demethylation-linked activation of urokinase plasminogen activator is involved in progression of...

Demethylation-Linked Activation of Urokinase Plasminogen

Activator Is Involved in Progression of Prostate Cancer

Sai Murali Krishna Pulukuri,1Norman Estes,

2Jitendra Patel,

3and Jasti S. Rao

1,4

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

Abstract

Increased expression of urokinase plasminogen activator(uPA) has been reported in various malignancies includingprostate cancer. However, the mechanism by which uPA isabnormally expressed in prostate cancer remains elusive.Here, we show that uPA is aberrantly expressed in a high per-centage of human prostate cancer tissues but rarely expressedeither in tumor-matched nonneoplastic adjacent tissues orbenign prostatic hyperplasia samples. This aberrant expres-sion is associated with cancer-linked demethylation of the uPApromoter. Furthermore, treatment with demethylation inhib-itor S-adenosylmethionine or stable expression of uPA shorthairpin RNA significantly inhibits uPA expression and tumorcell invasion in vitro and tumor growth and incidence of lungmetastasis in vivo . Collectively, these findings strongly suggestthat DNA demethylation is a common mechanism underlyingthe abnormal expression of uPA and is a critical contributingfactor to the malignant progression of human prostatetumors. [Cancer Res 2007;67(3):930–9]

Introduction

Prostate cancer is the most frequently diagnosed malignantdisease and the second leading cause of cancer death in men in theUnited States (1). Each year, f200,000 men are newly diagnosed,and 31,000 men will die from prostate cancer (2). When diagnosedearly, tumors confined to the prostate can be effectively treated byradical prostatectomy or radiotherapy (3, 4). However, a significantnumber of patients with clinically localized disease who are treatedwith radical prostatectomy may also develop metastases (5).Postsurgical residual disease requires radiation and/or hormonaltherapy, which may prevent tumor progression and metastasis.Nonetheless, no curative treatment currently exists for hormone-refractory, metastatic prostate cancer (6). Metastasis is frequently afinal and fatal step in the progression of prostate cancer. The bonesand lungs are frequent sites of prostate cancer metastasis;metastases to these sites signal the entry of the disease into anincurable phase (7). More effective therapies for prostate cancer arethus needed.

An understanding of the molecular basis of tumor cell metastasisis essential to the development of novel targeted therapies forprostate cancer. Metastasis is a complex, multistep process, duringwhich tumor cells spread from the primary tumor mass to distanttissues and organs of the body (8). The invasive ability of tumor

cells is crucial for cancer metastasis and is a major obstacle tosuccessful treatment (9). To date, several human metastasis-associated genes have been shown to regulate the metastaticcapacity of tumor cells (10). Among these genes, urokinaseplasminogen activator (uPA) plays a major role in cancer invasionand metastasis (11, 12). uPA converts plasminogen to plasmin,which facilitates matrix degradation and activates several matrixmetalloproteinases (13, 14). Additionally, binding of uPA with itsreceptor uPAR activates the Ras/extracellular signal-regulatedkinase pathway, which in turn, leads to cell proliferation, migration,and invasion (15).

Increased expression of the uPA gene has been reported invarious 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 isassociated with increased metastatic potential and poor survival(24–26). Moreover, studies using uPA inhibitors (27) or uPA genesilencing approaches (28, 29) have confirmed the important role ofuPA in the processes of tumor growth, invasion, and metastasis.However, little is known about the mechanism(s) by which uPAswitches from being a normally tightly controlled gene to one thatis deregulated in tumor cells.

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

The present study was designed to test the hypothesis thatdemethylation-linked activation of uPA is involved in the processesof tumor progression and metastasis of prostate cancer. We ana-lyzed the expression and the methylation status of the uPA gene inpatient samples of prostate cancer, nonneoplastic adjacent tissues(NNAT), and benign prostatic hyperplasia (BPH). We found thatuPA promoter demethylation plays a causal role in tumor growth,invasion, and metastasis of prostate cancer. Our results haveimplications in the progression of prostate carcinomas and themolecular diagnosis and treatment of prostate cancer metastases.

Materials and Methods

Human prostate tissues and immunohistochemistry. Human pros-

tate tumors with adjacent prostatic intraepithelial neoplasia (PIN) and

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

Requests for reprints: Jasti S. Rao, Department of Cancer Biology andPharmacology, University of Illinois College of Medicine, Box 1649, Peoria, IL 61656.Phone: 309-671-3445; E-mail: [email protected].

I2007 American Association for Cancer Research.doi:10.1158/0008-5472.CAN-06-2892

Cancer Res 2007; 67: (3). February 1, 2007 930 www.aacrjournals.org

Research Article

Research. on January 15, 2016. © 2007 American Association for Cancercancerres.aacrjournals.org Downloaded from

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NNAT samples were obtained from patients undergoing therapeutic orroutine 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, FosterCity, 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-specificPCR (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 sequencingwere 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 cellswere 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. TotalRNA 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 GreenPCR 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 smallinterfering RNA plasmid construct, complementary strands of oligonucleo-

tides 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) orcontrol 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 aTranswell 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 Amol/L Ado-Met or Ado-Hcy for 5 days, or PC3-RFP cells

stably expressing either control shRNA or uPA shRNA were injected into themouse 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 fluores-

cence intensity and areas were recorded once a week for a period of 35 daysafter cell implantation. At the end of the experiment, the mice were

sacrificed, and the lungs were removed and monitored for fluorescent

metastases.

Results

Expression of uPA in human prostate. To determine the effectof uPA expression on prostate cancer progression, we didimmunohistochemical staining of paraffin-embedded BPH andprostate cancer tissue specimens with an antibody against uPAprotein. We found that BPH tissues showed undetectable uPAprotein staining, whereas prostate cancer tissues were intenselystained for uPA protein (Fig. 1A). Using visual criteria, positiveuPA immunostaining was observed in 96.8% of the prostatecancer samples (31 of 32 samples). We also analyzed the intensityof immunostaining using NIH Image J software as quantitative

criteria. These results indicate that the intensity of uPA immunos-taining was significantly higher in prostate cancer samples than inBPH samples (P < 0.001; Fig. 1A).

The differential uPA expression between BPH and prostatecancer tissues prompted us to examine uPA expression systemat-ically in human prostate tumor tissues and the matched NNATusing immunohistochemistry. Either no or undetectable uPAprotein staining was observed in 90% tumor-matched NNAT(18 of 20 samples). We also detected either no or weak uPAimmunostaining in PIN lesions (9 of 13 samples), which representprecursors of prostate cancer. In contrast, moderate to strong uPAprotein staining was observed in 100% of the prostate cancertissues (all 20 samples). Examples of statistically significantdifferences in uPA staining intensity among NNAT, PIN, andprostate tumor tissues are illustrated in Fig. 1B and SupplementaryFig. S1. Real-time reverse transcription-PCR (RT-PCR) analysis wasused to further examine the expression of uPA mRNA in humanprostate tumors and matched NNAT samples. These results showthat uPA mRNA was significantly up-regulated in the majority ofprostate tumor samples when compared with their adjacentnormal tissue (Fig. 1C). Real-time RT-PCR analysis confirmed theimmunostaining results. A similar trend in uPA activity wasobserved as assessed by fibrin zymography (Fig. 1D). These BPHversus cancer and normal versus cancer comparisons suggest thatas prostate cancer progresses, there is a trend towards increasedexpression of uPA protein. They also suggest that uPA expressionlevels might be an effective molecular indicator of prostate cancerprogression, given that the highest expression was observedsignificantly in prostate cancers but not in their normal counter-parts.

Aberrant expression of uPA in prostate cancers by promoterdemethylation. To understand why uPA is aberrantly expressed inprostate cancers but not in their normal counterparts, we firstexamined the methylation status of the uPA promoter by perfor-ming MSP in the 32 prostate cancer samples and in the 24 BPHsamples. MSP distinguishes between unmethylated and methylatedCpG islands by using two sets of primers that amplify eitherunmethylated or methylated sequences after bisulfite treatment,which specifically converts unmethylated cytosines to uracils.Figure 2C (top) shows a schematic representation of the CpGisland and corresponding regions of MSP products using methy-lated and unmethylated uPA primers. Representative results ofBPH and prostate cancer tissues are shown in Fig. 2A . In prostatecancers, which express uPA aberrantly, the PCR products wereonly visible in the unmethylated lane representing demethylationat the promoter CpG regions of uPA . However, in the BPH samples,prominent PCR products were obtained specifically for methylatedCpG islands, with the methylated primer set indicating nodemethylation at the promoter CpG regions of uPA . There aretwo 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 inprostate cancers and tumor-matched NNAT. As shown in Fig. 2B ,the demethylated PCR product of uPA CpG island, either as thesole form or as the predominant form when compared with themethylated PCR product from the same DNA sample, was detectedin 100% of prostate tumor samples (all 20 samples), indicating lossof the epigenetic control of uPA gene in prostate cancers. Incontrast, uPA demethylation was detected only in 10% of tumor-matched NNAT (2 of 20 samples), which were positively stainedwith immunohistochemistry.

uPA Methylation Inhibits Metastasis

www.aacrjournals.org 931 Cancer Res 2007; 67: (3). February 1, 2007



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 uPAin human prostate tumor (red) compared with normal adjacent tissue of the same individuals (green , connected by a line). Significant up-regulation of uPA expressionin tumor tissue than in normal adjacent tissue samples. D, fibrin zymography of tumors (T ) and adjacent normal tissues (N ) corresponding to the RT-PCRsamples in (C ) showing activity of uPA in majority of prostate cancer samples.

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To confirm the methylation versus demethylation patterns ofuPA promoter CpG island in prostate cancer, NNAT, and BPH,bisulfite-modified DNA was amplified and cloned into TA-cloningvector for subsequent sequencing analysis. The methylationstatuses of 25 representative CpG sites are shown in Fig. 2C .Nearly all CpG sites within the CpG island of uPA weredemethylated in prostate cancer samples. In contrast, almost allof the CpG sites remained methylated in the matched NNATas wellas the BPH samples. From these sequencing results, we concludethat the uPA CpG island is fully methylated in normal prostatetissues. Tumors from these tissues contained completely demethy-lated uPA .uPA promoter demethylation correlates with high protein

levels. As shown in Supplementary Table S1, uPA promoter ishypermethylated 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 thatmethylation levels are significantly higher in BPH samplescompared with prostate cancer samples (Fig. 2D1). Likewise, wefound that the uPA promoter is hypermethylated in 90% of thetumor-matched NNAT (18 of 20 samples), but 100% of theseprostate tumor tissues (all 20 samples) showed uPA promoterdemethylation (Supplementary Table S1; Fig. 2D2). When theseresults were correlated with uPA expression, a statisticallysignificant association was found between these variables: 90% ofuPA negative cases were methylated, whereas 98% of positive caseswere demethylated (P < 0.001; Fig. 2D3 ; Supplementary Table S1).These results clearly show that cancer-linked demethylation of theuPA promoter CpG island is primarily responsible for the aberrantexpression of uPA in prostate cancer.uPA demethylation and protein expression in malignant and

normal prostate epithelial cell lines. To determine whether the

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 BPHsamples. U, unmethyl control; M, methyl control; NC, no template control; uPA-U, unmethylated PCR product; uPA-M, methylated PCR product. Positive and negativecontrols are described in Supplementary Methods. B, MSP analysis of the uPA gene promoter region in prostate cancers and in the matched NNAT of eachtumor. C, results of bisulfite DNA sequencing of the uPA promoter CpG island region. Top, schematic representation of the uPA gene promoter highlighting thepositions of the CpG islands relative to the transcription start site. Thin vertical lines, positions of CpG sites in the genomic sequence; bent arrow, transcriptionalstart site. Bottom, representative sequencing results of the MSP products. ., methylated CG dinucleotide; o, 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 genepromoter region. The majority of uPA-positive cases were demethylated on this promoter region (P < 0.001).





expression of uPA in prostate cancer cell lines is associated withdemethylation, we examined the methylation status of uPA andexpression in a panel of cell lines, including a human epithelial(RWPE1), tumorigenic (RWPE2), and three metastatic prostatecancer cell lines (LNCaP, DU145, and PC3). The highly metastaticprostate cancer cell lines DU145 and PC3 expressed the uPAprotein, and the uPA gene promoter CpG region was demethylated(Fig. 3A and B). Fibrin zymography for uPA activity supported theimmunoblotting findings (Fig. 3C). The remaining three cell linesRWPE1, RWPE2, and LNCaP did not express uPA, and MSP showedthat the promoter region of the uPA gene in these cell lines wasmethylated. Treatment of these cell lines with 20 Amol/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 biologicalactivity of the prostate cancer cell lines used, we measured theability of these cells to traverse a Matrigel-coated membrane with8-Am pores, a correlate of metastatic potential in vivo . The resultsof Matrigel invasion assay are shown in Fig. 3D . We found thatDU145 and PC3 cells, which express high levels of uPA protein bypromoter demethylation, displayed the greatest levels of invasivepotential, whereas LNCaP, RWPE2, and RWPE1 cells, which do notexpress uPA and have promoter methylation, exhibited low levels ofinvasive potential (Fig. 3D). These data also indicated that cancer-linked demethylation mainly leads to abnormal expression of theuPA gene in prostate cancer cell lines.

Effect of Ado-Met on uPA expression and activity inmetastatic prostate cancer cell lines. First, we reasoned that ifthe demethylation of the gene promoter plays a causal role inprostate cancer progression and metastasis, then an inhibition ofdemethylation of that gene would block the prostate cancerprogression into the aggressive and metastatic stages of thedisease. Prostate cancer cell lines DU145 and PC3, which expressuPA aberrantly by promoter demethylation, were used in theseexperiments. Several studies have shown that Ado-Met, a methyldonor of methyltransferase reaction, inhibits active demethylationin cell lines and tumors in animals (39, 40). To determine the

contribution of uPA promoter demethylation to the expression ofthis gene, we treated DU145 and PC3 cells with increasingconcentrations of Ado-Met from 25 to 150 Amol/L for 5 days.The effect of these treatments on uPA protein and mRNAexpression 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-dependentmanner. Fibrin zymography for uPA activity supported theimmunoblotting 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 silenceduPA expression in both the DU145 and PC3 cell lines, which hadhigh endogenous levels of uPA via promoter demethylation,indicating that Ado-Met effect was not confined to a single cellline model. In parallel experiments, cell viability was studiedin DU145 and PC3 cells treated with PBS (control), Ado-Met(150 Amol/L), or Ado-Hcy (150 Amol/L). These treatments hadno significant effect on cell survival of DU145 and PC3 cells(Fig. 4C).

Effect of Ado-Met on uPA promoter methylation andinvasive potential of the human metastatic prostate cancercell lines. To determine whether silencing of uPA by Ado-Met wasassociated with a change in the methylation pattern of the uPApromoter, genomic DNA was isolated from drug-treated DU145and PC3 cells and subjected to MSP analysis. We obtainedconsistent results in MSP analysis from three independent samplestreated with PBS, Ado-Met, or Ado-Hcy. As shown in Fig. 5A ,treatment of both DU145 and PC3 cell lines with Ado-Met resultedin visible PCR products only in the methylated primer setrepresenting hypermethylation at the promoter CpG regions ofuPA . In contrast, in DU145 and PC3 cells treated with PBS or Ado-Hcy, prominent PCR products remained specific for unmethylatedCpG islands with the unmethylated primer set, but no productswere detected with the methylated primer set. These results wereconfirmed by bisulfite sequencing analysis (data not shown).Because silencing of uPA by Ado-Met is a consequence of increasedDNA methylation, we then asked whether the DNA demethylating

Figure 3. Analysis of uPA promoter CpGmethylation in prostate cancer cell lines.A, MSP showing the methylation status ofprostate cancer cell lines. Positive andnegative controls are described inSupplementary Methods. B, immunoblotanalysis of prostate cancer cell linescorresponding to MSP samples in (A)showing expression of uPA protein inunmethylated samples. Glyceraldehyde-3-phosphate dehydrogenase (GAPDH ) wasused as a loading control. C, fibrinzymography of prostate cancer cell linescorresponding to MSP samples in (A)showing uPA activity in unmethylatedsamples. D, cells invading throughthe Matrigel were counted under amicroscope in three random fields at �200magnification. Columns, mean of threefields counted; bars, SD. *, P < 0.001,significant differences from normal prostatecancer cell line RWPE1.

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agent 5-aza would inhibit this effect. To this end, RNA wasextracted 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-azatreatments inhibited Ado-Met–dependent silencing of uPA. Asimilar trend was also observed by immunostaining with anti-uPA antibodies (Fig. 5C). These results indicate that Ado-Metsilencing of uPA is mediated by DNA methylation. Finally, weevaluated the effect of uPA silencing by Ado-Met on the invasiveability of the metastatic DU145 and PC3 cells using the Matrigelinvasion assay. As shown in Fig. 5D , silencing of uPA by Ado-Metsignificantly diminishes the invasive potential of metastatic DU145and PC3 cells. These observations suggest that reversal of uPApromoter demethylation by Ado-Met can inhibit uPA expressionand the invasive potential of metastatic DU145 and PC3 cells.These findings are also consistent with the hypothesis that theactivation of uPA is strongly associated with the demethylation ofuPA promoter.

uPA is essential for prostate cancer growth and metastasisin vivo . To further clarify the biological meanings of aberrantexpression of uPA in prostate cancer, we used both the Ado-Met–and shRNA-mediated knockdown experiments to examine whetherthe expression of uPA by promoter demethylation contributed to

the tumor growth and metastatic ability of PC3 prostate cancercells. We first derived PC3-RFP cells by stable transfection of aplasmid expressing the red fluorescence gene so that we couldtrack tumor cell growth and metastasis in vivo . We also derivedstable PC3-RFP cells expressing shRNA against uPA and verifiedspecific knockdown effects of uPA by immunoblotting (Supple-mentary Fig. S2A). Stable knockdown of uPA with plasmidexpressing uPA shRNA reduced invasion behavior dramatically,whereas proliferation was unaffected (Supplementary Fig. S2B–D).To test whether uPA knockdown in vitro would affect the abilityof PC3 prostate tumor growth or progression in vivo , weorthotopically introduced PC3-RFP cells treated with PBS aloneas control (mock), PC3-RFP cells treated with 150 Amol/L Ado-Met for 5 days, and PC3-RFP cells stably transfected with uPAshRNA or control shRNA into the prostate of immunodeficientmice. Tumor progression was monitored in mice by using in vivoimaging system (Xenogen). Representative data are shown inFig. 6. The mice injected with PC3-RFP cells treated with Ado-Metand PC3-RFP cells stably transfected with uPA shRNA developedprostate tumors of significantly smaller volume after 5 weekscompared with the mice inoculated with cells either treatedwith PBS or stably transfected with control shRNA (Fig. 6A, top).

Figure 4. Effect of Ado-Met treatmenton uPA expression in prostate cancer celllines. A, protein (top ), mRNA (middle ), andactivity (bottom ) levels of uPA in DU145and PC3 cells treated with differentconcentrations of Ado-Met for 5 dwere monitored by immunoblot, RT-PCR,and fibrin zymography, respectively.Glyceraldehyde-3-phosphatedehydrogenase (GAPDH ) was used as aloading control for RNA and proteinanalysis. B, immunoblot analysis showsthat treatment with 150 Amol/L Ado-Hcy for5 d did not lead to silencing of uPAprotein expression in the DU145 and PC3prostate cancer cells. GAPDH was usedas a loading control to check for equalloading of the gel. C, measure of viablecells after treatment with Ado-Met,Ado-Hcy, or vehicle control in DU145and PC3 prostate cancer cells aredescribed in Supplementary Methods.





The presence of significant growth difference in tumors ofthe prostate between control and uPA knockdown cells injectedmice groups was confirmed by autopsy after imaging (Fig. 6A,bottom).

We also assessed whether the uPA knockdown in PC3-RFPcells affected the ability of these cells to metastasize in vivo . Thelung was dissected from each mouse, and photon counts wererecorded. The in vivo imaging data and the quantification of thesignal from the lungs are shown in Fig. 6B (left and right ,respectively). The lungs derived from control mice injected withPC3-RFP and PC3-RFP+control shRNA cells showed high photoncounts by imaging system. In contrast, there was a markedreduction (f5.1-fold) in the incidence of lung metastasis whenprimary tumor cells experiencing uPA knockdown were seededin host mice. Analysis by RT-PCR showed that there was aprominent expression of uPA mRNA in the prostate tumorsisolated from cells treated with PBS or cells stably expressingunrelated control shRNA, whereas RT-PCR analysis determinedlong-term selective knockdown of uPA in tumors developed fromthe cells treated with Ado-Met or cells stably expressing uPAshRNA (Fig. 6C). Immunohistochemical analysis of the tumorsshowed 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 thetumors isolated from the animals in both the Ado-Met or uPAshRNA groups (Fig. 6D). These results indicate that knockdownof uPA in prostate cancer cells significantly suppresses bothprimary tumor growth and the in vivo incidence of lungmetastasis.

Discussion

uPA function has been implicated in tumorigenesis for over adecade, and recent uPA gene knockdown approaches have enabledexperimental confirmation that uPA does indeed play a key role inthe metastasis of solid tumors as well as mediating tumorangiogenesis both directly by promoting endothelial migrationand indirectly via release of proangiogenic molecules from theextracellular matrix (28, 29, 41). Numerous studies in tumor celllines have correlated the metastatic potential of tumor cells withuPA activity and protein expression (28, 42–44). Furthermore,several studies of clinical tumor samples have correlated high uPAexpression with tumor progression and, in some cases, poorpatient postoperative survival (45, 46). Clearly, there is significant

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 genepromoter 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 aredescribed in Supplementary Methods. B, RT-PCR analysis shows that Ado-Met–treated DU145 and PC3 cells did not express uPA, although treatment with Ado-Metplus 5-aza results in uPA expression. Glyceraldehyde-3-phosphate dehydrogenase (GAPDH ) was used as a loading control. C, immunostaining for uPA proteinexpression 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 h-actin expression(green by fluorescent isothiocyanate), and those having both uPA (red by Texas Red) and h-actin expression show yellow fluorescence. Ado-Met–treated DU145and 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 describedin Fig. 3D . *, P < 0.001, significant difference from controls (i.e., vehicle-treated DU145 and PC3 cells).

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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 witheither control PC3-RFP cells (mock ), PC3-RFP cells treated with 150 Amol/L Ado-Met for 5 d, or PC3-RFP cells stably transfected with uPA shRNA or controlshRNA. Top, nude mice carrying an orthotopic prostate tumor, established from either mock or stable control shRNA PC3-RFP cells, showed substantial fluorescencesignal as indicated by photon counts. In contrast, the orthotopic prostate tumors developed from PC3-RFP cells either treated with Ado-Met or stably transfected withuPA shRNA showed significantly low fluorescence signal. Bottom, surgical examination at autopsy confirmed the inhibition of orthotopic prostate tumor growthestablished from PC3-RFP cells either treated with Ado-Met or stably transfected with uPA shRNA (black arrows ). Right, a comparison of PC3 tumor photon countswith 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 orcontrol shRNA). B, fluorescence signal in the lung, representative of lung metastasis, was recorded for each mouse. Lung images from different mice (left ). Lungmetastasis 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 orstably transfected with uPA shRNA relative to the control groups. C, RNA samples extracted from PC3-RFP prostate tumors of six animals per group were analyzedusing 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 thatthe 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 whencompared with that in control groups.





interest in identifying the molecular mechanisms that control uPAgene expression in normal and pathologic settings. A recent studyon breast cancer tissues suggests that hypomethylation is one ofthe mechanisms contributing to the abnormal expression of uPA(47). However, such studies in relation to prostate cancer arelacking.

In the present study, we examined the relationship betweendemethylation of uPA promoter CpG island and the expression ofthis prometastatic gene in prostate cancer. We found that uPAexpression was undetectable in BPH and NNAT of prostate,whereas there was a dramatic increase of uPA expression inprostate cancer tissues at a high frequency (Fig. 1). These findingssuggest that increased expression of uPA contributes to prostatecancer development and progression. This notion has beensupported by numerous lines of evidence. For example, VanVeldhuizen et al. (17) reported that f70% of the prostate tumorswith extracapsular extension highly express uPA, whereas only 27%of tumors without capsular invasion do. Gaylis et al. (16) alsoreported a strong correlation between uPA expression andaggressive phenotype in a human PC3 prostate cancer model.Moreover, Hienert et al. (48) showed that uPA expression isincreased in the plasma of patients with prostate cancer whencompared with those with BPH.

The mechanism by which uPA is abnormally expressed inprostate cancer remains unclear. The proximal promoter of uPAcontains a CpG island spanning 1,600 bp around the transcrip-tional start site, which could be demethylated and thus activate theexpression of this gene. We used a series of prostate tumors toinvestigate whether demethylation of the uPA promoter could bea mechanism of gene activation in prostate cancer. We foundthat CpG sites within the uPA promoter region are completelymethylated in BPH and tumor-matched NNAT, whereas those inprostate cancer tissues are demethylated (Fig. 2). Thus, these datastrongly suggest that in BPH and NNAT, uPA is methylated at thepromoter-associated CpG sites, resulting in blocking of mRNAtranscription and, consequently, its protein expression. Here, weshow a statistically significant correlation between the methylationpatterns of the uPA promoter region and its aberrant proteinexpression levels in prostate cancer tissues (SupplementaryTable S1; Fig. 2D). A similar trend was observed in prostate cancercell lines (Fig. 3). Collectively, our results indicate that uPApromoter demethylation contributes to the process of prostatecarcinogenesis and metastasis.

Although hypomethylation was the first epigenetic alterationcharacterized in cancer, little is known about the potential roleof promoter CpG demethylation in the activation of tumor- ormetastasis-promoting genes. To address this issue, we used theuPA gene as a model because the promoter CpG region of thisgene is a frequent target of DNA demethylation in prostate cancer.This is the case in the highly metastatic prostate cancer cell linesDU145 and PC3, where uPA is demethylated and active (Fig. 3).Consistent with these results, a previous report has shown thatthe induction of the uPA gene expression in uPA-negative celllines LNCaP by 5-aza displayed a concurrent switch from hyper-methylation to hypomethylation of the CpG island in the uPAgene promoter region (49). At present, the enzymes responsiblefor hypomethylation or demethylation in tumors are unknown. Inrecent years, the methyl donor Ado-Met has been used frequentlyas a DNA-methylating agent. In this study, Ado-Met treatmentinhibited DNA demethylation either by enhancing DNA methyl-transferase 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, ithas been shown that Ado-Met treatment leads to hypermethyla-tion and silencing of uPA in breast cancer cell lines (40). Weinvestigated the effect of Ado-Met on uPA expression inmetastatic prostate cancer cell lines PC3 and DU145. Our datashow that Ado-Met treatment results in uPA gene silencing viapromoter methylation (Fig. 5). These results suggested that Ado-Met affects both demethylation of DNA and gene expression.This association of inhibition of uPA promoter demethylationand silencing of gene expression prompted us to rule out thepossibility that Ado-Met has a general, methylation-independentinhibitory effect on gene expression, which also might result ininhibition of gene expression.

Several findings presented here provide further evidence thatthe uPA gene silencing mechanism of Ado-Met involves DNAmethylation. First, treatment of DU145 and PC3 cells with Ado-Met, but not with its unmethylated analogue Ado-Hcy, inhibiteddemethylation and uPA expression. Notably, this Ado-Met–dependent silencing of uPA significantly inhibits tumor cellinvasion in vitro (Fig. 5D) and tumor growth and metastasisin vivo (Fig. 6). Second, the effects of Ado-Met on uPA expression(Fig. 5B and C) and tumor cell invasion in vitro (Fig. 5D) werereversed by the demethylating agent 5-aza. Third, treatment ofDU145 and PC3 cells with Ado-Met had no significant toxiceffect (Fig. 4C). Furthermore, we observed that Ado-Mettreatment had long-term effects in vivo on uPA expression andmetastasis (Fig. 6). This prolonged silencing provides additionalsupport for the conclusion that the mechanism of uPAknockdown by Ado-Met is dependent on methylation. shRNA-directed knockdown of uPA also strongly interfered with prostatetumor and metastases formation in mice after orthotopictransplantation of stably transfected PC-RFP cells. All thesefindings show that Ado-Met reverses hypomethylation andsilences the demethylation-associated uPA activity. The resultsare also consistent with the hypothesis that demethylation-linkeduPA activation is responsible for prostate cancer progression andmetastasis.

Based on our observations, we propose the following modelof activation of uPA in prostate cancer. During the neoplasticprocess, the metastatic prostate cancer cells would undergo a lossof epigenetic control leading to demethylation of the uPA genepromoter. Transcriptional activators would then be able to bind tothe demethylated promoter region and activate uPA expression.Under normal circumstances, uPA is methylated at the promoter-associated CpG sites, resulting in blocking of mRNA transcriptionand, consequently, its protein expression. This model may explainhow demethylation-linked abnormal expression of uPA contributesto prostate cancer progression and metastasis (SupplementaryFig. S3).

Thus far, most therapeutic strategies aimed at the reactivationof epigenetically silenced genes in human cancer cells havetargeted DNA methyltransferases or histone deacetylases. In thisstudy, uPA gene reactivation by cancer-linked demethylation hasbeen observed. It is noteworthy that during the course of thisinvestigation, several independent research groups have discov-ered the gene-specific hypomethylation during the progressivestages of many cancers (32–36). These studies have negativeimplications for the therapeutic use of demethylating agents, suchas 5-aza, in the treatment of cancer. Further studies need to focuson identifying the different factors involved in methylation/

Cancer Research




demethylation equilibrium shifts, which in turn should increaseour understanding of cancer progression and develop moreeffective therapies for this life-threatening disease. It will beinteresting to investigate the candidate factors, such as methyl-CpG binding proteins, chromatin modifying enzymes, anddemethylases involved in the loss of uPA methylation mark inprostate cancers. Perhaps, these investigations can provideinsights to uncover the molecular factors that are responsiblefor the DNA demethylation process in tumors.

AcknowledgmentsReceived 8/4/2006; revised 10/4/2006; accepted 10/23/2006.

Grant support: National Cancer Institute grants CA 75557, CA 92393, CA 95058, andCA 116708; National Institute of Neurological Disorders and Stroke grants NS47699 andNS57529; 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 pagecharges. This article must therefore be hereby marked advertisement in accordancewith 18 U.S.C. Section 1734 solely to indicate this fact.

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



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2007;67:930-939. Cancer Res Sai Murali Krishna Pulukuri, Norman Estes, Jitendra Patel, et al. Activator Is Involved in Progression of Prostate CancerDemethylation-Linked Activation of Urokinase Plasminogen

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