CCN3 (With Endnote)

8/3/2019 CCN3 (With Endnote)

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CCN3 regulates motility of prostate cancer cells through v3 integrin, ILK, Akt

and NF-B pathway

Po-Chun Chen1, Hsu-Chen Cheng1* and Chih-Hsin Tang2,3*

1Department of Life Sciences, National Chung Hsing University, Taichung, Taiwan

2Department of Pharmacology, School of Medicine, China Medical University,

Taichung, Taiwan

3Graduate Institute of Basic Medical Science, China Medical University, Taichung,

Taiwan

*Corresponding authors

Chih-Hsin Tang, PhD

Department of Pharmacology, School of Medicine, China Medical University

No. 91, Hsueh-Shih Road, Taichung, Taiwan

Tel: 886-4-22052121-7726; Fax: 886-4-22053764

E-mail: [email protected]

Or

Hsu-Chen Cheng, PhD

Department of Life Sciences, National Chung Hsing University

250 Kuo Kuang Rd., Taichung, Taiwan

E-mail: [email protected]

Tel: (886) 4-22840416, Ext. 505; Fax: (886) 4-22874740


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Abstract

Nephroblastoma overexpressed (NOV or CCN3) is a secreted, matrix-associated

protein that belongs to the CCN gene family and is involved in many cellular

functions, including growth, differentiation, and adhesion. The effect of CCN3 on

human prostate cancer cells, however, is unknown. Here, we have shown that CCN3

and intercellular adhesion molecule 1 (ICAM-1) affected the mobility of prostate

cancer cells. In addition, expression of CCN3 was positively correlated with both cell

migration and ICAM-1 expression in human prostate cancer cells. CCN3 activated a

signal transduction pathway that included v3 integrin, integrin-linked kinase (ILK),

Akt, and NF-B. Reagents that inhibit specific components of this pathway each

diminished the ability of CCN3 to effect cell migration and ICAM-1 expression.

Moreover, CCN3 increased binding of p65 to an NF-B binding element in the

ICAM-1 promoter. Finally, knockdown of CCN3 expression markedly inhibited cell

migration, tumor growth in bone, and bone metastasis. Taken together, our results

indicate that CCN3 enhances the migration of prostate cancer cells by increasing

ICAM-1 expression through a signal transduction pathway that involves v3 integrin,

ILK, Akt, and NF-B. CCN3 thus represents a promising new target for treating

prostate cancer.

Running title: CCN3 induces prostate cancer metastasis

Keyword: CCN3; ICAM-1; prostate cancer; migration; bone


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Introduction

Prostate cancer is the most commonly diagnosed malignancy in the U.S. and other

western countries (1). In early stages of prostate cancer, surgery is the most frequent

therapeutic intervention. In advanced disease states, however, more systemic

interventions are required to inhibit the growth and spread of secondary metastases.

Bone metastasis is a common complication associated with advanced prostate cancer,

often causing acute pain and bone fractures. Bone metastasis has prognostic value in

prostate cancer, because the extent of disease in the bone significantly affects survival

(2).

Cancer metastasis is a critical step in tumor progression and the major cause of

mortality for patients with cancer. This process is composed of several steps by which

cells detach from the primary tumor and form a secondary tumor at a distant site (3).

Environmental signals serve as cellular attractors, thereby promoting cancer cell

motility. To invade nearby tissues, tumor cells must change their cell-cell adhesion

properties, rearrange the extracellular matrix of their environment, suppress anoikis,

and reorganize their cytoskeleton (4). Cell adhesion molecules such as integrin,

cadherin, and immunoglobulin superfamilies have been studied extensively in the

context of tumor progression and metastasis (5). Intercellular adhesion molecule 1

(ICAM-1, also called CD54) is an inducible surface glycoprotein that belongs to the

immunoglobulin supergene family and mediates cell-cell adhesion (6-8). The surface

domain of ICAM-1 is crucial for transendothelial migration of leukocytes from the

capillary bed into the surrounding tissue (9), but ICAM-1 may facilitate migration of

other cell types as well (10-12). ICAM-1 plays a key role in breast and lung cancer

cell invasion (13,14), and knockdown ofICAM-1 expressionreducesthe invasiveness

of breast cancer cells (13). Give the critical role that ICAM-1 plays in tumorigenesis,

disruption of ICAM-1 may prove useful for preventing cancer cell metastasis.

Nephroblastoma overexpressed (NOV, also known as CCN3) belongs to the

CCN gene family, which includes CYR61 (cysteine-rich, angiogenic inducer, 61),

CGTF (connective tissue growth factor), and NOV. This gene family encodes secreted


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proteins that interact with the extracellular matrix and thereby regulate many cellular

functions, including cell division, chemotaxis, apoptosis, adhesion, motility, and ion

transport (15-17). In recent years, CCN3 has been shown to promote mesenchymal

cell differentiation, angiogenesis, and cell adhesion (18-20). Elevated CCN3

expression is also associated with osteosarcoma and poor prognosis in this context

(20,21), and with higher rates of metastasis in melanoma (22). Upregulation of CCN3

in prostate cancer cells suggests that it has a role in prostate tumorigenesis (23) .

Previous studies have shown that CCN3 modulates the migration and

invasiveness of human cancer cells (24). The molecular mechanism by which CCN3

affects human prostate cancer cells, however, is essentially unknown. Here, we

provide the first evidence that CCN3 increases both migration and ICAM-1

expression through v3 integrin in cultured human prostate cancer cells.

Furthermore, knockdown of CCN3 decreased cell migration in cultured human

prostate cancer cells and bone metastasis in vivo. In addition, a signal transduction

pathway including v3 integrin, integrin-linked kinase (ILK), Akt, and NF-B

mediated the response to CCN3 stimulation in prostate cancer cells. The elucidation

of this CCN3-responsive signaling pathway provides valuable clues toward

understanding the mechanisms of human prostate cancer metastasis and presents an

opportunity to develop more effective clinical therapies in the future.

Materials and methods

Materials

Protein A/G beads, rabbit polyclonal antibodies specific for NOV were purchased

from Santa Cruz Biotechnology (Santa Cruz, CA, USA). Mouse monoclonal antibody

specific for v3 integrin was purchased from Chemicon (Temecula, CA). KP-392,

Akt inhibitor, PDTC, TPCK were purchased from Calbiochem (San Diego, CA, USA).

The recombinant human CCN3 was purchased from R&D Systems (Minneapolis, MN,

USA). The Akt (Akt K179A) dominant-negative mutant was gift from Dr. W.M. Fu

(National Taiwan University, Taipei, Taiwan),the IKK(KM) and IKK(KM) mutants


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were gifts from Dr. H. Nakano (Juntendo University, Tokyo, Japan).

pSV--galactosidase vector and luciferase assay kit were purchased from Promega

(Madison, MA). All other chemicals were purchased from Sigma-Aldrich (St. Louis,

MO, USA).

Cellculture

Human prostate cancer cell lines (PC-3, DU145, and LNCaP) were obtained from the

American Type Culture Collection. Cells were maintained at 37 C, 5% CO2, in

RPMI-1640 medium supplemented with 20 mM HEPES, 10% heat-inactivated fetal

calf serum, 2 mM glutamine, 100 U/ml penicillin, and 100 Qg/ml streptomycin. CCN3

shRNAs plasmids were purchased from National RNAi Core Facility Platform (Taipei,

Taiwan). CCN3 shRNAs plasmids were transfected with Lipofectamine 2000

(Invitrogen, CA) and CCN3 shRNA-expressing cells were puromycin selected.

Surviving cells were picked and expanded to make clonal cell populations. For

monolayer growth curves, 104

cells were plated in 6 well plates and grown for 1 to 6

days. Cells were trypsinized, and cell numbers was counted.

Migration assay

The migration assay was performed using Transwell inserts (Costar, NY; 8-mm pore

size) in 24-well dishes. Before performing the migration assay, cells were pretreated

for 30 min with different concentrations of inhibitors (KP-392, Akti, PDTC or TPCK)

or vehicle control (0.1% DMSO). Approximately 1 104 cells in 200 Ql of serum-free

medium were placed in the upper chamber, and 300 Ql of the same medium

containing different concentrations of CCN3 were placed in the lower chamber. The

cells were incubated for 24 h at 37 C in 5% CO2, then fixed in 3.7% formaldehyde

for 5 min, and stained with 0.05% crystal violet in PBS for 15 min. Cells on the upper

side of the filters were removed with cotton-tipped swabs and the filters were washed

with PBS. Cells on the underside of the filters were examined and counted under a

microscope. Each experiment was performed in triplicate and repeated at least three


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times. The number of invading cells in each experiment was corrected for

proliferation effects using a cell viability assay (corrected invading cell number =

counted invading cell number/percentage of viable cells) (25).

siRNA transfection

Small-interfering RNAs (siRNAs) were purchased from Santa Cruz Biotechnology

(Santa Cruz, CA). Cells were transfected with siRNAs (0.4 nmol) using

Lipofectamine 2000 (Invitrogen, CA).

Flow cytometric analysis

Human prostate cells were grown in 6-well dishes and then washed with PBS and

detached using trypsin (Gibco, CA) at 37 C. Cells were fixed for 10 min in PBS

containing 3.7% paraformaldehyde, rinsed in PBS, and incubated with mouse

anti-human-ICAM-1 (1:100) (BD biosciences, CA) for 1 h at 4 C.Cells were then

washed in PBS and incubated with fluorescein isothiocyanate-conjugated goat

anti-mouse secondary IgG (1:100; Leinco Technologies, St Louis, MO) for 45 min at

4 rC. After a final rinse, cells were analyzed using a FACSCalibur flow cytometer

and CellQuest software (BD Biosciences, CA).

Western blotanalysis

Cellular lysates were prepared and proteins were resolved by SDS-PAGE. Proteins

were transferred to Immobilon polyvinylidene fluoride membranes. The blots were

blocked with 4% bovine serum albumin for 1 h at room temperature and probed with

rabbit anti-human antibodies against p-GSK3, GSK3ILK, p-Akt, Akt, p-IKK, IKK,

p-IB, IB, p-p65, p65, ICAM-1, CCN3 or -actin (1:1000) for 1 h at room

temperature (Santa Cruz, CA). After three washes, the blots were incubated with

peroxidase-conjugated donkey anti-rabbit secondary antibody (1:1000) for 1 h at

room temperature. The blots were visualized with enhanced chemiluminescence using

X-OMAT LS film (Eastman Kodak, Rochester, NY).


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ILK kinaseactivityassay

ILK enzymatic activity was assayed in PC-3 cells lysed in Nonidet P-40 buffer (0.5%

sodium deoxycholate, 1% Nonidet P-40, 50 mM HEPES, pH 7.4, 150 mM NaCl) as

reported (26). Briefly, ILK was immunoprecipitated from 250 Qg of lysate using an

ILK antibody overnight at 4 C. After immunoprecipitation, beads were resuspended

in 30 Ql kinase buffer containing 1 Qg recombinant substrate (a GSK3 fusion protein)

and 200 QM ATP. The reaction was allowed to proceed for 30 min at 30 C.

Phosphorylated substrate was visualized by western blot analysis using an antibody

against phospho-GSK3. Total GSK3 was also detected using the appropriate

antibody. Anti-ILK was used as a loading control.

Quantitativereal-time PCRTotal RNA was extracted from prostate cancer cells using a TRIzol kit (MDBio,

Taipei, Taiwan). Reverse transcription was performed using 1 g total RNA and an

oligo(dT) primer. Quantitative real-time PCR (qPCR) was carried out using a TaqMan

One-step PCR Master Mix (Applied Biosystems, CA, USA). Total cDNA (100 ng)

was added to each 25 l reaction with sequence-specific primers and TaqManprobes.

All target gene primers and probes were purchased commercially, including those for

(GAPDH as an internal control (Applied Biosystems). qPCR was carried out in

triplicate with a StepOnePlus (Applied Biosystems) sequence detection system. The

cycling conditions were 10 min at 95 C, followed by 40 cycles of 95 C for 15 s and

60 C for 60 s. To calculate the cycle number at which the transcript was detected

(CT), the threshold was set above the non-template control background and within the

linear phase of target gene amplification.

Reportergeneassay

Human prostate cancer cells were co-transfected with 0.8 Qg B-luciferase plasmid

and 0.4 Qg -galactosidase expression vector. PC-3 cells were grown to 80%


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confluent in 12-well plates and were transfected the following day by Lipofectamine

2000 (LF2000; Invitrogen). DNA and LF2000 were premixed for 20 min and then

applied to the cells. After a 24 h transfection, the cells were then incubated with the

indicated agents. After further 24 h incubation, the media were removed, and cells

were washed once with cold PBS. To prepare lysates, 100 Ql reporter lysis buffer

(Promega) was added to each well, and cells were scraped from dishes. The

supernatant was collected after centrifugation at 13,000 rpm for 2 min. Aliquots of

cell lysates (20 Ql) containing equal amounts of protein (2030 Qg) were placed into

wells of an opaque black 96-well microplate. An equal volume of luciferase substrate

was added to all samples, and luminescence was measured in a microplate

luminometer. The value of luciferase activity was normalized to transfection

efficiency monitored by the co-transfected -galactosidase expression vector.

Chromatin immunoprecipitation

Chromatin immunoprecipitation (ChIP) was performed as described (27). DNA was

immunoprecipitated using an anti-p65 antibody, then extracted, purified, and

resuspended in H2O. Immunoprecipitated DNA was amplified by PCR using the

following primers: 5d-AGACCTTAGCGCGGTGTAGA-3d and

5d-GCGACTCGAGGAGACGATGA-3d (28). PCR products were resolved by

1.5% agarose gel electrophoresis and visualized by UV light.

Electrophoretic mobilityshiftassay

Electrophoretic mobility shift assay was performed using an EMSA gel shift kit

(Panomics, Redwood City, CA) and an oligonucleotide corresponding to the NF-B

binding sequence (5d-AGCTTGGAAATTCCGGA-3d,(29)). Nuclear extracts of

PC-3 cells (3 Qg) were incubated with poly [d(I-C)] at room temperature for 5 min.

The nuclear extract was incubated with biotin-labeled probes at room temperature for

30 min. After electrophoresis on a 6% polyacrylamide gel, the samples were

transferred onto a presoaked Immobilon-Nyt membrane (Millipore, Billerica, MA).


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The membrane was baked at 80C for 1 h, cross-linked in an oven for 3 min,

incubated with blocking buffer followed by streptavidin-horseradish peroxidase

conjugate, and then subjected to western blot analysis.

Intratibialinjection ofPC-3 cells in nude mice

CCN3 shRNA-expressing PC-3 cells were cultured with fresh culture medium for

24 h before intratibial injection. Controls included untransfected PC-3 cells and cells

transfected with vector control plasmid. Cells were harvested with trypsin-EDTA

(Invitrogen, Carlsbad, CA), resuspended in PBS, and kept at 4 C before injection.

Male CB17-SCID mice (4 weeks old) were deeply anesthetized using

trichloroacetaldehyde monohydrate (0.4 mg/g body weight; KANTO Chemical Co.,

Tokyo, Japan). Intratibial injection of cells was performed using polyethylene tubing

(Recorder No. 427401, Becton Dickinson) fit around a 30-G needle. This design was

used to ensure the depth (1.5 mm) of the needle as it was inserted into the proximal

tibia, thereby preventing the cells from spilling out of the injection site. The cell

suspension (150 Ql containing 2 105

cells) was slowly injected into the bone marrow

cavity of the tibia. A tumor mass was visualized around the proximal tibia 28 days

after injection. To make sure of bone osteolysis, radiographs were taken using a soft

X-ray generating unit. Animals were deeply anesthetized with trichloroacetaldehyde

monohydrate, placed in a prone position on a Kodak Scientific Imaging film (13

18 cm), and exposed to X-rays (45 kV) for 5 s.

Statisticalanalysis

Data are presented as mean standard error of the mean (SEM). Statistical analysis

between two samples was performed using the Students t test. Statistical comparisons

of more than two groups were performed using one-way analysis of variance with

Bonferronis post-hoc test. In all cases, P < 0.05 was considered significant.


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Results

CCN3expression ispositively correlated withprostate cancercellmotility,andthis

effectrequires v3 integrin receptor

CCN3 has been shown to increase cell migration and metastasis in a variety of human

cancers (20,30). Here, we examined whether CCN3 affects cell migration and

metastasis in prostate cancer cells. As a first step, we examined the levels of CCN3

protein and mRNA expression in three human prostate cancer cell lines: PC-3, DU145,

and LNCaP. CCN3 protein and mRNA expression were significantly elevated in the

highly metastatic PC-3 cell line compared to both DU145 and LNCaP cell lines (Fig.

1A&B). To determine whether CCN3 affects migration of prostate cancer cells, a

Transwell assay was used. PC-3 cells had a higher rate of migration compared to the

other cell lines (Fig. 1C). Moreover, treatment with CCN3 (10100 ng/ml)

dramatically increased migration in all three cell lines (Fig. 1D). Therefore, the

expression of CCN3 is positively correlated with motility of prostate cancer cells.

Previous studies have shown that CYR61, another member of the CCN family,

affects cell migration by binding to integrin receptors on the cell surface (17,31). We

hypothesized, therefore, that v3 integrin signaling pathway may be involved in

CCN3-induced prostate cancer cell migration. Pretreatment of cells with an anti-v3

integrin monoclonal antibody (5 Qg/ml) for 30 min markedly inhibited CCN3-induced

cancer cell migration (Fig. 1E). Similarly, CCN3-induced migration was essentially

abolished by pretreatment with 100 nM cyclic RGD peptide (which is known to block

v3 function (32)), but not by pretreatment with cyclic RAD peptide (a negative

control). To confirm the positive correlation between integrin v3 and

CCN3-induced cell migration, we measured integrin v and 3 mRNA expression by

qPCR following CCN3 treatment. As expected, CCN3 up-regulated both v and 3

integrin expression in a dose-dependent manner (Fig. 1F). Collectively, these data

indicated that CCN3 expression was positively correlated with a higher metastatic

potential (i.e., rate of migration) in prostate cancer cells, and that this effect may have

been mediated by integrin v3 receptor.


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ICAM-1 is involved in CCN3-induced cellmigration

Recently, it has been reported that ICAM-1 plays a key role in cancer cell migration

and tissue invasion (10,33). We examined, whether ICAM-1 was involved in

CCN3-induced migration of prostate cancer cells. Western blot and qPCR analyses

indicated that ICAM-1 level was elevated in highly metastatic PC-3 cells (Fig.

2A&B). We next asked whether CCN3-induced cell migration correlated with

ICAM-1 expression. Results showed that CCN3 increased ICAM-1 protein and

mRNA expression in a dose-dependent manner (Fig. 2C&D). Finally, we asked

whether loss of ICAM-1 affects CCN3-induced cell migration. PC-3 cells were

transfected with ICAM-1 siRNA (100 nM) to knock down ICAM-1 levels. After 24 h,

CCN3 was added and cell migration was analyzed using the Transwell assay.

Transfection of cells with ICAM-1 siRNA markedly inhibited CCN3-induced cell

migration (Fig. 2E). To improve integrin v3 signaling pathway was involved in

ICAM-1 expression. We used anti-v3 integrin monoclonal antibody (5 Qg/ml) and

cyclic RGD peptide (100 nM) to abolish integrin v3 signaling pathway. In the

result, ICAM-1 mRNA expression was inhibited by anti-v3 integrin antibody and

RGD peptide but not RAD peptide (negative control) (Fig. 2F). These data clearly

show that CCN3-induced cancer cell migration may occur via upregulation of

ICAM-1 and through v3 integrin receptor.

CCN3promotes cellmigration andICAM-1expression viaan ILK-and

Akt-dependentpathway

Integrin-linked kinase (ILK) is a downstream regulator of integrin signaling (34). To

determine whether ILK participates in CCN3-dependent effects in prostate cancer

cells, we first treated PC-3 cells with CCN3 (30 ng/ml) and measured ILK activity. As

shown in Fig. 3A, GSK3 was used as substrate to measure ILK activity. Following

CCN3 stimulation, ILK activity increased in a time-dependent manner, reaching a

maximum level between 15 and 60 min. Treatment with an ILK-specific inhibitor


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(KP-392, 1 QM) or transfection with an ILK siRNA (100 nM) decreased

CCN3-induced migration (Fig. 3B&C). In addition, CCN3-dependent increases in

ICAM-1 expression were reduced by KP-392 treatment or ILK siRNA transfection

(Fig. 3D&E). Therefore, ILK is involved in CCN3-mediated migration and ICAM-1

expression.

It is well established that Akt is regulated by ILK signaling (26,35). We asked,

therefore, whether CCN3 stimulation could activate the Akt signaling pathway. CCN3

treatment increased the level of phosphorylated Akt (Fig. 4A). CCN3-induced

migration of prostate cancer cells was greatly reduced by addition of Akt inhibitor

(Akti) (Fig. 4B) or expression of a dominant-negative mutant of Akt (Fig. 4C). Akt

inhibition similarly reduced ICAM-1 expression in CCN3-treated cells (Fig. 4D&E).

Taken together, our results indicated that the Akt pathway was involved in

CCN3-induced cell migration and ICAM-1 upregulation in human prostate cancer

cells.

NF-B is involved in CCN3-induced cellmigration andICAM-1expression

NF-B is a primarily cytosolic transcription factor whose activity is often correlated

with cancer cell migration and invasion (25,36). Therefore, we used western blot

analysis to investigate whether components of the NF-B signaling pathway are

involved in the CCN3-dependent effects on prostate cancer cells. Levels of

phosphorylated IKK/, IB and NF-B subunit p65 were higher after 15 min of

CCN3 treatment and diminished after ~120 min (Fig. 5A). Moreover, cell migration

in response to CCN3 was decreased by pretreatment with the NF-B inhibitor PDTC

(10 QM) or the IB protease inhibitor TPCK (1 QM; Fig. 5B). Furthermore,

expression of dominant-negative mutant of IKK or IKK markedly inhibited

CCN3-induced cell migration (Fig. 5B). Similarly, each of these treatments reduced

ICAM-1 expression (Fig. 5C). It is clear, therefore, that CCN3-mediated effects on

cell migration and ICAM-1 expression are mediated by the NF-B signaling pathway.


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CCN3 increases binding ofNF-Bto the NF-Belementon theICAM-1promoter

The promoter region of human ICAM-1 contains an NF-B binding site (28);

therefore, we asked whether CCN3 stimulation caused NF-B to bind to the ICAM-1

promoter in prostate cancer cells. To this purpose, the electrophoretic mobility shift

assay was used. As shown in Fig. 5D, the p65 subunit of NF-B bound to the NF-B

element was increased by CCN3 treatment (0-100 ng/ml). We confirmed that NF-B

activity was increased by B-luciferase activity assay (Fig. 5E). In addition,

CCN3-mediated binding of p65 to the NF-B element was repressedby KP-392, Akti,

PDTC, and TPCK (Fig. 5F). Based on these results, activation of the ILK, Akt, and

NF-B signaling pathways wasrequired for the CCN3-induced increase in ICAM-1

expression in humanprostate cancer cells.

Knockdown ofCCN3expression inhibits cellmigration and osteolytic metastasis in a

mouse modelofprostate cancer

To confirm the role of CCN3 in prostate tumor metastasis, we took advantage of PC-3

cells that stably express CCN3 shRNAs. Following puromycin (1 Qg/ml) selection,

individual stable clones (CCN3 sh1, CCN3 sh2 and CCN3 sh3) were collected for

analysis.Empty vector plasmid was used as a negative control. Levels of both CCN3

and ICAM-1 were decreased in all three CCN3 shRNA stable clone, and CCN3 sh3

showed most decreased (Fig. 6A). CCN3 knockdown did not alter the rate of cell

proliferation (Fig. 6B), but significantly reduced migration (Fig. 6C).

The PC-3 cell line is an androgen-independent human prostate cancer line that

frequently metastasizes to bone (37,38). To examine the effect of CCN3 on prostate

tumor growth in vivo, PC-3 cells (2 105) were locally injected into the tibial bone

marrow cavity of 4-week-old severe combined immunodeficiency (SCID) mice. In

each mouse, one tibia was injected with negative PC-3 cells and the contralateral tibia

was injected with CCN3 sh3 stable clone. Mice were sacrificed after 28 days later. To

analyze bone osteolysis in these mice, radiographs were taken using a soft X-ray

generating unit. Radiographs which were taken 28 days post-injection revealed


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osteolytic lesions in tibia injected with negative PC-3 cells. In contrast, osteolytic

lesions were dramatically reduced in mice injected with CCN3 sh3 stable clone (Fig.

6D). Tumor weight was recorded after the animals were sacrificed. Quantitative

assessment of tumor weight and osteolytic lesions confirmed that knockdown of

CCN3 inhibited both tumor growth and formation of osteolytic lesions (Fig. 6E&F).

Discussion

In recent years, correlations between CCN3 and tumorigenesis have been identified in

a variety of cancers, but the mechanism of CCN3 activity in these contexts remains

unclear (20). In both osteosarcoma and Ewing sarcoma, high levels of CCN3

expression indicate a poor prognosis for patients (21,39). In contrast, reduced levels

of CCN3 expression are thought to promote cancer progression in childhood

adrenocortical tumors and melanomas (22,40). A correlation between prostate cancer

and CCN3 has been reported previously (23), but the mechanism by which CCN3

affects prostate cancer remains unclear. In this study, we have demonstrated for the

first time that CCN3 (an extracellular matrix-associated protein) increased migration

and metastasis of prostate cancer cells by transcriptionally upregulating ICAM-1

expression. CCN3 increased ICAM-1 expression by activating the v3 integrin,ILK,

Akt, and NF-B signaling pathways.

Prostate cancer is prevalent in developed countries worldwide. Most prostate

tumors remain confined to the prostate gland and adjacent soft tissue and cause little

to no harm. However, nearly one in eight cases leads to metastasis, typically to bone

(41). The role of androgenic hormones in prostate cancer progression and survival has

been reported previously, and is supported by the ability of androgen ablation therapy

to cause regression of both primary and metastatic disease (42,43).Here, we found a

correlation between CCN3 expression and the rate of prostate cancer cell migration.

The highest level of CCN3 expression and the greatest ability to migrate were seen in

the most malignant prostate cancer cell line (PC-3, Fig. 1A-C). However, CCN3 was

found to support the chemo-migration of both androgen-dependent (LNCaP) and


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androgen-independent (PC-3 and DU145) prostate cancer cells (Fig. 1D). Therefore,

CCN3 induced cancer migration in androgen-dependent and independent prostate

cancer cells.

Cell adhesion molecules have been implicated in tumor progression and

metastasis (44). In solid tumors such as prostate cancer, the level of ICAM-1

expression has been shown to dictate metastatic potential, which in turn determines

cancer lethality (45). Here, we found that ICAM-1 expression was upregulated by

CCN3 treatment and was directly responsible for the increase in prostate cancer cell

migration by CCN3 (Fig. 2). This result is similar to previous studies involving

prostate cancer (46). In addition, ICAM-1 levels were reduced following knockdown

of CCN3. Therefore, ICAM-1 appeared to mediate CCN3-induced cell migration.

Integrins, which link the extracellular matrix to intracellular signaling molecules,

regulate a number of cellular processes, including adhesion, signaling, motility,

survival, gene expression, growth, and differentiation.Integrins are known to serve as

receptors for CCN3 (17). CCN3 has been shown to bind v3 integrin and increase

cell migration (20,47). In this report, v and 3 integrin mRNA expression was

elevated following CCN3 treatment. Moreover, CCN3-induced cell migration was

inhibited by pretreatment with a neutralizing antibody against integrin v3 or with

an RGD peptide. This indicates that integrin v3 receptor played an important role in

CCN3-induced migration and ICAM-1 expression in prostate cancer cells.

ILK is activated by integrins, growth factors, and chemokines (34). In bone, ILK

promotes chondrocyte proliferation, adhesion, and spreading (48), but its role in

prostate cancer differentiation is still unclear. In this study, we presented the first

evidence to indicate that ILK plays a critical role in prostate cancer progression.

Treatment of prostate cancer cells with CCN3 increased the activity of ILK, and

CCN3-induced ICAM-1 expression and cell migration were both inhibited by the ILK

inhibitor KP-392 and by ILK-specific siRNA. These data suggest that ILK activation

is an obligatory step in CCN3-induced prostate cancer cell migration. ILK may

regulate these cellular functions by promoting phosphorylation of Akt on Ser473,


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thereby activating this important downstream pathway (49). Akt has been shown to

regulate cancer progression (50,51). We found that CCN3 treatment increased the

level of Akt phosphorylation in a time-dependent manner, and that inhibition of Akt

reduced the effects of CCN3 on cell migration and ICAM-1 expression (Fig. 4). Taken

together, our results provide evidence that CCN3 upregulated ICAM-1 expression and

promoted cell migration via the integrin/ILK/Akt signaling pathway in prostate cancer

cells.

Previous reports have indicated that NF-B is involved in tumor metastasis (52).

Here, we demonstrated that the NF-B inhibitors PDTC and TPCK reduced

CCN3-induced cell migration, indicating that NF-B acts downstream of CCN3. In its

inactivated state, NF-B is held in the cytoplasm by the inhibitory protein IB. Upon

stimulation (for example, by tumor necrosis factor , IB proteins become

phosphorylated by the multisubunit IKK complex. This targets IB for ubiquitination

and subsequent degradation by the 26S proteasome. Free NF-B can then translocate

to the nucleus, where it regulates the transcription of target genes (53). The NF-B

pathway has also been linked to CCN3 signaling (54). Treatment of PC-3 cells with

CCN3 led to increased levels of phosphorylated IKK, IB, and p65 (Fig. 5A). Using

transient transfection of a B-luciferase reporter construct that indicates NF-B

activity, we found that CCN3 increased NF-B activity. Moreover, chromatin

immunoprecipitation and electrophoretic mobility shift assays clearly demonstrated

that p65 binds to the NF-B element in the ICAM-1 promoter in response to CCN3

stimulation, and that p65 binding to the NF-B element was attenuated by KP-392,

Akti, PDTC, and TPCK (Fig. 5D-F). Taken together, these results indicate that CCN3

acted through the v3 integrin, ILK, Akt, and NF-B pathways to induce ICAM-1

expression in human prostate cancer cells.

Finally, to directly determine the effect of CCN3 on prostate cancer progression,

we knocked down CCN3 expression in PC-3 cells using shRNA (Fig. 6). CCN3

knockdown significantly reduced ICAM-1 expression and inhibited migration in PC-3

cells. We then injected negative PC-3 cells and PC-3 cells expressing CCN3 shRNA


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into the contralateral tibia of SCID mice to investigate the in vivo effects of CCN3 on

tumor growth and bone resorption (i.e., osteolysis). Tumor growth in the bone was

clearly decreased in PC-3 cells that lacked CCN3, as was bone metastasis. These data

indicated that CCN3 play an import role in prostate cancer metastasis to bone in vivo.

The rate-limiting step in metastasis, and a critical stage in cancer progression, is

the acquisition of motility by a tumor cell. Bone metastasis is the major cause of

mortality for patients with prostate cancer. Here, we have revealed critical new

insights into CCN3 function and its role in prostate cancer progression. CCN3

expression is upregulated in prostate cancer cells, promotes cell migration in vitro,

and promotes tumor growth in vivo. Although the mechanisms involved in

CCN3-induced bone metastasis are not yet complete, we have provided evidence that

CCN3 works via the integrin v3, ILK, Akt, and NF-B signaling pathways. Our

data demonstrate the importance of CCN3 in the growth and metastasis of prostate

cancer, and identify CCN3 as a novel therapeutic target for the clinical treatment of

prostate cancer.


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Figure legends

Fig. 1CCN3-induced migration of human prostate cancer cells. (A) Total protein was

extracted from PC-3, DU145, and LNCaPcells and subjected to western blot analysis

using anti-CCN3. Actin was used as a loading control. (B) Total mRNA was collected

from PC-3, DU145, and LNCaP cells, and CCN3 expression was determined by

qPCR. (C) In vitro migration activity of PC-3, DU145, and LNCaP cells was

measured using the Transwell assay. (D) PC-3, DU145, and LNCaP cells were

incubated with CCN3 (0, 10, 30, or 100 ng/ml) for 24 h, and in vitro migration was

measured using the Transwell assay. (E) PC-3 cells were pretreated with anti-v3

mAb (5 Qg/ml), cyclic RGD (100 nM), or cyclic RAD (100 nM) for 30 min followed

by stimulation with CCN3 (30 ng/ml) for 24 h (Control = no CCN3 treatment). In

vitro migration was measured after 24 h using the Transwell assay. (F) PC-3 cells

were incubated with CCN3 for 24 h, and v- and 3-integrin mRNA levels were

determined by qPCR. Results are expressed as mean SEM of triplicate samples. In

(B, C), * P < 0.05 compared with PC-3. In (D-F), * P < 0.05 compared with control; #

P < 0.05 compared with CCN3 treatment.

Fig. 2CCN3-directed migration of human prostate cancer cells involves upregulation

of ICAM-1. (A) Total protein was extracted from PC-3, DU145, and LNCaP cells,

and western blot analysis was used to detect ICAM-1. Actin was used as a loading

control. (B) Total mRNA was collected from PC-3, DU145, and LNCaP cells, and

ICAM-1 mRNA expression was determined by qPCR. (C)PC-3 cells were incubated

with CCN3 (0, 10, 30, or 100 ng/ml) for 24 h and the cell lysates were collected. The

level of ICAM-1 in cell lysates was determined by western blot analysis. Anti-actin

was used as a loading control. (D) PC-3, DU145, and LNCaP cells were incubated

with CCN3 (0, 10, 30, or 100 ng/ml) for 24 h, and ICAM-1 mRNA level was

measured by qPCR. (E)PC-3 cells were transfected with ICAM-1 or negative control

siRNA (Neg) for 24 h, followed by treatment with CCN3 for 24 h, and ICAM-1

protein level was examined by western blot analysis. Inset: PC-3 cells were


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transfected with ICAM-1 or control siRNA for 24 h. After 24 h, CCN3 was added and

cell migration was analyzed using the Transwell assay. Actin was used as a loading

control. (F) PC-3 cells were pretreated with anti-v3 mAb (5 Qg/ml), cyclic RGD

(100 nM), or cyclic RAD (100 nM) for 30 min followed by stimulation with CCN3

(30 ng/ml) for 24 h (N = no CCN3 treatment). Results are expressed as mean SEM

of triplicate samples. In (B, D), * P < 0.05 compared with PC-3. In (E, F), * P < 0.05

compared with control; # P < 0.05 compared with CCN3 treatment.

Fig. 3 ILK signaling affects the CCN3 response in human prostate cancer cells. (A)

PC-3 cells were incubated with CCN3 (30 ng/ml) for the indicated times. Cell lysates

were prepared and immunoprecipitated with anti-ILK. Immunoprecipitated proteins

were subjected to western blot analysis using anti-pGSK3, anti-GSK3, and

anti-ILK. (B) PC-3 cells were pretreated with the ILK inhibitor KP-392 (3 QM) or

vehicle (Control) for 30 min, followed by stimulation with CCN3 (30 ng/ml) for 24 h.

In vitro migration was measured using the Transwell assay. (C) PC-3 cells were

transfected with ILK siRNA or negative control siRNA (Neg) and stimulated with

CCN3 (30 ng/ml). In vitro migration was measured using the Transwell assay after 24

h. (D, E) PC-3 cells were treated as in (B, C) and ICAM-1 mRNA levels were

determined by qPCR. In (B-E), results are expressed as mean SEM of triplicate

samples. *P< 0.05 compared to control;#P< 0.05 compared to CCN3 treatment.

Fig. 4 Akt affects the response of human prostate cancer cells to CCN3. (A) PC-3

cells were incubated with CCN3 (30 ng/ml) for the indicated times and p-Akt or Akt

levels were determined by western blot analysis. (B) PC-3 cells were pretreated for

30 min with the Akt inhibitor Akti (1 M) or vehicle (Control), followed by

stimulation with CCN3 (100 ng/ml) for 24 h.In vitro migration was measured using

the Transwell assay. (C) PC-3 cells were transfected with a dominant-negative (DN)

Akt mutant for 24 h followed by stimulation for with CCN3 (30 ng/ml) for 24 h.

Vector represents a control transfection. In vitro migration was measured using the


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Transwell assay. (D, E) PC-3 cells were treated as in (B, C) and ICAM-1 mRNA

levels were determined by qPCR. Results in (B-E) are expressed as mean SEM of

triplicate wells. * P< 0.05 compared to control; # P< 0.05 compared to CCN3

treatment.

Fig. 5 NF-B mediates the response of human prostate cancer cells to CCN3

stimulation. (A)PC-3 cells were incubated with CCN3 (30 ng/ml) for the indicated

times, and phosphorylation of IKK/ IB and p65 was determined by western blot

analysis. (B)PC-3 cells were pretreated for 30 min with PDTC (10 QM), TPCK (1

QM), or vehicle (Control), followed by stimulation with CCN3 (30 ng/ml) for 24 h.

PC-3 cells were transfected with dominant-negative (DN) mutant IKK or IKK for

24 h, followed by stimulation with CCN3 (30 ng/ml) for 24 h. Vector represents a

control transfection.In vitro migration was measured using the Transwell assay.(C)

PC-3 cells were treated as described in (B) and ICAM-1 mRNA levels were

determined by qPCR. (D) Cells were stimulated with CCN3 (0-100 ng/ml) for 120

min and an electrophoretic mobility shift assay was performed. (E) PC-3 cells were

transfected with an NF-B promoter reporter plasmid for 24 h. Cells were then

incubated with CCN3 (0-100 ng/ml) for 24 h and luciferase activity was measured. (F)

Cells were pretreated with KP-392 (1 QM), Akti (3 QM), PDTC (10 QM), or TPCK (1

QM) for 30 min, followed by stimulation with CCN3 (30 ng/ml) for 120 min.

Chromatin immunoprecipitation was performed with anti-p65. One percent of

immunoprecipitated chromatin was assayed to verify equal loading (input). In (B, C,

E), results are expressed as mean SEM of triplicate samples. *P< 0.05 compared to

control;#P< 0.05 compared to CCN3 treatment.

Fig. 6 CCN3 knockdown decreases cell migration and osteolytic metastasis in a

mouse model. (A)Total protein was collected from untransfectedPC-3 cells and PC-3

cells stably expressing shRNAs directed against CCN3. A vector-only control is also

shown (Neg). Western blot analysis was used to detect CCN3 and ICAM-1. Actin was


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used as the loading control. (B) PC-3 cells stably expressing shRNA constructs were

seeded as monolayers and counted daily. Cells (104) were reseeded after each count,

and the cell numbers were plotted. (C) In vitro migration of PC-3 cells stably

expressing shRNA constructs was measured using the Transwell assay. (D) SCID

mice were treated by intratibial injection of CCN3 sh3 stable clone in one leg and

negative cells in the other leg, and photographed after 28 days. Radiographs taken at

28 days show an osteolytic lesion on the side injected with negative PC-3 cells (left,

n=9) but not on the side injected with CCN3- knockdown cells (right, n=9). (E)

Quantification of tumor weights following intratibial injection. (F) Quantification of

the bone resorption area following intratibial injection. In (C, F), results are expressed

as mean SEM of triplicate samples. *P< 0.05 compared to control.

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