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Cancer Research 67, 930-939, February 1, 2007. doi: 10.1158/0008-5472.CAN-06-2892
© 2007 American Association for Cancer Research
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Molecular Biology, Pathobiology, and Genetics

Demethylation-Linked Activation of Urokinase Plasminogen Activator Is Involved in Progression of Prostate Cancer

Sai Murali Krishna Pulukuri1, Norman Estes2, Jitendra Patel3 and Jasti S. Rao1,4

Departments of 1 Cancer Biology and Pharmacology, 2 Surgery, 3 Pathology, and 4 Neurosurgery, University of Illinois College of Medicine at Peoria, Peoria, Illinois

Requests for reprints: Jasti S. Rao, Department of Cancer Biology and Pharmacology, University of Illinois College of Medicine, Box 1649, Peoria, IL 61656. Phone: 309-671-3445; E-mail: jsrao{at}uic.edu.


   Abstract
Top
 Abstract
Introduction
 Materials and Methods
 Results
 Discussion
 References
 
Increased expression of urokinase plasminogen activator (uPA) has been reported in various malignancies including prostate cancer. However, the mechanism by which uPA is abnormally expressed in prostate cancer remains elusive. Here, we show that uPA is aberrantly expressed in a high percentage of human prostate cancer tissues but rarely expressed either in tumor-matched nonneoplastic adjacent tissues or benign prostatic hyperplasia samples. This aberrant expression is associated with cancer-linked demethylation of the uPA promoter. Furthermore, treatment with demethylation inhibitor S-adenosylmethionine or stable expression of uPA short hairpin RNA significantly inhibits uPA expression and tumor cell invasion in vitro and tumor growth and incidence of lung metastasis in vivo. Collectively, these findings strongly suggest that DNA demethylation is a common mechanism underlying the abnormal expression of uPA and is a critical contributing factor to the malignant progression of human prostate tumors. [Cancer Res 2007;67(3):930–9]


   Introduction
Top
 Abstract
 Introduction
Materials and Methods
 Results
 Discussion
 References
 
Prostate cancer is the most frequently diagnosed malignant disease and the second leading cause of cancer death in men in the United States (1). Each year, ~200,000 men are newly diagnosed, and 31,000 men will die from prostate cancer (2). When diagnosed early, tumors confined to the prostate can be effectively treated by radical prostatectomy or radiotherapy (3, 4). However, a significant number of patients with clinically localized disease who are treated with radical prostatectomy may also develop metastases (5). Postsurgical residual disease requires radiation and/or hormonal therapy, which may prevent tumor progression and metastasis. Nonetheless, no curative treatment currently exists for hormone-refractory, metastatic prostate cancer (6). Metastasis is frequently a final and fatal step in the progression of prostate cancer. The bones and lungs are frequent sites of prostate cancer metastasis; metastases to these sites signal the entry of the disease into an incurable phase (7). More effective therapies for prostate cancer are thus needed.

An understanding of the molecular basis of tumor cell metastasis is essential to the development of novel targeted therapies for prostate cancer. Metastasis is a complex, multistep process, during which tumor cells spread from the primary tumor mass to distant tissues and organs of the body (8). The invasive ability of tumor cells is crucial for cancer metastasis and is a major obstacle to successful treatment (9). To date, several human metastasis-associated genes have been shown to regulate the metastatic capacity of tumor cells (10). Among these genes, urokinase plasminogen activator (uPA) plays a major role in cancer invasion and metastasis (11, 12). uPA converts plasminogen to plasmin, which facilitates matrix degradation and activates several matrix metalloproteinases (13, 14). Additionally, binding of uPA with its receptor uPAR activates the Ras/extracellular signal-regulated kinase pathway, which in turn, leads to cell proliferation, migration, and invasion (15).

Increased expression of the uPA gene has been reported in various malignancies, including prostate (16, 17), glioblastoma (18, 19), melanoma (20), breast (21), colon (22), and lung (23) cancers. Notably, in most of these cases, its increased expression is associated with increased metastatic potential and poor survival (24–26). Moreover, studies using uPA inhibitors (27) or uPA gene silencing approaches (28, 29) have confirmed the important role of uPA in the processes of tumor growth, invasion, and metastasis. However, little is known about the mechanism(s) by which uPA switches from being a normally tightly controlled gene to one that is deregulated in tumor cells.

Changes in the status of DNA methylation at the CpG islands of gene promoters are some of the most common molecular alterations found in human cancers (30). Indeed, imbalance in DNA methylation has been frequently reported in prostate cancer (31). In recent years, it has become increasingly obvious that DNA hypermethylation is not the only mechanism by which tumor-associated genes are altered during tumorigenesis. Growing evidence now indicates that demethylation also plays an important role in carcinogenesis, and indeed, that it may be as significant as DNA hypermethylation. For example, the heparanase (32), cyp1b1 (33), synuclein-{gamma} (34), p-cadherin (35), r-ras (36), and c-myc (37) genes are activated by DNA demethylation in various tumors. In human prostate cancer tissues, however, the epigenetic regulation of uPA has not been investigated.

The present study was designed to test the hypothesis that demethylation-linked activation of uPA is involved in the processes of tumor progression and metastasis of prostate cancer. We analyzed the expression and the methylation status of the uPA gene in patient samples of prostate cancer, nonneoplastic adjacent tissues (NNAT), and benign prostatic hyperplasia (BPH). We found that uPA promoter demethylation plays a causal role in tumor growth, invasion, and metastasis of prostate cancer. Our results have implications in the progression of prostate carcinomas and the molecular diagnosis and treatment of prostate cancer metastases.


   Materials and Methods
Top
 Abstract
 Introduction
 Materials and Methods
Results
 Discussion
 References
 
Human prostate tissues and immunohistochemistry. Human prostate tumors with adjacent prostatic intraepithelial neoplasia (PIN) and NNAT samples were obtained from patients undergoing therapeutic or routine surgery, respectively (see Supplementary Methods). Many prostate cancer and BPH tissues were also obtained as paraffin-embedded, formalin-fixed blocks. For immunohistochemistry, tissue sections were labeled with mouse monoclonal anti-human uPA antibody (V10196, Biomeda, Foster City, CA), and we graded the expression levels as outlined in Supplementary Methods.

DNA methylation analysis. We extracted genomic DNA and treated it with sodium bisulfite as previously described (38). For methylation-specific PCR (MSP), sodium bisulfite–treated DNA was amplified with primers specific to methylated or unmethylated sequences. For bisulfite sequencing, sodium bisulfite–treated DNA was amplified with primers common to methylated and unmethylated DNA sequences. PCR products for sequencing were cloned into the TOPO TA cloning vector (Invitrogen, Carlsbad, CA) and sequenced with the M13R primer. MSP and bisulfite sequencing primer sequences and PCR conditions are given in the Supplementary Methods.

Cell culture, fibrin zymography, immunoblotting, and real-time reverse transcription-PCR. The prostate cancer cell lines RWPE1, RWPE2, LNCaP, DU145, and PC3 were obtained from the American Type Culture Collection (Manassas, VA) and cultured as directed. DU145 and PC3 cells were treated with either S-adenosylmethionine (Ado-Met; New England Biolabs, Ipswich, MA) or S-adenosylhomocysteine (Ado-Hcy; Sigma, St. Louis, MO) for 5 days. Tissue and cell lysates were prepared as previously described (28) and processed as outlined in Supplementary Methods. Total RNA was extracted from tissues and cell lines DU145 and PC3 using RNeasy kit (Qiagen, Valencia, CA). Expression analysis for uPA mRNA was measured using real-time quantitative PCR (Bio-Rad, Hercules, CA) with SYBR Green PCR Mastermix (Bio-Rad). See Supplementary Methods for all primer sets and detailed methods.

RNA interference, Matrigel invasion, and 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide analysis. To create the small interfering RNA plasmid construct, complementary strands of oligonucleotides specifically targeting uPA (5'-AAGAAATTCGGAGGGCAGCAC-3') was synthesized and cloned into pSilencer-U6 vector (Ambion, Austin, TX). Stable transfection of cells with either uPA short hairpin RNA (shRNA) or control shRNA (scramble) vector using LipofectAMINE transfection reagent (Life Technologies, Rockville, MD) was carried out as recommended by the manufacturer. Invasion of cells through Matrigel was conducted using a Transwell apparatus (Corning Costar, Acton, MA) as described previously (28). Proliferation of cells was assayed using the 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide reagent (Chemicon, Temecula, CA) as described (28).

Orthotopic mouse prostate tumor/metastasis model. Orthotopic implantation was carried out as previously described (28). PC3-RFP cells treated with 150 µmol/L Ado-Met or Ado-Hcy for 5 days, or PC3-RFP cells stably expressing either control shRNA or uPA shRNA were injected into the mouse prostate lateral lobe (106 cells per mouse). Primary tumor and metastasis fluorescence was detected noninvasively using an in vivo optical imaging system (IVIS-200, Xenogen Corp., Alameda, CA). Tumor fluorescence intensity and areas were recorded once a week for a period of 35 days after cell implantation. At the end of the experiment, the mice were sacrificed, and the lungs were removed and monitored for fluorescent metastases.


   Results
Top
 Abstract
 Introduction
 Materials and Methods
 Results
Discussion
 References
 
Expression of uPA in human prostate. To determine the effect of uPA expression on prostate cancer progression, we did immunohistochemical staining of paraffin-embedded BPH and prostate cancer tissue specimens with an antibody against uPA protein. We found that BPH tissues showed undetectable uPA protein staining, whereas prostate cancer tissues were intensely stained for uPA protein (Fig. 1A ). Using visual criteria, positive uPA immunostaining was observed in 96.8% of the prostate cancer samples (31 of 32 samples). We also analyzed the intensity of immunostaining using NIH Image J software as quantitative criteria. These results indicate that the intensity of uPA immunostaining was significantly higher in prostate cancer samples than in BPH samples (P < 0.001; Fig. 1A).


Figure 1
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  		Figure 1. Expression of uPA in human prostate tissue samples. A, representative immunohistochemical staining for uPA protein in human prostate cancer (PC) and in BPH samples. The average intensity of uPA immunostaining is significantly increased in prostate cancer samples compared with BPH samples (P < 0.001). B, representative immunohistochemical staining for uPA protein in human prostate cancer and matched NNAT. Compared with that in the matched adjacent NNAT, the overall average intensity of uPA immunostaining in the prostate cancer tissues was significantly higher (P < 0.001). C, quantitative mRNA expression of uPA in human prostate tumor (red) compared with normal adjacent tissue of the same individuals (green, connected by a line). Significant up-regulation of uPA expression in tumor tissue than in normal adjacent tissue samples. D, fibrin zymography of tumors (T) and adjacent normal tissues (N) corresponding to the RT-PCR samples in (C) showing activity of uPA in majority of prostate cancer samples.

 
The differential uPA expression between BPH and prostate cancer tissues prompted us to examine uPA expression systematically in human prostate tumor tissues and the matched NNAT using immunohistochemistry. Either no or undetectable uPA protein staining was observed in 90% tumor-matched NNAT (18 of 20 samples). We also detected either no or weak uPA immunostaining in PIN lesions (9 of 13 samples), which represent precursors of prostate cancer. In contrast, moderate to strong uPA protein staining was observed in 100% of the prostate cancer tissues (all 20 samples). Examples of statistically significant differences in uPA staining intensity among NNAT, PIN, and prostate tumor tissues are illustrated in Fig. 1B and Supplementary Fig. S1. Real-time reverse transcription-PCR (RT-PCR) analysis was used to further examine the expression of uPA mRNA in human prostate tumors and matched NNAT samples. These results show that uPA mRNA was significantly up-regulated in the majority of prostate tumor samples when compared with their adjacent normal tissue (Fig. 1C). Real-time RT-PCR analysis confirmed the immunostaining results. A similar trend in uPA activity was observed as assessed by fibrin zymography (Fig. 1D). These BPH versus cancer and normal versus cancer comparisons suggest that as prostate cancer progresses, there is a trend towards increased expression of uPA protein. They also suggest that uPA expression levels might be an effective molecular indicator of prostate cancer progression, given that the highest expression was observed significantly in prostate cancers but not in their normal counterparts.

Aberrant expression of uPA in prostate cancers by promoter demethylation. To understand why uPA is aberrantly expressed in prostate cancers but not in their normal counterparts, we first examined the methylation status of the uPA promoter by performing MSP in the 32 prostate cancer samples and in the 24 BPH samples. MSP distinguishes between unmethylated and methylated CpG islands by using two sets of primers that amplify either unmethylated or methylated sequences after bisulfite treatment, which specifically converts unmethylated cytosines to uracils. Figure 2C (top) shows a schematic representation of the CpG island and corresponding regions of MSP products using methylated and unmethylated uPA primers. Representative results of BPH and prostate cancer tissues are shown in Fig. 2A. In prostate cancers, which express uPA aberrantly, the PCR products were only visible in the unmethylated lane representing demethylation at the promoter CpG regions of uPA. However, in the BPH samples, prominent PCR products were obtained specifically for methylated CpG islands, with the methylated primer set indicating no demethylation at the promoter CpG regions of uPA. There are two BPH samples that were positive for immunostaining with anti-uPA antibody, which contained a partial demethylated uPA gene. We did additional MSP using bisulfite-modified genomic DNAs in prostate cancers and tumor-matched NNAT. As shown in Fig. 2B, the demethylated PCR product of uPA CpG island, either as the sole form or as the predominant form when compared with the methylated PCR product from the same DNA sample, was detected in 100% of prostate tumor samples (all 20 samples), indicating loss of the epigenetic control of uPA gene in prostate cancers. In contrast, uPA demethylation was detected only in 10% of tumor-matched NNAT (2 of 20 samples), which were positively stained with immunohistochemistry.


Figure 2
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  		Figure 2. Cancer-linked demethylation of the uPA promoter. A, MSP analysis of the uPA gene promoter region in human prostate cancer samples and in BPH samples. U, unmethyl control; M, methyl control; NC, no template control; uPA-U, unmethylated PCR product; uPA-M, methylated PCR product. Positive and negative controls are described in Supplementary Methods. B, MSP analysis of the uPA gene promoter region in prostate cancers and in the matched NNAT of each tumor. C, results of bisulfite DNA sequencing of the uPA promoter CpG island region. Top, schematic representation of the uPA gene promoter highlighting the positions of the CpG islands relative to the transcription start site. Thin vertical lines, positions of CpG sites in the genomic sequence; bent arrow, transcriptional start site. Bottom, representative sequencing results of the MSP products. bullet, methylated CG dinucleotide; {circ}, demethylated CG dinucleotide. In prostate tumor samples, CpG sites of the uPA promoter region were demethylated, whereas in BPH and NNAT of each tumor sample, most CpG sites were methylated. D1 and D2, percentage of cases methylated at uPA promoter-associated CpG islands. D3, correlation between uPA expression and the demethylation status of the analyzed gene promoter region. The majority of uPA-positive cases were demethylated on this promoter region (P < 0.001).

 
To confirm the methylation versus demethylation patterns of uPA promoter CpG island in prostate cancer, NNAT, and BPH, bisulfite-modified DNA was amplified and cloned into TA-cloning vector for subsequent sequencing analysis. The methylation statuses of 25 representative CpG sites are shown in Fig. 2C. Nearly all CpG sites within the CpG island of uPA were demethylated in prostate cancer samples. In contrast, almost all of the CpG sites remained methylated in the matched NNAT as well as the BPH samples. From these sequencing results, we conclude that the uPA CpG island is fully methylated in normal prostate tissues. Tumors from these tissues contained completely demethylated uPA.

uPA promoter demethylation correlates with high protein levels. As shown in Supplementary Table S1, uPA promoter is hypermethylated in 91.6% of the BPH samples (22 of 24 samples), but >96.8% of the prostate cancer samples (31 of 32 samples) showed uPA promoter demethylation. These results show that methylation levels are significantly higher in BPH samples compared with prostate cancer samples (Fig. 2D1). Likewise, we found that the uPA promoter is hypermethylated in 90% of the tumor-matched NNAT (18 of 20 samples), but 100% of these prostate tumor tissues (all 20 samples) showed uPA promoter demethylation (Supplementary Table S1; Fig. 2D2). When these results were correlated with uPA expression, a statistically significant association was found between these variables: 90% of uPA negative cases were methylated, whereas 98% of positive cases were demethylated (P < 0.001; Fig. 2D3; Supplementary Table S1). These results clearly show that cancer-linked demethylation of the uPA promoter CpG island is primarily responsible for the aberrant expression of uPA in prostate cancer.

uPA demethylation and protein expression in malignant and normal prostate epithelial cell lines. To determine whether the expression of uPA in prostate cancer cell lines is associated with demethylation, we examined the methylation status of uPA and expression in a panel of cell lines, including a human epithelial (RWPE1), tumorigenic (RWPE2), and three metastatic prostate cancer cell lines (LNCaP, DU145, and PC3). The highly metastatic prostate cancer cell lines DU145 and PC3 expressed the uPA protein, and the uPA gene promoter CpG region was demethylated (Fig. 3A and B ). Fibrin zymography for uPA activity supported the immunoblotting findings (Fig. 3C). The remaining three cell lines RWPE1, RWPE2, and LNCaP did not express uPA, and MSP showed that the promoter region of the uPA gene in these cell lines was methylated. Treatment of these cell lines with 20 µmol/L 5-azacytidine (5-aza) for 6 days resulted in uPA protein expression (data not shown). To determine whether these methylation-associated protein expression findings correlate with the biological activity of the prostate cancer cell lines used, we measured the ability of these cells to traverse a Matrigel-coated membrane with 8-µm pores, a correlate of metastatic potential in vivo. The results of Matrigel invasion assay are shown in Fig. 3D. We found that DU145 and PC3 cells, which express high levels of uPA protein by promoter demethylation, displayed the greatest levels of invasive potential, whereas LNCaP, RWPE2, and RWPE1 cells, which do not express uPA and have promoter methylation, exhibited low levels of invasive potential (Fig. 3D). These data also indicated that cancer-linked demethylation mainly leads to abnormal expression of the uPA gene in prostate cancer cell lines.


Figure 3
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  		Figure 3. Analysis of uPA promoter CpG methylation in prostate cancer cell lines. A, MSP showing the methylation status of prostate cancer cell lines. Positive and negative controls are described in Supplementary Methods. B, immunoblot analysis of prostate cancer cell lines corresponding to MSP samples in (A) showing expression of uPA protein in unmethylated samples. Glyceraldehyde-3-phosphate dehydrogenase (GAPDH) was used as a loading control. C, fibrin zymography of prostate cancer cell lines corresponding to MSP samples in (A) showing uPA activity in unmethylated samples. D, cells invading through the Matrigel were counted under a microscope in three random fields at x200 magnification. Columns, mean of three fields counted; bars, SD. *, P < 0.001, significant differences from normal prostate cancer cell line RWPE1.

 
Effect of Ado-Met on uPA expression and activity in metastatic prostate cancer cell lines. First, we reasoned that if the demethylation of the gene promoter plays a causal role in prostate cancer progression and metastasis, then an inhibition of demethylation of that gene would block the prostate cancer progression into the aggressive and metastatic stages of the disease. Prostate cancer cell lines DU145 and PC3, which express uPA aberrantly by promoter demethylation, were used in these experiments. Several studies have shown that Ado-Met, a methyl donor of methyltransferase reaction, inhibits active demethylation in cell lines and tumors in animals (39, 40). To determine the contribution of uPA promoter demethylation to the expression of this gene, we treated DU145 and PC3 cells with increasing concentrations of Ado-Met from 25 to 150 µmol/L for 5 days. The effect of these treatments on uPA protein and mRNA expression was monitored by immunoblot analysis and RT-PCR, respectively. Both uPA protein (Fig. 4A, top ) and mRNA (Fig. 4A, middle) were inhibited by Ado-Met treatment in a dose-dependent manner. Fibrin zymography for uPA activity supported the immunoblotting and RT-PCR analyses (Fig. 4A, bottom). In contrast, treatment with Ado-Hcy, an unmethylated analogue of Ado-Met, did not affect the expression of uPA (Fig. 4B). Ado-Met silenced uPA expression in both the DU145 and PC3 cell lines, which had high endogenous levels of uPA via promoter demethylation, indicating that Ado-Met effect was not confined to a single cell line model. In parallel experiments, cell viability was studied in DU145 and PC3 cells treated with PBS (control), Ado-Met (150 µmol/L), or Ado-Hcy (150 µmol/L). These treatments had no significant effect on cell survival of DU145 and PC3 cells (Fig. 4C).


Figure 4
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  		Figure 4. Effect of Ado-Met treatment on uPA expression in prostate cancer cell lines. A, protein (top), mRNA (middle), and activity (bottom) levels of uPA in DU145 and PC3 cells treated with different concentrations of Ado-Met for 5 d were monitored by immunoblot, RT-PCR, and fibrin zymography, respectively. Glyceraldehyde-3-phosphate dehydrogenase (GAPDH) was used as a loading control for RNA and protein analysis. B, immunoblot analysis shows that treatment with 150 µmol/L Ado-Hcy for 5 d did not lead to silencing of uPA protein expression in the DU145 and PC3 prostate cancer cells. GAPDH was used as a loading control to check for equal loading of the gel. C, measure of viable cells after treatment with Ado-Met, Ado-Hcy, or vehicle control in DU145 and PC3 prostate cancer cells are described in Supplementary Methods.

 
Effect of Ado-Met on uPA promoter methylation and invasive potential of the human metastatic prostate cancer cell lines. To determine whether silencing of uPA by Ado-Met was associated with a change in the methylation pattern of the uPA promoter, genomic DNA was isolated from drug-treated DU145 and PC3 cells and subjected to MSP analysis. We obtained consistent results in MSP analysis from three independent samples treated with PBS, Ado-Met, or Ado-Hcy. As shown in Fig. 5A , treatment of both DU145 and PC3 cell lines with Ado-Met resulted in visible PCR products only in the methylated primer set representing hypermethylation at the promoter CpG regions of uPA. In contrast, in DU145 and PC3 cells treated with PBS or Ado-Hcy, prominent PCR products remained specific for unmethylated CpG islands with the unmethylated primer set, but no products were detected with the methylated primer set. These results were confirmed by bisulfite sequencing analysis (data not shown). Because silencing of uPA by Ado-Met is a consequence of increased DNA methylation, we then asked whether the DNA demethylating agent 5-aza would inhibit this effect. To this end, RNA was extracted from both DU145 and PC3 cells treated with PBS, Ado-Hcy, Ado-Met, or Ado-Met plus 5-aza. As shown in Fig. 5B, 5-aza treatments inhibited Ado-Met–dependent silencing of uPA. A similar trend was also observed by immunostaining with anti-uPA antibodies (Fig. 5C). These results indicate that Ado-Met silencing of uPA is mediated by DNA methylation. Finally, we evaluated the effect of uPA silencing by Ado-Met on the invasive ability of the metastatic DU145 and PC3 cells using the Matrigel invasion assay. As shown in Fig. 5D, silencing of uPA by Ado-Met significantly diminishes the invasive potential of metastatic DU145 and PC3 cells. These observations suggest that reversal of uPA promoter demethylation by Ado-Met can inhibit uPA expression and the invasive potential of metastatic DU145 and PC3 cells. These findings are also consistent with the hypothesis that the activation of uPA is strongly associated with the demethylation of uPA promoter.


Figure 5
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  		Figure 5. Effect of Ado-Met treatment on uPA promoter CpG methylation and invasive potential of the prostate cancer cell lines. A, MSP analysis of the uPA gene promoter region in DU145 (top) and PC3 (bottom) cells treated with Ado-Met, Ado-Hcy, or vehicle alone (control) for 5 d. Positive and negative controls are described in Supplementary Methods. B, RT-PCR analysis shows that Ado-Met–treated DU145 and PC3 cells did not express uPA, although treatment with Ado-Met plus 5-aza results in uPA expression. Glyceraldehyde-3-phosphate dehydrogenase (GAPDH) was used as a loading control. C, immunostaining for uPA protein expression in DU145 and PC3 cells treated with Ado-Met, Ado-Hcy, or vehicle alone for 5 d. Note that the cells with green fluorescence represent ß-actin expression (green by fluorescent isothiocyanate), and those having both uPA (red by Texas Red) and ß-actin expression show yellow fluorescence. Ado-Met–treated DU145 and PC3 cells substantially changed the cell staining profiles of uPA compared with Ado-Hcy, Ado-Met plus 5-aza, and control cells. Nuclear counterstaining (blue) was obtained with 4',6-diamidino-2-phenylindole. D, the invasive potential of the DU145 and PC3 cells with the indicated treatments is examined as described in Fig. 3D. *, P < 0.001, significant difference from controls (i.e., vehicle-treated DU145 and PC3 cells).

 
uPA is essential for prostate cancer growth and metastasis in vivo. To further clarify the biological meanings of aberrant expression of uPA in prostate cancer, we used both the Ado-Met– and shRNA-mediated knockdown experiments to examine whether the expression of uPA by promoter demethylation contributed to the tumor growth and metastatic ability of PC3 prostate cancer cells. We first derived PC3-RFP cells by stable transfection of a plasmid expressing the red fluorescence gene so that we could track tumor cell growth and metastasis in vivo. We also derived stable PC3-RFP cells expressing shRNA against uPA and verified specific knockdown effects of uPA by immunoblotting (Supplementary Fig. S2A). Stable knockdown of uPA with plasmid expressing uPA shRNA reduced invasion behavior dramatically, whereas proliferation was unaffected (Supplementary Fig. S2B–D). To test whether uPA knockdown in vitro would affect the ability of PC3 prostate tumor growth or progression in vivo, we orthotopically introduced PC3-RFP cells treated with PBS alone as control (mock), PC3-RFP cells treated with 150 µmol/L Ado-Met for 5 days, and PC3-RFP cells stably transfected with uPA shRNA or control shRNA into the prostate of immunodeficient mice. Tumor progression was monitored in mice by using in vivo imaging system (Xenogen). Representative data are shown in Fig. 6 . The mice injected with PC3-RFP cells treated with Ado-Met and PC3-RFP cells stably transfected with uPA shRNA developed prostate tumors of significantly smaller volume after 5 weeks compared with the mice inoculated with cells either treated with PBS or stably transfected with control shRNA (Fig. 6A, top). The presence of significant growth difference in tumors of the prostate between control and uPA knockdown cells injected mice groups was confirmed by autopsy after imaging (Fig. 6A, bottom).


Figure 6
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  		Figure 6. Ado-Met– and uPA shRNA– directed knockdown of uPA inhibits PC3 tumor growth and metastasis in nude mice. A, representative nude mice injected with either control PC3-RFP cells (mock), PC3-RFP cells treated with 150 µmol/L Ado-Met for 5 d, or PC3-RFP cells stably transfected with uPA shRNA or control shRNA. Top, nude mice carrying an orthotopic prostate tumor, established from either mock or stable control shRNA PC3-RFP cells, showed substantial fluorescence signal as indicated by photon counts. In contrast, the orthotopic prostate tumors developed from PC3-RFP cells either treated with Ado-Met or stably transfected with uPA shRNA showed significantly low fluorescence signal. Bottom, surgical examination at autopsy confirmed the inhibition of orthotopic prostate tumor growth established from PC3-RFP cells either treated with Ado-Met or stably transfected with uPA shRNA (black arrows). Right, a comparison of PC3 tumor photon counts with the indicated groups. Columns, mean photon counts of six animals per group; bars, SD. *, P < 0.001, significant differences from control groups (i.e., mock or control shRNA). B, fluorescence signal in the lung, representative of lung metastasis, was recorded for each mouse. Lung images from different mice (left). Lung metastasis signals were analyzed by photon counts (right). P < 0.001, significant inhibition of lung metastasis is seen in PC3-RFP cells either treated with Ado-Met or stably transfected with uPA shRNA relative to the control groups. C, RNA samples extracted from PC3-RFP prostate tumors of six animals per group were analyzed using RT-PCR for uPA expression levels. Ado-Met and uPA shRNA groups showed the most prominent and specific knockdown of uPA, whereas glyceraldehyde-3-phosphate dehydrogenase (GAPDH) was unchanged. D, tumors isolated from these mice were also examined for uPA protein expression by immunostaining. Note that the cells with red fluorescence represent uPA expression (red by Texas Red). Nuclear counterstaining (blue) was obtained with 4',6-diamidino-2-phenylindole. The level of uPA protein expression was significantly decreased in the Ado-Met–treated cells or uPA shRNA stably expressing cells implanted in tumor group when compared with that in control groups.

 
We also assessed whether the uPA knockdown in PC3-RFP cells affected the ability of these cells to metastasize in vivo. The lung was dissected from each mouse, and photon counts were recorded. The in vivo imaging data and the quantification of the signal from the lungs are shown in Fig. 6B (left and right, respectively). The lungs derived from control mice injected with PC3-RFP and PC3-RFP+control shRNA cells showed high photon counts by imaging system. In contrast, there was a marked reduction (~5.1-fold) in the incidence of lung metastasis when primary tumor cells experiencing uPA knockdown were seeded in host mice. Analysis by RT-PCR showed that there was a prominent expression of uPA mRNA in the prostate tumors isolated from cells treated with PBS or cells stably expressing unrelated control shRNA, whereas RT-PCR analysis determined long-term selective knockdown of uPA in tumors developed from the cells treated with Ado-Met or cells stably expressing uPA shRNA (Fig. 6C). Immunohistochemical analysis of the tumors showed the intensive expression of uPA in the tumors isolated from the animals in the mock and control shRNA groups, whereas almost no positive staining for uPA was observed in the tumors isolated from the animals in both the Ado-Met or uPA shRNA groups (Fig. 6D). These results indicate that knockdown of uPA in prostate cancer cells significantly suppresses both primary tumor growth and the in vivo incidence of lung metastasis.


   Discussion
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
References
 
uPA function has been implicated in tumorigenesis for over a decade, and recent uPA gene knockdown approaches have enabled experimental confirmation that uPA does indeed play a key role in the metastasis of solid tumors as well as mediating tumor angiogenesis both directly by promoting endothelial migration and indirectly via release of proangiogenic molecules from the extracellular matrix (28, 29, 41). Numerous studies in tumor cell lines have correlated the metastatic potential of tumor cells with uPA activity and protein expression (28, 42–44). Furthermore, several studies of clinical tumor samples have correlated high uPA expression with tumor progression and, in some cases, poor patient postoperative survival (45, 46). Clearly, there is significant interest in identifying the molecular mechanisms that control uPA gene expression in normal and pathologic settings. A recent study on breast cancer tissues suggests that hypomethylation is one of the mechanisms contributing to the abnormal expression of uPA (47). However, such studies in relation to prostate cancer are lacking.

In the present study, we examined the relationship between demethylation of uPA promoter CpG island and the expression of this prometastatic gene in prostate cancer. We found that uPA expression was undetectable in BPH and NNAT of prostate, whereas there was a dramatic increase of uPA expression in prostate cancer tissues at a high frequency (Fig. 1). These findings suggest that increased expression of uPA contributes to prostate cancer development and progression. This notion has been supported by numerous lines of evidence. For example, Van Veldhuizen et al. (17) reported that ~70% of the prostate tumors with extracapsular extension highly express uPA, whereas only 27% of tumors without capsular invasion do. Gaylis et al. (16) also reported a strong correlation between uPA expression and aggressive phenotype in a human PC3 prostate cancer model. Moreover, Hienert et al. (48) showed that uPA expression is increased in the plasma of patients with prostate cancer when compared with those with BPH.

The mechanism by which uPA is abnormally expressed in prostate cancer remains unclear. The proximal promoter of uPA contains a CpG island spanning 1,600 bp around the transcriptional start site, which could be demethylated and thus activate the expression of this gene. We used a series of prostate tumors to investigate whether demethylation of the uPA promoter could be a mechanism of gene activation in prostate cancer. We found that CpG sites within the uPA promoter region are completely methylated in BPH and tumor-matched NNAT, whereas those in prostate cancer tissues are demethylated (Fig. 2). Thus, these data strongly suggest that in BPH and NNAT, uPA is methylated at the promoter-associated CpG sites, resulting in blocking of mRNA transcription and, consequently, its protein expression. Here, we show a statistically significant correlation between the methylation patterns of the uPA promoter region and its aberrant protein expression levels in prostate cancer tissues (Supplementary Table S1; Fig. 2D). A similar trend was observed in prostate cancer cell lines (Fig. 3). Collectively, our results indicate that uPA promoter demethylation contributes to the process of prostate carcinogenesis and metastasis.

Although hypomethylation was the first epigenetic alteration characterized in cancer, little is known about the potential role of promoter CpG demethylation in the activation of tumor- or metastasis-promoting genes. To address this issue, we used the uPA gene as a model because the promoter CpG region of this gene is a frequent target of DNA demethylation in prostate cancer. This is the case in the highly metastatic prostate cancer cell lines DU145 and PC3, where uPA is demethylated and active (Fig. 3). Consistent with these results, a previous report has shown that the induction of the uPA gene expression in uPA-negative cell lines LNCaP by 5-aza displayed a concurrent switch from hypermethylation to hypomethylation of the CpG island in the uPA gene promoter region (49). At present, the enzymes responsible for hypomethylation or demethylation in tumors are unknown. In recent years, the methyl donor Ado-Met has been used frequently as a DNA-methylating agent. In this study, Ado-Met treatment inhibited DNA demethylation either by enhancing DNA methyltransferase activity or by inhibiting active demethylation (Fig. 4). Several lines of evidence support the notion that exogenous Ado-Met causes hypermethylation of DNA (39, 50). More importantly, it has been shown that Ado-Met treatment leads to hypermethylation and silencing of uPA in breast cancer cell lines (40). We investigated the effect of Ado-Met on uPA expression in metastatic prostate cancer cell lines PC3 and DU145. Our data show that Ado-Met treatment results in uPA gene silencing via promoter methylation (Fig. 5). These results suggested that Ado-Met affects both demethylation of DNA and gene expression. This association of inhibition of uPA promoter demethylation and silencing of gene expression prompted us to rule out the possibility that Ado-Met has a general, methylation-independent inhibitory effect on gene expression, which also might result in inhibition of gene expression.

Several findings presented here provide further evidence that the uPA gene silencing mechanism of Ado-Met involves DNA methylation. First, treatment of DU145 and PC3 cells with Ado-Met, but not with its unmethylated analogue Ado-Hcy, inhibited demethylation and uPA expression. Notably, this Ado-Met–dependent silencing of uPA significantly inhibits tumor cell invasion in vitro (Fig. 5D) and tumor growth and metastasis in vivo (Fig. 6). Second, the effects of Ado-Met on uPA expression (Fig. 5B and C) and tumor cell invasion in vitro (Fig. 5D) were reversed by the demethylating agent 5-aza. Third, treatment of DU145 and PC3 cells with Ado-Met had no significant toxic effect (Fig. 4C). Furthermore, we observed that Ado-Met treatment had long-term effects in vivo on uPA expression and metastasis (Fig. 6). This prolonged silencing provides additional support for the conclusion that the mechanism of uPA knockdown by Ado-Met is dependent on methylation. shRNA-directed knockdown of uPA also strongly interfered with prostate tumor and metastases formation in mice after orthotopic transplantation of stably transfected PC-RFP cells. All these findings show that Ado-Met reverses hypomethylation and silences the demethylation-associated uPA activity. The results are also consistent with the hypothesis that demethylation-linked uPA activation is responsible for prostate cancer progression and metastasis.

Based on our observations, we propose the following model of activation of uPA in prostate cancer. During the neoplastic process, the metastatic prostate cancer cells would undergo a loss of epigenetic control leading to demethylation of the uPA gene promoter. Transcriptional activators would then be able to bind to the demethylated promoter region and activate uPA expression. Under normal circumstances, uPA is methylated at the promoter-associated CpG sites, resulting in blocking of mRNA transcription and, consequently, its protein expression. This model may explain how demethylation-linked abnormal expression of uPA contributes to prostate cancer progression and metastasis (Supplementary Fig. S3).

Thus far, most therapeutic strategies aimed at the reactivation of epigenetically silenced genes in human cancer cells have targeted DNA methyltransferases or histone deacetylases. In this study, uPA gene reactivation by cancer-linked demethylation has been observed. It is noteworthy that during the course of this investigation, several independent research groups have discovered the gene-specific hypomethylation during the progressive stages of many cancers (32–36). These studies have negative implications for the therapeutic use of demethylating agents, such as 5-aza, in the treatment of cancer. Further studies need to focus on identifying the different factors involved in methylation/demethylation equilibrium shifts, which in turn should increase our understanding of cancer progression and develop more effective therapies for this life-threatening disease. It will be interesting to investigate the candidate factors, such as methyl-CpG binding proteins, chromatin modifying enzymes, and demethylases involved in the loss of uPA methylation mark in prostate cancers. Perhaps, these investigations can provide insights to uncover the molecular factors that are responsible for the DNA demethylation process in tumors.


   Acknowledgments
 
Grant support: National Cancer Institute grants CA 75557, CA 92393, CA 95058, and CA 116708; National Institute of Neurological Disorders and Stroke grants NS47699 and NS57529; Caterpillar, Inc., Peoria, IL; and OSF Saint Francis, Inc., Peoria, IL (J.S. Rao).

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.

We thank Dr. Hnilica (Department of Pathology at the University of Illinois College of Medicine, Peoria, IL) for kindly providing normal and tumor tissues of human prostate, Shellee Abraham for preparing the article, Diana Meister and Sushma Jasti for reviewing the article, and Noorjehan Ali for technical assistance.


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

Received 8/ 4/06. Revised 10/ 4/06. Accepted 10/23/06.


   References
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
 References
 

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