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Experimental Cell Research

journal homepage: www.elsevier.com/locate/yexcr

The laminin-derived peptide C16 regulates GPNMB expression andfunction in breast cancer

Basilio Smuczeka, Emerson de S. Santosa,b, Adriane S. Siqueiraa,d, Joao J.V. Pinheiroc,Vanessa M. Freitasa, Ruy G. Jaegera,⁎

a Department of Cell and Developmental Biology, Institute of Biomedical Sciences, University of Sao Paulo, Sao Paulo, Brazilb School of Pharmaceutical Sciences of Ribeirao Preto, University of Sao Paulo, Ribeirao Preto, Sao Paulo, Brazilc School of Dentistry, Federal University of Para, Belem, Para, Brazild School of Dentistry, Positivo University, Curitiba, Parana, Brazil

A R T I C L E I N F O

Keywords:Breast cancerExtracellular matrixBasement membraneLaminin, C16GPNMB

A B S T R A C T

Breast cancer is an important public health problem, and its progression may be related to the extracellularmatrix (ECM), which acts as a structural scaffold and instruction source for neoplastic cells. Laminins are ECMproteins regulating tumor biology. The laminin-derived peptide C16 regulates different properties of tumorcells. Here we analyzed C16-induced differential gene expression in MDA-MB-231 breast cancer cells. MCF-10Anormal-like breast cells served as control. Among different cancer-related genes, C16 induced overexpression ofGPNMB. This gene encodes a transmembrane protein GPNMB (glycoprotein non-metastatic B), involved withmalignant phenotype of breast cancer cells. Immunoblot validated microarray results. To correlate gene andprotein expression with cellular function, we investigated whether C16 would regulate invasion in breast cancercells. siRNA experiments strongly suggested that C16 and GPNMB cooperate to regulate invasion of highlyaggressive MDA-MB-231 cancer cells. We addressed regulatory mechanisms involved in C16-mediated increaseof GPNMB protein levels in MDA-MB-231 cells, and observed that C16 stimulates β1 integrin and Srcphosphorylation. Furthermore, Src inhibition decreases peptide-induced GPNMB expression levels. Tocontextualize in vivo our results in vitro, we addressed GPNMB immunostaining in breast cancer humantissue microarrays. Quantitative immunohistochemistry showed that GPNMB is significantly more expressed inbreast cancer compared to normal tissue. We concluded that laminin-derived peptide C16 regulates gene andprotein expression of GPNMB in breast cancer cells. C16 and GPNMB may cooperate to regulate invasion ofhighly aggressive MDA-MB-231 cells, probably through Src signaling. GPNMB presented increased expressionin breast cancer in vivo compared to normal breast tissue.

1. Introduction

Breast tumors are the second most common type of cancer world-wide, affecting mostly women [1–3]. In Brazil, the National CancerInstitute (lNCA) estimates that, in 2016, 57.960 new cases of breasttumors would be diagnosed; representing the second most frequentlydiagnosed non-skin cancer type [4]. Certain reproductive factors, age,weight and hormone levels (especially in menopause) have significantinfluence on breast cancer occurrence [1–3,5–8].

Like other types of tumors, breast cancer originates from a singlenormal cell that acquires genetic or epigenetic changes, undergoesmonoclonal expansion, accumulates molecular alterations, and finallyprogresses to an invasive stage [9,10]. The complex pattern of

alterations acquired by the tumor cells can influence a variety ofcellular functions, including proliferation, migration, invasion, survi-val, and capacity to induce angiogenesis [9,10]. Besides molecularalterations acquired by tumor cells, during tumor progression cells areengaged in a complex interplay with the surrounding microenviron-ment, which can also influence tumor behavior [5,11]. The extracel-lular matrix (ECM) plays an important role in this context. Composedof collagens, proteoglycans, elastin and structural glycoproteins, ECMis a three-dimensional network that may act both as structural scaffoldand instruction source for cells [12–17]. The basement membrane is aspecialized ECM sheet-like structure predominantly composed bylaminin, type IV collagen, nidogen and perlecan [18–21].

Laminins are basement membrane glycoproteins, formed by three

http://dx.doi.org/10.1016/j.yexcr.2017.07.005Received 21 January 2017; Received in revised form 2 July 2017; Accepted 4 July 2017

⁎ Correspondence to: Departamento de Biologia Celular e do Desenvolvimento, Universidade de São Paulo, Instituto de Ciências Biomédicas, Av. Prof. Lineu Prestes 1524, EdBiomédicas 1 sala 410, São Paulo, SP 05508-000, Brazil.

E-mail address: rgjaeger@usp.br (R.G. Jaeger).

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Available online 05 July 20170014-4827/ © 2017 Elsevier Inc. All rights reserved.

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different polypeptide chains, termed α, β, and γ [18,22–24]. It has beenshown that laminin modulate different cellular processes such as celladhesion, migration, growth, and differentiation [18]. A crucial step intumor development is the proteolytic cleavage of basement membranecomponents, including laminin, through the action of specific proteasessecreted by both tumor and stromal cells [25]. This process not onlyremoves physical barriers to cell invasion but also unmasks crypticbioactive peptides [26]. An increasing number of evidences have shownthat peptides derived from laminin cleavage may control tumorbehavior [27–38]. Among them, C16 (KAFDITYVRLKF), derived fromlaminin-111 γ–1 chain, is a cell-adhesive peptide related to migration,metastatic activity, and angiogenesis [37,39–41]. Previous works fromour group also demonstrated that C16 is a major regulator ofinvadopodia formation and activity [34,42].

Although these evidences suggest that C16 plays a relevant part intumor progression, its influence in genetic programs of breast cells hasnot yet been investigated. This prompted us to analyze C16-induceddifferential gene expression in MDA-MB-231 breast cancer cells (highlyaggressive, triple negative for estrogen, progesterone and HER-2receptors). Normal-like MCF-10A cell line served as control. Ourresults showed that C16 induced overexpression of GPNMB gene(glycoprotein non-metastatic B or osteoactivin) in neoplastic cells.GPNMB is a potential biomarker associated with increased cellmigration, invasion, angiogenesis and bone metastasis of breast cancercells [43–47]. We also found that MDA-MB-231 cells present highlevels of GPNMB protein.

To correlate gene and protein expression with cellular function, weinvestigated whether C16 and GPNMB would cooperate to regulate theinvasive phenotype of breast cancer cells. Regulatory mechanismsinvolved in C16-mediated increase of GPNMB protein levels wereanalyzed. Furthermore, to contextualize in vivo our results in vitro, wehave addressed GPNMB immunostaining in breast cancer humantissue microarrays (TMAs).

2. Material and methods

2.1. Peptides

EZ Biolab (Westfield, IN, USA) synthesized peptide C16(KAFDITYVRLKF) and scrambled control peptide C16SX(FKLRVYTIDFAK). Peptides purity was 98% (RP-HPLC), and mole-cular mass was confirmed by mass spectrometry. Peptides C16 andC16SX were diluted in culture medium without serum or supplementsfor all experiments.

2.2. Cell culture

MDA-MB-231 cells were purchased from the Cell Bank of Rio deJaneiro, Brazil. MCF-10A cells were kindly provided by Prof. NathalieCella (Department of Cell and Developmental Biology, Institute ofBiomedical Sciences, University of São Paulo, São Paulo, Brazil). Thiswork did not involve direct manipulation of humans or animals, andwas approved by ICB Ethics Committee (Protocol CEP-ICB 524/12).

Cells derived from invasive ductal breast carcinoma (MDA-MB-231,"basal-like", triple-negative for estrogen, progesterone and HER-2receptors) were cultured in Dulbecco's modified Eagle medium(DMEM, Sigma Chemical Co., St. Louis, MO, USA) supplemented with10% fetal bovine serum (FBS, Life Technologies, Carlsbad, CA, USA).Normal-like breast epithelial cells (MCF-10A) were grown in DMEMplus Ham's F12 nutrient (DMEM: F12, Sigma) supplemented with10 mg/ml insulin, 0.1 mg/ml cholera toxin, 0.5 mg/ml hydrocortisone,5% FBS and 10 ng/ml EGF (all from Sigma). Cells were maintained in75 cm2

flasks in an atmosphere of 5% CO2 at 37 °C.MDA-MB-231 and MCF-10A were cultured in 6-well plates and

treated with peptide C16 (100 μg/ml) diluted in serum free medium.Control groups were treated with scrambled peptide C16SX diluted in

serum-free medium. Non-peptide controls were used. Positive controlwas media with 10% FBS. Negative control was serum-free media.

2.3. Total RNA extraction

After treatment of cells with C16 or C16SX (100 µg/ml) for 24 h,total RNA was extracted using PureLink RNA Mini kit (LifeTechnologies), following manufacturer's instructions. Total RNA con-centration was determined by spectrophotometry at 260/280 nm(NanoDrop Technologies, Wilmington, DE, USA), and its structuralintegrity was confirmed by visual inspection of ribosomal RNA bandsin 1% agarose gel stained with ethidium bromide.

2.4. cDNA microarray

We used the Human Gene 1.0 ST Array (Affymetrix, Santa Clara,CA, USA) to analyze the expression of 28,869 human genes in MDA-MB-231 and MCF-10A cells treated with C16 in comparison totreatment with scrambled control peptide (C16SX). Ambion WTExpression Kit (Life Technologies) was used to generate sense-strandcomplementary DNA (cDNA) from total RNA. Briefly, 100 ng of totalRNA underwent cDNA synthesis using random primers containing aT7 promoter sequence. Afterwards, complementary RNA (cRNA) wasgenerated through an in vitro transcription, and then used to synthe-size sense-strand cDNA. Sense-strand cDNA was then fragmented andend-labeled with Biotin Allomide Triphosphate. Labeled cDNA (2.5 µg)was hybridized to the Human Gene 1.0 ST Array for 17 h at 45 °C and60 rpm. GeneChips were then washed, stained with streptavidin-phycoerythrin, and scanned. Conversion of the acquired fluorescenceintensities to numerical values was performed using the ExpressionConsole software (Affymetrix). Data was summarized using theiterPlier algorithm with background adjustment (PM - GCBG method),and quantile normalization. Normalized gene expression values wereimported into the TIGR Multi-Experiment Viewer 4.6 software (http://www.tm4.org/mev/) for statistical analysis. Student's t-test wasperformed to assess gene expression differences between treatmentswith C16 and C16SX. Genes whose expression levels were increased ordecreased by more than 1.2-fold and showed statistical significance (p≤ 0.05) were considered differentially expressed. Heatmap ofdifferentially expressed genes (DEG) was generated using the freelyavailable software Heatmap Builder 1.1 (Stanford University, Stanford,CA, USA) [25]. Gene Ontology term annotation was provided by web-based software Molecule Annotation System (MAS) 3.0 (CapitalBioCorporation, Changping District, Beijing, China).

2.5. Immunoblot

MDA-MB-231 and MCF-10A cells were grown on 6-well plates andincubated with either C16 or C16SX scrambled control peptide fordifferent time intervals. All samples were subjected to immunoblot, asdescribed elsewhere [48]. Samples were lysed in RIPA buffer (150 mMNaCl, 1% NP-40, 0.5% deoxycholate, 0.1% SDS, 50 mM Tris pH 8.0)containing protease and phosphatase inhibitors when necessary (NaF,Ortovanadate Sodium, pepstatin A, PMSF, and E-64, Sigma). Sampleswere centrifuged at 10,000g for 10 min at 4 °C and the supernatantcollected. Quantitation of proteins was carried out using the BCAmethod (Thermo, Rockford, IL USA). Samples were resuspended inLaemmli buffer containing 62,5 mM Tris–HCl pH 6.8, 2% sodiumdodecyl sulphate (SDS), 10% glycerol, 5% mercaptoethanol, 0.001%bromophenol blue. Equal amounts (30 µg) of cell lysates were electro-phoresed in 10% polyacrylamide gels. Proteins were transferred to aHybond ECL nitrocellulose membrane (Amersham), blocked in TBSwith 5% non-fat milk or BSA 3% for one hour. The following primaryantibodies were used: GPNMB (1:250, sc-271415, Santa CruzBiotechnology), and β-actin (1:10.000, A5316, Sigma). Membraneswere revealed by ECL protocol.

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2.6. Invasion assay

Invasion assays were carried out in Boyden chambers coated with13 mg/ml Matrigel (BD Biosciences, Franklin Lakes, NJ, USA).Membrane pore size was 8 μm-diameter. After a starvation period of1 h, MDA-MB-231 and MCF-10A cells (15 × 104) were plated in theupper chamber wells in serum-free media. Lower chamber wells werefilled with media containing either C16 or C16SX (100 µg/ml), or withnon-peptide positive or negative controls. Cells were cultured in theseconditions for 48 h, and then fixed with 4% paraformaldehyde. Cells onthe upper side of the polycarbonate membrane were removed, whileinvading cells, located on the lower side of the membrane, were stainedwith crystal violet, photographed at a final magnification of 200x, andcounted. On each well, 7 random fields were evaluated. Invasion assayswere conducted in triplicates.

2.7. Invasion assay in cells with depleted GPNMB by siRNA

In order to assess the involvement of GPNMB in the invasionprocess possibly triggered by C16, MDA-MB-231 cells were transfectedwith commercially available siRNA targeting GPNMB (80 nM, SantaCruz Biotechnology, sc-60721) following the manufacturer's instruc-tions. One day before transfection, sub confluent MDA-MB-231 cellswere cultured in Dulbecco's modified Eagle's medium, supplementedby 10% fetal bovine serum. Cells were incubated with a complexformed by the siRNA, transfection reagent (Lipofectamine 2000;Invitrogen), and transfection medium (Opti-MEM I; Invitrogen) for48 h at 37 °C. Control siRNA (80 nM, Santa Cruz Biotecnology, sc-37007), a non-targeting siRNA, served as negative control. Cells weresubmitted to invasion assays in presence of C16 as described above.Additionally, cells were subjected to invasion assays in the presence ofscrambled peptide C16SX.

At least three siRNA oligos were used in these experiments yieldingsimilar results.

2.8. Effect of C16 in β1 integrin expression

GPNMB is described as pro-growth and pro-metastatic moleculedue to its ability to bind α5β1 integrin and increase downstreamsignaling in breast cancer cells [49]. This prompted us to evaluatewhether C16 would increase β1 integrin levels in breast cancer cells.MDA-MB-231 and MCF-10A cells were treated by either C16 or C16SXand subjected to immunoblot to detect β1 integrin (1:1000, MAB2079Z Millipore).

We also addressed C16-induced colocalization of GPNMB and β1integrin in MDA-MB-231 cells. Treated and control samples were fixedin 4% paraformaldehyde in PBS for 15 min, followed by blocking with3% bovine serum albumine (BSA, Sigma). Cells were stained withantibodies against β1 integrin-conjugated with Alexa Fluor 555(MAB2079-AF555, Millipore) and GPNMB (sc-271415, Santa Cruz).GPNMB was revealed by Alexa-488 secondary antibody. Samples weremounted with Pro Long Gold with DAPI (Life Technologies).

2.9. Effect of Src in C16-induced GPNMB expression

GPNMB recruitment into integrin complexes activates Src signalingpathways in an RGD-dependent manner [49]. Furthermore, we pre-viously demonstrated that C16 stimulates β1 integrin and Src phos-phorylation in different tumor cell lines [42]. We then decided to assessC16-induced Src activation in breast cancer cells. MDA-MB-231 cellswere grown on 6-well plates and incubated with either C16 or C16SXscrambled control peptide for different time points. All samples weresubjected to immunoblot, as described before, using primary antibo-dies against Src-phospho-Tyr 416 (1:1000, #2113, Cell SignalingTechnology) and Src (1:1000, #2109, Cell Signaling). Cells wereserum-starved for 24 h prior to peptide treatment.

To correlate C16 peptide, Src phosphorylation status, and GNPMBprotein levels, MDA-MB-231 cells were treated by the Src inhibitorPP2, followed by C16 treatment. Cells cultured overnight in mediumwith 10% FBS were serum-starved for 24 h and then incubated with Srcinhibitor PP2 (5 μM, Sigma). As a control, cells were incubated withvehicle (methanol). Both PP2 inhibited and control cells were treatedby C16, followed by detection of GNPMB, Src and phospho-Src byimmunoblot.

2.10. Immunohistochemistry

To contextualize in vivo our results in vitro, we studied thepresence of laminin γ1 chain and GPNMB in breast cancer and normalbreast tissue, using Tissue Microaarray Slides (TMAs, IMGENEX Co,San Diego, CA, USA). TMA contained 35 infiltrating duct carcinomas, 9normal breasts, 1 ductal carcinoma in situ, 1 atypical medullarycarcinoma, 1 intraductal papillary carcinoma, 1 metaplastic carcinoma,1 sarcomatoid carcinoma, and 10 lymph node metastatic carcinomatissue samples. Sections were deparaffinized in xylene and hydrated indecreasing concentrations of ethanol. Antigen retrieval was carried outwith citrate buffer (10 mM citric acid, 0.05% Tween 20, pH 6) in waterbath (95–100 °C) for 30 min. Sections were blocked for 1 h with 1%bovine serum albumin (BSA, Sigma) in phosphate-buffered saline(PBS). Laminin γ1 chain was stained by a rabbit polyclonal antibody(Santa Cruz Biotechnology, sc-17763, 1:600). GPNMB was labeled by amouse monoclonal antibody (Santa Cruz Biotechnology, sc-60721,1:100). Antibodies were incubated overnight at 4 °C. Endogenousperoxidase blocking was carried out for 20 min, followed by incubationwith NovolinkTM Polymer Detection Systems (Leica BiosystemsNewcastle Ltd, Upon Tyne, UK). Diaminobenzidine (Sigma) was usedas chromogen and sections were counterstained with Mayer's hema-toxylin (Sigma) and mounted with Permount (Fisher Scientific, FairLawn, NJ, USA). Non-immune serum served as negative control.

Immunostaining was assessed by measuring GPNMB-labeled areafraction (%). Bright field images of at least five arbitrarily selected areasin each sample were acquired in a Primostar microscope equipped withan AxioCam HRc color CCD camera (Carl Zeiss, Oberkochen,Germany), using a 40X objective. The areas stained with diaminoben-zidine were separated and segmented using the color deconvolutionplug-in (ImageJ software).

2.11. Statistical analysis

Gene Microarrays statistical analysis was carried out in TIGR Multi-Experiment Viewer 4.6 software. Additional statistical analyses wereperformed using Graph Pad Prism 5.0 software (Graph Pad, San Diego,CA, USA). Differences between groups were assessed by either ANOVA(parametric) or Kruskal-Wallis (non-parametric) followed by appro-priated multiple comparison tests. Differences were considered sig-nificant when p ≤ 0.05.

3. Results

3.1. C16 regulates gene expression in MDA-MB-231 and MCF-10Acells

Microarray analysis revealed that the C16 peptide regulates geneexpression in both breast carcinoma (MDA-MB-231) and normal-likebreast cells (MCF-10A) (Fig. 1A). Gene annotation provided by GeneOntology and the literature allowed us to classify the differentiallyexpressed genes according to their biological function. We found genesdirectly associated with processes related to cancer, including genesinvolved in 1) cell migration, invasion and metastasis; 2) cell adhesion;3) angiogenesis; 4) cell proliferation; 5) apoptosis (Fig. 1A). Nearly50% of genes were involved with cell adhesion, migration, invasion andmetastasis.

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Thirty-three genes were upregulated and forty-seven genes were down-regulated by C16 in MDA-MB-231 cells (Fig. 1B). Among these genes,GPNMB was upregulated in both cell lines studied here (Fig. 1B). SinceGPNMB is involved in cell adhesion, migration, invasion, metastasis andangiogenesis we decided to investigate C16-induced GPNMB increase andits consequences in MDA-MB-231 cells. C16 clearly increased GPNMBexpression in tumor cell line MDA-MB-231. However, the highest effect ofC16 on GPNMB expression occurred in MCF-10A cells.

Supplementary Tables I and II illustrated differentially expressedgenes in MDA-MB-231 and in MCF-10A cells treated by C16 peptide.

3.2. C16 increases GPNMB protein levels in breast cells

C16 significantly regulated GPNMB gene expression in normal andbreast cancer cells. This prompted us to investigated whether C16would modulate GPNMB protein levels in the highly aggressive MDA-MB-231 breast cancer cell line compared to normal-like MCF-10Acells.

MDA-MB-231 and MCF-10A were treated by either C16 orscrambled peptide control C16SX (100 µg/ml). Immunoblot demon-strated that the peptide stimulated GPNMB expression in MDA-MB-

Fig. 1. C16 peptide regulates expression of cancer-related genes in MDA-MB-231 and MCF-10A cells compared to C16SX control peptide. Pie chart shows the relative number(percentage) of genes associated with different biological processes related to cancer (A). Representative graphs show different values of relative expression of genes regulated by C16compared to C16SX and related to cancer in the breast carcinoma line MDA-MB-231 (B, upper graph) and normal-like cell line MCF-10A (B, lower graph). Numbers in front of the genesymbol indicate biological functions of each gene. GPNMB overexpression is indicated in red. Thirty-three genes were upregulated and forty-seven genes were downregulated by C16.Color intensity was scaled for each gene so that the lowest expression value corresponds to light green and the highest to light red. Columns display individual samples.

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231 cells (Fig. 2A), compared to C16SX. Discrete GPNMB increase wasdetected in MCF-10A cells treated by C16 peptide (Fig. 2A). GPNMBdetection was moderate in cells treated by non-peptide controls.

Immunoblot (Fig. 2A) also showed different forms of the GPNMB.Fast migrating band were more intense in MDA-M-231 cells comparedto MCF-10A. This result would raise the possibility that GPNMB ispost-translationally processed in the cancer cells but not in the controlcells.

In MDA-MB-231 cells, time course analysis demonstrated that C16significantly increased GPNMB levels 24 h after C16 treatment, whileC16SX control peptide did not stimulate a significant GPNMB increaseover time (Fig. 2B).

3.3. GPNMB regulates C16-induced invasion in triple negative,highly aggressive MDA-MB-231 breast cancer cell line

GPNMB has already been associated with invasive phenotype inother tumor cell lines [47]. Therefore, we decided to assess whetherC16 would stimulate invasion in breast cancer cells lines. Invasionassays in Matrigel-coated Boyden chambers showed that C16 stimu-lated invasion activity in the highly aggressive MDA-MB-231 cell line.Cells treated by C16 showed a two-fold increase in invasive activitycompared to C16SX peptide control (Fig. 3A, left graph). Furthermore,C16-induced invasion increased 5-fold compared to non-peptidenegative control. C16 failed to modulate invasion rate of MCF-10A

Fig. 2. C16 increases expression of GPNMB. MDA-MB-231 breast cancer cells treated with C16 peptide show increased GPNMB compared to control peptide C16SX (A, left blots).Lower GPNMB expression is observed in MCF-10A normal-like breast cells compared to tumor cells (A, right blots). Immunoblot (A) also shows different forms of the GPNMB. Fastmigrating band are more intense in MDA-MB-231 cells compared to MCF-10A. GPNMB levels are discrete in MDA-MB-231 and in normal-like MCF-10A cells treated by non-peptidecontrols. In MDA-MB-231 cells, time course analysis demonstrates that C16 significantly increases GPNMB levels 24 h after C16 treatment (B). Results in B represent mean ± standarderror of three experiments. Asterisk indicates statistically significant differences (p < 0.001).

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control cells (Fig. 3A, right graph). Interestingly, the control cell lineMCF-10A showed an increased invasive phenotype in presence of FBS(Fig. 3A, right panel). This behavior has been previously reported [50].

To correlate protein expression and cell function, we investigatedwhether GPNMB would be related to C16-induced invasion in MDA-MB-231 cells. For this purpose, we carried out invasion assays using MDA-MB-231 cells with siRNA-depleted GPNMB, followed by treatment with C16peptide. Cells with reduced GPNMB expression exhibited significantdecrease in C16-mediated invasion compared to control (Fig. 3B). Nosignificant differences were observed between C16SX-treated samples.Immunoblot confirmed transfection efficiency (Fig. 3B).

3.4. Peptide C16 increases β1 integrin expression and inducesGPNMB and β1 integrin colocalization

GPNMB binds α5β1 integrin and increase downstream signaling inbreast cancer cells [49]. MDA-MB-231 and MCF-10A treated bypeptide C16 increased β1 integrin levels after 24 h (Fig. 4A).Immunofluorescence addressed C16-induced colocalization ofGPNMB and β1 integrin. The peptide induced colocalization ofGPNMB and β1 integrin at lamellipodia of tumor cells (Fig. 4B, upperpanel) compared to cells treated by the scrambled peptide controlC16SX (Figure 4B, lower panel).

3.5. Src signaling pathway is involved in C16-induced GPNMBexpression

GPNMB cooperate with integrins to activate Src signaling pathway[49]. Furthermore, we previously demonstrated that C16 stimulates β1integrin and Src phosphorylation in different tumor cell lines [42].C16-induced Src phosphorylation was investigated in breast cancer

cells. Here we correlated C16, Src phosphorylation, and GNPMBprotein levels in MDA-MB-231 cells.

C16 enhanced Src phosphorylation in MDA-MB-231 cells (Fig. 5A).Src phosphorylation increase was significant 6 h after peptide additioncompared to C16SX scrambled control peptide. Moreover, a decreasein C16-mediated GPNMB protein levels was observed in MDA-MB-231cells treated with Src inhibitor PP2, compared to cells treated with theinhibitor vehicle (methanol) and C16 (Fig. 5B). No differences inGPNMB levels were detected in Src-inhibited cells incubated withscrambled peptide C16SX.

Taken together our results strongly suggest that Src signalingpathway modulates C16-induced GPNMB protein levels in breastcancer cells.

3.6. Immunostaining of laminin γ1 chain in breast cancer comparedto normal breast

To contextualize in vivo our results in vitro, we assessed laminin γ1chain and GPNMB immunostaining in breast cancer samples comparedto normal breast tissue.

Strong γ1 laminin reactivity was detected in normal mammaryglands (Fig. 6A). On the other hand, GPNMB labeling was discrete orabsent in similar regions (Fig. 6B). Tumor breast tissue showed diffuseand discontinuous γ1 laminin staining (Fig. 6C, arrows). Interestingly,increased GPNMB detection was seen in corresponding areas (Fig. 6D).Negative controls exhibited no staining (not illustrated).

These results suggest that γ1 laminin disruption may correlate withincreased GPNMB detection.

Fig. 3. C16 and GPNMB cooperate to regulate invasion of MDA-MB-231 cells. Invasion assays carried out in Boyden chambers coated with Matrigel (A) show that C16 peptidesignificantly increases invasion of MDA-MB-231 cells by two-fold compared to C16SX peptide control, and five-fold compared to serum free group (A, left graph). MCF-10A cells fail toinvade in presence of C16 (A, right graph). However, significant result was observed in the positive control FBS group (A, right graph). Invasion assays were also carried out using cellswith siRNA-depleted GPNMB, followed by treatment with C16 peptide (B). Cells with reduced GPNMB expression exhibit significant decrease in C16-mediated invasion compared tocontrols (B). Immunoblot analysis confirmed transfection efficiency (B). The siRNA control is the group transfected with the scrambled non-depleting siRNA. Results represent mean ±standard error of three experiments. Asterisk indicates statistically significant differences (p < 0.05).

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3.7. GPNMB staining is prominent in human breast cancer

Area fraction analysis showed that GPNMB staining was signifi-cantly increased in lymph node metastatic carcinoma compared tonormal breast tissue (Fig. 7A and B). Following breast cancer mole-cular classification [51,52], increased GPNMB immunostaining wasobserved in more aggressive subtypes basal-like and HER2 neoplasmscompared to normal breast (Fig. 7C). Considering tumor stage,augmented GPNMB labeling was observed in IIA, IIIA and IIIC stagingcompared to normal breast (Fig. 7D).

4. Discussion

We have demonstrated that peptide C16, derived from laminin γ1chain, increases the expression of GPNMB gene and protein levels inMDA-MB-231 and MCF-10A cells. This peptide also stimulated inva-sion of MDA-MB-231 cells. We also found that GPNMB knockdowndecreases C16-induced invasive phenotype cell of MDA-MB-231 cells.β1 integrin and Src signaling pathway are related to C16-inducedGPNMB expression in breast cancer cells. GPNMB has a clearrelevance in human breast tumors, as shown by TMA analysis. Toour knowledge, this study demonstrates for the first time that a

Fig. 4. Peptide C16 increases β1 Integrin expression and induces colocalizaton of β1 Integrin and GPNMB at lamellipodia of MDA-MB-231 cells. MDA-MB-231 and MCF-10A treatedby peptide C16 increased β1 integrin expression after 24 hours compared to controls (A). β1 Integrin (B, red) and GPNMB (B, green) staining are distributed throughout cell cytoplasm.However, C16-induces colocalization of β1 Integrin and GPNMB at the lamellipodia of MDA-MB-231 cells (B, top panel). This feature is clearly observed at higher magnification (boxedarea). Moreover, in the Linescan chart, Red (β1 Integrin) and Green (GPNMB) lamellipodia peaks are clearly overlapped. Cells treated by with reverse C16SX fail to show colocalizationareas at lamellipodia (B bottom panel). At least 30 cells were observed and these results repeated consistently. Scale bar: 10 µm.

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laminin-derived peptide and a putative breast cancer biomarkercooperate to regulate the invasive phenotype of a highly aggressivebreast cancer cell line.

Tumor development involves a direct relationship between cancercells and extracellular matrix. In this context, controlled ECM proteo-lysis may have an important impact regulating tumor biology [26,53–56]. Laminin is a major ECM protein and presents cryptic sites in itsstructure, which may be exposed by protease activity [54,58]. C16(KAFDITYVRLKF), located at the γ1 chain, could be one of theseexposed sites. The γ1 chain of laminin is found in breast epitheliumand blood vessels basement membranes [18], thus being prone toproteolysis. Additionally, mass spectrometry studies showed that MCF-10A cells treated by proteases generated an amino acid sequencesimilar to C16 (except for the absence of the two last amino acids in thecarboxy-terminal end), strongly suggesting that the peptide is aproduct of cell-induced proteolysis [57]. C16 has been associated withcell adhesion [41], angiogenesis [37] and secretion of metalloprotei-nases [59].

Microarray analysis revealed that C16 peptide regulates expressionof genes associated with breast cancer cells. This peptide increased theexpression of genes involved with different aspects of cancer biology,among them adhesion, migration, invasion and metastasis. TheGPNMB (glycoprotein non-metastatic melanoma protein B) gene wasa strong hit in both normal and breast cancer microarrays.

Microarray results were confirmed by immunoblot, showing in-creased GPNMB protein levels in breast cancer cells compared tonormal-like cells. No correlation was found between gene expressionand protein levels in normal-like MCF-10A breast cells. Biologicalreasons for this discrepancy include post-transcriptional and post-translational modifications [59–61]. It has also been suggested thatprotein concentrations correlate with the corresponding mRNA levelsby only 20–40% [61].

GPNMB is a type I single-pass transmembrane protein with a largeextracellular domain composed by an integrin-binding (RGD) motifand a polycystic kidney disease (PKD) domain [62]. Cytoplasmic tail isshort with 53 amino acids [63]. This molecule is expressed in

Fig. 5. Src signaling pathway is involved in C16-induced GPNMB expression. C16 enhances Src phosphorylation in MDA-MB-231 cells (A). Src phosphorylation increase is significant6 h after peptide addition (A). A decrease in C16-mediated GPNMB protein levels is observed in MDA-MB-231 cells treated with Src inhibitor PP2, compared to cells treated with theinhibitor vehicle (methanol) and treated with C16 (B). No significant differences are detected in Src-inhibited cells incubated with scrambled control peptide C16SX (B). Resultsrepresent mean ± standard error of three experiments. Asterisk indicates statistically significant differences (p < 0.05).

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approximately 40–75% of breast cancers and promotes migration,invasion and metastasis [45,46,49,62,63].

Our immunoblot results showed different forms of GPNMB. Fastmigrating band were more intense in MDA-MB-231 cells compared toMCF-10A. This result would raise the possibility that GPNMB is post-translationally processed in the cancer cells but not in the control cells.It is known that proteases such as ADAM10 release a solubleectodomain of this protein [63]. We addressed whether GPNMB fastbands would be the results of MMP-mediated proteolysis(Supplementary Fig. 1). MMP-inhibition by GM6001 clearly decreasedfast migratory bands in treated and control cells. However, nodifference was found in either C16 or C16SX control samples. On theother hand, GPNMB was not detected in the conditioned media. Wemay infer that C16 induced no GPNMB shedding, since no solublefraction of this molecule was detected in the conditioned media.

We have already demonstrated that C16 increases invasion andinvadopodia activity in cancer cell [34,42]. Here, we showed that C16increased invasion rate of MDA-MB-231 cells. Our results showed thatC16 increased GPNMB gene and protein expression in breast cancercells. Furthermore, C16 stimulated invasion of a highly aggressivetriple negative MDA-MB-231 cells. We then decided to investigatewhether GPNMB would interact with C16 regulating invasion of MDA-MB-231 cells. GPNMB depletion diminished C16-induced invasion,showing that this protein and the peptide C16 may cooperate toregulate breast cancer invasiveness. Integrin-binding properties mayexplain GPNMB and C16 interaction. GPNMB exhibits a RGD motif[62,63]. It is well known that this RGD motif binds integrins, mostlyα5β1 and αvb3 heterodimers [64]. Moreover, integrins α5β1 and αvβ3

integrins have been reported as C16 partners [65]. We furtheraddressed C16-induced colocalization of GPNMB and β1 integrin.MDA-MB-231 cells treated by C16 exhibited colocalization ofGPNMB and β1 integrin at lamellipodia. Thus, we may conclude thatintegrins are involved in the cooperation between C16 and GPNMB.

C16 interactions with integrins receptors may activate differentsignaling pathways [34,42]. We may infer that β1 integrin would bindGPNMB, generating signals transduced by Src signaling pathway inbreast cancer [44]. We observed an increase of β1 integrin byimmunoblot, a significant Src phosphorylation at 6 h and an increaseof GPNMB at 24 h in time-course experiments; all triggered by C16peptide in MDA-MB-231 cells. Furthermore, cells treated with Srcinhibitor decreased C16-induced GPNMB protein levels. Taken to-gether these results strongly suggest that C16 would interact with β1integrin, generating signals to be transduced by the Src pathway, thusregulating GPNMB expression and the invasive phenotype of a highlyaggressive breast cancer cell line. Diagram portrayed in Fig. 8 sum-marizes our current model for the effect of C16 on GPNMB expressionand how it modulates cancer cell invasion.

GPNMB is associated with bone metastasis of breast carcinoma and isoverexpressed in aggressive breast cancer (basal-like or triple-negative)[43]. Our results indicate that GPNMB expression is related to lamininbreakdown. We showed laminin γ1 chain in breast cancer and normalbreast. Laminin γ1 chain, containing the peptide C16, is disrupted in breastcancer. Furthermore, our data suggests that γ1 laminin disruption appearsto correlate with GPNMB increased immunostaining. We may assume thatlaminin disruption in breast cancer would release bioactive peptides,among them C16, thus stimulating GPNMB levels. TMA analysis confirmed

Fig. 6. Laminin γ1 chain disruption may correlate with increased GPNMB detection. Strong γ1 laminin reactivity is detected in normal mammary glands (A). GPNMB labeling isdiscrete or absent in similar regions (B). Tumor tissue shows diffuse and discontinuous γ1 laminin staining (C, arrows). Increased GPNMB detection is observed in corresponding areas(D). Scale bars: 50 µm.

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that GPNMB immunostaining was significantly increased in breast cancercompared to normal tissue. These differences were consistently observedtaking into account different parameters such as tumor type, molecularclassification and staging, and in all parameters analyzed here GPNMB wasprominent in more aggressive subtypes.

We have attempted to elucidate mechanisms involved in C16effects, to better understand the role played by this peptide in breast

cancer. Based on our experimental findings we propose that C16enhances GPNMB gene expression and protein levels in cells derivedfrom breast cancer. Furthermore C16 and GPNMB may cooperate toregulate cellular invasiveness of breast cancer cells, probably throughintegrins and Src signaling pathway. The putative interaction betweenGPNMB and the laminin-derived peptide C16 regulating tumor biologywould be the target of our future investigations.

Fig. 7. GPNMB staining is prominent in human breast cancer. Area fraction analysis shows that GPNMB staining is significantly increased in lymph node metastatic carcinomacompared to normal breast tissue (A and B). GPNMB immunostaining is significantly increased in basal-like and HER2 neoplasms compared to normal breast (C). Augmented GPNMBis also observed in IIA, IIIA and IIIC staging, compared to normal breast (D). Asterisks indicate significant results (p < 0.05). IDC = infiltrating (p < 0.05). IDC = infiltrating ductcarcinoma. LMC = Lymph LMC = Lymph node metastatic carcinoma. Scale bars: 20 µm.

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Conflict of interest

The authors declare that they have no conflict of interest.

Acknowledgments

The author want to especially thank Prof. Nathalie Cella(Department of Cell and Developmental Biology, Institute ofBiomedical Sciences, University of São Paulo, São Paulo, Brazil) forkindly provided MCF-10A cells. This investigation was supported byFAPESP (2015/03393-9, 2015/02498-1) and CNPq (471411/2013-2).BS received a MSD fellowship from FAPESP (2012/13045-0) and iscurrently funded by a CAPES PhD fellowship. The funders had no rolein study design, data collection and analysis or preparation of themanuscript.

Appendix A. Supporting information

Supplementary data associated with this article can be found in theonline version at doi:10.1016/j.yexcr.2017.07.005.

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