AntagonistsofIGF:VitronectinInteractionsInhibit IGF-I ... · Abhishek S. Kashyap1,5, Gary K. Shooter1, Ali Shokoohmand1, Jacqui McGovern1, Manaswini Sivaramakrishnan2,Tristan I. Croll1,Ga€elle

Large Molecule Therapeutics

Antagonists of IGF:Vitronectin Interactions InhibitIGF-I–Induced Breast Cancer Cell FunctionsAbhishek S. Kashyap1,5, Gary K. Shooter1, Ali Shokoohmand1, Jacqui McGovern1,Manaswini Sivaramakrishnan2, Tristan I. Croll1, Ga€elle Cane3, David I. Leavesley1,Ola S€oderberg3, Zee Upton1, and Brett G. Hollier1,4

Abstract

We provide proof-of-concept evidence for a new class oftherapeutics that target growth factor:extracellular matrix (GF:ECM) interactions for the management of breast cancer. Insulin-like growth factor-I (IGF-I) forms multiprotein complexes withIGF-binding proteins (IGFBP) and the ECM protein vitronectin(VN), and stimulates the survival, migration and invasion ofbreast cancer cells. For the first time we provide physical evidencefor IGFBP-3:VN interactions in breast cancer patient tissues; theseinteractions were predominantly localized to tumor cell clustersand in stroma surrounding tumor cells. We show that disruptionof IGF-I:IGFBP:VN complexes with L27-IGF-II inhibits IGF-I:IGFBP:VN-stimulated breast cancer cell migration and prolifer-ation in two- and three-dimensional assay systems. Peptide arrays

screened to identify regions critical for the IGFBP-3/-5:VN andIGF-II:VN interactions demonstrated IGFBP-3/-5 and IGF-IIbinds VN through the hemopexin-2 domain, and VN bindsIGFBP-3 at residues not involved in the binding of IGF-I toIGFBP-3. IGFBP-interacting VN peptides identified from thesepeptide arrays disrupted the IGF-I:IGFBP:VN complex, impededthe growth of primary tumor-like spheroids and, more impor-tantly, inhibited the invasion of metastatic breast cancer cells in3D assay systems. These studies provide first-in-field evidence forthe utility of small peptides in antagonizing GF:ECM-mediatedbiologic functions and present data demonstrating the potentialof these peptide antagonists as novel therapeutics.Mol Cancer Ther;15(7); 1602–13. �2016 AACR.

IntroductionThe insulin-like growth factors IGF-I and IGF-II form multi-

protein complexes with the extracellular matrix (ECM) proteinvitronectin (VN), with IGF-II binding VN directly (1) whereasIGF-I binds VN via IGF-binding proteins (IGFBP) -2, -3, -4, and-5 (2). Although biochemically validated in vitro, the physicalinteraction of VN with IGF family members remains to beinvestigated in human tissues. In breast tumor cells, IGF-I:

IGFBP:VN complexes have been demonstrated to coactivatethe IGF-I receptor (IGF-1R) and av integrins (VN receptors;refs. 3, 4) to stimulate cell migration and proliferation andpromote the transition of cancer cells to a therapy-resistant andmetastatic phenotype (5).

Current strategies targeting the IGF system in disease states suchas cancer include monoclonal antibodies directed against thereceptor, tyrosine kinase inhibitors or anti-ligand (anti–IGF-I)antibodies (6). These, however, have proven to be unsuccessful inlarge-scale clinical trials (7).We contend these strategies, althougheffectively targeting and blocking the IGF-1R, may fail becausethey overlook the critical contribution of the tumor ECM on IGFfunction and on tumor behavior. For instance, ligand occupancyof integrins (receptors for ECM proteins) promotes phosphory-lation and activity of the IGF-1R (8) and affects IGF and integrinsignaling mediated by IRS-1, Shc, PI3K, and MAPK (9). There isalso significant evidence that growth factors and ECM proteinsinteract (10), facilitating local storage of growth factors (11) in a"bioavailable" form. This localizes ligands in close proximity totheir cognate cell surface receptors, thereby prolonging the acti-vation of downstream signaling cascades (12), and in turn,enhancing growth factor-induced biologic functions. Destabili-zation of this enhancement may be critical for successful thera-peutic approaches.

Because IGFBPs (13), IGFs (14), and the matrix protein VN(10, 15) are all present and overexpressed in the microenvi-ronment of many tumor types, ECM-associated VN may formcomplexes with soluble IGFBPs/IGFs, exposing cells to concen-trated foci of growth factors (16). Interestingly, deposition ofVN into the ECM is enriched in areas surrounding cancers(17). This suggests that targeted disruption of the interactionbetween IGFBPs/IGFs and ECM-deposited VN may confer ther-apeutic benefit in IGF-responsive cancer types. This approach

1Queensland University of Technology, Tissue Repair and Regenera-tion Program, Institute of Health andBiomedical Innovation, Brisbane,Queensland, Australia. 2Roche Innovation Center, Pharma Researchand Early Development, Basel, Switzerland. 3Department of Immu-nology, Genetics and Pathology Science for Life Laboratory, BMC,Uppsala University, Uppsala, Sweden. 4Australian Prostate CancerResearch Centre - Queensland, Institute of Health and BiomedicalInnovation, Translational Research Institute, Brisbane, Queensland,Australia. 5Cancer Immunology, Department of Biomedicine, Univer-sity Hospital Basel, Basel, Switzerland.

Note: Supplementary data for this article are available at Molecular CancerTherapeutics Online (http://mct.aacrjournals.org/).

Current address for A.S. Kashyap: Cancer Immunology, Department of Biomed-icine, University Hospital Basel, Switzerland; and current address for D.I. Lea-vesley and Z. Upton, Institute of Medical Biology, Agency for Science, Technol-ogy and Research (A�STAR), Singapore.

Corresponding Authors: Abhishek S. Kashyap, Department of Biomedi-cine, University Hospital Basel, Hebelstrasse 20, Basel 4056, Switzerland.Phone: 0041612652355; E-mail: [email protected]; and Brett G.Hollier, Australian Prostate Cancer Research Centre-Queensland, TranslationalResearch Institute, 37 Kent St, Woolloongabba QLD 4102, Australia. Phone:0061734437000; Email: [email protected]

doi: 10.1158/1535-7163.MCT-15-0907

�2016 American Association for Cancer Research.

MolecularCancerTherapeutics

Mol Cancer Ther; 15(7) July 20161602

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may be target-specific given that reduced VN immunoreactivityis evident in healthy tissues relative to tumors or peritumoralstroma (18).

We provide herein, for the first time, in vivo evidence of theIGFBP-3:VN interaction in breast cancer patient tissues.Moreover,these interactions were predominantly localized to tumor cellclusters and the stroma surrounding tumor cells. We also provideevidence that small molecular peptides can antagonize the for-mation of the IGF-I:IGFBP-3:VN complex and its stimulation ofdownstreameffects such as cell proliferation andmigration. Thesedata suggest that this novel strategy, of using peptide antagoniststo disrupt the association of IGFs with ECM-deposited VN,warrants further examination in neoplasia where the IGF systemis implicated.

Materials and MethodsStudy design

Studies were designed firstly to detect the presence and local-ization of VN:IGFBP-3 complexes in vivo. Upon confirmation,whole protein or peptide mimics were used to disrupt the IGF-I:IGFBP-3:VN complexes and their effects on IGF-I-stimulated cellfunctions were investigated in vitro. The in situ Proximity LigationAssay (PLA) was used to detect VN:IGFBP-3 interactions in breastcancer patient samples and in xenograft tissues. To detect thespecific IGFBP-3:VN interaction regions, peptide arrays coveringthe entire VN as well as the IGFBP-3 sequence were utilized. Onthe basis of the data acquired from the peptide arrays, in silicomodeling was performed to map the VN-binding sites on IGFBP-3. Peptides were designed on the basis of the IGFBP-3:VN inter-action sequences and their ability to competitively displace IGF-I:IGFBP-3 from VN was assessed, in addition to testing their effectson IGF-I:IGFBP-3:VN-stimulated cell proliferation, migration,and invasion in 2D and 3D assay systems. Experiments werereproduced in three independent experiments as indicated in thefigure legends. Each of these independent experiments tested eachtreatment in triplicate. One-way ANOVA followed by the Tukey'spost hoc test was performed to determine statistically significantdifferences. This study was not randomized.

MaterialsPlasma purified human VN was purchased from Promega.

Recombinant human IGF-I, IGFBP-5, IGFBP-3, IGF-II, L27-IGF-II, and rabbit anti-IGFBP-5 antibody were purchased from Gro-Pep. Growth factor-reduced (GFR), phenol red-free Matrigel wasobtained from BD Biosciences. Phalloidin and DAPI stains andAlamarBlue reagent were purchased from Life Technologies. Rab-bit anti–IGF-II antibody and rabbit anti-NuMA antibody werefrom Abcam and rabbit anti–IGFBP-3 antibody was obtainedfrom Prof Robert Baxter (Kolling Institute of Medical Research).Antibodies for phospho-AKT (Ser473; clone D9E), phospho-ERK1/2 (Thr202/Tyr204; clone E10), phospho-IGF-1Rb (Tyr1135/1136; clone 19H7), AKT and ERK 1/2 were purchased from CellSignaling Technology. Rabbit anti–IGF-1Rb and mouse anti-VNantibody were from Santa Cruz Biotechnology. Rabbit anti-vWFantibodywas obtained fromDAKO. Peptides were obtained fromMimotopes.

Cell linesThe MCF-7 weakly metastatic human breast carcinoma, the

MDA-MB-231 highly metastatic breast cancer and the MCF-10A

transformed normal mammary epithelial cell lines wereobtained from the ATCC in 2009 and were maintained asdescribed before (3). The identity of these cell lines wasauthenticated in 2013 using short tandem repeat profiling (byCellBank Australia).

Tissue samples and in situ PLAFormalin-fixed paraffin-embedded (FFPE) tissues obtain-

ed from primary tumor xenograft of immortalized humanmammary epithelial V12H-Ras transformed cells (HMLER)overexpressing FOXC2 (HMLER-FOXC2) were generouslygifted by Dr Sendurai Mani (M.D. Anderson Cancer Center).Briefly, these HMLER-FOXC2 cells were prepared as describedpreviously (19) and one million cells were injected sub-cutaneously in the flank of nude mice. Once tumors were1,000 mm3, mice were euthanized and tumors collected asdescribed previously (19). Tissue slices were cut to 5-mmthickness and stored. All mouse procedures were approvedby the Animal Care and Use Committees of the M.D. Ander-son Cancer Center and performed in accordance with Insti-tutional policies. Human tissue microarrays (TMA) incorpo-rating 14 breast cancer and 4 normal breast patient tissueswere purchased from US Biomax Inc. (Cat# T086c, T087b).TMA information is outlined in Supplementary Fig. S1. Xeno-graft and TMA slides were deparaffinized and subjected toantigen retrieval for 20 minutes at 98�C in 10 mmol/L Tris-EDTA, buffer pH 9. The VN:IGFBP-3 interactions weredetected with in situ PLA, performed as outlined previously(20) using anti-VN (1:200) and anti–IGFBP-3 (1:200) anti-bodies. Sections were incubated with secondary antibodiesconjugated with oligonucleotide probes (rabbit secondaryantibody conjugated with a plus oligonucleotide and mousesecondary antibody conjugated with the minus oligonucleo-tide; purchased from Olink Bioscience) and the circulariza-tion oligonucleotide to facilitate the template for subsequentin situ rolling circle amplification. The amplified productswere detected using complementary Cy3-labelled detectionoligonucleotide probes. PLA signals were then analyzed withconfocal microscopy. A detailed description is contained inthe Supplementary Methods.

ImmunohistochemistryAntigen retrieval was performed on 5-mm-thick paraffin-

embedded sections of HMLER-FOXC2 xenografts in a decloak-ing chamber (Biocare Medical). Incubation at 95�C for 4minutes in 10 mmol/L Sodium Citrate buffer (pH 6.0; NuMAand vWF), or at 98�C for 20 minutes in Tris-EDTA buffer (pH9.0; VN and IGFBP-3), were used to unmask the antigens.Immunohistochemistry was performed using the MACH 4Universal Detection System (Biocare Medical) as describedpreviously (21). Briefly, primary antibodies against NuMA(1:200), vWF (1:400), VN (1:300), and IGFBP-3 (1:150) werediluted in Da Vinci Green Diluent (Biocare Medical), andincubated overnight on the sections at 4�C. Positive immu-noreactivity was visualized using the 3,3-diaminobenzidine(DAB; Biocare Medical) substrate, resulting in formation of abrown pigment. The sections were then counterstained withHematoxylin-G1 (HD Scientific), dehydrated and mounted,before the images were captured on an SCN400 Slide Scanner(Leica Microsystems).

Antagonists of the IGF-I:IGFBP-3:VN Complexes

www.aacrjournals.org Mol Cancer Ther; 15(7) July 2016 1603




Treatment approach for in vitro studies investigatingfunctional responses of IGF:VN complexes

The previously used substrate-bound approach (3, 5, 22) wasadopted to investigate the effects of L27-IGF-II and candidatepeptides on: the formation of IGF-I:IGFBP:VN complexes; com-plex-induced cell signaling; as well as monolayer cell viabilityand Transwell migration assays. IGF-I, IGFBP-3 and VN mixed1:1:1 at 10 nmol/L each (IGF-I:IGFBP-3:VN complex), with orwithout the indicated concentrations of L27-IGF-II or peptides,were added to the culture-ware and incubated for 3 hours toallow complex formation. Unbound proteins were removedfrom the wells by washing and the cells were then seeded intothe wells containing these immobilized proteins.

Solid-plate ELISAVN (10 nmol/L) was immobilized onto 96-well plates by

incubation at 37�C for 3 hours. IGFBP-3 (10 nmol/L) was addedto thesewells and incubated overnight at 4�C to enable formationof IGFBP-3:VN complexes, before the addition of the indicatedconcentrations of the competitive binders. The unbound proteinswere removed by washing and the remaining IGFBP-3 was mea-sured using an anti–IGFBP-3 antibody. HRP-labeled secondaryantibody was added and quantified colorimetrically using a TMB(3,30,5,50-tetramethylbenzidine) substrate solution (Millipore).The plates were incubated for 20 minutes before addition of thestop solution (0.25 mol/L HCl). The aborbance was recorded at450 nm.

Protein:peptide docking and modelingA homology model of the N- and C-terminal domains of

IGFBP-3 bound to IGF-I was generated using the PHYRE2 server(23) based upon a crystal structure of a hybrid IGFBP-4 (N-terminal domain)/IGFBP-1 (C-terminal domain) - IGF-I com-plex (PDB ID 2DSQ; ref. 24), and the residues identified fromIGFBP-3 peptide array screening were mapped out on its surfacein blue. The somatomedin B domain of vitronectin obtainedfrom PDB ID 1OC0 (25) and a de novo model of the VN P8peptide (RVNLRTRRVDTVDPPYPRS covering VN residues 425to 443) were aligned with these regions based on shape andcharge complementarity, and then docked in interactive molec-ular dynamics simulations. In each simulation, the backbone ofIGFBP was fixed and the side chains in the vicinity of thedocking peptide were free to move, whereas the VN peptideswere gently steered into place with the aid of a haptic interface.After docking, the entire complex was solvated in TIP3P water,neutralized with 0.15 mol/L NaCl, and equilibrated for 100nanoseconds in NAMD (26) at 300 K under standard NPTconditions.

Transwell cell migration assayMigration was assessed as described previously (5, 22). Briefly,

subconfluent cells were serum-starved for 4 hours and 6 � 104

cells suspended in serum-free medium (SFM) were seeded ontothe upper-surface of 8-mm-pore Transwell inserts (Corning Inc.)precoated on the under-surface with VN and IGF-I:IGFBP-3:VN,either alone or in combination with L27-IGF-II/peptides. After14 hours, the migrated cells were fixed using 3.7% paraformal-dehyde and stained with 0.01% crystal violet. The stain wasextracted using 10% acetic acid and absorbance was measuredat 595 nm using an optical plate reader.

Cell viability assayCell viability was assessed as described before (3, 5). Briefly,

log phase cells were serum-starved for 4 hours and 5 � 103 cellssuspended in SFM were seeded into 96-well plates precoatedwith VN and IGF-I:IGFBP-3:VN, either alone or in combinationwith L27-IGF-II/peptides. The cells were incubated for 72 hoursat 37�C, 5% CO2 and cell viability was assessed using theCellTiter 96 AQueous One Solution (MTS; Promega) as per themanufacturer's instructions.

Cell signaling and Western immunoblottingTotal protein was extracted from cells (following previously

published protocols; refs. 3, 5, 22), resolved on NuPAGE 4% to12% gradient SDS-PAGE gels (Life Technologies) and trans-ferred onto nitrocellulose membranes (Pall Corporation). Themembranes were probed with anti-phospho ERK1/2 (1:1,000),anti-phospho AKT (1:5,000) and anti–phospho IGF-1Rb(1:1,000) antibodies. The membranes were subsequently incu-bated with HRP-linked secondary antibody (1:5,000) anddeveloped using an enhanced chemiluminescence (ECL)ChemiDoc XRS detector system (Bio-Rad). The membraneswere stripped using Restore Plus stripping buffer (Pierce) andthe total protein levels were validated for equal loading usingthe respective anti-ERK1/2, anti-AKT or anti-IGF-1Rbantibodies.

3D On-Top Matrigel assayOne thousand MCF-7 or MDA-MB-231 cells suspended in the

3D growth medium (3D-GM; DMEM/F12 þ 2.5% growth factorreduced (GFR) Matrigel þ 1% FBS) were seeded onto a 100%GFR-Matrigel layer. After two days the spent medium for MCF-7cells was replaced with 3D-GM containing 10 nmol/L each of VN,IGFBP-3, IGF-I and L27-IGF-II or peptide combinations (indicatedconcentrations). Over the following 12 days the medium wasreplaced every three days with fresh 3D-GM containing theprotein treatments. MDA-MB-231 cells were treated from the startwith the protein and peptide treatments and assessed over 7 days,with treatments replaced every 2 days. The 3D-GMwithout addedproteinswas the negative control. Cell viability was assessed usingAlamarBlue (Life Technologies), colony size and shape was cal-culated using ImageJ software (NIH, Bethesda, MD), and colonymorphology of fluorescein diacetate (FDA)-stained live cells orfixed cells was assessed using fluorescence and confocal micro-scopy, respectively.

Analysis of 3D cell survival using AlamarBlue assaySpent medium was removed from the cells grown in 3D

Matrigel and replaced with fresh 3D growth medium containing5% (v/v) AlamarBlue solution. AlamarBlue (5%) added to wellswith 3Dgrowthmediumwithout cells served as blanks. The plateswere incubated for 4 hours at 37�C, 5%CO2before being assayed.Fluorescent signals (excitation 544 nm, emission 590 nm) weredetected using a fluorescence plate reader (BMG PolarStar; BMGLabtech).

Immunofluorescence of 3D cell spheroidsThe 3DMatrigel cultures were stained using fluorescent dyes.

All incubations were performed at room temperature. Cellsgrowing on the 100% Matrigel layer were fixed with 4%paraformaldehyde, washed with PBS:Glycine and then per-meabilized with 0.4% Triton-X. Cells were then blocked with

Kashyap et al.

Mol Cancer Ther; 15(7) July 2016 Molecular Cancer Therapeutics1604




5% BSA, washed with 0.1% PBS-Tween-20 (PBS-T) and incu-bated for 40 minutes in the dark with fluorescently conjugatedAlexaFluor 594 (red) or 488 (green) phalloidin (1:40, in PBS)to stain actin cytoskeleton and DAPI (1:10,000 in PBS)

for 10 minutes to stain nuclei. Images were recorded usingeither a fluorescence microscope (Nikon Eclipse TS100,Nikon) or a confocal microscope (Leica SP5 TCS X, LeicaMicrosystems).

Figure 1.

Ex vivo visualization of VN:IGFBP-3 interactions in breast cancer tissues. The in situ PLA was used to detect and visualise VN:IGFBP-3 interactions in primarytumors of HMLER-FOXC2 human xenograft tissue sections (A and B), as well as in human samples (C–F), including 2 cases of invasive ductal breastcarcinoma (C and D), medullary breast carcinoma (E), and normal breast tissue (F). The red fluorescence signal in Ai–Fi indicates locations where anti-VNand anti–IGFBP-3 bound in sufficiently close proximity to allow formation of a fluorescent PCR product in the in situ PLA. Each red spot represents asingle protein:protein interaction. DNA was stained with DAPI (blue). Serial sections were H&E stained (Aii–Fii) for histologic examination of the correspondingtissue sections probed for VN:IGFBP-3. The HMLER-FOXC2 tissue sections were immunohistochemically probed with anti-NuMA antibody to specificallydetect human cells (Aiii and Biii) and with anti-vWF antibody to detect endothelial cells from blood and lymphatic vessels (Aiv and Biv). Arrowheadsand arrows represent blood clot and tumor cell clusters, respectively. In all cases, scale bars, 50 mm.






Figure 2.

L27-IGF-II affects the binding of IGFBP-3:VN and IGF-I:IGFBP-3:VN-induced cell signaling and tumor cell functions. A, experimental model, indicating that IGF-I:IGFBP:VN (TRI) stimulates the IGF-1R and downstream functions. However, in the presence of L27-IGF-II, IGF-I:IGFBP-3 would be unable to bind VN, hence unable togain access to the receptor. B, the IGFBP-3 remaining in VN-coated wells post L27-IGF-II was measured using anti–IGFBP-3 antibody. The asterisks indicatetreatments significantly different to the VNþBP3 control. C, the levels of phosphorylated proteins at indicated times in MCF-7 cells were determined by immunoblotanalyses. The membranes were re-probed to determine total protein levels. D, MCF-7 cell viability was assessed over 72 hours using MTS. � , treatmentssignificantly different to VN controls. Hashes indicate treatments significantly different to TRI (#, P < 0.05). E, Transwell cell migration assessed over 15 hoursin response to TRI � increasing doses of L27-IGF-II was represented as the percentage of stimulation over VN control. (Continued on the following page.)

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ImageJ analysis for spheroid size and shape calculationsOnce the 3D cultures were fluorescently stained (with FDA)

and images recorded, they were subjected to analyses viaImageJ. The threshold value above/below which the spheroidsare considered were set to include small spheroids but to omitdebris accumulated within the 3D culture (detected as smalldots within the image). The scale was set in mm, after which thesoftware converted pixels into length (in mm). The number ofspheroids and the size of each spheroid (in mm2) within theimage were calculated. In addition to the spheroid size andnumber, the shape-factor ranging from 0–1 was also measured,with value of 1 assigned to the most circular spheroids. Threesuch images from 3 different quadrants within the well wererecorded for each treatment, with treatments tested in dupli-cates in each experiment.

Peptide arraysVN and IGFBP-3 peptide arrays were obtained from Kinexus

Corp. For the VNpeptide array, the VNprotein sequence (UniProt#P04004), excluding the signal peptide (residues 1–19), wasdivided into 16-mer overlapping peptides with 3-residue frame-shifts and spotted onto cellulose membranes. Similarly, theIGFBP-3 protein sequence (UniProt #P17936), excluding thesignal peptide (residues 1–27), was divided into 12-mer over-lapping peptideswith 3 amino acid frameshifts. For details refer toSupplementary Figs. S2 and S3. Replicate VN peptide arrays wereincubated overnight at 4�C with one of the bait ligands, IGFBP-3,IGFBP-5, or IGF-II (1 mg/mL) diluted in casein blocking buffer(MBS; Sigma-Aldrich), whereas the IGFBP-3 peptide array wasincubated with 1 mg/mL VN. Binding of ligands to immobilizedpeptideswas detected using anti-IGFBP-3 (1:10,000), anti-IGFBP-5 (1:10,000) or anti-IGF-II (1:5,000) antibodies (for VN peptidearrays) or with anti-VN antibody (1:5,000; for the IGFBP-3peptide array). Membranes were incubated with horseradishperoxidase (HRP)–conjugated secondary antibody (1:15,000)and developed using the ECL ChemiDoc XRS system. Molecularinteractions were identified using TotalLab Quant software(TotalLab Ltd.) and signal intensities were quantified as fold-change in spot intensity over the average intensity of the negativecontrols.

ResultsVisualization of VN and IGFBP-3 interactions in breasttumor tissues

Although strong evidence exists in vitro for the interactionof VNwith IGFBP-3:IGF-I, limited in vivo evidence is available forsuch an interaction. To address this question, we probed fixedhuman breast cancer and primary tumor xenograft tissues usingthe in situ PLA. In situ PLA produces a fluorescent spot only wherethe two targetmolecules are within 30 nmof each other. BecauseIGF-I is known to bind VN through IGFBP-3 (2) we elected to

investigate the IGFBP-3:VN interaction. Human TMAs derivedfrom 14 breast cancer and 4 normal patient tissues (details inSupplementary Fig. S1), as well as tumor tissues collected fromHMLER-FOXC2 xenografts (19), were analyzed with in situ PLA.In the xenograft tissues, the PLA signal for VN:IGFBP-3 wasobserved in the peri-cellular stroma (Fig. 1A, i), adjacent to theHMLER-FOXC2 human tumor cells which react positively tohuman-specific nuclear mitotic apparatus (NuMA) antibody(Fig. 1A, iii). VN:IGFBP-3 was also detected around NuMA-positive human tumor cells (Fig. 1B, i and iii) proximal tovWF-positive blood vessels (Fig. 1B, iv) and in blood clots(arrowhead in Fig. 1B, i and iv). The distribution of VN:IGFBP-3 PLA immunoreactivity correlated well with the indi-vidual immunohistochemical reactivity of VN and IGFBP-3(Supplementary Fig. S4). Various breast cancer tissue samples,including invasive ductal, invasive lobular, medullary andmucinous carcinoma, immuno-reacted positively for VN:IGFBP-3. The PLA immunoreactivity and its association withtumor/tumor stroma is outlined in Supplementary Fig. S1.Comparison of the PLA immunoreactivity with hematoxylinand eosin (H&E) staining indicated that the VN:IGFBP-3 com-plex was tumor associated (Fig. 1D and E) and interestingly, wasmore strongly associated with tumor cell clusters invading intothe stroma (Fig. 1C and D; arrows). Evidence for stromalimmunoreactivity is also present in these tumor samples. Inthe normal breast tissue samples, VN:IGFBP-3 immunoreactiv-ity was localized primarily around blood vessels (Fig. 1F). NoPLA signal was seenwhen single-antibody controls were applied(Supplementary Fig. S5G and S5H), thereby validating the PLAapproach. Single-channel images for each section assessed usingPLA are reported in Supplementary Fig. S5.

L27-IGF-II prevents the association and functional effects ofthe IGF-I:IGFBP:VN complex

Unlike IGF-I, IGF-II is able to directly bind to VN with asubstantially higher affinity than IGFBP-3 or -5 (2), making it apromising model for use in competition assays probing theeffects of preventing IGF-I:IGFBP:VN association. Because wild-type IGF-II can also activate the IGF-1R (Supplementary Fig. S6;ref. 27), we used the Y27L-mutant (L27-IGF-II), which hasapproximately 100-fold reduced affinity for the IGF-1R (28)with undiminished affinity for VN (strategy outlined inFig. 2A). Dose-dependent displacement of IGFBP-3 from pre-formed IGFBP-3:VN complexes was observed in the presenceof L27-IGF-II (Fig. 2B). Similar responses were observed whenL27-IGF-II was added simultaneously to IGFBP-3 and VN (datanot shown). This implies that L27-IGF-II disrupts pre-formedIGFBP-3:VN complexes and may also prevent new IGFBP-3:VNassociations. The disruption of the IGFBP-3:VN interaction wasassociated with a reduction in IGF-I:IGFBP:VN-stimulated acti-vation of the IGF-1R, AKT and ERK1/2 in MCF-7 breast cancercells (Fig. 2C). The presence of L27-IGF-II (VN þ L27-IGF-II) did

(Continued.) � , significant differences compared to TRI; n.s., not significant. F, at day 14, MCF-7 cells in 3D Matrigel were either fixed and probed foractin (green) and nucleus (blue) for confocal microscopy (left) or incubated with FDA (green) for fluorescence microscopy (middle). Representativeimages are shown. Fluorescence microscopy images were converted to single color images (right) for size quantification using ImageJ; scale bar, 100 mm.G, cell viability was assessed at day 14 using AlamarBlue dye. H, the cross-sectional area of spheroids is represented as box plots depicting mediansand 80% of the population. Extremities represent minimum and maximum values, with the spheroid size distribution represented under each treatment.In all cases, � , P < 0.05; �� , P < 0.01; and n ¼ 3 biologic replicates. Error bars, SEM. 3D-GM, DMEM/F12 growth medium containing 1% serum and 2.5%Matrigel; FCS, medium containing 10% FCS; SFM, serum-free medium.






Figure 3.

Identification and functional characterization of VN peptides covering the VN:IGFBP-3/-5 and VN:IGF-II interaction motif. A, VN peptide arrays incubatedwith recombinant IGFBP-3, IGFBP-5, or IGF-II ligands are depicted. Membranes were spotted with positive (A1-4, J13-16) and negative (A5-6, J11-12) controls forantibody detection. B, IGFBP-3 peptide array was incubated with plasma purified VN and probed with anti-VN antibody (A1-3, I3-5 ¼ positive and A4-5, I6-7 ¼negative controls). Positive peptide:ligand interactions are indicated by dark opaque circles. The VN and IGFBP-3 primary sequence schematic details the approximatelocation of the interaction motifs (numbered 1–8 on VN and 1–5 on IGFBP-3). C, VN peptides (identified from A) docked to an IGFBP-3:IGF-I complex (modeled fromPDB ID: 2DSQ) and equilibrated for 100 nanoseconds. The surface representation of N- and C-terminal domains of IGFBP-3 (BP-N, BP-C) is depicted in red, with blue-colored regions indicating IGFBP-3 residues experimentally identified (in B) to be binding to VN (IGFBP-3 motifs 1 and 5). (Continued on the following page.)

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not enhance signaling. L27-IGF-II also caused a dose-dependentdecrease in IGF-I:IGFBP:VN-stimulated proliferation (Fig. 2D)and migration (Fig. 2E) in MCF-7 cells. Real-time assessment ofcell migration (xCELLigence analyzer, Roche) revealed similarreductions in migration of MCF-7 and the non-tumorigenicMCF-10A cells (Supplementary Fig. S7). These data suggestthat L27-IGF-II competes with the binding of IGF-I:IGFBP-3 toVN, and alters IGF-I-mediated cell signaling and downstreambiologic functions. When tested in combination with VN,L27-IGF-II had no effect on cell signaling or cell function (Fig.2C and D).

To better represent the in vivo microenvironment the 3DMatrigel On-Top assay (29) was used. Here, the unboundproteins remain in the medium throughout the assay, incontrast with the 2D "substrate bound" studies in whichIGF-I:IGFBP-3 displaced by L27-IGF-II is removed by washing.MCF-7 cells formed tumor spheroids in this assay system asreported previously (30). In the presence of IGF-I:IGFBP-3:VNcomplexes larger spheroids developed (average cross-sectionalarea of 2.2 � 104 mm2) and exhibited increased cell viabilitycompared to VN alone (average of 4.6 � 103 mm2). This IGF-I:IGFBP-3:VN-induced gain in spheroid size and viability wassignificantly reduced (P < 0.05) by the addition of L27-IGF-II(30 nmol/L; average of 1.6 � 104 mm2; Fig. 2F–H). Thisindicates that within the composite in vivo-like microenviron-ments the effects of IGF-I:IGFBP:VN complexes are amenable toinhibition via antagonism of the IGFBP:VN interaction.

Identification and functional characterization of VN peptidesthat inhibit the binding of IGFBPs to VN

To overcome challenges associated with the use of wholeproteins as therapeutics, and to avoid potential residual acti-vation of the IGF-1R by L27-IGF-II, short peptides replicatingthe antagonistic functions of L27-IGF-II were designed. Thepeptide design was guided by the identification of amino acidsequences critical for the IGFBP:VN and IGF-II:VN protein:protein interactions.

Arrays of 16-mer peptides covering the entire VN amino acidsequence (UniProt #P04004) were incubated with one of thefollowing "bait" proteins: IGF-II, IGFBP-3 or IGFBP-5 (detailsfor VN peptide array in Supplementary Fig. S2). The capture ofbait proteins by full-length VN (Promega) spotted onto themembrane was first confirmed using bait-specific antibodies(Supplementary Fig. S8). In addition to the unique bindersidentified for each bait ligand (Fig. 3A, dark spots), a cluster ofpeptides (circled blue in Fig. 3A) common in all three "bait"ligands (IGF-II, IGFBP-3, and IGFBP-5) was identified from thepeptide array. This was localized to the C-terminal of VN,spanning residues 428–443 within the hemopexin-like (HX)domain. In addition, a unique IGF-II–binding site (circledorange in Fig. 3A), adjacent to the previous motif, was detectedspanning residues 389–403 within the heparin-binding domain

(HBD) of VN (also for L27-IGF-II, Supplementary Fig. S9). Toinvestigate the complementary sequences of IGFBP-3 involvedin the binding to VN, a similar set of peptide arrays spanning theIGFBP-3 sequence (IGFBP-3 peptide array sequence details inSupplementary Fig. S3) were incubated with VN. VN was foundto bind to 5 different regions within IGFBP-3 (Fig. 3B andSupplementary Fig. S10). Taking into account the data fromboth the VN and IGFBP-3 peptide arrays, a tentative in silicomodel was created for the binding of VN to the IGFBP-3:IGF-Icomplex (Fig. 3C and D). We generated a homology model ofthe IGFBP-3:IGF-I complex based upon the crystal structure of ahybrid IGFBP-4:IGFBP-1:IGF-I complex (31), and applied inter-active molecular dynamics simulations to find favorable dock-ing sites for the identified VN peptides in proximity to theresidues highlighted in the IGFBP-3 peptide array. To assessstability of the proposed interactions, we equilibrated thestructure for 100 ns in explicit solvent under standard NPTconditions. In the resultant model (Fig. 3C and D) both VNpeptides (blue and purple ribbons) bind to a composite sur-face formed by the N- and C-terminal domains of IGFBP-3,well outside of the IGF-binding region. Informed by the in silicoand the peptide array data, VN peptides LFSSEESNLGANNYD-DYRMDWL (referred to hereafter as P7) and RVNLRTRRV-DTVDPPYPRS (P8) covering the IGFBP-3 consensus motifs weresynthesized. A non-binding negative control peptide KTYLFK-GSQYWRFEDG (Pneg) was also designed from a VN regiondistant to the binding of the ligands tested.

Functional studies revealed P7 and P8 to reduce the amount ofIGFBP-3 bound to VN in a dose-dependent manner (Fig. 3E,complex disruption), and with a concomitant reduction in IGF-I:IGFBP:VN-induced breast cancer cell migration (Fig. 3F, cellmigration). No significant effect was observed from the Pnegcontrol (Fig. 3E and F). These data indicate that the P7 and P8peptides inhibit the enhanced cell migration elicited by IGF-I:IGFBP:VN complexes over that of VN alone by competitivelydisplacing IGF-I:IGFBP from VN.

P7 and P8peptides inhibit growth and invasionof breast cancercells in a 3D matrix

The effects of the P7 and P8 peptides (90 nmol/L dose) onIGF-I:IGFBP-3:VN-induced tumor-spheroid growth of MCF-7cells and the invasion of the highly metastatic MDA-MB-231cell model was evaluated within a 3D ECM environment, usingthe approach described for the L27-IGF-II studies above.

The addition of the P7 or P8 peptide to MCF-7 cells culturedwith the IGF-I:IGFBP-3:VN complex significantly reducedtumor spheroid size (Fig. 4A and B). Although the P7 peptideyielded a significant reduction (P < 0.05) in spheroid size(average cross-sectional area of 13.3 � 103 mm3) comparedwith the controls (average of 16.9 � 103 mm2), the effect of P8was markedly stronger (average of 7.4 � 103 mm2; P < 0.01). Inthe P8 treatment, >80% of the spheroids were �10,000 mm2,

(Continued.) IGF-I is in yellow. VN motifs 1 (purple) and 8 (blue) are represented as ribbons. D, the residues from the VN motifs that made stableinteractions with IGFBP-3 are depicted. Blue dotted lines indicate hydrogen bonds. VN residues are italicized and IGFBP-3 residues arenonitalicized. E, the amount of IGFBP-3 remaining in VN-coated wells after peptide treatments was assessed using anti–IGFBP-3 antibody.Data were curve fit using the non-linear log(inhibitor) versus the response model. � , comparison of treatment versus VNþBP-3 control. F,increasing concentrations of peptides (P7, P8, and Pneg) were supplemented to wells pre-bound with IGF-I:IGFBP-3:VN (TRI; 10 nmol/L) andcell migration was assessed over 15 hours using the Transwell assay. � , comparison of treatment versus TRI. � , P < 0.05; ��, P < 0.01 and n ¼ 3 biologicreplicates; error bars, SEM.






compared to the majority of the control spheroids being�20,000 mm2 (Fig. 4B). Combining the P7 and P8 peptidesdid not result in additional inhibitory effects (data not shown).

In contrast with the spherical morphology of MCF-7 cells, theMDA-MB-231 cell line (a prototypic model for the metastaticbreast cancer subtype) displayed highly invasive dendritic net-works in 3D Matrigel On-Top assays in the presence of the IGF-I:IGFBP-3:VN complex (Fig. 4D), as previously described (30).The addition of the P8 peptide, however, caused a dramaticchange in the morphology of MDA-MB-231 colonies to morespherical, less invasive spheroids (Fig. 4D). The invasiveness ofthe individual colonies was estimated using shape factor anal-ysis in ImageJ, where a value of 1 indicates a perfect circle andthe value approaches zero as the perimeter/area ratio increases(Fig. 4E and F). The MDA-MB-231 cell viability was coinci-dently inhibited by the P8 peptide in a dose-dependent manner(Fig. 4F).

Discussion

IGF-I has been implicated in conferring increased motilityand survival advantage in certain tumor cells (27), whereas theexpression of activated IGF-1R has been reported in invasivebreast cancer (32). In addition, increased deposition of VN intothe ECM at the leading edge of tumors has been observed (15).Of relevance, IGF-I has been shown to indirectly bind VN viaIGFBP-2, -3, -4, and -5 (2) to form heterotypic protein com-plexes that promote biologic functions associated with thetransition of breast cancer cells to a therapy-resistant (5) andmetastatic phenotype (3, 5). Although biochemically validated,before this study the formation of these IGF-I:IGFBP:VN com-plexes in vivo remained undocumented. Using the PLA wereport for the first time the presence of IGFBP-3:VN complexesin vivo in human tissue sections (Fig. 1). Given that IGF-I ispredominantly bound to IGFBPs (especially IGFBP-3) in vivo(33), and that IGF-I:IGFBP binding is unaffected by complex-ation to VN (2), it is reasonable to presume that IGF-I is boundto the IGFBP-3:VN complexes. Of note, we localized the IGFBP-3:VN complexes to the invasive front of tumor cell clusters, aswell as near blood vessels within the tumor. This is in concor-dance with evidence that VN and IGFBP-3 are abundant in thecirculation and become deposited in tumor tissues (16, 33).Interestingly, the VN receptor, avb3, is expressed at high levelsin endothelial cells during angiogenesis (34) and in addition toproviding a provisional ECM for invading tumor cells, VNlocalizes functional MMPs to the tumor–ECM boundary (35)allowing tumor cells to penetrate constraining interstitial tissueECM. We propose that in tumors, particularly in regions inclose proximity to blood vessels, the increased accumulation ofVN/IGFBP/IGF complexes may promote primary tumor dis-semination and invasion into the perivascular tissue.

The failure to demonstrate therapeutic benefit of existingIGF-1R inhibitors in clinical trials, together with the paucity ofbiomarkers able to predict their efficacy, has prompted a re-evaluation of strategies to target the IGF axis (36, 37). Wetherefore explored a novel approach directed at inhibitingaccessibility of IGF ligands to the IGF-1R by antagonizing theassociation of IGFs with components of the ECM. BecauseIGFBP-3 tethers IGF-I within the ternary IGF-I:IGFBP:VN com-plex, we hypothesized that inhibition of the IGFBP-3:VN inter-action could be used as a strategy to reduce the reservoir of

ECM-bound IGF-I (schematic in Fig. 2A). As we have previouslyreported that IGF-II binds to VN with a higher affinity thanIGFBP-3 (2), we used the non–IGF-1R-binding mutant IGF-IIanalogue L27-IGF-II as a proof-of-concept antagonist to assessthe feasibility of this approach.

Data presented herein for the first time demonstrates that L27

-IGF-II can displace IGFBP-3:IGF-I bound to VN, and therebyantagonize IGF-I:IGFBP:VN-induced breast cancer cell prolifera-tion and migration (Fig. 2D–H). In our monolayer cell cultureassays, all unbound protein (including the IGFBP-3:IGF-I dis-placed by L27-IGF-II) is removed by washing before cell culture.This is reflected in our observation of reduced activation of theIGF-1R and the downstreamMAPK/ERK and PI3-K/AKT signalingintermediates in thepresence of L27-IGF-II (Fig. 2C). Because it hasalready been shown that all three components of the complex(IGF-I:IGFBP:VN) are required to stimulate enhanced cell func-tions (3, 5, 38), VNþIGF-I or VNþIGFBP-3 controls were notincluded. In contrastwithmonolayer assays, theprotocol adoptedfor the 3D Matrigel assays allowed displaced proteins to remainin the culture system during the entire assay period. Despite theconstant presence of IGF-Iwithin thewells, L27-IGF-II significantlyinhibited IGF-I:IGFBP:VN-stimulated growth of MCF-7 breastcancer cells. This suggests that preventing access of IGF-I to theECM may hinder its ability to activate the IGF-1R and supportsthe concept that antagonism of ternary IGF-I:IGFBP:VN comp-lex formation can inhibit breast cancer cell migration andproliferation.

In contrast with whole proteins or antibodies, bioactivepeptides represent a rich class of pharmaceuticals as they aresmall, less immunogenic, and easily modified to increase sta-bility in vivo (39). Short peptides were therefore designed tomimic the antagonistic functions of the full L27-IGF-II protein.Because of the lack of well resolved crystal structures andliterature defining the binding sites on VN for IGFBPs (and/orIGF-II) and vice versa, we used a peptide array strategy similar tothat used by others (40, 41) to identify the key protein:proteininteraction sites. We report for the first time that IGFBP-3,IGFBP-5 and IGF-II appear to preferentially bind VN near itsC-terminus (residues 428–443) within the hemopexin-2 (HX-2)domain (Fig. 3). A tentative homology model of the completeVN protein in its active state (10) suggests that the HX-2 domainis highly accessible to other proteins. Indeed, this fits well withthe observation that several other proteins, including PAI-I,collagen, heparin (42), and now IGFBP-3, IGFBP-5 and IGF-II,bind to this region of VN. The differences in their affinities forVN and the tissue specificity of these ligand:VN interactionsremain to be investigated.

When short peptides spanning the key VN residues identifiedfrom the peptide array weremodeled in silico and docked onto theIGFBP-3:IGF-I complex, a set of core interactions remained stablefor at least 100 ns. In this model, VN binds a composite surfaceformed by the N- and C-terminal domains of IGFBP-3, with nointeraction of VN with the bound IGF-I. Gui and colleagues (22),who first described IGFBP-3 interacting with fibronectin (FN),reported a similar finding where they demonstrated no effect ofexcess IGF-I on IGFBP-3:FN binding. Moreover, the bindingaffinity of the immobilized IGFBP-3:FN complex for [125I]IGF-Iwas similar to that of IGFBP-3 alone. All the same, mutationalanalyses are required to confirm that these IGFBP-3:VN interac-tion regions we propose are indeed correct. Nevertheless, thisnovel information provides intriguing targets for therapeutic

Kashyap et al.





intervention and suggests that antagonists designed at targetingthe VN:IGFBP-3 interaction may not alter IGF-I:IGFBP-3associations.

Similar to our results with L27-IGF-II, rationally designedpeptides encompassing the consensus sequences of high-bind-ing regions from the peptide array were shown to displaceIGFBP-3 from VN (Fig. 3D) and inhibit IGF-I:IGFBP-3:VN-stimulated growth of the MCF-7 cell line (Fig. 4A–C), aprototypical luminal breast cancer model, and the invasive-ness of the highly metastatic MDA-MB-231 cell model(Fig. 4D–F). These key findings from the 3D Matrigel assaysupport our hypothesis that matrix/VN-bound IGF-I contri-butes the majority of IGF-mediated responses observed in thebreast cancer model and that by perturbing these interactionsthe biologic effects elicited by IGFs are altered. This conceptnow needs to be examined in relevant in vivo preclinicalmodels where the peptides could be delivered intratumorally;this will ascertain the potential of these peptides as novel anti-IGF therapeutics. In vivo toxicity also needs to be investigated.This will enable assessment of the impact of these peptideantagonists on the "normal" physiologic activities of IGFBPsand IGF-I. However, we anticipate that these peptide antago-nists should not perturb normal IGFBP:IGF-I–binding inter-actions, because we have provided evidence herein (Fig. 3C)that these VN peptides bind IGFBP-3 at sites not involved inIGF-I binding. However, the impact of the IGFBP-3–bindingpeptides on IGF-I-independent effects of IGFBP-3 will need tobe investigated to fully understand the off-target effects ofthese peptides. In addition, modification to the peptide toimprove stability (cyclization, functionalization; ref. 43) andto increase target specificity, for instance antibody-peptidechimeras to direct them to tumors (Her2- or Folate receptormAb-P8 peptide conjugates; ref. 43), could be consideredbefore their entry into the clinical setting.

Ninety percent of breast tumors express the IGF-1R (44)which, when compared with the approximately 20% and 25%of breast cancers that are HER2þ (45), represents a large cohortof patients that may benefit from anti-IGF therapies. Recently,hyperactivated IGF-1R was reported in breast cancer stemcells (46) and has been associated with resistance to chemo-therapy and targeted molecular therapy (35, 43). Moreover,upregulation of an IGF-I gene signature has been reportedin the majority of luminal-B breast tumors [estrogen receptorpositive (ERþ), invasive and highly proliferative; ref. 47]. Hence,anti-IGF therapies, such as the one proposed herein, may haveparticular clinical utility in patients with IGF-1Rþ/ERþ tumors,

Figure 4.

Growth and phenotype of MCF-7 and MDA-MB-231 cells cultured in 3DMatrigel are altered in response to VN-peptides. A and D, growthcharacteristics of MCF-7 cells (A) and MDA-MB-231 cells (D) in 3DMatrigel cultures were assessed in the presence of peptides.Representative images for the 90 nmol/L peptide dose are depicted ofcultures incubated with FDA observed under phase contract (left) and

fluorescence microscope (middle). The right depicts the correspondingsingle-color images converted using ImageJ; scale bars, 100 mm. B,cross-sectional area of MCF-7 spheroids was calculated (using ImageJ)and data are represented as box plots depicting medians and 80% of thepopulation. Extremities represent minimum and maximum values. Sizedistribution of the spheroids is graphically depicted below the box-plot. E,values for circularity (Shape-factor values of 0 to 1) were assigned to theMDA-MB-231 colonies (using ImageJ) and data are represented as thepercentage of distribution of the colony circularity, with 0 being leastcircular. C and F, cell viability was assessed using the AlamarBlue dyeand the fluorescence intensities measured as a result of the metabolicactivity of the cells was plotted; error bars, SEM. In all cases, asterisksindicate treatments significantly different to the TRI control (� , P < 0.05;�� , P < 0.01; n ¼ 2 biologic replicates).






or tumors that exhibit resistance to current therapies where theIGF-1R is implicated. This new class of peptide-based therapeuticstargeting IGF-I:IGFBP:matrix interactions, as proposed herein,may be a feasible alternative to not only inhibit the progressionof breast cancer and other carcinomas, but also for hyperpro-liferative disorders where such interactions are implicated indisease progression.

Disclosure of Potential Conflicts of InterestZ. Upton and D.I. Leavesley have provided consulting services to Tissue

Therapies Ltd., a company spun out from the Queensland University ofTechnology. G.K. Shooter has an adjunct appointment at the QueenslandUniversity of Technology and is employed by Tissue Therapies Ltd. TheQueensland University of Technology holds shares in Tissue Therapies Ltd.The Queensland University of Technology has filed a patent related to thepeptides outlined in this study (Patent#WO2014078896A1) and this has beenacquired by Tissue Therapies Ltd. A.S. Kashyap, G.K. Shooter, D.I. Leavesley,Z. Upton and B.G. Hollier are named inventors on this patent. D.I. Leavesley,Z. Upton, and B.G. Hollier have purchased shares in Tissue Therapies Ltd. Nopotential conflicts of interest were disclosed by the authors.

Authors' ContributionsConception and design: A.S. Kashyap, G.K. Shooter, D.I. Leavesley, Z. Upton,B.G. HollierDevelopment of methodology: A.S. Kashyap, G.K. Shooter, M. Sivaramakrish-nan, D.I. Leavesley, Z. Upton, B.G. HollierAcquisition of data (provided animals, acquired and managed patients,provided facilities, etc.): A.S. Kashyap, A. Shokoohmand, J. McGovern,G. Cane, O. S€oderbergAnalysis and interpretation of data (e.g., statistical analysis, biostatistics,computational analysis): A.S. Kashyap, T.I. Croll, Z. Upton, B.G. HollierWriting, review, and/or revision of themanuscript: A.S. Kashyap, G.K. Shooter,A. Shokoohmand, J. McGovern, M. Sivaramakrishnan, T.I. Croll, D.I. Leavesley,Z. Upton, B.G. Hollier

Administrative, technical, or material support (i.e., reporting or orga-nizing data, constructing databases): A.S. Kashyap, M. Sivaramakrishnan,B.G. HollierStudy supervision: A.S. Kashyap, O. S€oderberg, Z. Upton, B.G. Hollier

AcknowledgmentsThe authors wish to thank: Gemma A. Forgeard for assistance with solid-

plate ELISA; Leonore de Boer (Cell Imaging Facility, Institute of Health andBiomedical Innovation) for help with confocal microscopy; Anna Tauben-berger (Dresden University of Technology) for assistance with ImageJ soft-ware; and the Central Analytical Research Facility-Histology Division at theQueensland University of Technology for assistance with tissue sectioningand staining. The authors also thank Robert Baxter (Kolling Institute ofMedical Research, University of Sydney) for providing the anti-IGFBP-3antibody and Sendurai Mani (MD Anderson Cancer Center, Houston, TX)for providing the HMLER-FOXC2 xenograft tissues and Prof. Adrian Her-rington (QUT) and Prof. Erik W Thompson (QUT) for providing construc-tive feedback on the article.

Grant SupportThis work was supported by the Tissue Repair and Regeneration Program at

the Institute of Health and Biomedical Innovation, as well as Tissue TherapiesLtd. A.S. Kashyap received funding from a John Williams Cancer Fellowship(Queensland University of Technology) to support the in situ PLA studies. B.G.Hollier was supported by funding received from the Smart Futures Fund(Queensland State Government) and the Australian Government Departmentof Health.

The costs of publication of this article were defrayed in part by thepayment of page charges. This article must therefore be hereby markedadvertisement in accordance with 18 U.S.C. Section 1734 solely to indicatethis fact.

Received November 20, 2015; revised April 7, 2016; accepted April 28, 2016;published OnlineFirst May 9, 2016.

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Correction

Correction: Antagonists of IGF: VitronectinInteractions Inhibit IGF-I–Induced BreastCancer Cell Functions

In this article (Mol Cancer Ther 2016;15:1602–13), which appeared in the July 2016issue of Molecular Cancer Therapeutics (1), the School of Biomedical Sciences wasinadvertently omitted from the first and fourth listed affiliations in the article. Thecorrected affiliations are listed below. The authors regret this error.

1. Queensland University of Technology, Tissue Repair and Regeneration Program,Institute of Health and Biomedical Innovation, School of Biomedical Sciences,Brisbane, Queensland, Australia.

4. Australian Prostate Cancer Research Centre - Queensland, Institute of Health andBiomedical Innovation, School of Biomedical Sciences, Translational ResearchInstitute, Brisbane, Queensland, Australia.

Reference1. Kashyap AK, Shooter GK, Shokoohmand A, McGovern J, Sivaramakrishnan M, Croll TI, et al.

Antagonists of IGF: vitronectin interactions inhibit IGF-I–induced breast cancer cell functions. MolCancer Ther 2016;15:1602–13.

Published online December 2, 2016.doi: 10.1158/1535-7163.MCT-16-0643�2016 American Association for Cancer Research.

MolecularCancerTherapeutics

Mol Cancer Ther; 15(12) December 20163120

2016;15:1602-1613. Published OnlineFirst May 9, 2016.Mol Cancer Ther Abhishek S. Kashyap, Gary K. Shooter, Ali Shokoohmand, et al. Breast Cancer Cell Functions

Induced−Antagonists of IGF:Vitronectin Interactions Inhibit IGF-I

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AntagonistsofIGF:VitronectinInteractionsInhibit IGF-I ... · Abhishek S. Kashyap1,5, Gary K. Shooter1, Ali Shokoohmand1, Jacqui McGovern1, Manaswini Sivaramakrishnan2,Tristan I. Croll1,Ga€elle