RAGE overexpression confers a metastatic phenotype to the WM115 human primary melanoma cell line

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Biochimica et Biophysica Acta xxx (2014) xxx–xxx

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BBADIS-63895; No. of pages: 11; 4C: 4, 7

Contents lists available at ScienceDirect

Biochimica et Biophysica Acta

j ourna l homepage: www.e lsev ie r .com/ locate /bbad is

RAGE overexpression confers a metastatic phenotype to the WM115human primary melanoma cell line

FVarsha Meghnani, Stefan W. Vetter, Estelle Leclerc ⁎Department of Pharmaceutical Sciences, North Dakota State University, PO Box 6050, Fargo, ND 58108-6050, USA
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Abbreviations: RAGEs, receptor for advanced glycationglycation end products; HMGB1, high mobility group boxenchymal transition; cdk, cyclin dependent kinase⁎ Corresponding author at: North Dakota State Univer

Fargo, ND 58108, USA. Tel.: +1 701 231 5187; fax: +1 70E-mail address: [email protected] (E. Leclerc).

http://dx.doi.org/10.1016/j.bbadis.2014.02.0130925-4439/© 2014 Published by Elsevier B.V.

Please cite this article as: V. Meghnani, et al.,line, Biochim. Biophys. Acta (2014), http://d

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Article history:Received 25 July 2013Received in revised form 16 February 2014Accepted 26 February 2014Available online xxxx

Keywords:RAGES100BMelanomap53 cancerMetastatic switch
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The formation of melanoma metastases from primary tumor cells is a complex phenomenon that involves theregulation of multiple genes. We have previously shown that the receptor for advanced glycation end products(RAGEs) was up-regulated in late metastatic stages of melanoma patient samples and we hypothesized thatup-regulation of RAGE in cells forming a primary melanoma tumor could contribute to the metastatic switchof these cells. To test our hypothesis, we overexpressed RAGE in the WM115 human melanoma cell line thatwas established from a primary melanoma tumor of a patient. We show here that overexpression of RAGE inthese cells is associatedwithmesenchymal-likemorphologies of the cells. These cells demonstrate highermigra-tion abilities and reduced proliferation properties, suggesting that the cells have switched to ametastatic pheno-type. At themolecular level, we show that RAGE overexpression is associatedwith the up-regulation of the RAGEligand S100B and the down-regulation of p53, ERK1/2, cyclin E and NF-kB. Our study supports a role of RAGE inthe metastatic switch of melanoma cells.

© 2014 Published by Elsevier B.V.

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The receptor for advanced glycation end products (RAGEs) isexpressed during early development, but repressed in most adulttissues, except in the lungs, and is found up-regulated in a large numberof pathologies such as diabetes, Alzheimer's disease and in manycancers [1,2]. RAGE is activated by structurally unrelated ligands suchas the advanced glycation end products (AGEs), HMGB1, or membersof the S100 protein family, that are associated with tissue damage andpathological conditions [3–10]. Due to the diversity of its ligands,RAGE is classified as a pattern recognition receptor. RAGE signaling iscomplex and depends on ligands and cell types [11]. For instance, incolon, gastric, breast, pancreatic and liver cancer tissues RAGE is foundup-regulated and suppression of RAGE expression or RAGE signaling re-duces cellular proliferation and/or migration [12–17]. However, in lungcarcinomas and rhabdomyosarcoma, it is the down-regulation of RAGEthat results in increased cellular proliferation and/or migration [18,19],suggesting that the role of RAGE is cancer specific and depends of thetissue and tumor environment [20–22].

The goal of this studywas to elucidate the role of RAGE upregulationin melanoma. In melanoma, RAGE has been detected at high levels in a

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end products; AGEs, advanced1 protein; EMT, epithelial mes-

sity, Dept. 2665, PO BOX 6050,1 231 8333.

RAGE overexpression confersx.doi.org/10.1016/j.bbadis.201

subset of metastatic tumor samples, both at the transcription and proteinlevels [12,23], suggesting that RAGE might contribute to tumor develop-ment only in certain melanoma tumors [23]. It was also shown that themetastatic humanmelanoma cells G361 showed higher levels of cellularproliferation and migration in the presence of RAGE activating AGEligands [24] and that cellular proliferation could be blocked by anti-RAGE antibodies [24]. Similarly, anti-RAGE antibodies have been shownto reduce the growth of melanoma tumor xenografts in mice implantedwith G361 melanoma cells [24]. In a different study, RAGE was shownto promote the formation of B16F10 melanoma metastases in mice [25].

Based on these data, we hypothesized that RAGE may contributesignificantly to melanoma tumor cell proliferation and/or metastasesformation in the population of melanoma tumors where RAGE is up-regulated. To test our hypothesis, we overexpressed RAGE in a melano-ma cell line (WM115) that originates from a primary melanoma tumorand compared the cellular proliferation and migration of populations ofWM115 cells that differed only by their level of RAGE.We observed thatthe cells expressing high levels of RAGE presented an altered morphol-ogy with an elongated mesenchymal-like shape that is a typical featureof metastatic cells. Interestingly, we observed that the RAGE overex-pressing cells migrated at higher levels and showed reduced cellularproliferation compared to control cells suggesting that RAGE up-regulation led to a metastatic switch [26]. At the molecular level, weshowed that the RAGE overexpressing cells produced higher levels ofS100B, which is used as a prognostic marker in melanoma patients[27–29]. In addition, we found lower levels of the tumor suppressorp53 protein, cyclin E as well as of ERK1/2 in the cells overexpressingRAGE, which supports the reduced cellular proliferation observed.

ametastatic phenotype to theWM115 human primarymelanoma cell4.02.013

http://dx.doi.org/10.1016/j.bbadis.2014.02.013

mailto:[email protected]


http://www.sciencedirect.com/science/journal/09254439


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2. Materials and methods

2.1. Cell lines and transfection

The human melanoma cell lines WM115 and WM266 were pur-chased from ATCC and grown in Opti-MEM (Invitrogen, Carlsbad, CA)supplemented with 4% FBS (Invitrogen) in the presence of penicillinand streptomycin (JR Scientific, Woodland, CA), 5% CO2 and at 37 °C.The cells were stably transfected with pcDNA3 expressing full-lengthRAGE (a gift from Prof. C.W. Heizmann, Children's Hospital, Zürich,Switzerland) using the SatisFection (Stratagene, Santa Clara, CA)or the X-tremeGENE 9 (Roche Applied Science, Indianapolis, IN)transfection reagents according to the manufacturer's protocols. Cellstransfected with the empty pcDNA3 vector were used as negative con-trol cells (WM115-MOCK andWM266-MOCK). Prior to transfection, allplasmids were linearized with MfeI. The transfected cells were selectedby limited dilution in the presence of 1 mg/ml G418 (WM-115-RAGEand WM115-I) or 0.5 mg/ml G418 (WM115-MOCK).

Transfection ofWM115-RAGE andWM115-MOCKwith RAGE siRNA(sc-37007) or S100B siRNA (sc-43356) (Santa Cruz Biotechnology,Dallas, TX) was performed according to procedures recommended bythe manufacturer. Briefly, cells were seeded and grown to 60%–80%confluence, in 6 well plates in Opti-MEM (Invitrogen) and 4% FBS. Thetransfection was performed in the absence of serum. The cells were col-lected after 48 h and cell lysates were prepared for RT-PCR analysis(Section 2.2) or ELISA (Section 2.5).

2.2. Real-time PCR (RT-PCR)

RNAs were extracted using a commercial kit (Ambion, Invitrogen)according to the manufacturer's instructions. The quality of the RNAwas assessed by absorbance spectroscopy and by agarose gel electro-phoresis. The RNAwas reverse transcribed into cDNA using the ReverseTranscription System from Promega (Madison, WI).

The real time PCR (RT-PCR) was run with 20 ng cDNA per well on aStratagene Mx3000p thermocycler using the Brilliant II SYBER GreenQPCR Master mix (Stratagene). The genes of β-actin and glyceraldyde-3-phosphate dehydrogenase (GAPDH) were used as housekeepinggenes. The primers used to detect the transcripts of RAGE, S100B,S100A2, S100A6, S100A10 and β-actin are listed in a previous publica-tion [23]. The other primers were as follows: GAPDH_Forward: GAAGGTGAAGGTCGGAGTC; GAPDH_Reverse: GAAGATGGTGATGGGATTTC;p53_Forward: CCAGGGCAGCTACGGTTTC; and p53_Reverse: CTCCGTCATGTGCTGTGACTG. The following RT-PCR program was used: 10 min at95 °C followed by 40 cycles of 30 s at 95 °C, 30 s at 58 °C, and 30 s at72 °C. A melting curve was recorded at the end of the cycles to evaluatethe quality of the amplified products.

The Ct values for each gene were calculated and normalized toβ-actin and GAPDH (ΔCt = Ctgene − Ctactin or GAPDH). Since similar ΔCtvalues were obtained with the housekeeping genes β-actin andGAPDH, only the delta Ct normalized with β-actin are shown. The ΔCtobtained from RAGE transfected cells were then compared toMOCK transfected cells (Δ(ΔCt) = ΔCtRAGE − ΔCtMOCK). For eachgene, the fold of change (Fgene) of gene expression was calculatedusing F = 2Δ(ΔCt). The experiments were run in triplicate using cDNAsobtained from three independent RNA preparations. The standard devi-ations were calculated from the folds of changes in gene expression.

2.3. Immunofluorescence

For staining the actin filaments, cells were seeded on microscopechamber slides (BD Biosciences, San Jose, CA) overnight, fixed with3.7% paraformaldehyde followed by permeabilization with 0.1% TritonX-100. The cells were then blocked with 3% BSA in PBS prior to incuba-tion with sulforhodamine 101 conjugated phalloidin (1/40 dilution)(Biotium, Hayward, CA). Nuclear staining was performed by adding

Please cite this article as: V. Meghnani, et al., RAGE overexpression confersline, Biochim. Biophys. Acta (2014), http://dx.doi.org/10.1016/j.bbadis.201

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Hoechst 33342 (Invitrogen) in PBS. The microscope slides were exam-ined using a Zeiss AxioObserver Z1 inverted microscope with a 40×magnification.

The ellipticity of the cells and nuclei of the WM115-MOCK andWM115-RAGE cells was assessed by calculating the ratio of the lengthover the width of at least 30 cells and their corresponding nuclei.

2.4. Flow cytometry

Flow cytometric measurements were performed on a C6 Accuri flowcytometer (Accuri, Ann Arbor, MI). The cells were gently detached witha cell scrapper and incubated on ice with a serial dilution of a mouseanti-RAGE antibody (Mab 1145, R&D Systems, Minneapolis, MN). AfterPBS washes, the cells were further incubated with a FITC-conjugated sec-ondary anti-mouse antibody (10 μg/ml, Jackson ImmunoResearch, WestGrove, PA). Themeanfluorescence of 5000 events per samplewas plottedas a function of the antibody concentration. The data were fitted witha 1:1 binding model using the KaleidaGraph software (Synergy). The ex-periments were performed in triplicate.

Cell cycle analysis was performed on the same instrument usingpropidium iodide (PI) (Calbiochem) and an antibody against cyclin E(H12, Santa Cruz). Briefly, 60–80% confluent WM115-MOCK andWM115-RAGE cells were fixed with 70% alcohol, and permeabilizedwith Triton X-100 prior to incubation with cyclin E antibody and PI, ac-cording to the published procedures [30]. A FITC-conjugated secondaryantibody (Jackson ImmunoResearch) was used to detect the bound pri-mary anti-cyclin E antibody. The fluorescence of PI was used to identifythe G1, S and G2/M phases of the cell cycle and to gate the cells. To com-pare the level of cyclin E in the different phases of the cell cycle, themean fluorescence of FITC was reported for each population of gatedcells. The experiment was performed independently twice using tripli-cate samples in each experiment.

2.5. ELISA

Cell extracts were prepared using a commercial kit (Paris, Invitrogen)and the protein content was determined with the Pierce BCA proteinassay kit (Pierce/Thermo Scientific, Rockford, IL). RAGE protein levels inthe cell extracts were determined using the Quantikine human RAGEImmunoassay kit (R&D Systems) according to the manufacturer's proce-dure, and expressed in picogram of RAGE per mg of total protein.

S100B levels in the cell extracts and the conditionedmedia were de-terminedwith the help of a calibration curve obtained from a sandwichELISA. For the sandwich ELISA, we used a polyclonal anti-S100B anti-body (DakoCytomation, Denmark) to capture S100B. A calibrationcurve was performed using recombinant human S100B (0.01 nM to150 nM),whichwas purified according to the standard procedures [31].To detect bound S100B, we used a second anti-S100B antibody(MAB1820, R&DSystems). The detectionwas performedusing an alkalinephosphatase-conjugated (AP) secondary antibody and para-nitrophenylphosphate as substrate. The cell lysate and conditioned media samplesfrom two independent experiments were run in duplicate.

2.6. Western blots

Cell extracts were prepared using a commercial kit (Paris, Invitrogen)and the protein concentration in the cell extracts was determined as de-scribed above. The proteins (20–100 μg) were resolved on 10 or 15% SDSPAGE and blotted against nitro-cellulose membranes. The blots wereblockedwith 3% BSA in TBS (50mMTris pH 7.4, 150mMNaCl) and incu-bated with the primary antibody diluted in 1% BSA in TBS. HRP conjugat-ed secondary antibodies (donkey anti-rabbit, goat anti-mouse and rabbitanti-goat) were all from Jackson ImmunoResearch and used at dilutionsrecommended by the manufacturer. The primary antibodies againstβ-actin (#4970), Akt (#4691), phospho-Akt (Ser473, #4060S), SAPK/JNK(#9258), phospho-SAPK/JNK (#4668, Thr183/Tyr185), p44/42 (#4695)



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and phospho-p44/42 (#9101, Thr202/Tyr204) were all from Cell Sig-naling (Danvers, MA). Antibodies against p53 (sc-47698) and Dia-1(sc-373807) were from Santa-Cruz Biotechnology (Dallas, TX). Anti-S100B (#Z0311) was from DakoCytomation (Denmark) and anti-S100A10 (#ab52272) was from Abcam (Cambridge, MA). The cyclin Eantibody (#630701) was from BioLegend (San Diego, CA). Rabbit seradirected against S100A2, A6 and A4 were generous gifts from Prof.Heizmann (Children's Hospital, Zurich, Switzerland). The blots weredeveloped using a chemoluminescent substrate (Pierce ECL WesternBlotting Substrate, Thermo Scientific). The blots were scanned and theintensities of the scanned bandswere determined using the ImageJ soft-ware [32]. Following the staining for S100 proteins, p53 and cyclin E, theblots were stripped using standard conditions, which consisted of 45min incubation at 50 °C in the presence of 200 mM glycine buffer atpH 2.2, and re-stained for actin. For the staining of AKT/P-AKT, JNK, p-JNK, and ERK/p-ERK, the blots were first stained with the antibodiesrecognizing the phosphorylated form of the proteins and, when indi-cated in the figure legend, the blot was stripped and re-stained withthe antibody recognizing the total forms (phosphorylated and non-phosphorylated) of the protein. Alternatively, samples from the samecellular extracts were loaded onto different wells of SDS-PAGE gels.After electroblotting, the blot was cut into parts that were used forseparate Western blot analysis.

2.7. Cell assays

2.7.1. Alamar Blue assayThe cells were seeded (4 × 104 cells/well) in 24 well plates and

incubated in Opti-MEM as described above. When the cells reached70–80% confluency, Alamar Blue (1/10 dilution, Nalgene) was addedto the wells and the plate was further incubated for 3 h or 4 h at37 °C. The reduced form of Alamar Blue was detected by fluorescencespectroscopy (Ex: 540 nm; Em: 590 nm) [33] and corrected for thefluorescence emission of AB in the cell culture medium only. The exper-iments were performed in triplicate.

2.7.2. Migration assayThe cells were added (104 cells/well) to the top of 8 μm filter inserts

(24 well plate, Greiner Bio-One). The inserts were then added into thewells of a 24 well plate containing Opti-MEM and 4% FBS. After 24 h in-cubation at 37 °C, the cells on top of the filter were gently removedusing cotton swabs. Alamar Blue (1/10 dilution, Nalgene) was addedto the wells and its fluorescence emission (Ex: 540 nm; Em: 590 nm)was measured after 3 h or 4 h further incubation. The wells containingeither the cell culture media alone or the seeded cells without insertwere used as negative and positive controls, respectively.When indicat-ed, a polyclonal anti-S100B antibody (DakoCytomation) or polyclonalanti-RAGE antibody (100 nM) was added to the cells at the time pointof seeding into wells. The polyclonal anti-RAGE antibody consistedof an equimolar mixture of anti-V, anti-C1 and anti-C2 antibodies asdescribed in our previous publications [34,35].

2.7.3. Invasion assayThe assay was performed similarly to the migration assay with the

exception that the insert filters were coated with a solution of bovinecollagen I (Santa Cruz Biotechnology, Santa Cruz, CA) prior to theaddition of the cells. Themigration and invasion experiments were per-formed in triplicate.

2.7.4. Soft agar colony formation assayCell suspensions (1 × 104 cells/well) were mixed (1:1) with 1.4%

agarose in Opti-MEM containing 8% FBS and added on top of the wellscontaining solidified 1% agarose. After solidification of the soft agarose,the cell culture medium (Opti-MEM containing 4% FBS) was added tothe wells. The plate was then incubated for 4 to 5 weeks at 37 °C andfresh medium was added to the plate every 3 to 4 days. The colonies


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formed were visualized by staining with crystal violet. The colonies inten different fields were counted.

2.7.5. Wound healing assayThe cells were seeded in 96well plates and incubated until reaching

90% to 100% confluence. Awoundwas performed using the tip of sterileP10pipet tip. Thewellswere imaged at the indicated timepoints using aCCD camera attached to an Olympus Fluoview microscope.

2.8. NF-κB assay

The cells were seeded (104 cells/well) in 24 well plates and incubat-ed until they reached 60–70% confluence. The cells were then co-transfected with the NF-κB cis-Reporting System (# 219077; Strata-gene) containing a luciferase encoding gene and the monster GFPplasmid (SABiosciences) that was used to estimate the transfection effi-ciency. The transfectionwas performed using the X-tremeGENE 9 trans-fection reagent (Roche Applied Science, Indianapolis, IN) according tothe manufacturer's protocols. The pFC-MEKK plasmid was used in co-transfection experimentswith theNF-κB plasmid and served as positivecontrol.

Following transfection (24 h–48 h), the cells were lysed using theReporter lysis buffer (Promega,Madison,WI). The activity of the lucifer-ase was determined using a commercial luciferase detection system(# E1500; Promega) on a Quick-Pak Quantifier luminometer.

2.9. Statistical analysis

The migration and invasion assays were replicated at least threetimes using 6 wells for each condition. All other experiments werealso performed in duplicate or triplicate, as indicated. Themean± stan-dard deviation is reported. Statistical significances between groupswere determined using the Student's t-test or one-way ANOVA usingBonferroni Post-Hoc test.

3. Results

3.1. Overexpression of RAGE in WM115 cells and characterization of thetransfected cell lines

To determine the effect of RAGE up-regulation in melanoma cells,we stably transfected theWM115melanoma cell linewith a vector cod-ing for full-lengthRAGE.WM115 cells stably transfectedwith the emptyvector (WM115-MOCK) were used as negative control. To characterizethe transfected cells, we determined the levels of RAGE mRNA and pro-tein by RT-PCR and ELISA, respectively. From two independent transfec-tion procedures, we selected two clones WM115-RAGE and WM115-RAGE-I, which overexpressed RAGE, at the protein levels, 94-fold and7-fold, respectively, when compared to the WM115-MOCK controls.WM115-MOCK cells expressed 11.5 ± 0.1 pg RAGE per mg of total pro-tein and WM115-RAGE cells expressed 1081.3 ± 53.0 pg RAGE per mgtotal protein (Fig. 1A). The second transfected cell line WM115-RAGE-Iexpressed 81.0 ± 2.0 pg RAGE per mg total protein. The expression ofRAGE at the protein level reflected the levels of transcripts thatwere de-termined by RT-PCR. We also measured the level of RAGE in WM-266cells. This cell-line (WM266) originates from the same patient as theWM115, but was established from a metastatic tumor [36]. RAGE pro-tein levels in WM266-MOCK were 2.4 higher than in WM115-MOCKcells and were equal to 24.4 (±5.4) pg per mg of protein.

To ensure that in the transfected cells RAGE was properly processedand exported to the cell surface, wemeasured the binding of anti-RAGEantibodies to cell surface RAGE by flow cytometry (Fig. 1B). We foundthat the antibodies bound to larger numbers of RAGE receptors inRAGE overexpressing cells than in control cells.



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Fig. 1.Q2 A) Levels of RAGE in RAGE transfected WM115 cells as determined by ELISA. Levels are expressed in pg RAGE protein per mg of total protein. WM115-RAGE andWM115-RAGE-Iexpressed 94 fold and 7 fold higher RAGE protein than theMOCK control cells, respectively. B) Binding of the anti-RAGE antibodyMAB1145 toWM115-RAGE (filled circles) andWM115-MOCK (filled squares)measured by flow cytometry. The binding curve ofMAB1145 toWM115-RAGEwasfitted using a 1:1 bindingmodel and showed an affinity of 1.5 (±0.3) nM. RAGEoverexpressed in themelanoma cells is properly processed and translocated to the cell-surface, as demonstrated by their recognition by specific antibodies. The experimentwasperformedin triplicate and the standard deviation is indicated. C–F) Morphology of WM115-MOCK (C), WM115-RAGE-I (D), WM115-RAGE (E) and WM266-MOCK (F) by bright field microscopy.G–H)Differences inmorphology betweenWM115-MOCK (G) andWM115-RAGE (H) transfected cells, as shown by actin staining. Actinwas stainedwith PE conjugated phalloidin and thenuclei were stained with Hoechst 33342. (20× magnification).


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3.2. RAGE overexpression is accompanied with changes in cell morphologyand actin remodeling

We observed large differences in morphologies between theWM115-MOCK,WM115-RAGE-I andWM115-RAGE cell lines. Whereasthe WM115-MOCK cells presented the classical epithelial morphologyas previously described for this cell line (Fig. 1C) [37,38], the RAGEtransfected cells showed a more elongated morphology resemblingthat of fibroblasts (Fig. 1E). We also measured the length and width ofthe cells and found that the ratio (R) of the length over the width wassignificantly larger (p b 0.001) in RAGE transfected cells than in MOCKcontrol cells (RMOCK=1.77±0.68 versus RRAGE=9.85± 3.8). Interest-ingly, theWM115-RAGE-I cells that expressed RAGE at an intermediatelevel also showed an “intermediate” morphology between those ofWM115-MOCK and WM115-RAGE (Fig. 1D). We also compared themorphologies of WM115-RAGE and WM266-MOCK, the “sister” cellline of WM115 [36]. We observed that WM266-MOCK presented simi-lar elongated shape thanWM115-RAGE (Fig. 1F). Since the architectureof cells is under the control of actin filaments, we also investigated thedistribution of F-actin filaments in the transfected cells by fluorescencemicroscopy, using sulforhodamine conjugated phalloidin. We observeddifferences in organization of the actin filaments between the two celllines. In the WM115-MOCK cells, many structures resembled shortcortical bundles (Fig. 1G), these structures appeared to be absent inthe WM115-RAGE cells (Fig. 1H). Instead in the RAGE-overexpressingcells, longer parallel fibers were observed as well as structures that re-semble collapsed F-actin (Fig. 1H). These changes in actin organizationresemble the changes occurring during the epithelial mesenchymaltransition (EMT) of cancer cells [39,40].

Fig. 2. Comparison of the cellular proliferation of WM115-MOCK andWM115-RAGE cellsusing Alamar Blue. Excitation was performed at 540 nm and the fluorescence emissionwas recorded at 590 nm. The experiment was performed at least three times with 4–6replicates each time.

3.3. RAGE overexpression causes changes in cell proliferation andmigration

In order to determine the effect of RAGE overexpression on thecellular behavior of theWM115melanoma cells, we compared the pro-liferation and migration properties of these cells compared to MOCK


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controls. To our surprise, we did not observe an increase but rather asignificant 30% decrease in cellular proliferation in the WM115-RAGEcells compared to the MOCK cells (Fig. 2).

We tested several metastatic properties of the transfected cells andas a first step, we studied the growth of the cells in the absence of sup-port or anchor, a phenomenonwhich is also referred as anoikis [41–44].In these conditions, we observed that the WM115-RAGE cells formed asignificantly larger number of colonies (19 ± 5 colonies; p b 0.01) thanthe WM115-MOCK cells (7 ± 3 colonies) (Fig. 3A, B).

We next compared the migration properties of the melanoma cellsusing standard Boyden chamber assays. We found that after 24 h, a sig-nificantly larger number of WM115-RAGE cells had migrated throughthe filter (33 ± 4.5%; p b 0.01) compared to the WM115-MOCK cells(14± 1.9%) (Fig. 4A).We also observed that the percentage ofmigratedWM115-RAGE cells was not significantly different from the percentageof migrated WM266-MOCK cells (27.9 ± 4.2%).



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Fig. 3.Q3 A: Soft agar assaywithWM115-MOCK (a–c) andWM115-RAGE cells (d–f). Three representativefields are shown for each type of cells. The colonieswere stainedwith crystal violet.B: Averaged number of colonies per field. Three independent experiments were performed, **p b 0.01.

Fig. 4. Q4A–B: Migration and invasion assays. A: Percentage of migrated cells (WM115-MOCK,WM115-RAGE, WM115-RAGE-I and WM266-MOCK). B: Percentage of invaded cells(WM115-MOCK, WM115-RAGE-I and WM115-RAGE). Alamar blue was used to determinethe percentage of migrated and invaded cells. The mean values of three independentexperiments performed in triplicates are indicated with their standard deviations(*p b 0.05; **p b 0.01).


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We also investigated whether the migration abilities of the WM115cells correlated with the level of RAGE in these cells. We indeed ob-served that the WM115-RAGE-I cells migrated to a level (19 ± 3.2%)that was intermediate between that of the WM115-MOCK cells(14 ± 1.9%) and that of theWM115-RAGE cells (33± 4.5%), the differ-ence of percentage of migrated cells being statistically significantbetween the three groups (p b 0.05) (Fig. 4A).

In addition, we investigated the invasion properties of these cellsand observed that a significantly larger number of WM115-RAGE cellshad invaded collagen-coated filters (18.5% ± 1%, p b 0.05) than theWM115-MOCK cells (8% ± 4.5%) (Fig. 4B).

To complement our findings, we compared the migration abilitiesof the melanoma cells after the formation of a wound on a layer ofconfluent cells (Fig. 5). In agreement with the Boyden chamber assayresults, we observed that theWM115-RAGE cells (Fig. 5, lower row) re-covered the wounded area significantly faster than the MOCK controlcells (Fig. 5, upper row).

To further support our hypothesis that RAGE overexpression in theWM115 cell line was responsible for the observed changes in cellularmigration, we tested whether RAGE suppression by siRNA in theWM115-RAGE cells could revert the increased migration. We observedthat suppression of RAGE by specific siRNA significantly reduced(p b 0.05) the percentage of migrated cells from 49% ± 1% (controlsiRNA) to 39% ± 5% (RAGE siRNA) (Fig. 6A). The level of suppressionat the protein levelwasmeasured by ELISA and showed a 3.2 fold reduc-tion (±1.5) after transfection with siRNA (Fig. 6B). In addition, weobserved that RAGE suppression resulted in a significant (p b 0.01)increase in cellular proliferation (Fig. 6C).

3.4. RAGE overexpression is accompanied with the up-regulation of S100B

Sincewe showed that the RAGE overexpressing cells acquired sever-al metastatic properties, our next goal was to interrogate the levels ofRAGE ligands in the transfected cells. We chose to compare the levelsof several S100 proteins that are relevant to melanoma and thereforemeasured the levels of S100A2, S100A4, S100A6, S100A10 and S100Bin cell extracts of the different cell-lines. Among those S100 proteins,

Please cite this article as: V. Meghnani, et al., RAGE overexpression confers ametastatic phenotype to theWM115 human primarymelanoma cellline, Biochim. Biophys. Acta (2014), http://dx.doi.org/10.1016/j.bbadis.2014.02.013


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Fig. 5. Wound healing assay. Top row: Assay with WM115-MOCK cells. Bottom row: Assay with WM115-RAGE cells. After wounding the confluent cells with a pipet tip, the RAGEtransfected cells (bottom row) recovered the wounded area faster than theMOCK transfected cells (top row). Three independent experiments were performed and show similar results.The figure shows a representative experiment. The pictures of the wounded area were taken at t = 0 h; t = 14 h and t = 24 h.


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we could only detect significant changes in S100B levels. S100B mRNAlevels inWM115-RAGE cells were (4.6± 0.7) higher than in the controlWM115-MOCK cells. At the protein level, S100B levels were also signif-icantly higher in WM115-RAGE cells than in WM115-MOCK cells asdetermined by Western blot (2.5 ± 0.7) and ELISA (2.7 ± 1.4) (Fig. 7).In addition, we found that WM115-RAGE cells secreted higher levelsof S100B in the media (8.2 ± 1.5 fold) than the WM115-MOCK cells(Fig. 7B).

Interestingly, suppression of RAGE by siRNA in the WM115-RAGEcells did not result in a significant reduction in the levels of intracellularS100B protein (data not shown). To test the hypothesis that S100B par-ticipates in the increased migration of WM115-RAGE cells, we com-pared the migration of these cells after suppressing extracellularRAGE/S100B interaction with anti-S100B antibodies (DakoCytomation)or suppressing intracellular S100B using siRNA (Fig. 8). S100B suppres-sion by siRNA resulted in a 8.5 (±3.3) fold decrease in S100B transcriptlevels. We observed no changes in migration in theWM115-RAGE cellsafter treatment with either S100B antibodies or S100B specific siRNAsuggesting that the changes in migration in theWM115-RAGE cells oc-curred in a S100B independent manner (Fig. 8). To test the hypothesisthat other RAGE ligands might stimulate the migration of the WM115-RAGE cells, we compared the migration of these cells after treatmentwith anti-RAGE antibodies. We could not detect changes in migration

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Fig. 6. Effect of RAGE blockage by siRNA on cellularmigration and proliferation ofWM115-RAGEsiRNA. B) Protein RAGE levels following suppression with RAGE siRNA. C) Changes in cellular p


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polyclonal anti-RAGE antibodies (data not shown), suggesting that theincrease in migration of the WM115-RAGE cells occurred in a RAGE-ligand independent manner.

3.5. Overexpression of RAGE results in decreased levels of the tumorsuppressor p53 and cyclin E

Following the observation that S100B levels were up-regulated in theWM115-RAGE cells, we next investigated the levels of possible intracellu-lar targets of S100B relevant in cancer and melanoma. One of thesetargets is the tumor suppressor protein p53 and the mechanism of inter-action of S100B with p53 has been well documented [28,45,46]. Weobserved that the level of p53 was significantly lower in the WM115-RAGE cells than in the MOCK control cells, both at the transcription(2.6 ±0.3 fold) andprotein levels (1.9±0.4 fold) (Fig. 9A).We alsomea-sured the levels of the cell cycle regulator cyclin E, and found significantlylower protein levels of cyclin E (4.0 ± 1.2 fold) in RAGE overexpressingcells than in MOCK controls (Fig. 9B). To further support our findings,we analyzed the level of cyclin E during the different phases of thecell cycle (G1, S andG2/M) by flow cytometry. Our data shows that cyclinE was significantly reduced (p b 0.01) in all phases of the cell cycle(Fig. 9C).

cells. A)Migration ofWM115-RAGE following transfectionwith control and RAGE specificroliferation of WM115-RAGE following transfection with control and RAGE specific siRNA.



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454Fig. 7.Q5 A. Detection of S100B, S100A6 and S100A10 in cell lysates by Western blot. Threeindependent cell lysateswere prepared and analyzed. Thefigure shows the representativeblot of one cell lysate. Thedetection of actinwasperformedusing the same lanes thatwereused for the detection of S100 proteins. B. Detection of S100B in cell lysates and superna-tant as determined by ELISA.

Fig. 9. A. Levels of p53 protein in WM115-MOCK and WM115-RAGE as determined byWestern blot. Antibodies against β-actin were used as loading control, in the same lanesthat were used for p53 staining. B. Levels of cyclin E in cell lysates of WM115-MOCK andWM115-RAGE as determined by Western blot. Antibodies against β-actin were used forthe loading control and the actin staining was performed on the same lanes that wereused for the cyclin E staining. C. Level of cyclin E in the different phases G1, S and G2/Mof the cell cycle. PI was used to gate the cells according to cell cycle phases. A FITC-labeled secondary antibody was used to detect bound anti-cyclin E.

Fig. 8. Effect of S100B blockage by siRNA and RAGE/S100B interaction by anti-RAGE anti-bodies on the migration of WM115-RAGE cells. Following transfection with S100B siRNA,RAGE levels were decreased by 8.5 fold (±3.3) as determined by RT-PCR. Ctrl (siRNA)indicates that the cells were transfected with a negative control siRNA.



E3.6. Overexpression of RAGE is accompanied by a decrease in NF-κB activity

The transcription factor NF-κB has been shown to promote mul-tiple processes in cancer such as proliferation, invasion, metastasis,angiogenesis, and chemo- and radioresistance [47–49]. We hencedetermined whether RAGE overexpression influenced the activity ofNF-κB. For this purpose, we transfected both WM115-MOCK andWM115-RAGE with a luciferase/NF-κB reporter system and observedthat the basal activity of NF-κB was significantly reduced in the RAGE-transfected cells and was only 37% of the level in the control cells(Fig. 10A).

Fig. 10.A. Activity of NF-kB inWM115-MOCK andWM115-RAGE cells. The activity of NF-kBwas determined using a luciferase/NF-kB reporter system. B. Detection of AKT, ERK1/2 andSAPK/JNK in cell lysates by Western blot. Three independent cell lysates were preparedand analyzed. The intensities of the bands corresponding to the non-phosphorylated andphosphorylated forms of each kinase (AKT, p-AKT; SAPK/JNK, p-SAPK/JNK and ERK1/2,p-ERK1/2) were compared by densitometric analysis. The figure shows a representativeblot of cell lysates. The same cellular extract was loaded onto different wells of the gel andblotted. After electroblotting, the blotwas cut into twoparts and eachpartwas stained eitherfor the total forms of AKT, JNK and ERK1/2 or for the phosphorylated forms of the proteins.

a metastatic phenotype to theWM115 human primarymelanoma cell4.02.013


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3.7. Overexpression of RAGE is accompanied by a decrease in ERK1/2phosphorylation

We next analyzed the activity of several signaling kinases that arerelevant in cancer and melanoma and we investigated the levels ofERK1/2, AKT and JNK/SAPK in cell extracts. We observed a 1.8 (±0.5)fold reduction of the levels of phosphorylated ERK1/2 in WM115-RAGE cells compared to the control cells (Fig. 10B). However, we didnot observe differences in phosphorylated Akt or JNK between thetwo cell lines (Fig. 10B).

4. Discussion

The mechanisms by which RAGE contributes to cancer are not clearand may differ between cancer types [20–22]. Indeed, although RAGEup-regulation correlates with increased cellular proliferation andmigration in many cancers (colon, gastric, breast, pancreatic and livercancer) [12–17], it is the down-regulation of RAGE that correlateswith increased proliferation and migration in lung carcinomas andrhabdomyosarcoma [18,19].

We previously showed that RAGE transcript levels varied greatlyamong stage III and IV melanoma tumor samples, suggesting that therole of RAGE in melanomamay be tumor specific [23]. To better under-stand the function of RAGE inmelanoma progression, we chose to com-pare the proliferation and migration properties of the WM115 primarymelanoma cell line following overexpression of RAGE. We chose theWM115 cell line because i) it was established from a primary tumorof a melanoma patient, ii) it has low basal levels of RAGE expressionand iii) the metastatic cell line WM266, which was established fromthe same patient, was also available. Therefore, we were able tocompare the metastatic properties of the WM115 cell line, after RAGEoverexpression, to those of the sister metastatic cell line WM266.

We first observed that RAGE overexpression in WM115 cells result-ed in changes in both cellmorphology and actin remodeling, resemblingthe epithelial to mesenchymal transition (EMT) characteristic of meta-static cells [39]. In addition to the change in cell morphology and theincrease in cell ellipticity (Fig. 1), we noticed that the nuclei of theRAGE overexpressing cells were also notably more elongated thanthose of the control MOCK cells. As observed with the cells, comparisonof the ratio (R) of the length over width of the nuclei showed a signifi-cant (p b 0.001) difference in ellipticity between the two cell lines.(RNuclei-MOCK = 1.4 ± 0.3 versus RNuclei-RAGE = 1.8 ± 0.5). Studieshave shown that the nuclear shape is tightly regulated by the cell mor-phology and that the nuclear morphology could reflect changes in cellhealth [50,51]. In addition, defects in nuclear shape are also used in clin-ical settings as markers of disease [51]. The changes in nuclear shapethat we observed following RAGE overexpression are thus consistentwith the observed changes in cell morphology and the accompaniedchanges in cellular proliferation and migration properties. We also ob-served that RAGE overexpression increased significantly the mobilityof the melanoma cells in all assays used by us, i.e. migration, invasion,anchorage independent colony formation and wound healing assays(Figs. 3 and 5). It thus appears that high levels of RAGE conferthe WM115 cells a metastatic phenotype by inducing an epithelial–mesenchymal like transition. Our results are in agreement with earlierstudies by Huttunen et al. who showed that B16 mouse melanomacells overexpressing full-length RAGE produced a larger number of me-tastases than the B16 cells overexpressing a signaling incompetent formof RAGE that lacked the intracellular signaling domain [52]. AlthoughRAGE overexpression in WM115 cells correlated with increased migra-tion properties, other genes and proteins are involved in this complexprocess as demonstrated by many studies aiming at identifying meta-static melanoma gene expression signatures [53–56]. Indeed, we ob-served that in our experimental conditions, the WM266-MOCK cellshad similar migration properties to the WM115-RAGE cells. However,in the WM266-MOCK cells, the level of RAGE is only 2.4 fold higher


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than in WM115-MOCK cells (24.4 ± 5.4 pg RAGE per mg of proteinversus 11.5+ 0.1 pg RAGE permg of total protein, respectively). Our re-sults demonstrate that overexpression of RAGE was sufficient to inducemetastatic properties in theWM115 cell line, however, theWM266 cellline displays a metastatic phenotype despite only minimal increase inRAGE expression, suggesting that other genes are also involved in thisprocess.

RAGE overexpression also resulted in decreased proliferationabilities of the cells, suggesting that RAGE triggered a phenomenondescribed as the metastatic switch, where the migration properties ofcells are increased at the expenses of the proliferative capabilities ofthe cells [26]. The observed reduction in cell proliferation in RAGE over-expressing cells correlated with a decrease in levels of cyclin E in all cellcycle phases (Fig. 9C). Cyclin E can regulate cell proliferation by cyclindependent kinase 2 (cdk2) dependent and independent processesresulting in cell cycle progression [57]. Overexpression of cyclin Ehas been previously observed in WM115 melanoma cells and wasassociated with the hyper-proliferative nature of this cell line [58]. Todemonstrate that RAGE overexpression correlated to reduced cellularproliferation of the WM115 cells, we investigated if these effects werereversible, using specific RAGE siRNA. We observed that suppressionof RAGE resulted in a significant (p b 0.01) increase in cellular prolifer-ation (Fig. 6C), supporting our hypothesis that in melanoma cells, RAGEoverexpression can control the metastatic switch.

One of the current prognostic markers for metastatic melanoma pa-tients is S100B [27–29]. We show here that theWM115-RAGE cells ex-press and release significantly larger amounts of S100B than the controlcells. Thisfinding is another indication that theWM115-RAGE cells havedeveloped metastatic features and led us to investigate the level of thetumor suppressor p53, which is expressed as wild type in WM115cells [59]. We observed lower levels of p53 in the WM115-RAGE cellsthan in control cells, in agreement with earlier studies that showedthat S100B down-regulates p53 inmelanoma cells [60–62]. Interesting-ly, a recent study has shown that p53 deficiency in the A375Pmetastaticmelanoma cells resulted in increased migration and invasion of thesecells and is in line with our results [63].

Downstream RAGE signaling is complex and not yet fully under-stood. Several intracellular proteins such as ERK1/2, Diaphanous-1(Dia-1), TIRAP and MyD88, have been suggested to play the role ofadaptor proteins of RAGE [64–66]. While Dia-1 was shown to promotecellular migration of C6 glioma through the activation of Rac-1 andcdc42 [65], it is not known if this interaction is relevant in melanomacells. Sakagushi et al. recently described that they could not immuno-precipitate Dia-1 from HEK-293 cells overexpressing RAGE [66]. Totest a possible effect of RAGE overexpression on Dia-1, we measuredDia-1 protein levels in cell lysates of WM115-MOCK and WM115-RAGE cells. Western blot analysis did not reveal differences in Dia-1expression levels between the two cell lines (data not shown).

The activation of the Ras/BRAF/ERK signaling pathways is a centralmechanism leading to cell proliferation and survival and promotinganchorage independent growth, migration and invasion [41,67–71]. Inmelanoma, the Ras/RAF/ERK signaling pathway is constitutively activat-ed in more than 60% of melanoma cells, includingWM115 andWM266cells, due to gain of functionmutations in B-RAF (BRAFV600E) [72–74]. Ouranalysis of signaling pathways that modulated upon overexpression ofRAGE showed a significant decrease in the phosphorylated form ofERK1/2. We propose that the decrease of ERK1/2 activity reflects the de-crease in proliferation of WM115-RAGE cells. Interestingly, a decrease inphosphorylated levels of ERK1/2 also correlated with a decrease in cellu-lar proliferation in MC3T3-E1 osteoblast cells overexpressing RAGE [75].

In melanoma, cellular proliferation is also under the control of PI3K/AKT [76,77]. In our experimental conditions, we could not detect chang-es in activity of the AKT kinase, suggesting that the PI3K/AKT pathway isnot involved in the changes of proliferation ormigration observedwhenRAGE is overexpressed. Since JNK has also been shown to be activated inmelanoma, at times through ERK signaling, we also determined the



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levels of activated JNK in the two cell lines but could not find significantchanges in the WM115-RAGE cells compared to the control cells [78].These data suggest that RAGE overexpression modulates the ERK path-ways without affecting the JNK and AKT pathways.

The observed increases in migration, invasion and anchorageindependent cellular proliferation of melanoma cells occurred in theabsence of exogenous ligand and correlated with the level of RAGE ex-pression. Recent evidence strongly suggests that RAGE oligomerizationis required for activation of the receptor [79–86], and for example, ithas been shown that on the surface of HEK293 and HeLa cells, RAGEexists as constitutive dimers [87,88] and that oligomerization of thereceptor could be further enhanced following stimulation with S100Bor AGEs [88]. We propose that when overexpressed in the melanomaWM115 cells, RAGE spontaneously forms higher order oligomerswhich are sufficient to trigger RAGE signaling. In this case, RAGE oligo-merization could thus result in ligand-independent RAGE activation.This hypothesis is also supported by our observation that the anti-RAGEantibodies, used in this study, were not able to suppress increases in cel-lularmigrationwhereas treatment of the cells with RAGE targeting siRNAdid reduce cellularmigration. RAGE oligomerization is currently an activearea of research and several mechanisms of oligomerization-dependentRAGE activation have been proposed: RAGE oligomerization could occureither through the V domains [84], C1 domains [84,89] or the C2 domainsof the receptor [85,86] in a ligand dependent manner [85]. We proposehere that RAGE oligomerization could also be a means for RAGE togenerate intracellular signaling in the absence of interaction with its li-gands. Although ligand-induced receptor oligomerization is a commonmechanism of signaling, as observedwith the epithelial growth factor re-ceptor [90], several studies have also reported ligand-independent recep-tor signaling caused by receptor oligomerization [91–93]. For example,overexpression of CD30 was found to be sufficient to mediate signalinginHodgkin–Reed–Sternberg cells through oligomerization of the receptor[92]. RAGE could therefore represent a new paradigm of receptor thatexhibits ligand-independent activation through receptor oligomerization.Further studies would be necessary to elucidate themechanisms of RAGEoligomerization in the RAGE overexpressing melanoma cells.

In conclusion, our data support the hypothesis that RAGE up-regulation in primary melanoma cells triggers a metastatic switchwith increasemigration and reduced proliferation.Our data also suggestthat RAGE overexpression can lead to ligand independent RAGE activa-tion, possibly through oligomerization of the receptor. We will furtherinvestigate the mechanisms of ligand independent RAGE activation inmodel cells.

Conflict of interest

The authors have no conflict of interest.

Acknowledgements

The authors thank the ND-EPSCoR program and the Department ofPharmaceutical Sciences and the College of Pharmacy, Nursing andAllied Sciences for financial support. This work was in part supportedby the NDSU Advance FORWARD program sponsored by NSF HRD-0811239 and ND-EPSCoR through NSF grant #EPS-081442, and by theInfrastructure Improvement Program-Doctoral Dissertation Assistant-ship accorded to V.M. The authors also would like to thank Dr. PawelPiotr Borowicz from the Department of Animal Sciences at NDSU forhis help with the fluorescence microscopy images.

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RAGE overexpression confers a metastatic phenotype to the WM115 human primary melanoma cell line