Tumor Biology and Immunology

XIAP Regulation by MNK Links MAPK andNFkB Signaling to Determine an AggressiveBreast Cancer PhenotypeMyron K. Evans1,2, Michael C. Brown3, Joseph Geradts2, Xuhui Bao1,Timothy J. Robinson5, Mohit Kumar Jolly6, Peter B. Vermeulen7, Gregory M. Palmer4,5,Matthias Gromeier3, Herbert Levine6, Michael A. Morse4,8, Steven J. Van Laere7,9,and Gayathri R. Devi1,2,4

Abstract

Hyperactivation of the NFkB pathway is a distinct feature ofinflammatory breast cancer (IBC), a highly proliferative and lethaldisease. Gene expression studies in IBC patient tissue have linkedEGFR (EGFR/HER2)-mediated MAPK signaling to NFkB hyperac-tivity, but the mechanism(s) by which this occurs remain unclear.Here, we report that the X-linked inhibitor of apoptosis protein(XIAP) plays a central role in linking these two pathways. XIAPoverexpression correlated with poor prognoses in breast cancerpatients and was frequently observed in untreated IBC patientprimary tumors. XIAP drove constitutive NFkB transcriptionalactivity, which mediated ALDH positivity (a marker of stem-likecells), in vivo tumor growth, and an IBC expression signature inpatient-derived IBC cells. Using pathway inhibitors andmathemat-

ical models, we defined a new role for the MAPK interacting(Ser/Thr)-kinase (MNK) in enhancing XIAP expression and down-stream NFkB signaling. Furthermore, targeted XIAP knockdownand treatment with a MNK inhibitor decreased tumor cell migra-tion in a dorsal skin fold window chamber murine model thatallowed for intravital imagingof local tumorgrowthandmigration.Together, our results indicate a novel role for XIAP in themolecularcross-talk between MAPK and NFkB pathways in aggressive tumorgrowth, which has the potential to be therapeutically exploited.

Significance: Signaling by the MNK kinase is essential ininflammatory breast cancer, and it can be targeted to inhibitXIAP–NFkB signaling and the aggressive phenotype of this malig-nancy. Cancer Res; 78(7); 1726–38. �2018 AACR.

IntroductionThe primary cause of breast cancer morbidity and mortality is

metastasis to surrounding tissues and distant organs, a processdependent on hyperproliferation and hypermotility of cellsderived from the primary tumor. Among all breast cancer types,inflammatory breast cancer (IBC) is a highly aggressive subtype

characterized clinically by extremely motile tumor cell clustersthat exhibit localized dermal invasion and frequent lymph nodeinvolvement (1–3). Despite an aggressive multimodal treatmentregimen, tumor recurrence and metastatic progression are unmetchallenges in IBC patients (4).

Comparative gene expression studies from preclinical modelsand pretreatment patient samples have attempted to definemolecular profiles specific to IBC. They reveal highly activatedMAPK and NFkB transcriptional profiles associated withincreased proliferation in IBC compared with other locallyadvanced breast cancers (5–8); however, the mechanism for thelinkage between these two pathways in IBC tumors has not beendescribed. We sought to determine how EGFR-mediated MAPKactivation and NFkB hyperactivity were coordinated to enhancecancer cell survival and proliferation with the goal of elucidatingtargets for therapeutic intervention. Our previous studies haveshown that IBC tumor cells escape from various cell death andoxidative stress stimuli, including EGFR inhibition, throughupregulation of the X-linked inhibitor of apoptosis protein (XIAP;refs. 9–13). XIAP, through its multiple domains, not only directlyinhibits the initiation and execution phases of the caspase cascadeduring programmed cell death, but also regulates, in a caspase-independent manner, a range of cellular activities that enhancesurvival signaling, including NFkB activity (14, 15). Recent stud-ies reveal that translational regulation of select survival proteins isregulated by MAPK signaling, protecting cancer cells duringcellular stress (16). However, the understanding of mechanismslinking MAPK signaling and survival signaling remains limited.

1Department of Surgery, Division of Surgical Sciences, Duke University MedicalCenter, Durham, North Carolina. 2Department of Pathology, Duke UniversityMedical Center, Durham, North Carolina. 3Department of Neurosurgery, DukeUniversityMedical Center, Durham, NorthCarolina. 4Duke Cancer Institute, DukeUniversity Medical Center, Durham, North Carolina. 5Department of RadiationOncology, Duke University Medical Center, Durham, North Carolina. 6Center forTheoretical Biological Physics, Rice University, Houston, Texas. 7TranslationalCancer Research Unit, Oncology Center, General Hospital Sint-Augustinus,Antwerp, Belgium. 8Department of Medicine, Duke University Medical Center,Durham, North Carolina. 9Center for Oncological Research (CORE), University ofAntwerp, Antwerp, Belgium.

Note: Supplementary data for this article are available at Cancer ResearchOnline (http://cancerres.aacrjournals.org/).

M.K. Evans and M.C. Brown are the co-first authors of this article.

X. Bao, T.J. Robinson, and M.K. Jolly contributed equally to this article.

Corresponding Author: Gayathri R. Devi, Duke University, 203 Research Drive,DUMCBox 2606,MSRB I, Durham,NC27710. Phone: 919-668-0410; Fax: 919-681-7970; E-mail: gayathri.devi@duke.edu

doi: 10.1158/0008-5472.CAN-17-1667

�2018 American Association for Cancer Research.

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In this study, we demonstrate that elevated XIAP expression inpatient IBC tumors is associated with aggressive biology and poorclinical outcome. Using IBC cell lines derived from previouslyuntreated primary tumors combined with modulation of XIAPexpression, we found that XIAP drives activation of NFkB and itstarget genes, leading to enhanced tumor growth. Furthermore, wediscover that MAPK interacting kinase (MNK) signaling, down-stream of EGFR/HER2 activation, promotes XIAP expression andNFkB activity. Collectively, our findings indicate a role for XIAP asa central regulatory node connecting MAPK and NFkB signals,which governs IBC tumor-specific gene signatures, survival, andtumorigenesis.

Materials and MethodsHuman breast tumor mRNA expression studies

Gene expression datasets previously published were used togenerate a combined total of 1032 breast cancer patients [GEOdatasets GSE6532, GSE9195, GSE16391, GSE16446, GSE17907,GSE20685, GSE20711, and GSE21653]. A total of 1018 patientshad nonzero event-free survival time and were available foranalysis for the expression of two probesets (206536_s_at and206537_at, Affymetrix), which targeted the XIAP ORF. Patientswere grouped into high or low expression of XIAP using the topquartile versus remaining patients by probe set expression valuesand compared using Kaplan–Meier plots with 95% confidenceintervals of event-free survival (earliest event provided withineach dataset) by log-rank test. For correlation of XIAP expressionwith lymph node involvement, two available GEO datasets,GSE6532 and GSE9195, were identified that included lymphnode status for a total of 164 patients with IBC.

Breast cancer tissue microarraysThe TMA sections used in this study, with prior patient consent

and approval from the Institutional Review Boards from eachcenter, and their clinical characteristics are available in refs. 17–19.

IHCFour-micron–thick paraffin sections were deparaffinized, rehy-

drated, and antigen retrieval performedusing EDTAbuffer at 95�Cfor 30minutes. Slides were incubated in a 1:60 dilution of mouseanti-human XIAP (BDBiosciences) overnight at 4�C,washed, andincubated in anti-mouse secondary (Dako anti-mouse Envisionkit) for 30minutes at room temperature. Imaging was performedonaZeiss AxioObserver A1microscope and images analyzedwithMetaMorph. Scoring of slides was carried out by a board-certifiedsurgical pathologist in a blinded manner. Staining intensity wasgraded on a qualitative scale [no staining (negative), very focal orvery weak staining (borderline), and positive]. For the purpose ofstatistical analysis, the data were dichotomized as negative orpositive (including borderline).

Cell linesSUM149 and SUM190 cells were obtained from Asterand, Inc.

and were cultured as previously described (9). The rSUM149 cellvariant is derived from SUM149 and cultured as described pre-viously (10, 20). SUM149 cells stably expressing wtXIAP, shXIAP,and shXIAPþwtXIAP were generated using a lentiviral expressionsystem (kindly provided by Dr. Colin Duckett, University ofMichigan, Ann Arbor, MI) and previously reported (13). HeLacells were grown in DMEM þ 10% FBS. For overexpression of

MNK1 T344D mutant, cells were transfected with 0.5 mg ofpcDNA3.1 (empty vector) or pcDNA3.1 MNK1 T344D (previ-ously described in ref. 21), and harvested 48 hours later forimmunoblot analysis. Characterization and authentication of thepurchased cell lines were done at Asterand. Additionally, shorttandem repeat polymorphism analysis was performed at regularintervals on all cell lines at the Duke Sequencing facility. Cellswere cultured at 37�C and 5% CO2.

In vivo tumor xenograft studiesAll animal experiments were performed in accordance with

protocols approved by the Duke University Institutional Ani-mal Care and Use Committee. IBC cells (5 � 106) weresuspended in 50-mL PBS and 50-mL Matrigel and injectedorthotopically into the fourth mammary fat pad of femaleSCID mice. Mice were monitored twice weekly and tumorvolume measured using the formula V ¼ (L � W2)/2, whereL is the longer measurement. Tumor doubling time was foundby fitting a nonlinear regression model to the tumor volumes.Mice were euthanized when tumors reached a humane end-point of �1,500 mm3, at the first sign of morbidity, or at end ofstudy. Tumors were removed and tissue harvested for RNA andWestern immunoblot analysis.

In vivo window chamber studiesA dorsal skin-fold window chamber was implanted in Nu/Nu

mice as described previously (22). Briefly, the dorsal skin wastented and sutured to a c-frame to hold it in position. Three�1 mm diameter holes were cut, through which the windowframe could be secured, and a 12-mmdiameter full-thickness skinpunch was removed from the superior skin fold. The titaniumwindow frame (Small Dorsal Kit, APJ Trading) was sutured inplace and a total of 1 � 105 tumor cells were injected into thefascia in the center of the window in a 20-mL volume, using a 30-gauge needle. Sterile saline was used to fill the window, overwhich a coverslip was placed and affixed by a retaining ring.

In vivo CGP57380 treatmentFemale nude mice (around 10 weeks old) were randomized

into two groups (vehicle and CGP57380 group, n ¼ 2 for eachgroup) after the installment of dorsal window chamber andimplantation of GFP-tagged SUM149 IBC cells. Tumor-bearingmice were treated with either CGP57380 (25 mg/kg, i.p.) orvehicle for 4 doses on days 0, 2, 4, 6 after the surgery. A stocksolution of CGP57380 in DMSO was made up and furtherdiluted into PBS for administration; vehicle solution containedthe same percentage of DMSO in PBS as the CGP57380 solution.The experiment was repeated twice.

In vivo imagingMice were anesthetized and mounted to a microscope stage

with a custom-made mouse window chamber slide mount. TheZeiss Axio Observer Z1 microscope was used for all imagingwith a 5� objective and Apotome (Carl Zeiss AG). GFP fluo-rescence was excited and acquired with a 488 nm/509 nmexcitation/emission filter set, as well as a bright-field trans-mission image, all recorded by a CMOS camera (C11440,HAMAMATSU photonics K.K). A whole window chamberimage was acquired using the tiling function within Zen Prosoftware. Tumor growth was monitored with fluorescent imag-ing at designated time points.

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RNA isolationTotal RNA isolation from adherent cells was completed using

the Ambion mirVana miRNA isolation kit (Invitrogen) followingmanufacturer's instructions. Tissue sampleswere homogenized inthe provided lysis buffer (mirVana kit) and total RNA isolatedfollowing instructions.

Affymetrix GeneChip analysisRNA quality was assessed using the Agilent 2100 Bioanalyzer

(Agilent Technologies) and total RNA profiled using the U133A2.0 Human Gene microarrays at the Duke Institute for GenomeSciences & Policy Microarray facility. Expression data were quan-tile-normalized and summarized using GCRMA express (23).Probe sets with a fluorescence intensity above log2(100) in atleast two samples were considered informative. Expression levelswere compared using generalized linear models on log2 expres-sion data andprobe setswith nominal P values less than0.05wereconsidered significant. Differentially expressed genes were trans-lated into pathways using Expression2Kinases (24). Transcriptionfactors and kinases with a combined enrichment score of at least10 were included for the protein–protein interaction (PPI) net-work construction. The PPI network was then analyzed forenriched pathways using the Reactome FI plugin from Cytoscape.Pathwayswith a prior probability of less than 1%were consideredrelevant. Unsupervised hierarchical cluster analysis of the geneexpression data was performed using the Manhattan distance asthe dissimilarity metric and the Ward linkage as the dendrogramdrawing method. Application of the IBC signature to the expres-sion data of SUM149, wtXIAP, and shXIAP tumors was done asdescribed before (7).

Gene-set enrichment analysisExpressiondata ofwtXIAP (XIAPhigh) cellswas comparedwith

the combined parental and shXIAP cell lines (XIAP low) usingdefault parameters with gene-set level permutations and signal2-noise used to rank genes. Gene-set enrichment visualization wasperformed using Cytoscape 2.8.3 and a P < 0.001, Q-value cutoff0.006, similarity cutoff of 0.5, and false discovery rate of 0.1.Gene sets examined were from the current molecular signature(MSigDB) versions 4.0.

Treatment of cells for viability and caspase activityCells were seeded and allowed to reach approximately 80%

confluence. Cell viability was determined by Trypan blue exclu-sion assay as described previously (9). Caspase-3/7 activity wasdetermined in cells untreated and treated with 50 ng/mL TRAIL,using the Caspase-Glo Assay (Promega) as per manufacturer'sinstructions.

Western immunoblot analysisWestern immunoblots were carried out as described previously

(10). Cells were harvested after indicated treatments and times.Tissue samples were homogenized in lysis buffer in a BulletBlender Storm 24 (Next Advance). Membranes were incubatedat 4�C overnight with antibodies against NFkB (P65), p-NFkB(P65), ERK, p-ERK, p-eIF4E, eIF4E, p-p38, p38, survivin, MNK1(Cell Signaling Technology, all 1:1,000), SOD2 (1:1,000), Bcl-2,(1:1,000), XIAP (1:2,000; BD Biosciences), c-Myc (Sigma-Aldrich), or GAPDH (Santa Cruz Biotechnology, 1:4,000). Den-sitometric analysis was performed using the NIH ImageJ software(25); for western quantitation measuring effects of MNK modu-

lation on XIAP and NFkB signaling a LI-COR Odyssey FC imagerwith Image Studio software (LI-COR) was used.

NRAGE peptide treatmentThe NRAGE peptide was purchased from NeoBioLab and used

as previously described by our lab (13). For all experiments,unpurified NRAGE peptide was added to cells for 24 hours with6 mmol/L EndoPorter delivery reagent (GeneTools LLC).

Anchorage-independent growth assayAnchorage-independent growth assay was performed as

described previously (26). Indicated treatments were applied for24 hours, after which cells were harvested and counted. Oncevisible colonies had formed, they were counted under a micro-scope, and colony counts were normalized to the untreatedsample. Images of representative fields were taken with 5�magnification using a Zeiss Axio Observer A1microscope (Zeiss),Hamamatsu Orca ER digital camera, and MetaMorph software(Molecular Devices).

ALDEFLUOR assayALDH enzymatic activity was evaluated using the ALDEFLUOR

kit (Stem Cell Technologies) according to the manufacturer'sinstructions. Cells were incubated with provided ALDH substratefor 35 minutes at 37 �C. The specific ALDH inhibitor diethyla-minobenzaldehyde (DEAB) was used as a negative control. Sort-ing gateswere established using 7-AAD for viabilityDEAB-treated,ALDEFLUOR-stained cells as negative controls.

Quantitative PCR analysisTotal RNA was subjected to reverse transcription using the

iScript Reverse Transcription SuperMix Kit (Bio-Rad) and oligod(T) primers as per manufacturer's instructions. cDNA and SYBRGreenwere added to a customPrimePCRplate (Bio-Rad) contain-ing primer pairs for the indicatedNFkB target genes and b-actin asa loading control. Further information onprimers canbe found inSupplementary Table S1. For MNK qRT-PCR studies, qRT-PCRwas performed using Applied Biosystems MYC, BIRC5, IL1B, andBCL2 primers and probes using the RNA to CT one step RT-PCRreagent kit (Invitrogen), following the manufacturer's instruc-tions. The PCRwas conducted on an iCycler instrument (Bio-Rad)using the following conditions: [95�C � 2 minutes, (95�C � 5seconds, 60�C � 30 seconds) � 40 cycles] and fold changescalculated by the 2(-DDCt) method, except for MNK qRT-PCRstudies where primer efficiency correction was deemed necessaryand the 2(-DDCP) method was used (27).

Mathematical model constructionSimulationswereperformed inMATLAB (Mathworks Inc.), and

bifurcation diagrams were drawn using MATCONT (28). Themodel formulation for the interactions of MNK, XIAP, and NFkBbased on this study and previous work is given by:

dNdt

¼ gNHs X;lX;N� �� kNN

dXdt

¼ gXHs N:lN;X� �

Hs M;lM;X� �� kXX;

where N, M, and X denote NFkB, MNK, and XIAP levels,respectively. gN and gX are the respective production rates for

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NFkB and XIAP, and kN and kX are their respective degradationrates. Shifted Hill functions, denoting the effect of X on Y, aredefined as:

Hs X; lX;Y� � ¼ H� Xð Þ þ lX;YHþ Xð Þ;

where H�ðXÞ is the inhibitory Hill function, Hþ (X) is theexcitatory Hill function, and lX;Y denote the equivalent of fold-change in production of Y due to X (29). The parameters usedhere are: kN ¼ 1:0; kX¼1:2; gN ¼ 120; gX ¼ 150; lN;X¼ 4; lX;N¼ 5,and lM;X¼ 2. For different Hill functions used in themodel, N0

X ¼ X0N ¼ M0

X ¼ 400; nN;X ¼ nX;N ¼ 4; nM;X ¼ 2, wherenX;Y nX;Y denotes the Hill coefficient for the Hill function corre-sponding to the effect of X on Y, and X0

Y represents the respectivehalf-maximal concentration. Degradation rates (represented inper unit hour) for XIAP and NFkB are estimated from exper-imental data on their half-lives (30, 31). The fold-change foreffect of MNK on XIAP has been estimated from our results,while the effect of XIAP on NFkB and vice versa were gatheredfrom existing data (15, 32). Production rate (represented in1,000 molecules per hour) estimation is based on total numberof protein molecules per cell reported for signaling molecules(�100,000; ref. 33). For considering the effect of NRAGEmimic, a shifted Hill function HsðNr; lNr;NÞ is included in thefirst term of equation for NFkB levels, since NRAGE is notsupposed to alter the levels of XIAP, but its interaction withNFkB. Parameters used are: lNr;N ¼ 0:5 (estimated from ourdata), and Nr0N ¼ 500; nNr;N ¼ 2.

Statistical analysisThe statistical analyses were conducted using GraphPad Prism

(GraphPad Software, Inc.) Student two-tailed t test, Fisher exacttest (IHC), and Mantel–Cox log-rank test (survival). Differenceswere considered significant at P < 0.05.When comparingmultiplegroups, ANOVA protected Tukey HSD test was performed. Com-parison of XIAP mRNA expression using the two probesets wereanalyzed with respect to their expression distribution using therank-based Mann–Whitney test.

ResultsHigh XIAP levels are associated with aggressive breast cancercharacteristics

To explore whether XIAP expression correlates with survivaloutcomes in breast cancer, we queried expression data from acollection of 1,032 breast cancers, using microarray probe setstargeting the open reading frame (ORF) of XIAP (SupplementaryFig. S1). Breast cancer patients with elevated expression (topquartile) of XIAP mRNA had decreased event-free survival com-pared with patients with lower XIAP mRNA levels (Fig. 1A). Wefurther examined whether expression of XIAP was independentlyassociated with disease-free survival adjusting for available cov-ariates, which included PAM50-based molecular subtype andlymph node status. High XIAP expression was associated withincreasedHRof recurrence (HR1.68, P < 0.001) after adjusting forPAM50 subtype and lymph node status, while expected trendswere observed with increased recurrence noted for nonluminalmolecular subtypes anddecreased recurrence risk noted for lymph

Figure 1.

High XIAP mRNA expression correlates withpoor event-free survival and lymph nodeinvolvement in advanced breast cancer patients.A, Event-free survival for patients with IBC(n ¼ 1018), separated as high (top quartile)versus low XIAP expression, as determined byprobe set 1 (left) 206536_s_at and probe set 2(right) 206537_at from the AffymetrixGeneChip U133A 2.0 Array [top quartile vs.remaining patients, log-rank test for probe set1 - 206536_s_at: P < 0.0001, HR 1.80 (95% CI,1.53–2.62) and probe set 2 - 206537_at:P ¼ 0.0006, HR 1.40 (95% CI, 1.11–1.87), bothP < 2e�6]. B and C, Increased XIAP expressionamong breast patient tumors (n ¼ 164) withlymph node (LN) involvement versus no lymphnode involvement at diagnosis. Box and whiskerplots (B) and histograms (probeset 1, P¼ 0.005;probeset 2, P ¼ 0.02; C) with expressiondistribution analyzed using the rank-basedMann–Whitney test.

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node–negative disease. Given the role of IAP proteins in metas-tasis (34), we hypothesized that XIAP expression would beincreased in breast cancer patients with lymph node–positivedisease (LNþ). We observed increased expression of XIAP amongpatient tumors with lymph node involvement versus no lymphnode involvement at diagnosis (Fig. 1B and C).

Lymph node involvement and the presence of tumor cellclusters (tumor emboli) in the dermal lymphatic vessels is aclassic feature of IBC presentation at diagnosis. Therefore, weconducted IHC analysis for XIAP protein expression in breasttissue microarrays, which included benign and malignant sam-ples (n ¼ 198) of different stages and grades from non-IBC andIBC patients (Table 1). Overall, positive cytoplasmic staining ofXIAP was only observed in invasive breast tumors and triple-negative samples. IBC samples characterized by tumor embolishowed strong staining for XIAP in >90% cells alongwith positivestaining in the identified tumor emboli. Representative images areshown in Fig. 2A–F. We performed a multivariate analysis todetermine whether XIAP overexpression in IBC is related to otherclinicopathologic features [e.g., histologic grade, hormone recep-tor status, HER2 status, and triple-negative breast cancer (TNBC)status - n ¼ 158]. In univariate analysis, XIAP expression wassignificantly associated with high histologic grade (grade 3 vs.grade 1; HR ¼ 1.305; P < 0.001), ER status (ERþ vs. ER�; HR ¼0.887;P¼0.036), PR status (PRþ vs. PR�; HR¼0.887;P¼0.010),TNBC status (TNBCþ vs. TNBC�; HR ¼ 1.178; P ¼ 0.012) andtumor phenotype status (IBC vs. non-IBC; HR ¼ 1.299; P ¼0.010). In multivariate analysis, including all parameters associ-ated with XIAP expression in univariate analysis, only histologicgrade (grade 3 vs. grade 1; HR ¼ 1.232; P ¼ 0.016) and tumorphenotype (IBC vs. non-IBC; HR ¼ 1.230; P ¼ 0.049) remainedsignificant. These data suggest that XIAP expression is correlatedwith breast cancer of higher histologic grade and that XIAP over-expression is specifically associated with IBC, independent of theclassical clinicopathologic determinants of IBC.

We next investigated whether XIAP upregulation contributes tothe hyperproliferative phenotype in IBC. To explore this, we usedSUM149 (basal-like, constitutively activated EGFR) and SUM190(luminal-like, HER2-overexpressing) tumor cells, which are con-sidered true IBC-like models derived from primary tumors of IBCpatients before treatment (35, 36). To assess the global effects ofmodulating XIAP expression in IBC cells we conducted transcrip-tome profiling of genetically modified derivatives with XIAPoverexpression (wtXIAP), depletion (shXIAP), and reconstitution(shXIAPþXIAP), along with appropriate vector controls. XIAPexpression and function in these variants was validated by immu-

noblot (Fig. 2G) and measurement of caspase activation and cellviability after treatment with TRAIL (Fig. 2H). GSEA analysis andGNF expression atlas ontologies revealed a network of relatedgene sets enriched in XIAP-overexpressing cells (SupplementaryFig. S2) reported to be associated with cell-cycle regulation andproliferation, response to cell stress and stem cell maintenance,and resistance to hypoxic and oxidative stress. The list of the top100 genes that are differentially expressed in XIAP high versus lowsamples are provided in Supplementary Table S2A and S2B. Ofinterest was a strong and positive correlation between XIAPoverexpression and gene sets (37) enriched for high-grade breastcancer (Fig. 2I), corroborating the observed increased XIAP stain-ing in IBC specimens, in particular IBC, which are already at stageIII or higher at diagnosis.

XIAP knockdown abrogates IBC-specific gene signatureTo further investigate the clinical relevance of XIAP in IBC, we

evaluated how the gene expression profile of the XIAP-overex-pressing versus knockdown cells compared with a published IBC-patient derived gene signature from a comparative analysis ofuntreated primary tumors from stage- and subtype-matched IBCand non-IBC patients, which includes the largest collection of IBCtumors from the World IBC Consortium (7). As expected, thepatient-derived SUM149 cell line is IBC-like with an averageposterior probability (similarity) of 44.7%; likewise, the XIAP-overexpressing (wtXIAP) cells also show a significant posteriorIBC probability (i.e. 51.5%). In contrast, we observed thatknockdown of XIAP (shXIAP) abolished the IBC-specificpatient gene expression profile (i.e., posterior IBC probabilityof 0.05%; Fig. 2J). Taken together, these results indicate thatXIAP maintains an IBC-like phenotype and associated geneexpression signatures, which are dominated by proliferativeand prosurvival gene network.

XIAP overexpression enhances in vivo IBC tumor growthDespite the correlation of XIAP overexpression with prolifer-

ative genes, modulating XIAP expression alone in vitro does nothave a significant effect on cellular proliferation (10). Therefore,to determine a possible in vivo role for XIAP overexpression, wecharacterized the tumor growth kinetics of the XIAP modulated(wtXIAP, shXIAP, and vector control) cells. Tumor cells wereimplanted in the mammary fat pad of nude mice and tumorgrowth measured over time (Fig. 3A). Initially all mice formedtumors with similar kinetics; however, the growth inmice bearingwtXIAP-overexpressing tumors was significantly increased com-pared with vector control tumors (doubling time of 6.9 days,wtXIAP; 10.3 days, vector controls). In contrast, although shXIAPtumors grew to palpability, most regressed (10/12) or plateauedin size compared with vector control tumors in the study period.Vector controls are shown combined in Fig. 3A as they had similargrowth kinetics (separate in Supplementary Fig. S3A). Demon-strating specificity of these phenotypes to XIAP, robust tumorgrowth similar to wtXIAP was observed in tumors expressing anshRNA-resistant XIAP construct (shXIAPþwtXIAP; Fig. 3A).

In addition to our exogenously XIAP-modulated cell lines, wealso compared parental SUM149 cells to rSUM149, a highlyapoptotic-resistant cell line with endogenous XIAP overexpres-sion dependent on XIAP IRES-mediated translation (10). HighXIAP levels in rSUM149 correlate with multidrug resistance tochemo-, immuno-, and targeted therapy–mediated apoptosis(20); however, the in vivo growth characteristics of this cell line

Table 1. Correlation of XIAP expression with clinicopathologic parameters ininvasive breast carcinomas (IHC analysis of tissue microarrays)

Grade Positive Negative

1,2 42 713 43 27P 0.0022

Stage1, 2 54 683, 4 13 2P 0.0021

Molecular subtypeTNBC 20 7Other types 60 73P 0.0103

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has not been studied. Similar to wtXIAP tumors, mice bearingrSUM149 tumors showed an aggressive growth pattern withformation ofmultiple tumor cell clusters (Fig. 3B). Representativepictures from tumor-bearing mice and related statistical analysisfor SUM149 and rSUM149 are shown in Fig. 3C and D, respec-tively; additional images are shown in Supplementary Fig. S3.

Collectively, these results reveal that XIAP overexpression(endogenous or exogenous) enhances tumor growth, possiblyexplaining why XIAP expression is associated with aggressivefeatures in patient tumors.

XIAP depletion reduces expression of ALDHþ, a cancer stem-like marker, and decreases IBC tumor cell motility

A pathologic hallmark of IBC tumors is the formation of tumoremboli enriched with ALDHþ cells [enzymatic marker of cancer

stem-like cells reported to be high in IBC cells and patient tumors(38) and postulated to reflect collective tumor cell migration ofIBC cells (39)]. On the basis of our evidence of a proliferativesignature corresponding with enhanced tumorigenicity in vivo,weinvestigated whether XIAP expressionmodulates cancer stem-likecharacteristics. Indeed, both wtXIAP and rSUM149 cells exhibitedincreased proportions of ALDHþ cells compared with significant-ly reduced ALDH positivity in XIAP-silenced (shXIAP) cells (Fig.3E). This effect was specific to XIAP, as reconstitution of XIAP inshXIAP cells enabled reemergence of ALDHþ cells.

To test whether XIAP inhibition affects IBC tumor cell motility,we employed a dorsal skin fold window chamber model in nudemice that allows for intravital imaging of local tumor growth andmigration (Fig. 3F and G). GFP-SUM149 (vector ctr) IBC tumorsgrew as multiple tumor cell clusters inside the window chamber

Figure 2.

XIAP protein expression in high-grade breast cancers and higher cellular levels associated with proliferative signature. A–F, XIAP levels were analyzed by IHCanalysis in TMAs of breast cancers from human patients. A, No expression in normal breast lobule. B, No expression in benign duct (right) and normal acini(left). C, Borderline staining in >50% of tumor cells. D, Positive staining in >90% of invasive tumor cells. E, Infiltrating IBC with tumor cell clusters showingstrong positive staining in >80%–90% of cells. F, Staining in a representative intralymphatic tumor emboli identified in IBC specimens. Magnification,�400. See Table 1 for full histopathology results. G, Immunoblot analysis for expression of XIAP in indicated parental and XIAP-modulated cell lines. H, Functionaleffects of XIAP expression or depletion evaluated by cell viability (left axis, white bars) and caspase activity (right axis, black bars) of indicated cell lines afteradministration of 50 ng/mL TRAIL; viability bars represent mean � SEM (n ¼ 3–4), caspase-3/7 activity bars represent mean � SEM fold change normalized tountreated (n ¼ 2–3). I, Enrichment plot showing correlation of XIAP overexpression with published features of high-grade breast cancer (37) from GSEA analysis.J, Application of the IBC-specific patient gene signature to the expression data from SUM149, wtXIAP, and shXIAP cells. The figure shows, in boxplotformat, the posterior IBC probability on the y-axis for all samples including patients with IBC (red, positive control), patients with non-IBC (moss green,negative control), SUM149 cells (green), wtXIAP (blue), and shXIAP (purple).

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(Fig. 3H), similar to that observed in IBC patients (39, 40), asopposed to single solid masses observed with other breast cancerlines (22). Using this approach, we compared the short-term(0–120 hours) growth and migration pattern of the SUM149-derived vector control and shXIAP-implanted cells, whichrevealed significant inhibition of motility in the tumors arisingfrom XIAP-depleted cells (Fig. 3I).

XIAP-overexpressing tumor cells exhibit high NFkB target geneexpression

Gene expression profiles identified 933 differentially expressedgenes between control and wtXIAP tumors (n¼ 3 each genotype,Supplementary Table S3). Those genes were enriched for biolog-ical processes of transcription, RNA biosynthesis, and proteinmetabolism among others. As IBC patient tumor profiles aredominated by NFkB target genes, we investigated whether IBCtumors generated with XIAP-overexpressing cells have increasedNFkB activity. Expression2Kinases (X2K) analysis revealed thatthis gene list was enriched for target genes of two transcriptionfactors in the NFkB family (RELA and NFkB1). In addition to

target gene enrichment analysis, X2K also builds a protein–pro-tein interaction (PPI) network that provides signal transductionpathways capable of explaining observed gene expression differ-ences. Analysis of this PPI network identified a subnetworkregulated by NFkB activity (Fig. 4A). These data were furtherconfirmed with qRT-PCR analysis: increased expression of severalknown NFkB target genes (NFKB1, MYC, and TNFAIP3) wasobserved inwtXIAP and shXIAPþXIAP tumors, while their expres-sion was reduced in shXIAP tumors (Fig. 4B). Immunoblotanalysis of both wtXIAP and shXIAPþXIAP tumors confirmedenhanced activation of the nuclear transcription factor NFkB(phospho-p65; Supplementary Fig. S3).

Targeting the XIAP–NFkB interaction inhibits anchorage-independent growth

To further investigate the role of XIAP-mediated NFkB activityin IBC cells, we employed a small peptide mimetic modeled afterthe NRAGE repeat domain (41), which blocks the XIAP–BIR1domain interaction with TAB1, interrupting XIAP-NFkB signaling(15). Treatment of cells with NRAGE peptide, delivered using

Figure 3.

XIAP depletion suppresses in vivo tumor growth and motility. A, Tumor growth curves of indicated XIAP-modulated tumor xenografts implanted orthotopically.B, Tumor growth curves of SUM149 (gold) and rSUM149 (black) xenografts. C and D, Representative images of mice implanted with tumors (C) andextracted tumor clusters (D). E, Flow cytometric analysis of ALDH activity in population of cells indicated. Bars represent mean � SEM of ALDEFLUOR-positivecells as a percentage of the total number of cells analyzed (n ¼ 2–3; �, P < 0.05). F and G, Representative image showing position of the window chamberimplanted in the dorsal skin of the nude mice and live imaging. H and I, Time-course imaging of the local tumor growth and migration in the window chamberof GFP labeled SUM149-vector control (H) and shXIAP (I) cells.

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Endoporter, led to decreased transcriptional activity of NFkB asmeasured by target gene expression (Fig. 4C). wtXIAP cells exhib-ited a modest increase in anchorage-independent growth relativeto control cell lines (Fig. 4D and E). Treatment with NRAGEpeptide reduced anchorage-independent growth in control andwtXIAP cells in a dose-dependent manner (Fig. 4D and E).Collectively, these results reveal that XIAP drives activation ofNFkB and its target genes, demonstrating a functional interplay inadvanced breast cancers like IBC, which are characterized by anincreased proliferative state. Furthermore, use of the NRAGEpeptide highlights the potential for developing BIR1 domainantagonists that can target the XIAP–NFkB interaction and/orsignaling to potentiate therapeutic apoptosis in IBC cells.

XIAP is regulated by the MAPK-interacting kinase, MNKOur results demonstrate a role for XIAP in the proliferative

phenotype of IBC through a functional partnership with NFkB,which we have shown to be caspase-independent (13).Although previous reports indicate pervasive NFkB activity inIBC and suggest that NFkB activity may be downstream of theEGFR/HER2–MAPK signaling (6), the link between the tworemains ill-defined. MAPK signaling is a critical regulatorof stress response, including control of protein synthesis

machinery driving cancer cell survival (42). To evaluate theeffects of ERK1/2 signaling on XIAP expression, we treatedSUM149 and SUM190 IBC cells with the MEK1/2 inhibitorUO126, which effectively reduced ERK1/2 MAPK phosphory-lation, but led to only modest decreases in XIAP levels (Fig. 5A).As expected, p38 MAPK phosphorylation, which is downstreamof MKK3/4/6, was relatively unchanged by UO126. Both p38and ERK1/2 MAPK signaling intersect translation machinerythrough the MNK, which phosphorylates the cap-binding pro-tein, eIF4E (43). Likely due to the maintenance of p38 MAPKsignaling during UO126 treatment, MNK signaling to eIF4Ewas also only modestly reduced (Fig. 5A). Given MNK's knownroles in oncogenesis and survival signaling (44), we nextinvestigated whether MNK functions downstream of MAPKsignaling to promote XIAP–NFkB signaling. Treatment withthe prototypical MNK inhibitor CGP57380, abolished eIF4E(S209) phosphorylation, indicating MNK signaling interrup-tion. Strikingly, MNK inhibition led to a significant decrease inXIAP protein levels in both cell lines, an effect more robust thanthat of UO126 (Fig. 5B). The intensity of XIAP reduction byMNK inhibition, relative to ERK1/2 inhibition, suggests thatMNK more directly controls XIAP than ERK1/2 MAPK. Con-firming this effect on XIAP expression, MNK depletion by RNAi

Figure 4.

Functional partnership of XIAP and NFkB signaling in IBC tumor cell survival. A, Subnetwork of the PPI network identified by E2K to regulate the geneexpression profile identified by comparing SUM149 and wtXIAP primary tumors (n ¼ 3). The subnetwork shows potential signaling mechanisms controllingNFkB transcriptional activity. In the network, transcription factors are color-coded red, activating kinases are color-coded green, and cytoplasmic signal transducesare color-coded yellow. The size of the nodes relates to the number of interactions each node has within the identified PPI network. B, Bar graphs showingquantitative PCR analysis of indicated NFkB target mRNAs in tumor samples from indicated xenografts. Bars represent mean � SEM in fold compared withFG9 (n ¼ 2; � , P < 0.05). C, NRAGE-treated cells subjected to quantitative PCR analysis for indicated NFkB target mRNAs. Bars represent average fold expression(compared with FG9 control) � SEM. � , P < 0.05; �� , P < 0.005 compared with EndoPorter (EP) alone. D, Anchorage-independent growth assay of cellstreated with EndoPorter alone or NRAGE peptideþEP. Bars represent mean � SEM colonies formed in soft agar as a percentage of untreated (n¼ 3; �� , P < 0.005compared with EndoPorter alone). E, Representative images of the indicated treatments in C and D.

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also led to a decline in XIAP protein levels (Fig. 5C), andoverexpression of a constitutively active MNK1 mutant(T344D) in HeLa cells induced XIAP expression concomitantwith increased eIF4E (S209) phosphorylation (Fig. 5D). Quan-titation of the effects of MNK modulation on XIAP proteinlevels (Fig. 5A–D) is shown in Fig. 5E. Taken together, theseresults suggest that MNK activity controls XIAP expression.

Targeting MNK inhibits NFkB activationWe next investigated whether MNK inhibition could target

NFkB (p65) activity. Indeed, using either CGP57380 (Fig. 5B)ordirectMNKdepletion (Fig. 5C) reduced the levels of bothp-p65andp65 in IBC cells, coincidentwith a decline in XIAP. Analysis ofRNA expression by qRT-PCR following MNK1 depletion revealeda decrease in RNA levels of several select NFkB target genes

Figure 5.

MNK signaling regulates XIAP and NFkB and facilitates SUM149 cell motility in vivo.A and B, Immunoblot analysis of indicated proteins in SUM149 and SUM190 cellstreated with U0126 (10 mmol/L; A) or CGP57380 (10 mmol/L; B) for the designated intervals. C, Analysis of SUM149 cells transfected with control or MNK1 targetingsiRNAs harvested 72 hours posttransfection. D, Immunoblots of HeLa cells transfected with vector control or constitutively active MNK1 (T344D). E,Quantitation of Western blot images in A–D correcting each respective control to 1; CGP57380 values were determined for 24-hour time point. F, qRT-PCRquantification of RNA in SUM149 cells that were transfected with siRNA as in C for select NFkB target genes.G andH, SUM149-GFP cells were implanted into a dorsalwindow chamber and imaged at the designated intervals after treatment with vehicle (G) or CGP57380 (H). Arrows, treatment schedule. Error bars,SEM; �, P < 0.05; �� , P < 0.001.

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including MYC and survivin (Fig. 5F). Together, these resultsindicate that modulating MNK signaling regulates XIAP proteinlevels and downstream NFkB target genes. Thus, MNK controlsXIAP–NFkB signaling and can be targeted to restrain the onco-genic effects of XIAP–NFkB activity.

MNK inhibition decreases IBC cell dissemination in vivoMNK inhibition has been shown to reduce in vivo tumor growth

in several cancer types (45, 46). However, the role of MNKsignaling in mediating tumor cell invasion, a characteristic ofIBCwe show is reliant uponXIAP expression (Fig. 3), has not beentested. Using the window chamber model with GFP-taggedSUM149, we tested infiltration of SUM149 cells within thewindow chamber in vehicle or CGP57380-treated mice. MNKinhibition with CGP57380 led to a marked reduction in celldissemination within the window chamber, suggesting that MNKsignaling enables IBC migration (Fig. 5G and H). Thus, MNKinhibition reduces XIAP levels and NFkB activity and mirrors theeffects of XIAP depletion in IBC cells in vivo (Figs. 3 and 5).

Mathematical modeling suggests how cells might maintainhigh levels of XIAP and NFkB

Integrating our quantitative immunoblot and qRT-PCR dataindicating XIAP regulation of NFkB in aMNK-responsive mannercombined with earlier reports that also propose XIAP as a tran-scriptional target of NFkB, we constructed a quantitative math-ematical model to decode the dynamics of the MNK/XIAP/NFkBaxis. Mutual activation between NFkB and XIAP leads to bist-ability in the system: cells display either low XIAP/low NFkB, orhigh XIAP/high NFkB protein levels (shown by two solid greencircles in Fig. 6A). To switch between these two states/phenotypes,cells must cross a "tipping point" or threshold (as shown byhollow green circle in Fig. 6A). Once cells have attained such athreshold (high XIAP, high NFkB), this mutual activation wouldmaintain that state (Fig. 6B and C). Only when a "brake" issignificantly applied on either NFkB or XIAP levels (e.g., usingan inhibitor like NRAGE peptide), can cells be postulated to exitthat state and eventually attain a low XIAP, low NFkB state (Fig.6D). These findings indicate a homeostatic relationship between

Figure 6.

Simulation of a mathematical model for MNK/XIAP/NFkB axis. A, Nullcline simulations for the mathematical model, where red curve represents the changeinNFkB levels on changingXIAP, andblack curve represents the change inXIAP levels as a function ofNFkB. The solid green circles indicate twopossible stable states(phenotypes) of the MNK/XIAP/NFkB network, high XIAP, high NFkB and low XIAP, low NFkB, whereas hollow circle indicates an unstable state.B and C, Bifurcation diagram showing how cells in low XIAP, low NFkB state switch to a high XIAP, high NFkB upon increasing MNK levels. D, Bifurcation diagramshowing how cells with high NFkB levels may switch to a lowNFkB state upon treatment with NRAGEmimics. InB–D, solid blue lines reflect stable states, dotted redlines denote unstable state, and different colored regions highlight the existence of different phenotype(s) at different values of MNK or NRAGE. E, Schemasummarizing the cross-talk between EGFR-mediated MAPK activation, XIAP, and NFkB activity. Activation of the EGFRs, EGFR, and HER2 by exogenous ligandultimately leads to activation of the ERK1/2 MAPK. ERK may also be activated by other receptor tyrosine kinases or microenvironment stresses, along withthe p38 MAPK. The MNK is an eIF4G-associated, eIF4E kinase activated by both p38 and ERK1/2 MAPKs. MNK activation leads to increased XIAP levels.The BIR1 domain of XIAP facilitates a physical interaction with the TGFb-associated binding protein, TAB1, and its cognate kinase, TAK1. This bindingevent leads to the phosphorylation of the NFkB-activating kinase, IKKb, allowing NFkB to translocate to the nucleus and to increase expression of genesthat can promote tumor cell proliferation, growth, migration, and disease progression.

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XIAP and NFkB and suggest that mutual activation of XIAP/NFkBstabilizes a hyperproliferative phenotype in cancer.

Collectively, our findings define XIAP as a signaling interme-diate linking the MAPK mitogenic cascade to NFkB prosurvivalsignaling (schema in Fig. 6E). In doing so, XIAP confers a prolif-erative signature and phenotype to IBC cells, enabling aggressivetumor growth in one of the most lethal subtypes of breast cancer.

DiscussionThe current study uncovers XIAP as an oncogenic signaling

intermediate, linking the MAPK and NFkB signaling pathways,with significant implications for locally advanced breast cancertumor growth. XIAP mRNA levels correlated with lymph nodeinvolvement and decreased event-free survival among patientswith IBC, and XIAP overexpression was observed in high-gradebreast cancers and IBC patient tumors, substantiating previousreports of XIAP overexpression in breast cancer tissue (47) andcorrelation of XIAP expressionwith tumor recurrence in basal-likebreast cancer patients (48).We show that XIAP is necessary for theconstitutive activation of the NFkB pathway in IBC, and demon-strate that the XIAP–NFkB axis directly correlates with the tumorgrowth rate in vivo. These findings reveal a functional necessity forXIAP expression in the progression of aggressive, locally advancedbreast cancers like IBC. Finally, we defined a critical role of XIAP intransducingMAPK signals toNFkBdownstreamofMNK, possiblyexplaining the survival andoncogenic phenotypes associatedwithMNK signaling (44).

It has been postulated that in aggressive tumors like IBC, adelicate balance exists between unabridged cellular proliferation,the requirement for cancer stemcell self-renewal, and the ability ofcancer stem cell progeny to "self-metastasize" and migrate away,freeing up space for continued tumor expansion (39, 49). Ourresults showing that XIAP expression directly correlates with thenumber of ALDHþ cells and cell motility in IBC cells warrantsfurther investigation of the role of XIAP in IBC metastatic pro-gression. Studies overwhelmingly show that XIAP antagonism inestablished tumors or in cell lines can sensitize tumor cells totherapy-mediated cell death, thereby implicating XIAP as a che-moresistance factor (50). However, other reports suggest thatXIAP expression correlates with favorable clinical outcome(51). Perhaps, contributing to these contradictions are theupstream signals regulating XIAP expression and broader cellularcontext of the signaling landscape.

MNK is well known for its role in regulating IRES-mediatedtranslation (21) and in our study, interruption of MNK signalingled to a significant reduction in XIAP protein expression. Intrigu-ingly, XIAP mRNA contains an IRES (52) and MNK regulationof eIF4G and eIF4E may function to facilitate XIAP translationin IBC, or MNK may act on XIAP through one of its more recent-ly described eIF4E-independent contexts (53). Interestingly,MNK1/2–null (44) like XIAP-null (54) animals are viable anddo not exhibit anymajor defects in growth and ability to undergoapoptosis. MNK1/2–null mice were reported to exhibit delays intumor development, suggesting a role for MNK1/2 and down-stream effectors in tumorigenesis (44). Our current study show-ing a strong correlation between XIAP expression and tumorgrowth in vivo in two MAPK-hyperactivated IBC models providessupport for XIAP as a possible downstream effector in potentiat-ing the mitogenic effects of MAPK/MNK signaling. Indeed,we found that MNK inhibition restricted IBC tumor invasion/

migration, suggesting therapeutic potential in targeting MNK inIBC as a means to target the XIAP–NFkB axis in cancer.

IBC cells overexpressing XIAP were shown to be resistant toimmunotherapy-mediated cell death (13). This work revealed acaspase-independent ability of XIAP to activate NFkB and dem-onstrated that direct targeting of caspase-binding domains maynot reverse resistance (unpublished data). The efficacy of theNRAGE peptide, which prevents the XIAP–BIR1 domain fromactivating NFkB, in inhibiting anchorage-independent growth inIBC cells underscores the recent mounting evidence for a non-apoptotic function of XIAP as a signaling intermediate in tumorgrowth (14, 55). However, the practical hurdles of delivering apeptide to tumor cells renders this approach clinically difficult,particularly as NRAGE peptide has a short half-life. Our findingsdemonstrate that inhibition of MNK signaling represents anothermechanismto targetXIAP–NFkB signaling in IBC. Thefinding thatMNK inhibition disrupts tumor dissemination is especially rele-vant for IBCandother subtypesof cancer that are highlymetastatic(Fig. 5). Importantly, MNK inhibitors are being developed andpursued clinically, making MNK a more practical target for thispathway. This work presents a new druggable pathway consistingof MNK, XIAP, and NFkB (Fig. 6) that can be used to enhance theefficacy of therapeutic agents by pushing the cells below thetipping point (Fig. 6A) and consequently constraining the prolif-erative advantage. Thus, XIAP serves as a link between MAPK andNFkB signaling to control IBCproliferation and tumor aggression.

Disclosure of Potential Conflicts of InterestNo potential conflicts of interest were disclosed.

Authors' ContributionsConception and design: M.K. Evans, M.C. Brown, M.A. Morse, S. Van Laere,G.R. DeviDevelopment of methodology: M.K. Evans, M.C. Brown, J. Geradts, X. Bao,T.J. Robinson, M.K. Jolly, G.M. Palmer, G.R. DeviAcquisition of data (provided animals, acquired and managed patients,provided facilities, etc.): M.K. Evans, M.C. Brown, J. Geradts, X. Bao,P. Vermeulen, G.M. Palmer, S. Van Laere, G.R. DeviAnalysis and interpretationofdata (e.g., statistical analysis, biostatistics, compu-tational analysis): M.K. Evans, M.C. Brown, J. Geradts, X. Bao, T.J. Robinson,P. Vermeulen, G.M. Palmer, H. Levine, M.A. Morse, S. Van Laere, G.R. DeviWriting, review, and/or revision of the manuscript: M.K. Evans, M.C. Brown,J. Geradts, X. Bao, T.J. Robinson, M.K. Jolly, P. Vermeulen, M. Gromeier,H. Levine, M.A. Morse, S. Van Laere, G.R. DeviAdministrative, technical, or material support (i.e., reporting or organizingdata, constructing databases):M.Gromeier, H. Levine, S.J. Van Laere, G.R. DeviStudy supervision: G.R. Devi

AcknowledgmentsThisworkwas supported byAmericanCancer Society Research Scholar Grant

(to G.R. Devi), P30 Cancer Center Support Grant NIH CA014236 develop-mental funds (to G.R. Devi), Department of Defense W81XWH-13-1-0047 and-0046 (to G.R. Devi and M.A. Morse); Department of Defense W81XWH-17-1-0297 (to G.R. Devi); Duke University Diversity Enhancement Fellowship (toM.K. Evans), and the Duke School of Medicine Interdisciplinary InflammatoryBreast Cancer Colloquium Funds (to G.R. Devi); Computational Cancer Bio-logy fellowship from Gulf Coast Consortia - CPRIT RP170593 (to M.K. Jolly);and NSF PHY-1427654 (to H. Levine). The authors would like to thank AmyAldrich, Courtney Edwards, Adrian Ramirez, Arianna Price, Ronnie Shammas Jr,Larissa M. Gearhart-Serna for technical assistance; Drs. Hengtao Zhang, YulinZhao, and Scott Sauer for technical support in the window chamber studies. Wethank Tao Wang for flow cytometry support, members of the Devi Lab,Drs. Mark Dewhirst, Ashley Chi, Jeffrey Marks, and Sally Kornbluth, for helpfuldiscussions during manuscript preparation, Dr. Donna Crabtree for editorialassistance, andDukeUniversity Core facilities (LightMicroscopy, Cancer Center

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Isolation Facility, Center for Genomic and Computational Biology, FlowCytometry, Optical Molecular Imaging and Analysis, Preclinical TranslationalResearch Unit).

The costs of publication of this article were defrayed in part by thepayment of page charges. This article must therefore be hereby marked

advertisement in accordance with 18 U.S.C. Section 1734 solely to indicatethis fact.

Received June 12, 2017; revised November 7, 2017; accepted January 16,2018; published OnlineFirst January 19, 2018.

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2018;78:1726-1738. Published OnlineFirst January 19, 2018.Cancer Res   Myron K. Evans, Michael C. Brown, Joseph Geradts, et al.   Determine an Aggressive Breast Cancer Phenotype

B Signaling toκXIAP Regulation by MNK Links MAPK and NF

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