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2005;65:1325-1334.Cancer ResUmadevi V. Wesley, Michelle McGroarty and Asal HomoyouniFactor Signaling PathwayProstate Cancer Cells by Blocking Basic Fibroblast GrowthDipeptidyl Peptidase Inhibits Malignant Phenotype of

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Dipeptidyl Peptidase Inhibits Malignant Phenotype of Prostate

Cancer Cells by Blocking Basic Fibroblast Growth

Factor Signaling Pathway

Umadevi V. Wesley, Michelle McGroarty, and Asal Homoyouni

Department of Microbiology and Molecular Genetics, Vermont Cancer Center, University of Vermont, Burlington, Vermont

Abstract

Dipeptidyl peptidase IV (DPPIV) is a serine protease with

tumor suppressor function. It regulates the activities of

mitogenic peptides implied in cancer development. Progres-

sion of benign prostate cancer to malignant metastasis is

linked to increased production of basic fibroblast growth

factor (bFGF), a powerful mitogen. In this study, using in vitro

model system we show that DPPIV loss is associated with

increased bFGF production in metastatic prostate cancer cells.

DPPIV reexpression in prostate cancer cells blocks nuclear

localization of bFGF, reduces bFGF levels, inhibits mitogen-activated protein kinase (MAPK)-extracellular signal-regulated

kinase (ERK)1/2 activation, and decreases levels of urokinase-

type plasminogen activator, known downstream effectors of

bFGF signaling pathway. These molecular changes were

accompanied by induction of apoptosis, cell cycle arrest,

inhibition of in vitro cell migration, and invasion. Silencing

of DPPIV by small interfering RNA resulted in increased

bFGF levels and restoration of mitogen-activated protein

kinase (MAPK)-extracellular signal-regulated kinase (ERK)1/2

activation. These results indicate that DPPIV inhibits the

malignant phenotype of prostate cancer cells by blocking

bFGF signaling pathway. (Cancer Res 2005; 65(4): 1325-34)

Introduction

Prostate cancer is the second leading causeof cancer deaths among

men. Recent evidence suggest that the development of androgen-

independent, metastatic prostate cancer is associated with increased

activities of basic fibroblast growth factor (bFGF), a powerful mitogen

and an angiogenic inducer (16). Although, the role of bFGF in cancer

progression is fairly well understood, very little is known about the

mechanisms that regulate bFGF signaling during the transition from

benign to highly malignant prostate cancer.

Dipeptidyl peptidase IV (DPPIV), a membrane glycoprotein,

regulates the activities of mitogenic growth factors and neuro-

peptides. Its proteolytic activity leads to inactivation or degrada-

tion of these peptides (7, 8). DPPIV is involved in diverse biologicalprocesses, including cell differentiation, adhesion, immunomodu-

lation, and apoptosis, functions that are critical for controlling

neoplastic transformation (713). DPPIV is expressed in normal

epithelial cells and melanocytes (1115). Its expression is lost in

various cancers, suggesting loss of DPPIV as an important event

during cancer progression (11, 12, 1418). We have previously

shown that reexpression of DPPIV in melanoma and lung cancers

leads to reemergence of dependence on exogenous growth factors

and suppresses their tumorigenic potential (11, 12).

bFGF, a multifunctional molecule regulates cell proliferation,

migration, cell survival, wound healing, angiogenesis, and tumor

progression (19, 20). It is expressed as a low molecular weight

cytoplasmic isoform (18 kDa) and high molecular weight nuclear

isoforms (21, 2.5, and 24 kDa). The high molecular weight isoforms

possess the NH2-terminal nuclear localization signal and are

exclusively localized to the nucleus (1921). Recent data indicate

that increased expression of bFGF promotes tumorigenic and

metastatic properties of various malignancies including prostate

cancer and is associated with inferior survival of prostate cancer

patients (2, 3, 19, 2126). Furthermore, loss of even one allele of

bFGF in the transgenic adenocarcinoma of the mouse prostate

model increases their survival by suppressing metastasis (27).

Several studies have linked bFGF signaling to activation of the

mitogen-activated protein kinase (MAPK)-extracellular signal-

regulated kinase (ERK)1/2 pathway that promotes cancer progres-

sion (2831). During cellular migration and angiogenesis, activation

of ERK1/2 by bFGF increases the production of urokinase-type

plasminogen activator (uPA), a serine protease that converts

plasminogen to plasmin. Plasmin, in turn promotes cancer

metastasis by breaking down the extracellular matrix, including

fibronectin and laminin (32, 33). Notably, the malignant phenotypeof prostatic tumor cells correlates with high expression of both uPA

and its receptor (uPAR), and plasma uPA has been identified as a

biomarker for prostate cancer progression in Dunning rats and

prostate cancer patients (3436).

In this study, we investigated the mechanism by which DPPIV

suppresses the malignant phenotype of prostate cancer cells. We

establish a correlation between DPPIV loss, increased bFGF

production, and malignant phenotype of prostate cancer cells.

We show that reexpression of DPPIV in metastatic prostate cancer

cells (DU-145), blocks nuclear accumulation of bFGF, decreases

bFGF leading to inhibition of MAPK-ERK1/2 activation and uPA

production, known downstream effectors of bFGF signaling

pathway. We also show that DPPIV expression is associated withinhibition of in vitro cell migration, invasion, induction of

apoptosis, and cell cycle arrest. These data identify a novel

mechanism for regulation of bFGF activity by DPPIV in prostate

cancer cells and provide new directions for therapeutic inter-

ventions aimed at blocking the mitogenic activities of cancer

promoting growth factors.

Materials and Methods

Antibodies and Reagents. The antibodies to bFGF, uPA, p27, and actin

were obtained from Santa Cruz Biotechnology (Santa Cruz, CA); the

polyclonal antibody against ERK1/2 from Cell Signaling Technologies

Requests for reprints: Umadevi V. Wesley, Department of Microbiology andMolecular Genetics Vermont Cancer Center, University of Vermont, 320B, StaffordHall, Burlington, VT 05405. Phone: 802-652-2164; Fax: 802-656-8749; E-mail:[email protected] American Association for Cancer Research.

www.aacrjournals.org 1325 Cancer Res 2005; 65: (4). February 15, 2005

Research Article

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(Beverly, MA); CY-3 goat anti-rabbit (red) secondary antibody, horseradish

peroxidase-sheep anti-mouse, goat anti-rabbit IgG were from Jackson

laboratories (Bar Harbor, ME). The mouse monoclonal antibody to DPPIV

was purified from the culture media of S27 hybridomas obtained from

American Type Culture Collection (Manassas, VA). The cyanine dimer

nucleic acid stain, YOYO-1 iodide, and Oregon Green were from Molecular

probes, (Eugene, OR); LipofectAMINE reagent from Invitrogen, (Grand

Island, NY); 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide

assay kit was from American Type Culture Collection. Cell invasion assay kit

was from Chemicon International, Inc. (Temecula, CA); APOPTAG kit wasfrom Intergen (Gaithersburg, MD); and pSilencer small interfering RNA

(siRNA) expression vectors were from Ambion, Inc. (Austin, TX).

Cells. The normal prostate epithelial cells (NPrEC) were obtained from

Cambrex Bio Science, Inc. (Baltimore, MD). RWPE-1, LNCaP, and DU-145

cells were obtained from the American Type Culture Collection. DU-145

and LNCaP cells were grown in RPMI 1640 supplemented with 10% fetal

bovine serum, 0.1 mmol/L nonessential amino acids, 100 units/mL

penicillin, and 0.1 mg/mL streptomycin. NPrEC cells were grown in media

provided by the supplier. RWPE-1 cells were grown in keratinocyte

medium containing bovine pituitary extract (0.05 mg/mL) and epidermal

growth factor (0.5 ng/mL; Invitrogen).

Flow Cytometry. Flow cytometric analysis was done using FACScan

(Becton Dickinson, San Jose, CA). Cells were stained using the S27

monoclonal antibody (anti-DPPIV) as the primary antibody. FITC-

conjugated rabbit anti-mouse IgG was used as the secondary antibody.

Cells stained with secondary antibody alone were used as controls.

DPPIV Enzyme Activity. DPPIV peptidase activities were measured in

presence and absence of doxycycline by colorimetric assay using Gly-Pro

nitroanilide as the substrate, following previously described procedures (11).

Total RNA Isolation and Northern Blot Analysis. Total RNAs were

isolated from cells using RNAeasy extraction kit (Qiagen, Valencia, CA).

Total RNA (20 Ag) from each cell line was size fractionated and transferred

to nylon membrane. The mRNA levels of DPPIV, glyceraldehyde-3-

phosphate dehydrogenase, uPA, and h-actin were assessed using the

probes generated by PCR. The primers used in PCR reactions are as follows:

5V-tcatatgacatttatgattta-3V and 5V-caaaatgaggaggcaagatcata-3V (800-bp

DPPIV); 5V-atcttccaggagcgagatcc-3V and 5V-accactgacacgttggcagt-3V (502-bp

glyceraldehyde-3-phosphate dehydrogenase); 5V-tcccggactatacagaccat-3V

and 5V-tctcttccttggtgtgactg-3V (387-bp uPA); 5V-gggaaatcgtgcgtaacattaag-3Vand 5V-tgtgttggcgtacaggtctttg-3V (275-bp h-actin). Membranes were

washed and visualiz ed by autoradio graphy.

Establishment of Prostate Cancer Cell Line DU-145 Reexpressing

DPPIV. Tetracycline inducible expression vectors carrying full-length

DPPIV (pTRE-DPPIV) was constructed as described (11). DU-145 cell line

was cotransfected at 60% confluency with pTRE-DPPIV or control vector,

along with the plasmid pTET-on using LipofectAMINE. Stably transfected

cells were selected using G418 (600 Ag/mL). To determine the growth rate,

cells were plated at a density of 104 cells per well in triplicate in 24-well

plates. Viable cells were counted daily for 10 days using trypan blue. Time of

doubling was determined from a least-squares regression fit of cell number

versus time during the logarithmic growth phase.

Immunofluorescence Microscopy. For assessing bFGF, uPA, fibronec-

tin, and ERK1/2 expression, cells grown on chamber slides for 48 hours,

without or with doxycycline (1 Ag/mL) were stained with the respectivemonoclonal antibodies and incubated with either Oregon green or CY-3

conjugated goat anti-rabbit (red) secondary antibody. Cells were counter

stained with nuclear DNA-specific stain, Yo-Yo 1 iodide. Stained cells were

viewed with a inverted microscope (model- ECLIPSE TE2000-U from Nikon,

Melville, NY) connected to a RT Slider Spot Digital camera (Diagnostic

Instruments, Sterling Heights, MI). Images were acquired and merged

images were generated using the software SPOT version 3.2.

Immunoprecipitation and Western Blot Analysis. For metabolic

labeling of proteins, cells were cultured in medium containing [35S]

methionine for 18 hours. Total cell lysates were immunoprecipitated with

the DPPIV-specific monoclonal antibody S27. The protein samples were

analyzed by SDS-PAGE and visualized by autoradiography. For Western

blot analysis, equal amounts of protein lysates (50 Ag) were separated by

SDS-PAGE, transferred to nitrocellulose membranes, and probed with the

respective antibodies, over night at 4C. Signals were developed with

chemiluminescence.

MTT Cell Proliferation Assay. Colorimetric MTT assays were done

using MTT assay kit on days 1, 2, 4, 6, 8, and 10. Cells (n = 5 103) per well

were plated in triplicates on 96-well plates and cultured in absence or

presence of doxycycline (1 Ag/mL). The absorbance of released purple

formazan was measured at 570 nm in a microtiter plate reader. The

experiment was repeated twice and the data are presented as mean F SD

of triplicates.Wound Induced Cell Migration Assay and Quantitative Cell Invasion

Assay. Cells were plated at 60% confluence and cell monolayers were

wounded 24 hours after plating, by scraping with a sterile micropipette tip.

Each well was then washed and daily fed with normal medium. Cells were

photographed at 2 and 24 hours after wounding using a phase contrast

microscope. Additionally, a quantitative cell invasion assay was done using

an invasion assay kit following the manufacturers instructions. Absorbance

values correlating with cell invasion were plotted. The experiment was

repeated twice. The data are presented as the mean of triplicate plates.

In situ Apoptotic Cell Detection by Terminal Deoxynucleotidyl

TransferaseMediated Nick End Labeling Assay. For apoptosis assays,

cells were grown (without or with doxycycline, 1 Ag/mL) in serum-free

media for 3 days. Fixed cells were washed and measurements of DNA nicks

were carried out by terminal deoxynucleotidyl transferasemediated nick

end labeling assay using the APOPTAG kit. Percent apoptosis was

calculated by FACScan analysis.

Cell Cycle Analysis. Equal number of DU-145, vector, and DPPIV

transfected DU-145 cells (2 106) were washed, resuspended in PBS

containing 2% fetal bovine serum, 5 mmol/L EDTA, and were stained with

20 mg/mL propidium iodide for 2 hours at room temperature. The cells

were then examined by flow cytometry (Becton Dickinson FACS Scanner,

Franklin Lakes, NJ) to determine cell cycle distribution. The percentage of

cells present in G0-G1, S, and G2-M phases were determined using ModFit

software package (Verity Software House, Topsham, ME).

RNA Interference Assay. Expression of DPPIV was silenced using the

vector, psilencer 3.1-H1 hygro. The 60-mer complementary oligonucleotides

encoding 19-mer hairpin sequences specific to the DPPIV (NM001935)

mRNA (nucleotide 1,219 to 1,237, AGTCATCGGGATAGAAGCT), a loop

sequence (TTCAAGAGA) separating the two complementary domains, anda polythymidine tract (TTTTTT) to terminate transcription were synthe-

sized and ligated into the psilencer 3.1-H1 hygro vector. Two sequential

transfections (days 1 and 4) of LNCaP and DU-145 cells reexpressing DPPIV

with 1 Ag of siRNA plasmid were carried out using the lipofectamine

reagent. Stably transfected cells were selected for further analysis by adding

hygromycin (200 Ag/mL) to culture media.

Results

DPPIV Loss Is Associated with Increased bFGF Production

in Metastatic Prostate Cancer Cells (DU-145). We analyzed the

expression pattern of DPPIV and bFGF in (a) NPrEC; (b)

nontumorigenic in vitro transformed RWPE-1 cells (37); (c)

androgen-sensitive, LNCaP cells that grow slow and are weaklytumorigenic (38, 39), and (d) androgen-independent, tumorigenic,

and moderately metastatic prostate cancer cell line DU-145 (40).

Flow cytometric analysis indicated high levels of cell surface

expression of DPPIV on NPrEC cells with decreased expression to

moderate levels on RWPE-1 and LNCaP cells, and very low levels on

DU-145 cells (Fig. 1A). Whereas DPPIV enzyme activities in NPrEC

cells ranged from 180 to 220 and 100 to 120 pmol per g protein per

minute in RWPE-1 and LNCaP cells, it was reduced to 20 to 40

pmol per g protein per minute in DU-145 cells (Fig. 1B). Northern

blot analysis showed that decreased or loss of DPPIV expression in

prostate cancer cells occurs at RNA level (Fig. 1C). Western blot

analysis showed low levels of bFGF in NPrEC, RWPE-1, and LNCaP

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cells, but high levels in DU-145 cells (Fig. 1D). These observationsindicate an inverse correlation between DPPIV and bFGF

expression during the emergence of malignant phenotype in

prostate cancer cells.

DPPIV Blocks Nuclear Localization and Reduces Expression

Levels of bFGF in Metastatic Prostate Cancer, DU-145 Cells. To

understand the role of DPPIV in prostate cancer progression, we

reexpressed DPPIV in DU-145 cells using the tetracycline inducible

expression system. Two independent clones expressing DPPIV

were randomly selected for further studies. Untransfected and

vector-transfected DU-145 cells were used as controls. The levels of

DPPIV enzyme activities in presence of doxycycline (1 Ag/mL)

ranged between 180 and 225 pmol per minute per Ag protein

in DPPIV transfected cells and were comparable to the levels inNPrEC (200-220 pmol per minute per Ag protein; Fig. 2A). The

DPPIV enzyme activities in DPPIV transfected cells ranged from

30 to 60 pmol per minute per Ag protein in the absence of

doxycycline, similar to control DU-145 cells (20-40 pmol per minute

per g protein; Fig. 2A). Furthermore, immunoprecipitation analysis

showed the expected 110-kDa protein corresponding to DPPIV

with its natural post-translational glycosylation in DPPIV trans-

fected DU-145 cells in presence of doxycycline (Fig. 2B).

Because DPPIV and bFGF levels were inversely correlated in

normal and metastatic prostate cancer cells, we investigated the

effect of DPPIV on bFGF levels in DU-145 cells. Western blot

analysis showed high levels of both nuclear (22, 22.5, and 24 kDa)

and cytoplasmic isoforms (18 kDa) of bFGF in DU-145 and vectortransfected DU-145 cells. Reexpression of DPPIV in DU-145 cells

greatly decreased the levels of nuclear bFGF isoforms; the levels of

low molecular weight cytoplasmic isoform were also decreased

(Fig. 2C). In the absence of DPPIV induction, the same DU-145 cells

showed bFGF levels comparable to control DU-145 cells, demon-

strating the specific effect of DPPIV on bFGF levels (Fig. 2D).

Consistent with Western blot results, immunofluorescence staining

revealed high levels of both nuclear and cytoplasmic form of bFGF

in control DU-145 cells (Fig. 2E, a and d). In DPPIV reexpressing

DU-145 cells, the nuclear accumulation of bFGF was greatly

reduced; most of bFGF was observed in a punctate pattern around

the perinuclear region (Fig. 2E, g and j). We confirmed altered

nuclear localization by costaining the cells with nuclear-specificYo-Yo 1 iodide (Fig. 2E, b , e , h , k; see also merged images in c, f, i, l).

The staining patterns of bFGF in individual cells are shown in

Fig. 2F. These observations indicated that DPPIV negatively

regulates bFGF expression and disrupts its subcellular localization.DPPIV Inhibits Activation of MAPK-ERK1/2, Decreases uPA

Expression, and Restores Fibronectin Matrix Assembly.

Expression of bFGF is associated with constitutive activation of

the MAPK-ERK1/2 signaling pathway. Interestingly, the levels of

phosphorylated p44/42 were greatly reduced in DPPIV expressing

DU-145 cells, compared with the control DU-145 cells (Fig. 3A). In

absence of doxycycline, levels of phosphorylated p44/42 were not

affected, indicating specific effects of DPPIV on ERK 1/2 kinase

Figure 1. DPPIV loss and increased bFGF productionare correlated in metastatic PCa cell lines. A, cell surfaceexpression of DPPIV in NPrEC and prostate cancer cells(RWPE-1, LNCaP, and DU-145) as determined by flowcytometry. Cells were stained with the DPPIV-specific mAbS27. Y-axis, relative cell number; X-axis, log of relativefluorescence intensity. Solid black line, DPPIV expression;filled histogram, control IgG1 antibody. B, DPPIVenzymatic activity in total cell lysates as measured bycolorimetric assay using p-nitroanilide as a substrate.Columns, mean values of DPPIV activity of triplicateexperiments; F1 SD. C, DPPIV mRNA levels as assessedby Northern blot analysis using the PCR-amplified cDNAfrangment of DPPIV as probe. GAPDH probe was usedto assess relative levels of total RNA in all samples. D,bFGF protein levels as assessed by Western blot analysis

using the antibody against bFGF. The monoclonal antibodyagainst actin was used to assess relative levels of totalprotein in all lanes.

Dipeptidyl Peptidase Blocks bFGF Signaling




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activities (Fig. 3A). The levels of total ERK1/2 remained the same in

all cell lines in the presence and absence of doxycycline (Fig. 3A).

Immunofluorescence staining showed the nuclear localization of

phosphorylated ERK1/2 in control DU-145 cells (Fig. 3B, a and b),

whereas in DPPIV-transfected cells, nuclear ERK1/2 were barely

detectable in the presence of doxycycline (Fig. 3B, c and d).

During prostate cancer progression, high levels of bFGF and

ERK1/2 activation are associated with increased uPA expression.

Our current study shows that control DU-145 cells express high

levels of uPA and reexpression of DPPIV leads to undetectable

levels of uPA mRNA in these cells (Fig. 3C). Immunofluorescence

staining further confirmed high levels of uPA in control DU-145

Figure 2. DPPIV blocks nuclear localization and reduces expression levels of bFGF. A, DPPIV enzyme activity in NPrEC, DU-145 clones transfected with vector,and DPPIV in the absence or presence of doxycycline (1 Ag/mL). Columns, mean values of triplicates; bars, F1 SD. B, immunoprecipitation analysis of DPPIVexpression in DU-145 cells transfected with DPPIV and one vector in the absence and presence of doxycycline (1 Ag/mL). C and D, Western blot analysis of the levelsof nuclear and cytoplasmic isoforms of bFGF. DU-145 and vector control cells showed very high levels of nuclear and moderate levels of cytoplasmic isoform of bFGFin untransfected DU-145 cells and DU-145 cells transfected with either vector or DPPIV in the presence of doxycycline (1 Ag/mL; C) or in the absence of doxycycline(D). E, immunofluorescence staining showing bFGF expression (red) in untransfected, vector, and DPPIV transfected Du-145 cells in the presence of doxyccline.Cells were counter stained with nuclear specific Yo-Yo 1 iodide (green). Stained cells were viewed with an inverted microscope. Merged images show either yellow(red and green) or green nucleus. (Magnification 400). F, staining patterns of bFGF in single cells for each group are shown (Magnification 600). Mergedimages (yellow) represent colocalization of bFGF (red) and nuclear-specific Yo-Yo 1 iodide (green).

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cells (Fig. 3D, 1 and 2) and low levels in DPPIV reexpressing DU-

145 cells. (Fig. 3D, 3 and 4). Western blot analysis showed increased

levels of fibronectin proteins in DPPIV expressing DU-145 cells

compared with the control DU-145 cells (Fig. 3E). Immunofluores-

cence staining confirmed decreased levels of fibronectin matrix on

the control DU-145 cells but with increased assembly of fibronectin

matrix that distributed in a fibrilar array on DU-145 cells

reexpressing DPPIV (Fig. 3F).DPPIV Induces Contact Inhibition and Inhibits Cellular

Proliferation, Migration, and Invasion of Prostate Cancer DU-

145 Cells. Restoration of DPPIV expression resulted in profound

morphologic changes in DU-145 cells. DU-145 cells and vector

transfected DU-145 cells showed small, rounded morphology and

grew in disorganized clumps. These cells grew at higher saturation

densities (Fig. 4A , 1 and 2). DU-145 cells reexpressing DPPIV in

presence of doxycycline were enlarged, assumed flat epithelial

morphology, and grew in an organized manner, and exhibited

contact inhibition (Fig. 4A , 3 and 4). The growth pattern of DU-145

cells expressing DPPIV was comparable to NPrEC (Fig. 4A , e) with

no change in absence of doxycycline (shown in insets of Fig. 4A ,

3 and 4). The alteration of cell shape and growth pattern supports

previous reports that DPPIV is a cell adhesion molecule and may

Figure 3. DPPIV decreases activation of ERK1/2, decreases uPA expression and increases cell surface FN matrix assembly. A, Western blot analysis of the levels oftotal and phosphorylated ERK1/2 proteins in control- or DPPIV-transfected DU-145 cells, in the presence or absence of doxycycline. B, immunofluorescence stainingof nuclear translocation of phosphorylated ERK1/2 in DU-145- and vector-transfected DU-145 cells ( a and b) and in DPPIV-transfected DU-145 cells (c and d). C,Northern blot analysis of uPA mRNA levels in untransfected, vector control, and DPPIV-transfected DU-145 cells. The blot was reprobed with 275-bp PCR fragment ofh-actin to assess for equal amount of RNA loaded from all samples. D, immunofluorescence staining of expression of uPA protein in DU-145- ( 1 ), vector- (2), andDPPIV-transfected DU-145 cells (3 and 4). E, Western blot analysis showing the levels of fibronectin in DU-145-, vector-, and DPPIV-transfected DU-145 cells. F,immunoflourescence staining showing very low levels of fibronectin matrix assembly on the cell surface of DU-145 and vector transfected DU-145 cells ( 1 and 2) andincreased assembly of fibronectin matrix on the cell surfaces of DPPIV-transfected DU-145 cells (3 and 4). Data shown in B-F are in the presence of doxycycline.





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play an important role in regulating cell motility. The doubling

time for DU-145 cells reexpressing DPPIV was increased to 48 from

36 hours. DPPIV expression also inhibited cell proliferation up to

50% of the control DU-145 cells in presence of doxycycline without

much change in the absence of doxycycline as indicated by MTT

assay (Fig. 4B).In vitro cell migration and invasion are indirect measures of the

in vivo metastatic potential of a transformed cell and these

functions are facilitated by bFGF and ERK1/2 activation. The effect

of DPPIV on prostate cancer cell migration was evaluated

qualitatively by a wound induced migration assay. At 2 hours,

DU-145, DPPIV expressing DU-145 cells, and control DU-145 cells

showed no visible migration (Fig. 4C, 1 , 2, 3, and 4). After 24 hours,

DU-145 and vector control cells migrated to fill the wounded area

completely, indicating their strong migratory potential (Fig. 4C, 5

and 6). However, the migratory potential of DU-145 cells

Figure 4. DPPIV expression results in contact inhibition and inhibits cellular proliferation, in vitro cell migration, and invasion of prostate cancer cells (DU-145).A, bright field photographs of morphologic changes in DU-145 cells transfected with either vector or DPPIV. Untransfected DU-145 (1) and vector-transfectedDU-145 (2) grew in a disorganized array of cells with clumps indicating lack of contact inhibition. DU-145 cells transfected with DPPIV ( 3 and 4) showed flatepitheloid and dendritic morphology with contact inhibited growth in the presence of doxycycline, similar to normal prostate epithelial cells ( 5). Morphology of DPPIVtransfected DU-145 cells remained similar to parental DU-145 cells in the absence of doxycycline ( insets 3a and 4a). Original magnification, 200. B, the ratesof cellular proliferation of DU-145, either vector or DPPIV transfected DU-145 cells were assessed in absence (A) or presence (B) of doxycycline by MTT assay,as described in Materials and Methods. The experiment was repeated twice. Columns, absorbance expressed as mean of triplicates; bars, F1 SD. C, woundhealing assay showing migration of DU-145, vector or DPPIV transfected DU-145 cells in the presence of doxycycline, after 2 and 24 hours of scraping.(Magnification 100). D, quantitative cell invasion assay as done using Boyden chamber cell invasion assay kit. Absorbances correlating with the number ofcells invaded were read at 540 nm. Columns, mean values of triplicates; bars, F1 SD.

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reexpressing DPPIV was greatly reduced as indicated by theunfilled wounded area (Fig. 4C, 7 and 8). Furthermore, the ability of

cells to invade through ECM coated filters was assessed

quantitatively by the Boyden Chamber assay. The number

of invading DU-145 cells reexpressing DPPIV in presence of

doxycycline was greatly reduced with inhibition up to 50% to 60%

when compared with control DU-145 cells (Fig. 4D). These results

indicated that DPPIV inhibits in vitro invasive and migratory

potential of prostate cancer cells.DPPIV Induces G2-M Arrest and Cell Death in Prostate

Cancer Cells (DU-145). To further understand the mechanism by

which DPPIV inhibits proliferation of DU-145 cells, we did cell

cycle analysis. The percentage of DPPIV expressing DU-145 cells

in G2-M stage increased to 24% to 34 % compared with the 9%to 10% in control DU-145 cells (Fig. 5A). Transformed cells are

typically released from dependence on exogenous growth factor

for survival during tumor progression. Parental-, vector-, and

DPPIV-transfected DU-145 cells were serum starved with or

without induction of DPPIV by doxycycline. Control DU-145 cells

continue to grow with low levels of detectable apoptotic cell

death (about 5% of cells showed DNA fragmentation over 3

days). However, apoptosis was evident at day 3, in 24% to 27% of

DU-145 cells reexpressing DPPIV in presence of doxycycline

(Fig. 5B). We further found out that DPPIV up-regulates the

expression of cycline D kinase inhibitor, p27(kip1) by 4- to 6-folds in

DPPIV transfected cells in the presence of doxycycline, compared

with the parental and vector transfected DU-145 cells (Fig. 5C).These results link DPPIV expression with the blocking of bFGF

function that is shown to decrease p27 levels, thereby increasing

proliferative and survival potential of cancer cells.Silencing of DPPIV by siRNA Restores bFGF Expression and

Increased ERK1/2 Phosphorylation. To confirm the direct

involvement of DPPIV in regulating bFGF expression and ERK1/2

activation, we used RNA interference (siRNA) to silence DPPIV

expression in LNCaP and DU-145 cells reexpressing DPPIV (DU-

DPPIV-1). The siRNA plasmid transfected LNCaP and DU-DPPIV-1

cells were CHANGE analyzed for DPPIV, bFGF, ERK1/2 levels, and

phenotypic changes. Flow cytometric analysis showed decreased

cell surface expression of DPPIV in siRNA transfected DU-DPPIV-1

cells (Fig. 6A). Furthermore, immunoprecipitation and enzymeactivity analysis confirmed the decreased levels of DPPIV protein

and enzyme activities in siRNA transfected DU-DPPIV-1 cells as

shown in Fig. 6B and C. The morphology and growth pattern of cells

transfected with DPPIV-specific siRNA reverted to malignant

phenotype and started to grow in disorganized colonies, indicating

loss of contact inhibition similar to the vector transfected DU-145

cells (Fig. 6D). Knock down of DPPIV expression resulted in

increased expression of bFGF (Fig. 6E) and restoration of increased

levels of phosphorylated ERK1/2 MAPK (Fig. 6F). Similar results

were obtained when DPPIV was silenced in LNCaP cells (Fig. 6G).

These results suggested that the inhibitory effects of DPPIV on the

malignant phenotype of prostate cancer cells are at least partly

Figure 5. DPPIV Induces G2-M cellcycle arrest and cell death in prostatecancer cells (DU-145). A, cell cycle analysiswas done by flow cytometry using DPPIVtransfected DU-145 cells in the presenceof doxycycline (1 Ag/mL). Untransfectedand vector transfected DU-145 cellswere analyzed in parallel. The results aredepicted as a cascade plot. Inset, %cells in various stages of cell cycle. B,% apoptosis of parental-, vector-, andDPPIV-transfected DU-145 cells inserum-free condition as indicated byterminal deoxynucleotidyl transferasemediated nick end labeling assay.Apoptosis was assessed in the absence or

presence of doxycycline at days 1 and 3.Columns, mean of triplicates; bars, F1 SD.C, Western blot analysis showing theexpression of cycline D kinase inhibitorp27 (kip1) and actin in DU-145-,vector-, and DPPIV-transfected DU-145cells in the presence and absence ofdoxycycline.





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mediated through the negative regulation of the bFGF-MAPK

signaling pathway.

Discussion

The results of our experiments show that DPPIV loss,

increased bFGF production and in vitro metastatic potential of

prostate cancer cells are correlated. DPPIV suppresses the

malignant phenotype of prostate cancer cells by decreasing the

levels and blocking nuclear localization of bFGF, thereby

resulting in reduced bFGF signaling as indicated by decreased

MAPK-ERK1/2 activation and uPA levels. This is the first

demonstration that bFGF, a powerful mitogen and an angiogenic

inducer is a target for a protease regulated growth suppressor

activity in prostate cancer cells.Progression of benign prostate cancer to fatal hormone

refractory disease is associated with over expression of growth

factors that mediate alternative mitogenic signaling (15). The cell

surface protease, DPPIV controls normal cellular growth and

differentiation by regulating the activity of peptide factors (7, 8).

Figure 6. Knockdown of DPPIV expression by siRNA restores bFGF expression and increases ERK1/2 phosphorylation. A, flow cytometric analysis showingcell surface expression of DPPIV in DU-145 cells transfected with vector or DPPIV (DU+DPPIV-1) with and without DPPIV specific siRNA treatment. Y-axis, relativecell number; X-axis, log of relative fluorescence intensity. Solid black line, DPPIV expression; filled histogram, control IgG1 antibody. B, immunoprecipitation analysisof the levels of DPPIV and actin protein in control and DU+DPPIV-1 cells with and without siRNA treatment. C, colorimetric enzyme assay of the levels of DPPIVenzyme activities in control and DU+DPPIV-1 cells with and without siRNA treatment. Columns, mean of triplicates; bars, F1 SD. D, morphology of DU-145, vector,and DU+DPPIV-1 cells before and after siRNA treatment. DU-145 cells transfected with control vector did not show any morphologic changes after transfectionwith DPPIV-specific siRNA (1 and 3). The DPPIV transfected DU-145 cells showed flat epitheliod morphology with contact inhibited growth ( 2). The morphologyand growth pattern of the DU-DPPIV-1 cells transfected with DPPIV-specific siRNA reverted back to malignant phenotype and grew in a disorganized coloniesindicating loss of contact inhibition similar to parental DU-145 cells ( 4). E and F, Western blot analysis of the effects of DPPIV silencing on levels of bFGF andphosphorylated ERK1/2. Knock down of DPPIV expression by siRNA resulted in increased expression of bFGF ( E) and increased levels of phosphorylated ERK1/2 (F).G, treatment of LNCaP cells with DPPIV specific siRNA resulted in decreased levels of DPPIV protein as indicated by immunoprecipitation analysis. Silencing of

DPPIV in LNCaP cells lead to increased levels of bFGF and phosphorylated ERK1/2 as shown by Western blot analysis.

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The present studies have shown that DPPIV is expressed at high

levels in NPrEC that were established from normal prostatic tissue

and its expression is decreased to moderate levels in RWPE-1 and

LNCaP cells, and to almost undetectable levels in DU-145 cells. The

three cell lines, RWPE-1, LNCaP, and DU-145 cells vary widely in

their phenotypic characteristics. The HPV-18 transformed and

immortalized RWPE-1 cells represent early stage of prostate cancer

progression because these cells neither grow in agar nor form

tumors when injected into nude mice. Although RWPE-1 cells

express cytokeratins 8 and 18, which are characteristic of luminal

prostatic epithelial cells, they also coexpress basal cell cytokeratins

(37). The DU-145 cell lines is AR negative and poorly differentiated,

whereas the LNCaP cell line that exhibit less aggressive phenotype

has functional AR, grows less rapidly, and is less tumorigenic than

the DU-145 cell line. Metastasis formation by LNCaP cells is

observed only when the cells were injected orthotopicaly into

prostate glands (38, 39, 41, 42). Our data suggest that loss of DPPIV

may be an important event during progression of prostate cancer

cells to aggressive phenotype. This observation is supported by

other studies conducted with clinical specimens, reporting loss of

DPPIV expression in >50% of the metastatic and poorly differen-tiated advanced prostate cancer samples (16, 17).

Several studies have also shown that bFGF is up-regulated in

prostate cancer cell lines and tumors tissues with increased me-

tastatic and invasive potential (16). Thus, the mutually exclusive

expression patterns of DPPIV and bFGF in metastatic cell lines

probably reflect their role in dedifferentiation from an epithelial to a

malignant phenotype. Supporting this notion, our data show that

reexpression of DPPIV in DU-145 cells induces the normal epithelial

phenotype, cell-cell contact, decreased cell proliferation rate, and

reduced anchorage-independent growth ability. We have previously

shown that DPPIV induces similar effects and abrogates the

tumorigenic potential of melanomas and lung cancer cells (11,

12). These data support the idea that DPPIV is associated withnonproliferative phenotype of normal prostate cells.

Although elevated levels of bFGF are often associated with

prostate cancer progression, the underlying mechanism leading

to dysregulated bFGF signaling is not known. Our studies

suggest that this dysregulation could be in part due to loss of

DPPIV leading to high levels of bFGF in the nucleus that

disrupts the normal cellular transcriptional program. We

speculate that in normal cells DPPIV cleaves the NH2 terminal

nuclear localization signal thereby confining bFGF to the

perinuclear region. This initial cleavage may be the first step

in the degradation of bFGF. Alternatively, it is possible that

DPPIV directly associates with bFGF and interferes with post-

translational modifications of bFGF such as NH2-terminalmethylation shown to affect its nuclear accumulation (1921).

DPPIV may also affect the stability of bFGF resulting in its

reduced levels. Indeed, high levels of bFGF are shown to be

required for promoting malignant transformation (13, 27).Loss of expression of other membrane associated peptidases

such as CD10/NEP, CD13/APN, and BP-1/6C3/APA has been

reported in several types of human cancers (4345). Recently,

neutral endopeptidase has been shown to interact directly with the

tumor suppressor gene, pTEN and block FAK signaling pathway

thereby functioning as a tumor suppressor gene for prostate cancer

cells (45). Thus, cell surface proteases seem to play important roles

in regulating the prostate cancer progression.

Several studies have linked bFGF to activation of the MAPK-

ERK1/2 signaling pathway that plays a critical role in survival and

migration of tumor cells (28, 29). Indeed, bFGF-deficient endothelial

cells lack ERK1/2 activation and loose their migratory potential

(28). Activation of ERK1/2 is shown to be associated with

progression of prostate cancer to the lethal androgen-independent

state (30, 31). However, the causal role of these downstream kinases

in androgen-independent prostate cancer is not completely

understood. Our data show that DPPIV expression leads todecreased levels of phosphorylated ERK1/2 resulting in inhibition

of migratory and invasive potential of prostate cancer cells (DU-

145). This effect was abrogated upon DPPIV silencing by siRNA

treatment of the same cells demonstrating the specific effects of

DPPIV on bFGF mediated ERK1/2 activation. Interestingly, silencing

of DPPIV in LNCaP cells also resulted in increased bFGF expression

and activation of ERK1/2. This indicates that regardless of androgen

receptor expression, DPPIV regulates bFGF and ERK1/2 signaling in

prostate cancer cells. This observation is of importance because

most clinical samples of hormone refractory disease still express

androgen receptor and continue to survive and proliferate probably

through activating alternative survival and proliferative signaling

pathways (15). There is evidence to show that, among otherfactors, polypeptide growth factors that are associated with

activation of MAP kinase signaling pathways play a critical role in

this phenomenon (4). Our present studies link DPPIV with blocking

of bFGF-mediated signaling pathway implicated in prostate cancer

progression.

DPPIV expression in DU-145 cells lead to an increase in the

expression of a cyclin D kinase inhibitor, P27 (kip1) leading to an

arrest in the G2-M phase, and induced apoptosis upon serum

withdrawal, indicating the emergence of dependence on exoge-

nous growth factors for their survival. It is shown that bFGF

promotes cell proliferation and protection from apoptosis by

decreasing p27 expression (46, 47). Also, disruption of FGF

signaling by the dominant-negative FGFR1 is shown to result in

cell cycle arrest of prostate cancer cells in G2-M stage (48). Takentogether, these observations suggest that DPPIV blocks the

signaling pathway coordinated by bFGF and ERK1/2 in prostate

cancer cells.

Tumor cell migration and invasion depends largely on an

enzymatic cascade of proteolysis that involves uPA and its receptor

(uPAR). This cascade is an essential step for malignant cell

transport. Growth factors including bFGF and activated ERK1/2

have been shown to promote uPA production in various tumoral

and normal cell types (3235). Elevated levels of uPA are found in

patients with advanced stages of prostate cancer and it is a strong

indicator of poor prognosis (3436). It is interesting to note that

uPA activates plasminogen to generate plasmin that degrades

extracellular matrix components including fibronectin, a requiredevent for malignant cell metastasis. We have shown that DPPIV

dramatically decreases uPA mRNA production and increases the

fibronectin assembly on the cell surface. Thus, increase in uPA

expression seen in DU-145 cells, may result from the cooperative

action of signaling provided by bFGF. Disruption of these signaling

events by DPPIV is probably involved in the assembly of fibronectin

matrix and increase in cell adhesion that serves as a barrier for cell

motility. This possibility is supported by data showing DPPIV

binds to fibronectin and functions as an adhesion molecule (10),

and that fibronectin on the cell surface disappears upon oncogenic

transformation. Furthermore, overexpression of fibronectin leads

to suppression of the transformed phenotype (49, 50). Thus, loss of





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DPPIV may render tumor cells less adhesive thereby promotingdetachment of cells from primary tumors initiating metastasis.However, in vivo studies will further strengthen the role of DPPIVin blocking metastatic potential of prostate cancer cells.

Given the ability of DPPIV to regulate the cell proliferation,differentiation, apoptosis, and cell motility through its regulationof bFGF provides a new and potentially significant approach forbetter understanding prostate cancer progression and develop-

ment of treatment strategies aimed at blocking mitogenic andangiogenic signaling.

Acknowledgments

Received 5/26/2004; revised 11/15/2004; accepted 12/3/2004.Grant support: Department of Microbiology and Molecular Genetics, Lake

Champlain Cancer Research Organization, Vermont Cancer Center at the University ofVermont.

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 Emma H ewes and Scott Tighe at shared Core Facility o f Vermont CancerCenter for technical help in construction of siRNA plasmids and flow cytometric

analysis and Drs. David Nanus, Cedric Wesley, Alan Houghton, Nick Heintz,Mark Plante, Brooke Mossman, and Susan Wallace for helpful discussions.

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