2 −25.98 
    H88540 Hs.187199 MALAT1 Metastasis associated lung adenocarcinoma transcript 1 (noncoding RNA) −16.50 
    R09561 Hs.527653 DAF Decay accelerating factor for complement (CD55, Cromer blood group system) −13.88 
    AA598817 Hs.30743 PRAME Preferentially expressed antigen in melanoma −13.75 
    N21573 Hs.514685 TWSG1 Twisted gastrulation homologue 1 (Drosophila−11.10 
    AI084613 Hs.56186 EGFL3 EGF-like domain, multiple 3 −9.36 
    AA400234 Hs.523789 TncRNA Trophoblast-derived noncoding RNA −7.81 
    AA464417 Hs.374650 IFITM3 IFN induced transmembrane protein 3 (1-8U) −7.65 
    AA424824 Hs.304192 DSTN Destrin (actin depolymerizing factor) −7.40 

We analyzed the array data based on the known osteogenic function of the genes by manual examination of published literature using PubMed. Genes involved in bone remodeling that show at least 3-fold changes are listed in Table 4. It is interesting to note that among the most significant changes induced by overexpression of PSA in SaOS-2 cells are the factors involved in bone remodeling (Table 4). These include an increase in some of the important genes involved in bone remodeling such as runx-2 (25-fold), cadherin 11 (10-fold), osteopontin (7-fold), TGF-β (5-fold), BMP4 (4-fold), and BMP8 (3-fold), and most significantly, decrease in the expression of osteoprotegerin (>95-fold decrease).

Table 4.

Changes on the expression of osteogenic associated genes

Accession no.Unigene IDSymbolDescriptionFold of change
Up-regulated (nine genes)     
    R55809 Hs.284122 WIF1 WNT inhibitory factor 1 31.12 
    AA858175 Hs.535845 RUNX2 Runt-related transcription factor 2 25.80 
    H96738 Hs.116471 CDH11 Cadherin 11, type 2, OB-cadherin (osteoblast) 9.82 
    AA775616  SPP1 Secreted phosphoprotein 1 (osteopontin, bone sialoprotein I, early T-lymphocyte activation 1) 7.45 
    N20203 Hs.471119 BMPR2 Bone morphogenetic protein receptor, type II (serine/threonine kinase) 5.35 
    TGF-β  TGFB Transforming growth factor-β 5.05 
    AA449300 Hs.481022 SFRP2 Secreted frizzled-related protein 2 4.71 
    AA463225 Hs.68879 BMP4 Bone morphogenetic protein 4 3.87 
    AA779480 Hs.494158 BMP8A Bone morphogenetic protein 8a 3.37 
Down-regulated (six genes)     
    AA194983 Hs.81791 TNFRSF11B Tumor necrosis factor receptor superfamily, member 11b (osteoprotegerin) −95.28 
    R38933 Hs.491582 PLAT Plasminogen activator, tissue −8.32 
    AA490172 Hs.489142 COL1A2 Collagen, type I, α2 −6.47 
    AA598601 Hs.450230 IGFBP3 Insulin-like growth factor binding protein 3 −5.96 
    AA143331 Hs.83169 MMP1 Matrix metalloproteinase 1 (interstitial collagenase) −5.66 
    AA936799 Hs.513617 MMP2 Matrix metalloproteinase 2 (gelatinase A, 72 kDa gelatinase, 72 kDa type IV collagenase) −5.46 
Accession no.Unigene IDSymbolDescriptionFold of change
Up-regulated (nine genes)     
    R55809 Hs.284122 WIF1 WNT inhibitory factor 1 31.12 
    AA858175 Hs.535845 RUNX2 Runt-related transcription factor 2 25.80 
    H96738 Hs.116471 CDH11 Cadherin 11, type 2, OB-cadherin (osteoblast) 9.82 
    AA775616  SPP1 Secreted phosphoprotein 1 (osteopontin, bone sialoprotein I, early T-lymphocyte activation 1) 7.45 
    N20203 Hs.471119 BMPR2 Bone morphogenetic protein receptor, type II (serine/threonine kinase) 5.35 
    TGF-β  TGFB Transforming growth factor-β 5.05 
    AA449300 Hs.481022 SFRP2 Secreted frizzled-related protein 2 4.71 
    AA463225 Hs.68879 BMP4 Bone morphogenetic protein 4 3.87 
    AA779480 Hs.494158 BMP8A Bone morphogenetic protein 8a 3.37 
Down-regulated (six genes)     
    AA194983 Hs.81791 TNFRSF11B Tumor necrosis factor receptor superfamily, member 11b (osteoprotegerin) −95.28 
    R38933 Hs.491582 PLAT Plasminogen activator, tissue −8.32 
    AA490172 Hs.489142 COL1A2 Collagen, type I, α2 −6.47 
    AA598601 Hs.450230 IGFBP3 Insulin-like growth factor binding protein 3 −5.96 
    AA143331 Hs.83169 MMP1 Matrix metalloproteinase 1 (interstitial collagenase) −5.66 
    AA936799 Hs.513617 MMP2 Matrix metalloproteinase 2 (gelatinase A, 72 kDa gelatinase, 72 kDa type IV collagenase) −5.46 

Validation of microarray results using Northern blot analysis. To validate the oligonucleotide array data, we did Northern blot analysis on a select number of osteogenic genes identified as being differentially regulated by PSA expression in osteosarcoma SaOS-2 cells. The Northern blot results for the selective genes relative to bone remodeling are shown in Fig. 2. Similar to the array data from runx-2, osteopontin expression is considerably elevated in PSA-producing clones, and osteoprotegerin is significantly reduced (Fig. 2). In general, there is a good agreement between array data and Northern blotting with regard to genes identified as being differentially expressed in SaOS-2 cells producing PSA.

Fig. 2.

Northern blot analyses of selective osteogenic genes in SaOS-2, vector (V), and PSA cDNA-transfected clones (C12, C14, and C21). OCN, osteocalcin. Glyceraldehyde-3-phosphate dehydrogenase (GAPDH) was used as a RNA loading control.

Fig. 2.

Northern blot analyses of selective osteogenic genes in SaOS-2, vector (V), and PSA cDNA-transfected clones (C12, C14, and C21). OCN, osteocalcin. Glyceraldehyde-3-phosphate dehydrogenase (GAPDH) was used as a RNA loading control.

Close modal

Overexpression of PSA enhances SaOS-2 cell calcium deposition and elevates alkaline phosphatase activity in vitro. The PSA-expressing SaOS-2 cells express elevated runx-2, osteocalcin, and bone sialoprotein compared with PSA-negative SaOS-2 cells. Because the increase in the expression of these genes is closely associated with osteoblastic differentiation, PSA-expressing SaOS-2 cells may result in the stimulation of differentiation and bone mineralization in culture. One of the characteristics of osteoblastic differentiation is bone formation. The von Kossa staining was used to detect the presence of mineral deposits of calcium in bone nodules formed during culture of osteoblasts. The mineralized bone nodule formation in SaOS-2 cells and SaOS-2 cells expressing PSA cultured for 11 days in medium supplemented with dexamethasone, ascorbic acid, and β-glycerophosphate was analyzed by von Kossa staining and is illustrated in Fig. 3A. The PSA-expressing SaOS-2 cells show a considerable increase in mineralized bone nodule formation compared with PSA-negative SaOS-neo cells. The activity of alkaline phosphatase was elevated in the PSA-expressing SaOS-2 cells compared with PSA-negative SaOS-neo control (Fig. 3B). These results suggest that overexpression of PSA induces SaOS-2 cells undergoing osteoblastic differentiation in vitro.

Fig. 3.

A, von Kossa staining of vector control (V) and PSA cDNA-transfected clones (C12, C14, and C21). Arrows, mineralized bone nodules. B, overexpression of PSA increased alkaline phosphatase activity in SaOS-2 cells.

Fig. 3.

A, von Kossa staining of vector control (V) and PSA cDNA-transfected clones (C12, C14, and C21). Arrows, mineralized bone nodules. B, overexpression of PSA increased alkaline phosphatase activity in SaOS-2 cells.

Close modal

Exogenously added PSA increases runx-2 expression and elevates alkaline phosphatase activity in SaOS-2 cells. To determine whether exogenously added PSA has similar effects as transfecting expressed PSA, SaOS-2 cells were treated with increasing doses of PSA. Cells were harvested and alkaline phosphatase activity was determined. PSA increased alkaline phosphatase activity in a dose-dependent manner (Fig. 4A). PSA also increased runx-2 and Wif-1 mRNA expression in SaOS-2 cells (Fig. 4B). These results suggested that exogenously added PSA has similar effects as transfecting expressed PSA on the induction of osteoblasts in SaOS-2 cells.

Fig. 4.

Exogenously added PSA affects bone remodeling in SaOS-2 cells. A, PSA increased the activity of alkaline phosphatase in SaOS-2 cells. B, PSA increased runx-2 and Wif-1 mRNA expression.

Fig. 4.

Exogenously added PSA affects bone remodeling in SaOS-2 cells. A, PSA increased the activity of alkaline phosphatase in SaOS-2 cells. B, PSA increased runx-2 and Wif-1 mRNA expression.

Close modal

Prostate cancer preferably metastasizes to bone and produces a primarily osteoblastic phenotype. Despite recent studies on the cellular and molecular interactions between prostate cancer and bone cells, which revealed several factors produced by prostate cancer cells that are involved in osteoblastic differentiation, the underlying mechanisms of prostate cancer–induced osteoblasts are poorly understood. PSA is the most commonly used biomarker for prostate cancer, but its role in bone metastasis is still unclear. In the present study, we show that PSA modulates the expression of genes involved in bone remodeling, including up-regulation of some of the most important osteoblastic genes such as runx-2, osteopontin, TGF-β, receptor activator of NF-κB ligand (RANKL), and down-regulation of osteoprotegerin. Furthermore, PSA overexpression increased mineralization in vitro using von Kossa staining. These results suggest that factors such as PSA produced by prostate cancer cells modulate bone remodeling, resulting in the induction of bone cells' osteoblastic differentiation.

We have identified 15 osteogenic associated genes which are dysregulated by the overexpression of PSA in SaOS-2 cells using 6K Cancer-Specific arrays. Many of those which are up-regulated by PSA are associated with osteoblast differentiation including runx-2, osteopontin, cadherin 11, and TGF-β. Overexpression of PSA enhances runx-2 mRNA expression by 25-fold. Runx-2 is a transcription factor essential for osteoblast differentiation (15, 16). Runx-2-deficient mice showed a complete lack of bone formation due to the absence of osteoblasts (17). Runx-2 has been identified as a key factor which mediates osteoblast differentiation induced by soluble factors produced by prostate cancer cells (18). Runx-2 is a transcription factor that regulates the expression of many bone matrix genes including osteopontin and osteocalcin (18, 19). Of those two genes, the expression of osteopontin was increased to ∼7-fold by PSA (Table 4). To examine whether overexpression of PSA also enhances osteocalcin expression in SaOS-2 cells, Northern blot analysis was done. Osteocalcin mRNA expression was considerably elevated in SaOS-2 cells expressing PSA, but not in the parental SaOS-2 or vector control cells (Fig. 2). Osteocalcin is expressed solely in osteoblasts (20), further suggesting that PSA induces osteoblast differentiation, possibly through up-regulation of runx-2 expression.

Besides runx-2, one of the well known factors which induce osteoblast differentiation is TGF-β. TGF-β is one of the most abundantly deposited growth factors in bone matrix that modulates proliferation and differentiation of osteoblastic cells, and enhances the accumulation of extracellular matrix components (21, 22). Direct injection of TGF-β in the periosteum increases bone volume in rodents (23). Prostate cancer cells produce TGF-β which can be further activated by PSA (11). In addition, TGF-β secretion in SaOS-2 cells can be elevated by PSA (12). Growth stimulation of SaOS-2 cells by PSA can be inhibited by TGF-β antibody, suggesting that PSA-induced growth of osteoblasts is mediated, at least in part, by TGF-β (12). These results are in agreement with the findings of our study that PSA enhances TGF-β mRNA expression in SaOS-2 cells. Collectively, these studies further show the involvement of TGF-β in osteoblast differentiation induced by prostate cancer cells.

Some of the lesser known genes involved in osteoblast differentiation regulated by PSA are cadherin 11, Wnt inhibitory factor-1 (Wif1), and secreted frizzled-related protein 2 (Sfrp2; Table 4). Cadherin 11 is a member of the cadherin family and can directly regulate the differentiation of mesenchymal cells into the cells of the osteo-lineage and the chondro-lineage, resulting in the formation of bone and cartilage tissues (24, 25). Wif1 and Sfrp2 are Wnt antagonists and their expressions are elevated during osteoblast differentiation (26). Wif1 is a secreted protein that binds Wnts and antagonizes their activity (27). Wif1 has been identified as a novel marker for osteoblast differentiation (28). It was hypothesized that Wnt maintains a proliferative signal for osteoblasts, which should be turned off by Wnt antagonists such as Wif1 and Sfrp2, allowing osteoblast differentiation to proceed (26). In this study, we showed that PSA enhances Wif1 and Sfrp2 mRNA expression in SaOS-2 cells by ∼31-fold and 5-fold, respectively. These data are in concordance with the role of Wif1 and Sfrp2 in osteoblast differentiation (26), suggesting that dysregulation of Wnt signaling pathways by PSA may contribute to osteoblast differentiation induced by prostate cancer cells.

It is interesting to note that osteoprotegerin is the most down-regulated gene by PSA (Table 2). Osteoprotegerin is a soluble member of the tumor necrosis factor receptor superfamily that prevents the association of RANKL with RANK by acting as a decoy receptor (29). The RANKL-osteoprotegerin system plays a very important role in bone remodeling. The binding of RANKL to the osteoclast surface receptor RANK induces osteoclastogenesis (29, 30). Osteoprotegerin is a soluble decoy receptor of RANKL, which neutralizes its interaction with RANK, resulting in reduction of osteoclast formation (30). Several studies show a role for RANKL and osteoprotegerin in regulating the bone remodeling in prostate cancer bone metastases (3133). In contrast to the reduction of osteoprotegerin, PSA enhances RANKL mRNA expression in SaOS-2 cells (Fig. 2). These data suggest that in addition to the induction of osteoblast differentiation, PSA also induces osteoclast formation. This is in concordance with clinical observations that prostate cancer metastases forms a heterogeneous mixture of osteolytic and osteoblastic lesions, although osteoblastic lesions are predominant (4, 34).

In summary, our results show that overexpression of PSA in SaOS-2 cells modulates genes involved in bone remodeling and induces osteoblast differentiation, suggesting that in addition to serving as a marker, PSA plays a functional role in bone metastases of prostate cancer. Further investigation of the biological effects of PSA and genes regulated by PSA in osteogenesis should enhance the current understanding of prostate cancer metastases in bone.

Grant support: NIH grants CA90271 and CA109441, and U.S. Army Medical Research Materiel Command, Prostate Cancer Research Program grant DAMD17-01-1-0089.

The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked advertisement in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

Note: Supplementary data for this article are available at Clinical Cancer Research Online (http://clincancerres.aacrjournals.org/).

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Supplementary data