biological effects induced by insulin-like growth factor binding protein 3 (igfbp-3) in malignant...

Biological effects induced by insulin-like growth factor bindingprotein 3 (IGFBP-3) in malignant melanoma

Geir Frode Øy1, Ana Slipicevic2, Ben Davidson2,3, Ragnar Solberg Faye1,3,4, Gunhild M. Mælandsmo1 and

Vivi Ann Flørenes2,5

1 Department of Tumor Biology, Institute for Cancer Research, Oslo, Norway2 Pathology Clinic, Norwegian Radium Hospital, Oslo University Hospital, Montebello, 0310 Oslo, Norway3 Faculty Division Radiumhospitalet, the Medical Faculty, University of Oslo, Oslo, Norway4 Department of Dermatology, Medical Division, Oslo University Hospital, 0027 Oslo, Norway5 Faculty of Health Sciences, Oslo University College 0130 Oslo, Norway

The insulin like growth factor (IGF) signaling pathway has been shown to contribute to melanoma progression, but little is

known about the role of the IGF binding protein 3 (IGFBP-3) in melanoma biology. The aim of the present study was to

characterize expression, function and regulation of IGFBP-3 in malignant melanomas and study its potential as a biomarker.

The expression of IGFBP-3 varied between different human melanoma cell lines and reintroduction of the protein in non-

expressing cells led to induction of apoptosis. Interestingly, in cell lines expressing endogenous IGFBP-3, siRNA silencing of

the protein led to a cell line-dependent decrease in proliferation, but had no effect on apoptosis and invasion. Examination of

patient material showed that IGFBP-3 is unexpressed in benign nevi while a slight increase in protein expression was seen in

primary and metastatic melanoma. However, expression of the protein was low and no correlation was found with circulating

levels of IGFBP-3 in serum, suggesting that IGFBP-3 has limited potential as a predictive marker in malignant melanoma. We

showed that promoter methylation of IGFBP-3 occurred in both melanoma cell lines and patient material, implicating

epigenetic silencing as a regulation mechanism. Furthermore, expression of the protein was shown to be regulated by the PI3-

kinase/AKT and MAPK/ERK1/2 pathways. In summary, our findings suggest that IGFBP-3 can exert dual functional effects

influencing both apoptosis and proliferation. Development of resistance to the antiproliferative effects of IGFBP-3 may be an

important step in progression of malignant melanomas.

Due to its irresponsiveness to current therapies malignantmelanoma is a highly lethal skin cancer. The molecularmechanisms underlying development and progression of mel-anoma are still not entirely understood, and there is a needfor novel diagnostic and prognostic markers and therapeutictargets.1

The insulin like growth factor (IGF) signaling pathway hasbeen shown to contribute to melanoma survival and progres-sion,2–4 and targeting the IGF signaling pathway is emergingas a promising strategy in anti-cancer therapy.5 The IGF sys-tem consists of multiple cell surface receptors, circulatingligands and ligand-binding proteins.6 Binding of IGF-1 to thetype 1 IGF receptor (IGF-R1) initiates phosphorylation ofadaptor proteins of the insulin receptor substrate (IRS) familyor src homologous and collagen (SHC) proteins. These adap-

tor proteins serve as docking sites for other signaling mole-cules, resulting in activation of the phosphoinositide 3-kinase(PI3-kinase) and mitogen-activated protein kinase/extracellu-lar signal-regulated kinases 1/2 (MAPK/ERK1/2) pathways.7

The IGF signaling pathway is regulated by a family of sixstructurally-related IGF binding proteins (IGFBP-1 to 6). Ofthese, IGFBP-3 has the highest affinity for IGF-1 and is alsothe most abundant IGFBP family member in the circulation.By being the primary transport protein for IGF-1, IGFBP-3sequesters the ligand, leading to prevention of IGF-1-inducedIGF-R1 autophosphorylation and signaling.8 In contrast, accu-mulation of membrane-bound IGFBP-3 has been reported toenhance the presentation of IGF-1 to its receptor and therebypotentiate IGF signaling.9,10 Furthermore, IGFBP-3 has beenshown to exhibit effects on proliferation, migration, and apo-ptosis that are independent of its effects on IGF signaling.11

The multifunctional roles reported for IGFBP-3 are likely tobe influenced by posttranslational modifications,12–14 suscepti-bility to proteases15 and/or interactions with several signalingpathways. Expression of IGFBP-3 has been shown to be regu-lated among others by interleukin-1, tumor necrosis factor-alpha (TNF-a), transforming growth factor-beta (TGF-b) andretinoic acid, as well as by IGF-1.8 Moreover, the PI3-kinase/AKT and MAPK/ERK1/2 pathways have been implicated inthe regulation of IGFBP-3.16

Additional Supporting Information may be found in the online

version of this article

DOI: 10.1002/ijc.24727

History: Received 19 Dec 2009; Accepted 25 Jun 2009; Online 8 Jul

2009

Correspondence to: Vivi Ann Flørenes, Pathology Clinic,

Norwegian Radium Hospital, Oslo University Hospital, Montebello,

0310 Oslo, Norway, E-mail: [email protected]

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Int. J. Cancer: 126, 350–361 (2010) VC 2009 UICC

International Journal of Cancer

IJC

Two recent reports have addressed the expression andfunction of IGFBP-3 in melanoma. While Xi et al. suggestedthat the protein could be used as a biomarker based onincreased expression in metastasis relative to primarytumors,2 Yu et al. reported small differences in proteinexpression between primary and metastatic tumors, and nodifferences in IGFBP-3 plasma levels.17 In order to clarifythese discrepancies we examined the expression level inpatient material and melanoma cell lines, and assessed differ-ent mechanisms by which its expression may be regulated.Furthermore, we studied the functional role of IGFBP-3 inmelanoma cell lines by reintroducing the protein in IGFBP-3negative cells and by down-regulating it in cells with highexpression. Our data suggest that IGFBP-3 has dual-func-tional effects on apoptosis and proliferation that are cell line-dependent. We also report that IGFBP-3 is not a biomarkerfor melanoma patients.

Material and MethodsCell lines and culture conditions

The Wistar Melanoma (WM) cell lines were kindly providedby Dr. Meenhard Herlyn (Wistar Institute, Philadelphia, PA)and have been described in detail elsewhere.18 The cell linesFEMX-I and LOX were established from lymph node meta-stasies obtained from melanoma patients treated at the Nor-wegian Radium Hospital, Rikshospitalet University Hospi-tal.19 All cells were cultured in RPMI 1640 medium (BioWhittaker, Verviers, Belgium) supplemented with 5-10% fetalbovine serum (FBS, PAA Laboratories, Linz, Austria) and2 mM L-glutamine (GibcoBRL, Paisley, UK). The cells weremaintained at 37�C in a humidified 5% CO2 atmosphere.Cell lines were routinely checked for mycoplasma infections.

Treatment of the cells with 500 ng/ml trichostatin A(TSA) and 10lM 50-aza-20-deoxycytidine (Aza) (both chemi-cals from Sigma-Aldrich, St. Louis, MO) was done for 24 and48 hours, respectively. Medium containing Aza was changedevery 24 hours. Conditioned medium from untreated andtreated cells were collected and stored at �20�C. Treatmentof the cells with 25 lM LY294002 (LY) and 25 lM PD98059(PD) (both from Cell Signaling Technology, Beverly, MA)was done for 24 hours.

Clinical samples

The collection and processing of patient plasma samples usedfor ELISA analysis have been described by Faye et al.20 For-malin-fixed, paraffin-embedded tissue sections were obtainedfrom 6 benign nevi, 17 primary malignant melanomas and40 metastases. Total genomic DNA used for methylation-spe-cific PCR was extracted from 11 snap-frozen metastatic mela-noma biopsies. The study was approved by the RegionalCommittee for Medical Research Ethics in Norway.

IGFBP-3 cDNA transfection

All cell lines were seeded 24 hours prior to transfection.WM35 and FEMX-1 cells were seeded in 6-well plates (2 �

105 cells per well) or in 96-well plates (2 � 103 cells perwell). Transfection in 6-well plates was performed using 2,5lg DNA/well in serum-containing medium using the Lipo-fectamin LTX transfection reagent (Invitrogen, Carlsbad, CA)according to the manufacturer’s instructions. 100 ng DNA/well was used in 96-well plates. Cells were harvested 72 hoursafter transfection for further analysis. The IGFBP-3 expres-sion vector pSF398 and its control pSF188 were a kind giftfrom Dr. Robert Baxter at the Kolling Institute for MedicalResearch in Sydney, Australia.

Small interfering RNA (siRNA) transfection

All cell lines were plated 24 hours prior to transfection.WM239 and WM9 cells were seeded in 24-well plates (5 �104 cells per well) or in 96-well plates (5 � 103 cells perwell) and transfected with 50 nM siRNA (Stealth RNAi) tar-geting IGFBP-3 or Stealth RNAi negative control duplexes(Medium GC Duplex) using LipofectamineTM RNAiMAXtransfection reagent (all reagents from Invitrogen). Cells wereharvested 48 hours after transfection for further analysis.

TUNEL assay

Detached cells were collected by centrifugation of culturedmedium and pooled together with adherent cells harvestedby EDTA (Cambrex, East Rutherford, NJ) treatment followedby fixation in 100% cold methanol. Fixated cells were washedwith PBS (Cambrex), incubated for 30 minutes at 37�C in50 ll terminal transferase (TdT) solution containing 5 unitsTdT (Roche, Basel, Switzerland), 10 ll 5� reaction buffer(supplied with TdT), 1.5 mM CoCl2, 0.5 nmol labeled biotin-16-dUTP (Roche), 0.1 mM dithiothreitol and distilled water.The cells were subsequently washed once in PBS containing0.1% Triton X-100 and incubated in 50 ll 1:50 streptavidin-FITC (Amersham, Buckinghamshire, UK) in PBS (containing0.1% Triton X-100) and 3% non-fat milk for 45 min at roomtemperature. After washing in PBS (0.1% Triton X-100) thepellet was resuspended in PBS (0.1% Triton X-100) contain-ing 2 lg/ml Hoechst 33258 (Roche) to a final concentrationof 1 � 106 cells/ml and incubated for 30 minutes at 4�C.Data acquisition and analysis were performed on BectonDickinson LARII (Becton Dickinson immunocytometry sys-tems, San Jose, CA) using Multifit software (FACSDiVaHouse inc., Tonsham, ME). Each experiment was repeated atleast three times.

IGFBP-3 ELISA

Blood plasma samples were analyzed for IGFBP-3 proteinlevel using Enzyme-Linked Immunosorbent Assay (ELISA)QuantikineV

R

IGFBP-3 Immunoassay DGB300 (R&D Systems,Minneapolis, MN) according to the manufacturer’s instruc-tions. Calibrations and analyses were carried out in dupli-cates. Optical densities were determined using a microtiterplate reader at 450 nm with correctional wavelength at540 nm (Victor,2 Perkin Elmer Life And Analytical Sciences,Inc. Waltham, Ma) The level of IGFBP-3 protein in the

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supernatants from the cell cultures was analyzed usingDuoSetVR ELISA Development System DY675 (R&D Systems).Samples and standards were assayed in singlets and the opti-cal densities were measured as described for plasma samples.

Immunoblotting

Cells were lysed in ice-cold NP-40 lysis buffer (1% NP-40,10% glycerol, 20 mM Tris–HCl, pH 7.5, 137 mM NaCl,100 mM sodium vanadate, 1 mM phenylmethylsulfonyl fluo-ride (PMSF), 0.02 mg/ml each of aprotinin, leupeptin, andpepstatin, and 10 ll/ml phosphatase inhibitor cocktail I andII (Sigma-Aldrich)). Protein quantification was done usingstandard Bradford protein assay, and 25 lg protein/lane wasresolved by SDS polyacrylamide gel electrophoresis (PAGE)and transferred to a PDVF immobilon membrane (Millipore,Bedford, MA). To ensure even loading, membranes werestained with naphthol-blue black (Sigma–Aldrich). The mem-branes were blocked with 5% non-fat milk in TBST(150 mM NaCl, 25 mM Tris-Cl pH 7.5, 0.01% Tween 20),and probed overnight (4�C, with gentle agitation) with ananti-IGFBP3 monoclonal antibody (clone 4) (1:200 dilution;BD Biosciences, San Diego, CA), anti-AKT (1:500; Cell Sig-naling Technology), anti-phospho-AKT (1:200; Cell SignalingTechnology), anti-phospho-ERK1/2 (1:1000; Promega), anti-ERK2 (1:5000; Santa Cruz Biothechnology, Santa Cruz, CA),anti-PTEN (1:1000; Cell Signaling Technology), anti-IGF-Ra(1:1000; Cell Signaling Technology) and anti-a-tubulin (Cal-biochem, San Diego, CA). Residual primary antibodies wereremoved by washing for 3 � 10 minutes in TBST. The mem-branes were thereafter probed for one hour at room tempera-ture (with gentle agitation) with the appropriate secondaryHRP-conjugated anti-rabbit or anti-mouse IgG antibodies(Promega, Madison, WI). Membranes were then washed for3 � 10 minutes in TBST before being incubated with ECL-plus (GE Healthcare, Chalfont St. Giles, UK) for 5 minutes.Protein bands were visualized by exposing the membranes toX-ray films.

Quantitative real-time RT-PCR analysis

Total RNA was extracted using the TRIZOL reagent accord-ing to the manufacturer’s instructions (Invitrogen). The highcapacity cDNA reverse transcription kit (Applied Biosystems,Foster city, CA) was used to reverse-transcribe total RNA(0.8 lg) in a 20 ll reaction mixture using random primers.The real-time PCR analyses were performed using TaqManFast Universal PCR Master Mix (2�) and TaqMan GeneExpression Assay (HS00426287_M1 IGFBP-3, HS99999908-ml GUS, Applied Biosystems). A total of 0.5 ll cDNA wasused in 25 ll PCR mixtures with 900 nM of each primer and250 nM TaqMan probe. The reactions were carried out in a7900HT Fast Real-time PCR system (Applied Biosystems)with the following program: 95�C for 20 seconds, followedby 40 cycles of 95�C for 1 second and 60�C for 20 seconds.Each sample was run in triplicate. The IGFBP-3 mRNAexpression level was normalized with respect to the beta-glu-

curonidase (GUS) gene, which had stable transcript levelsunder these experimental conditions and calibrated againstthe WM35 cell line. The data was analyzed with RQManagerSoftware (Applied Biosystems) and the mean from three in-dependent experiments was calculated.

[3H]-Thymidine incorporation assay

Cells were plated in 96-well plates and transfected withsiRNA, cDNA or treated with recombinant protein andcultured for 72 hours, the last 24 hours with the addition of3.7 � 104 Bq [3H]-Thymidine (ARC, St. Louis, MO). There-after, the cells were harvested using a Filtermate Harvester(Packard Instrument Co. Meriden, CT), and [3H]-Thymidineincorporation was assessed in a Packard Microplate Scintilla-tion Counter. Proliferation assays were performed in tripli-cate. Each experiment was repeated at least three times.

Methylation-specific PCR (MSP) assay

DNA extraction and bisulfite treatment. Total genomic DNAwas extracted from 11 snap-frozen melanoma biopsies andsix cell lines using a variation of the standard proteinase K/phenol:chloroform protocol.21 For bisulfite treatment, theextracted DNA (3 lg) was modified using the CpGenomeUniversal DNA Modification Kit (S7820, Millipore, BillericaMA) according to the manufacturer’s instructions. The finalsuspension volume was 50 lL of Tris-EDTA buffer.

MSP assay. The MSP assay was performed as described byTomii et al.,22 with modification of the annealing tempera-tures. Methylated alleles were amplified using the followingprimers: IGFBP-3-MSP-F (50-GACCCGAACGCGCCG-30)and IGFBP-3-MSP-R (50-AGGTGACGGGTTTCGGGC-30).The annealing temperature for the methylated allele primerswas 64�C. The unmethylated allele primers were as follows:IGFBP-3-USP-F (50-GATAAGGTGATGGGTTTTGGGT-30)and IGFBP-3-USP-R (50-ATCTAAACAACCCAAACA-CACCA-30). The annealing temperature for the unmethylatedallele primers was 62�C. PCR products were resolved by elec-trophoresis on a 7.5% PAGE gel stained with SYBR Gold(Invitrogen).

Matrigel invasion assay

[3H]-Thymidine incorporated WM239 and WM9 cells trans-fected with siRNA targeting IGFBP-3 and siRNA controlwere plated in BioCoat Matrigel invasion chambers (BD Bio-sciences, San Jose, CA) at a cell density of 2,5 � 104 perchamber in RPMI1640 supplemented with 5% fetal bovineserum (inner chamber) 48 hours post-transfection. Self-sup-plied fibroblast conditioned medium was used as chemoat-tractant in the outer chamber. The conditioned medium wasobtained from fibroblasts isolated as described by Costeaet al.23 cultivated in DMEM (Bio Whittaker) supplementedwith 10% fetal bovine serum. The medium was collectedwhen the cultures were 70% confluent. After 24 hours incu-bation at 37�C, non-invading cells remaining on the top

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352 (IGFBP-3) in malignant melanoma

surface of the chamber and the invading cells that hadadhered to the bottom surface of the chamber membraneswere collected by scrubbing with a cotton-tipped swab. Thetop and bottom fractions of cells were then transferred tocounting vessels and [3H]-Thymidine incorporation wasassessed using the 1211 rac-b liquid scintillation counter(PerkinElmer Life And Analytical Sciences, Inc). The invasionassay was performed in quadruplicate.

Immunohistochemistry

Formalin-fixed paraffin-embedded sections from all speci-mens were manually immunostained for IGFBP-3 using thetwo-step EnVisionTM system (DAKO A/S, Glostrup, Den-mark). PTEN staining was performed using EnVisionTM

FLEXþ system, (Dako Autostainer). All slides underwentpretreatment in a microwave oven for 20 minutes in 10 mMTris-1mM EDTA pH 8.0 buffer for IGFBP-3 staining and

High pH Flex (Dako) buffer for PTEN staining. Sectionswere then treated with 0.03% hydrogen peroxide (H2O2) for5 minutes. Following pretreatment, sections were incubatedfor 30 minutes at room temperature with an anti-IGFBP-3mouse (clone 4) antibody (BD Bioscience), diluted 1:50 andanti-PTEN mouse antibody (clone 6H2.1, Cascade Bioscien-ces) diluted 1:1000. The sections were further incubated withsecondary antibody for additional 30 minutes. Visualizationwas achieved using 303-diaminobenzidine tetrahydrochloride(DAB) substrate. Positive controls consisted of melanoma celllines WM9 (IGFBP-3) and WM35 (PTEN) tested for expres-sion by Western blot analysis. Four semiquantitative scoreswere used to describe the number of stained cells: negative,1–5%, 6–50% and >50%. Slides were scored by a surgical pa-thologist (BD).

Statistical analysis

All statistical analyses of in vitro experiments were performedusing two-tailed Student’s t test. Unless otherwise stated,treated cells were compared to untreated cells. A comparisonbetween IGFBP-3 and PTEN expression assessed by IHC wasperformed using the Fisher exact test. P-value less than 0.05was considered statistically significant.

ResultsIGFBP-3 is expressed in a subgroup of melanoma cell lines

Although the role of IGFBP-3 in proliferation and apoptosishave been characterized in a number of cancer types,6–8 littleis known regarding its role in human malignant melanomas.To study the impact of IGFBP-3 in melanoma we therefore,initially examined the mRNA expression level in three pri-mary and seven metastatic melanoma cell lines using qRT-PCR. As shown in Figure 1a, six of ten cell lines expressedIGFBP-3 mRNA at a higher level than the primary WM35cell line used as calibrator in the experiment. Using ELISAand Western blot analysis, IGFBP-3 protein was detected inWM9, WM239 and LOX, whereas weak or no expressionwas observed in the remaining cell lines (Fig. 1b and 1c).

Association between IGFBP-3 promoter methylation and

protein expression in melanoma cell lines

It has previously been reported that IGFBP-3 promotermethylation and gene silencing occur in several types ofhuman cancers.22,24,25 In order to examine whether this couldalso cause lack of gene expression in melanomas, methyla-tion-specific polymerase chain reactions (MSP) was per-formed on three expressing and three non-expressing celllines. The presence of a methylated band was interpreted as amethylated sample regardless of band intensities. The analysisrevealed that the IGFBP-3 promoter was unmethylated inWM9, WM239 and FEMX-1, while it was methylated inWM35 and WM1341 cells. In one cell line, LOX, both meth-ylated and unmethylated bands were detected (Fig. 2a).Except for the FEMX-1 cell line, good correlation was found

Figure 1. Characterization of IGFBP-3 expression in a panel of

human melanoma cell lines (a) IGFBP-3 mRNA expression

measured by qRT-PCR. The primary melanoma cell line WM35 was

used as calibrator. (b) ELISA analysis showing IGFBP-3 protein

levels in conditioned media. Bars represent mean values 6 SEM of

three independent experiments. (c) Western Blot analysis showing

the intracellular expression levels of IGFBP-3.

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between the MSP data and protein expression (Fig. 1b, 1cand 2a).

In order to exclude methylation as a mechanism responsi-ble for lack of IGFBP-3 expression in FEMX-1, and to con-firm the MSP data in WM35 cells, these cell lines weretreated with 10lM 5’-aza-2’deoxycytidine (Aza), and analyzedfor IGFBP-3 protein expression. An increase (70 fold) inIGFBP-3 protein level compared to untreated control cellswas observed in WM35, whereas the FEMX-1 cell line didnot respond to Aza exposure (Fig. 2b). In addition, to investi-gate whether histone acetylation was involved in regulationof IGFBP-3, FEMX-1 cells were treated with 500 ng/ml tri-chostatin A (TSA) for 24 hours. Only a minor increase inIGFBP-3 protein expression was observed (Supporting Infor-mation Fig. 1a and b), suggesting that mechanisms otherthan methylation and histone acetylation play a role in regu-lating IGFBP-3 expression in this cell line.

IGFBP-3 overexpression induces apoptosis

To further elucidate the biological role of IGFBP-3 in mela-nomas, we examined WM35 and FEMX-1 cells after transfec-tion with a wild type human IGFBP-3 cDNA construct andan empty vector as control. The concentration of IGFBP-3protein in the media of the transfected cells was found to bein the same range as the IGFBP-3 expressing cell linesWM239 and WM9 (Supporting Information Table 4).

By microscopic examination, cell detachment and deathwere the most prominent phenotypic changes 72 hours post-transfection (Supporting Information Fig. 2). Furthermore,

using the [3H]-Thymidine incorporation assay, a significantdecrease (40%, p ¼ 0.006 for FEMX-1 and p ¼ 0.017 forWM35) in DNA synthesis was observed in both cell linescompared to empty vector-transfected controls (Fig. 3a).

To elucidate whether the decrease in [3H]-Thymidineincorporation was due to decreased proliferation or increasedsusceptibility to apoptosis, a TUNEL-assay was performed(Fig. 3b). In both cell lines, induced expression of IGFBP-3caused apoptosis in approximately 30% of the cells (p ¼0.018 for FEMX-1 and p ¼ 0.011 for WM35), while noincrease in TUNEL-positive cells was observed in the emptyvector controls. Furthermore, an association between amountof endogenous expressed IGFBP-3 protein, apoptosis and cell

Figure 2. IGFBP-3 promoter methylation in melanoma cell lines.

(a) PCR products after methylation-specific PCR (MSP) performed

on bisulfite-treated DNA. M ¼ primers specific for methylated DNA,

U ¼ primers specific for unmethylated DNA. Commercially available

methylated or unmethylated DNA was used as positive controls.

(b)Relative IGFBP-3 expression measured by ELISA in supernatants

from WM35 and FEMX-1 cells after treatment with 50-aza 20-

deoxycytidine (10 lM) for 24 hours. Bars represent the mean value

of two representative experiments.

Figure 3. Biological effects of IGFBP-3 in FEMX-1 and WM35 cell

lines. (a and c) Relative proliferation measured by [3H]-Thymidine

incorporation. Proliferation in FEMX-1 and WM35 cells transfected

with an IGFBP-3 cDNA construct were related to empty vector CTR

transfected cells, while cells treated with recombinant IGFBP-3

were related to untreated control cells. (b) Induction of apoptosis

measured by the TUNEL-assay. Bars in a–c represent mean values

6 SEM of three independent experiments. P-values were

calculated using the two-tailed student t-test (*p < 0.05

and ** p < 0.01).

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number was found in WM35 cells (Supporting InformationFig. 6a, b and c).

In contrast, only marginal effects on proliferation and ap-optosis were obtained in both cell lines when treated withrecombinant human IGFBP-3 (250 ng/ml) (Fig. 3c and Sup-porting Information Table 1). Since the recombinant proteinmay lack post transcriptional modifications necessary for bio-logical activity, cells were also incubated with conditionedmedium collected from IGFBP-3-transfected cultures(IGFBP-3 concentration ranging from 40–600 ng/ml). Aswith the recombinant protein, a marginal decrease in prolif-eration, and no effect on apoptosis were observed (Support-ing Information Fig. 3, Table 3). These results suggest thatthe different biological effects of IGFBP-3 in melanoma cellsmay depend on its localization, and that only intracellularIGFBP-3 has apoptosis-inducing capabilities.

Biological effects of IGFBP-3 in melanoma cells with high

expression of IGFBP-3

Interestingly, even though IGFBP-3 over-expression inducedapoptosis in WM35 and FEMX-1 cells, a constitutively highexpression in WM239, WM9 and LOX cells suggests that thebiological function of IGFBP-3 is cell line-dependant. Inorder to examine the role of IGFBP-3 in these cells, apoptosisand proliferation were evaluated after siRNA-mediateddown-regulation of IGFBP-3 expression in WM239 andWM9 cells (Fig. 4a). No increase in TUNEL positive cellswas observed in either cell line (Supporting Information Ta-ble 3). Proliferation was reduced by 26% (p < 0.01) inWM239 cells, while no significant effect was observed in theWM9 cells (Fig. 4b).

Since it has been suggested that IGFBP-3 could also playa role in tumor invasion,2 siRNA transfected WM239 andWM9 cells were investigated using the Boyden Chamberinvasion assay. No effect in either of the cell lines wasobserved (Supporting Information Fig. 4a and b), suggestingthat IGFBP-3 do not influence the invasion capacity of theexamined cells.

IGFBP-3 is regulated by the PI3-kinase/AKT and MAPK/

ERK1/2 pathways

In an attempt to explain the different expression and biologi-cal effects in the IGFBP-3-expressing WM239 and WM9 andin the non-expressing FEMX-1 cell lines, downstream com-

ponents of the IGF signaling pathway were analyzed byWestern blot. The results revealed that the IGFBP-3-positivecell lines lacked expression of PTEN, demonstrated enhancedphosphorylation of AKT, and had lower levels of IGF-R1acompared to cells negative for IGFBP-3 (Fig. 5a). Further-more, all cell lines had constitutive activation of the MAPK/ERK1/2 pathway. Since the most predominant differencebetween IGFBP-3 positive and negative cell lines was loss ofPTEN, we examined how down-regulation of PTEN affectsIGFBP-3 protein level in FEMX-1 cells. As shown in Figure5b and c, PTEN down-regulation led to a 2.3 fold increase inIGFBP-3 protein expression (Fig. 5c) and minor up-regula-tion of AKT phosphorylation (Fig. 5b), without noteworthyeffect on proliferation (Supporting Information Fig. 5). Fur-thermore, inhibition of PI3-kinase/AKT activation in WM239and WM9 cells using 25 lM PI3-kinase inhibitor LY294002reduced IGFBP-3 protein expression by 75% (p < 0.01) and49% (p < 0.01), respectively (Fig. 5d). Although there was nodifference in activation level of MAPK/ERK1/2 in these celllines, previous studies have implicated this signaling pathwayin regulation of IGFBP-3.16 Therefore we examined whetherthis could be the case also in melanoma. When WM239 andWM9 cells were treated with 25 lM of the MEK1 inhibitor

Figure 4. siRNA-mediated down-regulation of IGFBP-3 affects

proliferation in IGFBP-3 expressing melanoma cells. (a) IGFBP-3

expression levels relative to siCTR transfected cells measured by

ELISA in WM239 and WM9 cells after IGFBP-3 siRNA transfection.

(b) Relative [3H]-Thymidine incorporation in WM239 and WM9 cells

after transfection with siRNA against IGFBP-3. Results are

expressed as mean 6 SEM of three independent experiments.

P-values were calculated using the two-tailed student t-test

(**p < 0.01).

Table 1. Expression of IGFBP-3 in melanoma biopsies as measuredby immunohistochemistry1

Immunohistochemical Score

Samples 0% 1–5% 6–50% 50% Total number

Nevi 6 0 0 0 6

Primary tumor 16 1 0 0 17

Metastasis 25 12 3 0 40

1% positive cells.

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PD98059, we observed a reduction in IGFBP-3 proteinexpression by 63% (p < 0.01) and 46%, respectively (Fig. 5d).Taken together, this suggests that both the PI3-kinase/AKTand MAPK/ERK1/2 pathways are involved in transcriptionalregulation of IGFBP-3, and may constitute an additional levelof regulation supplementary to epigenetic silencing of theIGFBP-3 promoter.

Expression of IGFBP-3 in clinical melanoma specimens

A previous study suggested that IGFBP-3 is an importantbiomarker in melanoma and that its expression is involved inmelanoma progression.2 To clarify the importance of IGFBP-3 in vivo, paraffin-embedded tissue from a panel of benign

nevi and primary and metastatic melanomas was analyzedfor protein expression using immunohistochemistry (IHC).As summarized in Table 1 and demonstrated in Figure 6 a–d, no expression of IGFBP-3 was found in benign nevi (0/6),while weak staining was observed (1–5% of the cells) in 1/17of the primary melanomas. No IGFBP-3 immunoreactivitywas seen in normal skin adjacent to the tumors. The metas-tases showed weak staining in 12/40 biopsies and moderatestaining (6%–50%) in 3/40 of the lesions, suggesting thatIGFBP-3 is up-regulated during disease progression. Further-more, we analyzed if circulating IGFBP-3 protein in plasmafrom melanoma patients was correlated with disease stage.Plasma samples from 18 healthy individuals, 16 patients withstage II and 57 patients with melanoma stage IV were

Figure 5. Regulation of IGFBP-3 by the PI3-kinase/AKT and MAPK/ERK1/2 pathways. (a) Expression of components in the IGF signaling

pathway in FEMX-1, WM239 and WM9 cells analyzed by Western Blot. (b) Western Blot showing up-regulation of p-AKT after down-

regulation of PTEN in FEMX-1 cells. (c) IGFBP-3 protein levels in FEMX-1 cells after siRNA-mediated down-regulation of PTEN relative of siCTR

transfected cells. (d) IGFBP-3 protein levels as measured by ELISA in supernatants from WM239 and WM9 cells after treatment with 25 lM

of the PI3-kinase inhibitor LY294002 and the 25 lM of the MEK1 inhibitor PD98059, relative to untreated control cells and corrected for

cell number. Results are expressed as mean 6 SEM of three parallel experiments, p-values was calculated using the two-tailed student

t-test. (*p < 0.05 and **p < 0.01).

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analyzed by ELISA. As shown in Figure 6e, no significant dif-ference was observed between samples from patients withstage II (p ¼ 0.22) and stage IV (p ¼ 0.14) disease as com-pared to the control group.

To examine if lack of IGFBP-3 expression in the patientsamples might be due to IGFBP-3 promoter methylation, as

observed in the melanoma cell lines, MSP was performed onfive IHC–positive and six IHC-negative melanoma biopsies.IGFBP-3 methylation was detected in two of five positive,and in five of six IGFBP-3 negative samples (Fig. 7) suggest-ing that promoter methylation is one of the possible mecha-nisms regulating IGFBP-3 expression also in vivo.

Figure 6. Expression of IGFBP-3 in patient material. (a) Representative IHC staining of IGFBP-3 in WM9 cell line (b) benign nevus (c) primary

and (d) metastatic melanoma. (e) Expression of IGFBP-3 protein level in plasma samples from melanoma patients with stage II and IV

disease as measured by ELISA. Results are expressed as mean 6 SD.

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In order to investigate if the association between IGFBP-3and PTEN observed in vitro is present also in the clinicalsamples we examined the same specimens for PTEN expres-sion using IHC. A strong trend for an inverse correlation(p ¼ 0.066) between IGFBP-3 and PTEN expression wasobserved (Table 2) suggesting that PTEN might also beinvolved in IGFBP-3 regulation in vivo.

DiscussionSeveral studies have discussed the involvement of IGFBP-3 indifferent cancer types,26–29 but little is still known about itsbiological significance in development and progression ofmalignant melanoma. In the present study, IGFBP-3 expres-sion was found to vary significantly in human melanoma celllines, and while unexpressed in benign nevi the protein wasinduced during melanoma progression. Furthermore, ourdata revealed that promoter methylation of IGFBP-3 occursin both melanoma cell lines and patient material, suggestingthat epigenetic silencing is a possible regulatory mechanism.In addition, we also showed that IGFBP-3 is regulated by thePI3-kinase/AKT and the MAPK/ERK1/2 pathways. Reintro-duction of IGFBP-3 in non-expressing cell lines led to induc-tion of apoptosis. Interestingly, in cell lines expressing endog-enous IGFBP-3, siRNA mediated down-regulation of theprotein led to cell line-dependent effect on proliferation, butwithout any effect on apoptosis and invasion capacity.

Analysis of IGFBP-3 protein expression in melanoma celllines and patient material showed that it is not ubiquitouslyexpressed. As measured by ELISA, three of ten cell lines, allestablished from metastatic lesions, expressed IGFBP-3 pro-tein. Similarly, in patient material, a tendency of IGFBP-3 tobe up-regulated in the metastatic lesions (37%) as comparedto benign nevi (0%) and primary melanomas (6%) was

observed. Our data is in agreement with a recent publicationby Xi et al.2 who reported IGFBP-3 expression in 5% primaryand 25% metastatic lesions, respectively, using immunostain-ing of tissue microarrays. However, recently Yu et al.17

observed less profound difference in IGFBP-3 expressionbetween primary and metastatic melanoma, although expres-sion was nevertheless slightly higher in metastasis.

High levels of IGFBP-3 in breast tumor specimens havebeen associated with unfavorable prognosis,30,31 while ele-vated serum IGFBP-3 levels were reported to reduce the rela-tive risk of developing breast cancer.32 Other studies onbreast,33 lung,27 and prostate34 cancer found no predictivevalue for serum IGFBP-3 levels. For this reason it was of in-terest to examine the IGFBP-3 level in plasma samples frommelanoma patients. However, our data did not reveal any dif-ferences in IGFBP-3 levels in the samples from normal con-trol individuals and patients with stage II and stage IV dis-ease. Furthermore, in most of the biopsies, IGFBP-3expression was seen in less than 5% of the tumor cells, afinding that may explain the lack of correlation between tu-mor progression and IGFBP-3 levels in the circulation, impli-cating that IGFBP-3 can not be used as a serum marker inpatients with malignant melanoma. In support of our find-ings, Yu et al. showed that IGFBP-3 serum levels in mela-noma patients and healthy adults were similar and had noclinicopathological relevance.17

Methylation of CpG islands in the promoter region ofIGFBP-3 has been shown to suppress expression in gastric,colorectal and breast carcinomas and in mesothelioma.22 Inthe present study, evidence is provided that methylation ofthe IGFBP-3 promoter also occurs in melanoma. Methylationwas more frequently observed in the IGFBP-3-negativepatient samples, but was also detected in two of five IGFBP-3positive cases. This can most likely be explained by contami-nation of normal cells, tumor heterogeneity or allele-specificmethylation. Nevertheless, these findings, together with theIHC results, suggest that IGFBP-3 promoter methylation is apossible mechanism for IGFBP-3 silencing in vivo. However,the presence of IGFBP-3 mRNA in the unmethylated FEMX-1 cell line, despite low protein level, suggests that regulationcan occur also at the translational or posttranslational level.In this regard it has been shown that proteases such as ca-thepsin D and plasmin can cause degradation and clearanceof IGF-binding proteins.35,36

Figure 7. Methylation-specific polymerase chain reaction (MSP) in

patient samples. MSP assay performed on 11 metastatic

melanoma patients biopsies. Samples 1-6 stained negative for

IGFBP-3 by IHC, while 7-11 stained positive for IGFBP-3. Five out of

six negative and two out of five IGFBP-3 positive samples

displayed a methylated PCR band. M ¼ primers specific for

methylated DNA, U ¼ primers specific for unmethylated DNA.

Commercially available methylated or unmethylated DNA, were

used as positive controls.

Table 2. Relationship between IGFBP-3 AND PTEN1 proteinexpression

PTEN

IGFBP3 Low2 High P

Low3 17 25 0.065

High 10 4

1Heterogenic PTEN cytoplasmic and/or nuclear expression wasobserved in 6/17 primary and 16/39 metastasis. 2Low proteinexpression was defined as immunoreactivity in 5% or less of the cells.3Low protein level defined as total lack of immunoreactivity.

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In addition to epigenetic regulation of IGFBP-3 expres-sion, many studies have shown that IGF-1 regulates theIGFBP-3 synthesis in a variety of cell types.37,38 Both theMAPK/ERK1/2 and the PI3-kinase signaling pathways havebeen implicated in mediating this effect,16 but little is knownabout the mechanisms behind these observations. Examina-tion of factors in these pathways revealed high constitutiveactivation of ERK1/2 and AKT, which can partially be attrib-uted to mutations in BRAF in WM239 and WM9 cells andHRAS in FEMX-1 cells.39,40 In addition, the IGFBP-3expressing cell lines WM239 and WM9 displayed low levelsof IGF-R1a and PTEN loss. Inhibition of the PI3-kinase/AKT and MAPK/ERK1/2 pathways in these cells reduced theIGFBP-3 protein levels. Furthermore, mimicking the changesof PI3-kinase/AKT pathway in the FEMX-1 cell line bydown-regulation of PTEN led to increased IGFBP-3 proteinexpression. Previously the opposite results have been reportedin gastric cancer41 where overexpression of PTEN or PI3-ki-nase inhibition was found to up-regulate IGFBP-3. However,our results are in agreement with Sivaprasad et al. whoshowed that stimulation of IGFBP-3 mRNA levels by mito-gens is regulated through both the PI3-kinase and MAPKpathways in mammary epithelial cells.16

Furthermore, a trend towards an inverse associationbetween IGFBP-3 and PTEN protein expression was observedin clinical samples suggesting that this regulation might alsooccur in vivo. Taken together, our data suggest that regula-tion of IGFBP-3 is cell-type specific and that in melanomatranscriptional regulation can be mediated through both thePI3-kinase/AKT and MAPK/ERK1/2 signaling pathways.

Re-expression of IGFBP-3 in WM35 and FEMX-1 cellsled to induction of apoptosis. These results are in agreementwith Rajah et al.42 who showed that IGFBP-3 is capable ofexerting proapoptotic effects independent of IGF-R1. Further-more, as reported by Butt et al., IGFBP-3 can induce apopto-sis by increasing the ratio of proapoptotic to antiapoptoticproteins in apoptotic breast cancer cells.43

The ability of exogenous IGFBP-3 to induce apoptosis inseveral cell systems,42,44 suggests that the apoptotic signalmay be initiated at the cell surface. However, in our study,exogenously applied IGFBP-3 did not have the same effectson proliferation or apoptosis as the endogenous protein tran-siently expressed by the cells. This suggests that in melanomacell lines only intracellular IGFBP-3 has apoptosis-inducingcapabilities, which may be independent of its IGF binding.

Interestingly, when IGFBP-3 was down-regulated in celllines expressing high levels of the protein, the only effectobserved was a decrease in proliferation in one of the twocell lines examined. It is possible that in the responsiveWM239 cell line, down-regulation of IGFBP-3 leads to lowerrecruitment of IGF-1, and thereby reduced receptor activa-tion, ultimately leading to decreased proliferation. However, aprevious study has demonstrated that IGF-1 do not have apredominant role in the proliferation of metastatic melanomacells,4 strengthening the possibility for IGF-independenteffects of IGFBP-3. Refractoriness of WM9 cells to IGFBP-3down-regulation shows that IGFBP-3 effects are cell line-dependent. Nonetheless, phenotypic similarities between thesecell lines regarding both levels of IGF receptor and activationof the PI3-kinase/AKT and MAPK/ERK1/2 pathways, makeus speculate that additional factors are important in media-ting the effects of IGFBP-3. Taken together, it is likely thataccumulation of malignant changes during disease progres-sion may explain how melanoma cell lines escape from thegrowth-inhibitory effects of IGFBP-3.

Conflicting data exist regarding IGFBP-3 and its involve-ment in invasion and metastasis. While the protein seems tosuppress invasion in ovarian,45 prostate46 and lung carcino-mas,47 Xi et al. reported the opposite effect in malignant mel-anoma.2 The latter study is not in agreement with our find-ings. However, Xi et al. did not address the effect of IGFBP-3on cell proliferation and for this reason it can not beexcluded that the reduction in the number of invading cellsreflects growth-inhibitory effects of IGFBP-3.

In conclusion, IGFBP-3 increases during melanoma pro-gression, but as reported for other cancers,26 IGFBP-3 expres-sion seems to have little diagnostic value in this cancer type.Furthermore, our data illustrate that IGFBP-3 may have adual role by influencing both apoptosis and proliferation.Mechanisms regulating the expression of IGFBP-3 in mela-noma include both epigenetic silencing by methylation andinvolvement of the PI3-kinase/AKT and MAPK/ERK1/2pathways. Before IGFBP-3 may be utilized in therapeutic set-ting in melanoma further investigation of its biological signif-icance and the regulatory mechanism will be needed.

AcknowledgementsThis work was supported by the Norwegian Cancer Society (AS) and the Pro-gram for functional genomics (FUGE) in the Norwegian Research Council(GFØ). The authors gratefully acknowledge the competent technical assistanceofMSc Anne Katrine Ree Rosnes, MSc Øystein Stakkestad and Ellen Hellesylt.

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biological effects induced by insulin-like growth factor binding protein 3 (igfbp-3) in malignant...

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