PlasminogenActivatorInhibitor1(PAI1)Promotes ... · PAI1 enhanced cytoskeletal features associated with migra-tion, actin-rich migratory structures, and reduced actin stress ﬁbers

Metabolism

PlasminogenActivator Inhibitor 1 (PAI1) PromotesActin Cytoskeleton Reorganization andGlycolyticMetabolism in Triple-Negative Breast CancerBrock A. Humphries1, Johanna M. Buschhaus1,2, Yu-Chih Chen3,4,5, Henry R. Haley1,Tonela Qyli1, Benjamin Chiang1, Nathan Shen1, Shrila Rajendran1, Alyssa Cutter1,Yu-Heng Cheng3, Yu-Ting Chen6, Jason Cong6, Phillip C. Spinosa7, Euisik Yoon2,3,Kathryn E. Luker1, and Gary D. Luker1,2,8

Abstract

Migration and invasion of cancer cells constitute funda-mental processes in tumor progression and metastasis. Migra-tory cancer cells commonly upregulate expression of plasmin-ogen activator inhibitor 1 (PAI1), and PAI1 correlates withpoor prognosis in breast cancer. However, mechanisms bywhich PAI1 promotes migration of cancer cells remain incom-pletely defined. Here we show that increased PAI1 drivesrearrangement of the actin cytoskeleton, mitochondrial frag-mentation, and glycolyticmetabolism in triple-negative breastcancer (TNBC) cells. In two-dimensional environments, bothstable expression of PAI1 and treatment with recombinantPAI1 increased migration, which could be blocked with thespecific inhibitor tiplaxtinin. PAI1 also promoted invasioninto the extracellular matrix from coculture spheroids withhuman mammary fibroblasts in fibrin gels. Elevated cellularPAI1 enhanced cytoskeletal features associated with migra-tion, actin-rich migratory structures, and reduced actin stress

fibers. In orthotopic tumor xenografts, we discovered thatTNBC cells with elevated PAI1 show collagen fibers alignedperpendicular to the tumor margin, an established marker ofinvasive breast tumors. Further studies revealed that PAI1activates ERK signaling, a central regulator of motility, andpromotes mitochondrial fragmentation. Consistent withknown effects of mitochondrial fragmentation on metabo-lism,fluorescence lifetime imagingmicroscopyof endogenousNADH showed that PAI1 promotes glycolysis in cell-basedassays, orthotopic tumor xenografts, and lung metastases.Together, these data demonstrate for the first time that PAI1regulates cancer cell metabolism and suggest targeting metab-olism to block motility and tumor progression.

Implications: We identified a novel mechanism throughwhich cancer cells alter their metabolism to promote tumorprogression.

IntroductionPlasminogen activator inhibitor 1 (PAI1, or serpin family E

member 1; SERPINE1) is a serine protease inhibitor and the mostprominent negative regulator of the proteolytic urokinase plas-minogen activator system. Although originally hypothesized tohave tumor suppressor effects, cancers typically upregulate PAI1with higher levels correlating with worse prognosis in breast and

other malignancies (1–3). Given the strong links between PAI1levels and prognosis in breast cancer, the American Society forClinical Oncology (ASCO) recommends analysis of PAI1 levels asa biomarker for risk assessment and treatment decisions in lymphnode–negative breast cancers (4).

We identified PAI1 as a key driver of cancer cell migration andchemotaxis in triple-negative breast cancer (TNBC) using a high-throughput, single-cell microfluidic device that separates migra-tory and nonmigratory subpopulations of cancer cells (5). Thisdevice enables recovery of migratory and nonmigratory cancercells for subsequent analysis, whichprovides a powerful approachto identify cells with highest capacity to migrate within a hetero-geneous bulk population. RNA sequencing revealed PAI1 as themost upregulated gene inmigratory cells from twodifferent TNBCcell lines. These data reinforce a recent screening study thatidentified PAI1 as a key gene driving epithelial-to-mesenchymaltransition and migration in TNBC (6).

Cells require a large amount of energy to coordinate cytoskel-etal reorganization and subsequent migration. Therefore, migrat-ing cancer cells must adopt a metabolic phenotype that allowsthem to generate energy near sites of rearrangement in the actincytoskeleton (7). Prior studies indicate cancer cells typically relyon glycolysis rather than oxidative phosphorylation to drivemigration and invasion, consistent with studies associating gly-colysis with more aggressive malignancies (7–9). Identifying

1Center for Molecular Imaging, Department of Radiology, University of Michigan,Ann Arbor, Michigan. 2Department of Biomedical Engineering, University ofMichigan, Ann Arbor, Michigan. 3Department of Electrical Engineering andComputer Science, University of Michigan, Ann Arbor, Michigan. 4Comprehen-sive Cancer Center, University of Michigan, Ann Arbor, Michigan. 5ForbesInstitute for Cancer Discovery, University of Michigan, Ann Arbor, Michigan.6Computer Science Department UCLA, Boelter Hall, Los Angeles, California.7Department of Chemical Engineering, University of Michigan, Ann Arbor,Michigan. 8Department of Microbiology and Immunology, University ofMichigan, Ann Arbor, Michigan.

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

Corresponding Author: Gary D. Luker, University of Michigan, Ann Arbor, MI.Phone: 734-763-5476; Fax: 734-763-5447; E-mail: [email protected]

doi: 10.1158/1541-7786.MCR-18-0836

�2019 American Association for Cancer Research.

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mechanisms promoting glycolytic metabolism in migrating can-cer cells offers the potential to block this key process in tumorprogression. Recent studies establishing correlations betweenPAI1 with obesity and metabolic syndrome suggest PAI1 as amolecular regulator of metabolism that may translate to cancerbiology (10, 11). In support of PAI1 as a regulator of cancermetabolism, secretion of this molecule by fibroblasts increasedmitochondrial mass in cocultured breast cancer cells (12). How-ever, a direct relationship between PAI1 and cancer metabolismremains to be established.

This study investigates mechanisms through which PAI1 drivesmigration and an aggressive phenotype in TNBC, focusing onactin dynamics andmetabolism. Increasing expression of PAI1 inbreast cancer cells causes dramatic actin reorganization and drivesfragmentation of mitochondria and glycolytic metabolism in 2Dand 3D culture environments. Overall, this research for the firsttime establishes PAI1 as a novel regulator of cancer cell metab-olism and highlights the critical interplay between metabolismand motility in breast cancer.

Materials and MethodsCell culture

We purchased MDA-MB-231 cells from the ATCC and culturedcells in DMEM supplemented with 10% FBS and 1% penicillin/streptomycin (Thermo Fisher Scientific). We obtained SUM159cells from Dr. Stephen Ethier (now at The Medical University ofSouth Carolina, Charleston, SC) and cultured cells in F-12 mediasupplemented with 10% FBS, 1% penicillin/streptomycin, 1%glutamine, 5 mg/mL hydrocortisone, and 1 mg/mL insulin. Weauthenticated all cells by short tandem repeats analysis andcharacterized cells as free ofMycoplasma at the initial passage. Weused all cells within 3 months after resuscitation, and we main-tained all cells at 37�C in a humidified incubator with 5% CO2.

Lentiviral vectorsWe cloned human full-length PAI1 fused to NanoBiT (Pro-

mega) with a Tev-cleavable linker into the pLVX-Puro vector(ClonTech; PAI1) and verified products by sequencing. We pro-duced recombinant lentiviral vectors and transduced target cells asdescribed previously (13). We first generated cells stably expres-sing click beetle green luciferase (SUM159-CBG and MDA-MB-231-CBG) as described previously through selection with blas-ticidin (14). We subsequently transduced cells with the PAI1 viralvector and used puromycin selection for cells stably expressingPAI1.We confirmed expressionof PAI1 in these cells throughqRT-PCR and luminescence of NanoBiT.

The pLenti.PGK.LifeAct-GFP.W vector was a gift from RustyLansford (Addgene plasmid # 51010; University of SouthernCalifornia, Los Angeles, CA). We transduced wild-type and PAI1cells with LifeAct-GFP viral vector and sorted cells by flow cyto-metry to obtain populations with homogeneous expression ofLifeAct-GFP. For in vivo fluorescence lifetime imaging microscopy(FLIM) studies, we transduced wild-type and PAI1 cells withmCherry Nuc-FUW viral vector and sorted cells by flow cytometrytoobtainapopulationexpressingnuclearmCherry. For3Dspheres,we transduced human mammary fibroblasts (HMF, providedby Daniel Hayes, University of Michigan, Ann Arbor, MI) withmCherry viral vector and sorted for stable cells by flow cytometry.

The pLentiCMV Puro DEST ERK KTRClover was a gift fromMarkus Covert (Addgene plasmid # 59150; ref. 15). We replaced

mClover fluorescent protein with mCitrine and added a nuclearH2B-mCherry and a puromycin selection marker through P2Alinker sequences using the NEB HiFi DNA Assembly Kit (NewEngland BioLabs). This vector allows us to visualize ERK activa-tion via a nucleocytoplasmic shuttling event of the mCitrine ERKreporter, while mCherry demarks the nucleus. We cloned theconstruct into the Piggyback transposon vector (Systems Bios-ciences) and transfected cells using FuGENE HD (Promega). Oneweek after transfection, we treated cells with puromycin to iden-tify stable integrants and confirmed expression by fluorescencefrom mCitrine and mCherry.

qRT-PCRTo analyze levels of PAI1, we performed qRT-PCR for PAI1

and GAPDH using SYBR Green detection as described previous-ly (16). Primers for PAI1 were 50-CGCAACGTGGTTTTCTC-30 and50-CATGCCCTTGTCATCAATC-30 and for GAPDH were 50-GA-AGGTGAAGGTCGGAGT-30 and 50-GAAGATGGTGATGGGATT-TC-30.

Whole transcriptome next-generation sequencingWe performed whole transcriptome next-generation sequenc-

ing as described previously (17). We deposited these data as GEOaccession number GSE125802.

Bioluminescence growth, migration, and cell adhesion assaysWe analyzed effects of PAI1 on cell growth using biolumines-

cence imaging for CBG with medium binning and 30-secondexposure as described previously (14).

We used our previously published microfluidic device andwound-healing assays to verify PAI1 as a regulator of cell migra-tion. We performed microfluidic migration assays and imagedcells as described previously (17). For wound-healing assays, weseeded 1� 105 cells into 35-mm dishes and allowed cells to formconfluent monolayers before creating a linear scratch with a 200-mL pipette tip. We washed dishes once with PBS and then addedfreshmediumcontaining the proliferation inhibitorMitomycinC(1 mg/mL). When indicated, we also added vehicle, the PAI1inhibitor tiplaxtinin (5 mmol/L) (Selleckchem), or recombinantPAI1 (rPAI1, Sigma-Aldrich, 40 nmol/L) at the time of wounding.Wemeasured the size of thewound in eachmonolayer of cells at 0and 17 hours. We used the following formula to calculate woundclosure over time: (1�(woundwidth at 17 hours/woundwidth at0 hours)) � 100.

We also analyzed the ability of PAI1 tomodulate cell adhesion.We seeded 1� 105 HMFs expressingmCherry into a 24-well plate48 hours before the assay. After 48 hours, we seeded 2.5 � 105MDA-MB-231 and SUM159 wild-type or PAI1 LifeAct-GFP–expressing cells onto the HMFs and incubated at 37�C for 15minutes. We removed nonadherent cells with PBS and visualizedadherent cells by fluorescence imaging. We present data as thenumber of adherent cells to total number of cells seeded relative tomatched wild-type cells.

Immunofluorescence stainingTo visualize the actin cytoskeleton, we performed immunoflu-

orescence staining. We seeded 2.5 � 104 cells on glass cover slipsand incubated overnight.Wefixed cellswith 4%formaldehyde for15 minutes at room temperature and then washed three timeswith PBS for 5minutes each.We permeabilized cells with ice-cold100% methanol for 10 minutes at �20�C; washed with PBS for

PAI1 Drives TNBC Cytoskeletal Reorganization and Glycolysis

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5 minutes; and blocked with 10% goat serum for 1 hour at roomtemperature. Next, we incubated cells for 1 hour at room tem-perature with an antibody against phosphorylated paxillin(p-paxillin, Cell Signaling Technology) in 5% goat serum. Afterincubation, we washed cells with PBS 3 times for 5 minutes eachand costained with Alexa Fluor 488–conjugated secondary anti-body (Jackson ImmunoResearch Laboratory) for p-paxillin andconjugated Texas Red-X phalloidin (Thermo Fisher Scientific) foractin for 1 hour at room temperature in the dark. We washed theslides andmountedwithmedium containing an anti-fade reagentand DAPI (ProLong Gold, Thermo Fisher Scientific). We acquiredimages on an Olympus IX73 microscope with a DP80 CCDcamera (Olympus) and we analyzed epifluorescence images withcellSens software (Olympus) and stress fibers with in-houseMATLAB code.

Three-dimensional assaysWe formed spheroids as described previously (14), coculturing

MDA-MB-231 or SUM159 wild-type or PAI1 LifeAct-GFP–expres-sing cells with human mammary fibroblasts (HMF) expressingmCherry. We embedded spheroids or single cells (1� 106/mL) infibrin gels (final concentration 4 mg/mL fibrin, 2.5 U/mL throm-bin, and 0.01 U/mL aprotinin; ref. 18) and imaged actin andfibroblasts by two-photon microscopy. We analyzed the perim-eter of spheroids using in-house MATLAB code. We mechanicallydissociated spheroids after 24 hours by pipetting up and down 5times with a micropipette in the bottom of a 96-well plate. Weenumerated dissociated cells by fluorescence microscopy.

Kinase translocation reporterTo quantify activation of ERK by PAI1, we used cells stably

expressing a validated kinase translocation reporter (KTR) for thiskinase. This reporter utilizes a known downstream substrate ofERK to drive reversible translocation of the reporter into and outof the nucleus (15).We seeded 1.2� 105MDA-MB-231 and 8.5�104 SUM159 wild-type cells containing the KTR for ERK and anuclear marker (H2B-mCherry) on to 35-mm dishes with a 20-mm glass bottom (Cellvis) in FluoroBrite DMEMmedia (ThermoFisher Scientific, A1896701). After 2 days, we changedmedium tolow (1%) serum in Fluorobrite DMEM and added tiplaxtinin (5mmol/L)where indicated. The next day,we treated cellswith eitherrPAI1 (40nmol/L) or vehicle control and acquired images at timeslisted in thefigure.We analyzed images for activationof ERKusingcustom MATLAB code.

FLIM and metabolic analysisWe used FLIM to quantify metabolic differences between wild-

type and PAI1 cells based on endogenous fluorescence fromNADH as described previously (ISS FastFLIM; refs. 19, 20). Forboth NADH and mCherry, we used 740 nm excitation, 25� NIRcorrected objective, 512�512matrix, 15% laser power, 4ms dwelltime, and 2.5� electronic zoom (Olympus FVMPE-RS uprightmicroscopewith Spectra-Physics InsightDSþ laser).We separatedlight emitted from NADH and mCherry with band pass filters of410–460 nm and 545–645 nm, respectively.

We exported modulation lifetime and direct counts (DC,intensity) data as ".tiff" files from ISS VistaVision into MATLABalongside red-channel intensity data obtained by two-photonmicroscopy from the Olympus software for analysis. We filteredimages to remove all data points with NADH lifetimes less than0.5 ns and greater than 5 ns, as well as points with DC's less than

15 as per themanufacturer's instructions.We created amask usingmCherry tomark locations of cancer cells in images. We analyzedNADH lifetimes of pixelswithin themask for each cell andpresentlifetimes on images based on a pseudo color scale.

To confirm our FLIM data, we performed metabolic flux anal-ysis to measure the glycolytic capacity of cells by measuring theextracellular acidification rate (ECAR) of cells using a SeahorseBioscience XFe96 Extracellular Flux Analyzer and XF Cell EnergyPhenotype test kit (Agilent) as described previously (19).

Mouse xenograft implantationThe University of Michigan Institutional Animal Care and Use

Committee approved all animal procedures (protocol00006795). The animals used in this study received humane carein compliance with the principles of laboratory animal careformulated by the National Society for Medical Research andGuide for theCare andUse of Laboratory Animals prepared by theNational Academy of Sciences and published by the NIH(Bethesda, MD; publication no NIH 85-23, revised 1996). Weestablished orthotopic tumor xenografts in the fourth inguinalmammary fat pads of 17–21-week-old female NSG mice (TheJackson Laboratory; ref. 16), implanting 2� 105MDA-MB-231 orSUM159 wild-type or PAI1 cells that express LifeAct-GFP. Wequantified tumor growth and metastasis by bioluminescenceimaging as described previously (13). We euthanized mice 7–8 weeks after injection (n ¼ 5 mice in each group) and thenimmediately visualized collagen and actin by two-photonmicros-copy. We used an 880 nm excitation wavelength and collectedsecond harmonic signal from fibrillary collagen and emissionfrom LifeAct-GFP with 410–460 nm and 495–540 nm filters,respectively (21). For FLIM experiments, we implanted 2 � 105MDA-MB-231 or SUM159 wild-type or PAI1 cells expressingmCherry Nuc-FUW to demark nuclei of cancer cells. Six weeksafter injection, we euthanized mice, removed the tumor andlungs, and immediately imaged metabolism of cancer cells inorthotopic tumors or lung metastases ex vivo by FLIM. We notethat previous research demonstrates NADH/FAD metabolism ofcancer cells maintained in intact tumors and organs ex vivoremains the same as in vivo for up to eight hours (22, 23). Tocalculate numbers of cells in an image, we used FIJI (24) tobinarize and analyze numbers of nuclei in a region. We thenpooled and plotted the density from each image.

MATLAB analysisMATLAB programs used to measure the kinase translocation

reporter (KTR), collagen directionality, stress fiber density, andspheroids are available through a Material Transfer Agreement(MTA).

Statistical analysisWe used a nonparametric Mann–Whitney U test for compar-

isons of cell migration and motility with a significance level of0.05 considered statistically significant. For sphere formation andother experiments, we used two-tailed, unpaired Student t tests.We prepared bar graphs (mean valuesþ SD or SEM as denoted infigure legends) and box plots andwhiskers using GraphPad Prism7 orOrigin 9.0. For box plots andwhiskers, the bottom and top ofa box define the first and third quartiles, and the band inside thebox marks the second quartile (the median). The ends of thewhiskers represent the 5th percentile and the 95th percentiles,respectively. The "þ" inside the box indicates the mean, dots

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outside thebox andwhiskers indicate outliers, and the "x" refers tothe maximum and minimum of all data.

ResultsPAI1 enhances cell migration

We recently used our migration-based microfluidic device toidentify critical positive and negative regulators of cell migration(ref. 17; Fig. 1A and B). RNA-sequencing data revealed plasmin-ogen activator inhibitor 1 (PAI1) as one of the most highlyupregulated genes (�8-fold relative to nonmigratory cells, P <0.0001) in both highly migratory SUM159 and MDA-MB-231triple-negative breast cancer (TNBC) cells. To investigate effects ofPAI1 on migration and motility of TNBC cells in vitro and in vivo,we stably expressed PAI1 in SUM159 and MDA-MB-231 cells.qRT-PCR analysis revealed higher expression of PAI1 in stablytransduced SUM159 andMDA-MB-231 PAI1 cells comparedwithwild-type control (Supplementary Fig. S1). RNA sequencing withgene set enrichment analysis (GSEA) identified significant asso-ciations with cell movement and organization of the cytoskele-ton/cell projections in PAI1 cells (Table 1), confirming the effectof PAI1 on cell migration. Stable expression of PAI1 significantlyincreased migration of both SUM159 and MDA-MB-231 cell inour migration device (Fig. 1C and D) and a wound-healing assay(Fig. 1E and F). Adding exogenous recombinant active PAI1

(rPAI1) also significantly increased migration of wild-type cellsfrom both cell lines, and a PAI1-specific inhibitor, tiplaxtinin,significantly reduced cellmigration (Fig. 1E and F; SupplementaryFig. S1). Together, these data show that elevated expression ofPAI1 enhances migration.

PAI1 promotes actin cytoskeleton reorganization and aninvasive phenotype

Cell migration critically depends on the actin cytoskeleton.Lamellipodia, membrane ruffles, filopodia, and stress fibers con-stitute the most important and studied actin-based structures formotility. A lamellipodium and membrane ruffles demark actin-rich sheet-like projections of a motile cell, while a filopodium

Figure 1.

PAI1 enhances migration of TNBCcells. A, Schematic of the migrationdevice. We initially loaded cells intothe left and right chambers of thedevice in serum-free media. Aftercells adhered, we loadedmediacontaining a chemoattractant(serum) into the middle channel tostimulate migration for 24 hours.B,We used trypsin to selectivelyrecover cells from the middlechannel for further analyses. C andD, Graphs show box and whiskerplot summaries (top) and frequencydistributions (bottom) for migrationof SUM159 (C) and MDA-MB-231 (D)WT and PAI1 cells toward serum inthe device (n¼ 600 cells per group;�� , P < 0.0001). E and F, Summaryof percentages of wound closurepresented as meanþ SD (n¼ 9).� , P < 0.05; �� , P < 0.01, as comparedwithWT control cells.

Table 1. Relevant GO biological processes of PAI1-expressing cells based onGSEA

GO Biological Process P

Biological adhesion 2.60E�31Movement of cell or subcellular component 4.43E�30Homophilic cell adhesion via plasmamembrane adhesionmolecules 2.22E�25Regulation of intracellular signal transduction 2.49E�25Cell projection organization 3.66E�25Cell–cell adhesion via plasma membrane adhesion molecules 9.20E�25Cytoskeleton organization 1.11E�24Positive regulation of gene expression 2.28E�23






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defines a finger-like projection extending from a lamellipodium.Stress fibers form from contractile actomyosin bundles. Effects ofPAI1 on each of these actin-based structures remain poorlydefined. Phalloidin staining revealed dramatic reorganization ofthe actin cytoskeleton in PAI1 cells compared with wild type.Stable expression of PAI1 or addition of rPAI to cells increased theformation of actin-rich migratory structures (lamellipodia andmembrane ruffles; Figs. 2A and 3A) and reduced stress fiberdensity (stress fiber area divided by total cell size; SupplementaryFig. S2) compared with control cells. In addition, we found thatPAI1 increases, and tiplaxtinin reduces, the aspect ratio (length/width) of cells (Figs. 2B and 3B), which suggests PAI1 supports amore mesenchymal morphology associated with cell migration.

To study actin dynamics in living cells, we stably expressedLifeAct-GFP, a 17-amino-acid peptide that binds to filamentousactin (F-actin) and does not interfere with actin dynamics (25), inbothwild-type andPAI1 cells. To confirmour phalloidin staining,we imaged SUM159PAI1 and control cells expressing LifeAct-GFPas theymigrated through ourmicrofluidic device. Consistent withour previous data, we found that PAI1 cells had greater actin-richmigratory structures compared with control cells (SupplementaryVideo S1 and S2). Next, we uniformly embedded single cellsexpressing LifeAct-GFP into fibrin gels and allowed them toproliferate. Fibrin promotes adhesion and migration of cells andprovides structural integrity for breast tumors (26). Aggregates ofSUM159 PAI1 cells displayed a more invasive phenotype withmore actin-rich structures extending into the surrounding gel(Fig. 2C). These projections contained concentrated actin-basedleading edges, suggesting an actin-richmigratory structure such aslamellipodia or membrane ruffles. We also noted that SUM159PAI cells formed smaller aggregates than wild-type cells. MDA-MB-231 cells did not form aggregates in gels, independent of PAI1expression. However, wild-type MDA-MB-231 cells did formgrape-like nonadherent structures, whereas PAI1 cells formedadherent structures that displayed actin-based projections(Fig. 3C).

We next formed spheroids using LifeAct-GFP cells andhuman mammary fibroblasts (HMF) and embedded spheroidsinto fibrin gels to investigate effects of PAI1 on cancer cells in aphysiologically relevant, coculture 3D model. While HMFsshowed similar phenotypes between groups, wild-type Life-Act-GFP cells displayed a compact, spherical shape with adefined edge (Fig. 2D and 3D). By comparison, both SUM159and MDA-MB-231 PAI1 cells demonstrated greater invasioninto the adjacent fibrin gel (Figs. 2D and 3D). We also discov-ered that cancer cells near the edge of PAI1 spheroids showedmore actin-rich migratory structures, suggesting actin reorga-

nization and enhanced motility. Using custom MATLAB codewe identified the margins of cancer cells in spheroids. Relativeto spheroids with control cells, PAI1 spheroids contained moreprojections from a spheroid, and these projections extendedfarther from the spheroid core into the adjacent gel (Figs. 2Dand 3D). Overall, these data establish that PAI1 promotes actincytoskeleton reorganization to display a more migratory phe-notype in 2D and 3D settings.

PAI1 enhances cellular adhesionIn addition to cell migration, GSEA also revealed multiple sets

with associations to cell adhesion or attachment (Table 1). Wenoted that cancer cells invading from a spheroid into the geltracked closely with HMF cells, suggesting that PAI1 promotesintercellular adhesion. To test this hypothesis, we mechanicallydissociated coculture spheroids of cancer cells andHMFs and thenenumerated single cancer cells. We found more single wild-typecancer cells following mechanical disruption of spheroids, con-sistent with reduced adhesion relative to cancer cells with elevatedPAI1 (Supplementary Fig. S3).We also identified greater adhesionof PAI1 cancer cells to HMFs in monolayer culture (Supplemen-tary Fig. S3). Together, these data demonstrate that PAI1promotesintercellular adhesion of breast cancer cells.

To investigate mechanisms through which PAI1 promotesadhesion, we focused on focal adhesions, structures of the actincytoskeleton that form anchor points to the extracellular matrix(ECM). Immunofluorescence staining for paxillin, a protein com-ponent of focal adhesions, showed reduced numbers of focaladhesions in breast cancer cells with elevated PAI1 relative tocontrol (Supplementary Fig. S4). This result suggests that PAI1increases adhesion of breast cancer cells independent of focaladhesions.

PAI1 activates ERK signaling and alters mitochondrialmorphology

MAPK signaling through ERK defines one prominent pathwaythat promotes reorganization of actin, turnover of focal adhe-sions, and cell motility (27). We investigated to what extent PAI1activates ERK in single cells using a kinase translocation reporter(KTR) system (15). Using cells stably expressing the ERK KTR, wefound that exogenous addition of rPAI1 to both wild-typeSUM159 and MDA-MB-231 cells increased kinase activity of ERKat 20 minutes (Fig. 4A; Supplementary Fig. S5). In addition,tiplaxtinin inhibited activation of ERK by rPAI1 (SupplementaryFig. S5). Activation of ERK provides one potential mechanismthrough which PAI drives cell migration.

Figure 2.Stable expression of PAI1 promotes actin cytoskeletal reorganization and amore invasive phenotype in SUM159 TNBC cells. A, Representative overlaid images ofimmunofluorescence staining of phalloidin (red) and nuclear DAPI (blue) ofWT cells; WT cells treated with rPAI1 (40 nmol/L); PAI1 cells; and PAI1 cells treatedwith tiplaxtinin (5 mmol/L; left). Arrows point to actin-rich migratory structures. Right, Frequency distribution of number of actin-rich migratory structures percell for each group (n¼ 50 cells). B, Box plot and whiskers for the aspect ratio (length/width) of cells in each group demonstrates a more elongated morphologyof PAI1 cells (n¼ 50 cells). �� , P < 0.0001. C, Representative images of spheres formed from single cells of SUM159WT and PAI1 cells that express LifeAct-GFP(left). Arrows point to projections. Right, Analysis of the mean number of projections per spheroidþ SD (top) and the mean circumference of spheroidsþ SD ineach group (bottom; n¼ 7 spheroids per group). � , P < 0.05; �� , P < 0.01. D, Representative images of spheroids composed of SUM159WT or PAI1 cells thatexpress LifeAct-GFP (green) and humanmammary fibroblasts (HMFs, red, i). Arrows in zoomed picture point to actin-rich migratory structures. Example imagesthat define the perimeter of cancer cells in a spheroid (green line), the center of the spheroid (red dot), and the spheroid core (red circle, circle from the center ofthe spheroid with a radius of the distance from the center to the closest point on the perimeter) for each of the groups (ii). Frequency distribution of the distanceof points on the perimeter of the spheroid from the spheroid core (n¼ 5 spheroids, iii). Graph showing the mean number of points on the perimeterþSD that falloutside of the spheroid core (n¼ 5 spheroids; iv). � , P < 0.05. These data show that spheroids made from cells that stably express PAI1 havemore projections,farther projections, and amore invasive phenotype than spheroids fromWT cells.






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Recent studies show that ERK promotes mitochondrial fis-sion to drive tumor growth (28) and cellular reprogram-ming (29), and more migratory cancer cells frequently exhibitgreater fission of mitochondria. Staining with MitoTrackerGreen showed greater fragmentation of mitochondria, indica-tive of mitochondrial fission, in both SUM159- and MDA-MB-231-PAI cells. Treatment of wild-type parental cells with rPAI1also shifted mitochondria from more fused, elongated struc-tures to a more fragmented morphology, characteristic offission (Supplementary Fig. S6).

PAI1 promotes glycolytic metabolism in vitroHaving established that PAI1 promotes mitochondrial fission,

we next investigated effects of PAI1 on cellmetabolism in vitro.Weused FLIM of fluorescence from endogenous NADH to measurerelative glycolytic versus oxidative metabolism in single TNBCcells (30). Cells predominantly relying on glycolysis have greateramounts of free NADH, which exhibits a shorter lifetime thanprotein-bound NADH in cells utilizing oxidative phosphoryla-tion (OXPHOS; ref. 31). We determined that PAI1 promotesglycolytic metabolism in both SUM159 and MDA-MB-231 cellsin culture as shown by a shorter lifetime for fluorescence fromNADH (Fig. 4B). We also found that PAI1 promotes glycolyticmetabolism in cells seeded infibrin gels (Fig. 4C). Consistentwithour FLIM data, we found that PAI1 increased the extracellularacidification rate (ECAR) in both SUM159 and MDA-MB-231cells (Supplementary Fig. S7), suggesting an increase in glycolyticcapacity in cells that express PAI1. Together, these data demon-strate that PAI1 shifts cellular metabolism to glycolysis.

PAI1 orthotopic tumors are smaller, but show a more invasivephenotype

To extend our studies to an in vivo setting, we orthotopicallyimplanted SUM159 or MDA-MB-231 wild-type or PAI1 cells thatexpress LifeAct-GFP into the fourth mammary fat pad of NSGmice. Consistent with our data showing that PAI1 cells form fewer(Supplementary Fig. S8) and smaller tumor spheres than wild-type cells (Figs. 2C and 3C), mice injected with PAI1 TNBC cellsproduced significantly smaller tumors (Fig. 5A). Even though wesaw significantly smaller orthotopic tumors, we did not observeany differences in growth of PAI1 versus wild-type cells in stan-dard culture (Supplementary Fig. S8). Interestingly, we foundreduced density of cancer cells in PAI1 tumors (number of cancercells divided by total tumor area; Fig. 5B and C). PAI1 TNBC cellsalso displayed more actin-rich migratory structures than cancercells in wild-type tumors (Fig. 5B). In addition to imaging cancercells, we analyzed the architecture of peritumoral collagen bysecond harmonic two-photonmicroscopy. Alignment of collagenfibers perpendicular to the margin of a tumor defines a moreinvasive phenotype with poor prognosis in breast cancer (32).

Tumors formed from SUM159- and MDA-MB-231-PAI cellsshowed greater alignment of collagen perpendicular to the tumormargin than wild-type, consistent with a more locally invasivephenotype (Fig. 5B and D; Supplementary Fig. S9). We note thatboth bioluminescent and two-photon imaging revealed no dif-ferences in metastasis between PAI and control tumors (data notshown), potentially because we euthanized mice at a relativelylate time point (8 weeks) after implantation to facilitate imagingby two-photon microscopy.

PAI1 promotes glycolytic metabolism in a mouse model ofbreast cancer

To determine effects of PAI on metabolism of cancer cellsin vivo, we used FLIM to quantify glycolysis versus OXPHOS inorthotopic tumors and lung metastases produced by SUM159 orMDA-MB-231 wild-type or PAI1 cells. For these studies, we usedcancer cells expressing nuclearmCherry to avoid interference fromGFP in detecting NADH and distinguish disseminated cancercells. Unlike studies that recover cancer cells from tumors andanalyze metabolism of bulk populations of dissociated cancercells in standard culture, FLIM allows quantitative analysis ofmetabolism in single cancer cells in intact tumors andmetastases.We analyzed mice six weeks after injection. Both SUM159 andMDA-MB-231 PAI1 cells in orthotopic tumors displayed moreglycolytic metabolism (Fig. 6A and B). In addition to orthotopictumors, we discovered enhanced glycolysis inMDA-MB-231 PAI1cells in lung metastases (Fig. 6C). We were unable to detectSUM159 metastases independent of PAI1 expression at thetime of imaging. These data establish that PAI1 promotes glyco-lytic metabolism at both the orthotopic tumor and the lungmetastatic site.

DiscussionDeveloping more effective treatments to block local invasion

and metastasis in cancer requires mechanistic understanding ofpathways driving cancer cell migration. While well-established asa prognostic and predictive biomarker in breast cancer (1), target-ingPAI1 for cancer therapy remains challenging due to limitationsof existing drugs and incomplete understanding of functions ofPAI1 in tumor progression (33). While other studies have iden-tified PAI1 as an important regulator of actin dynamics and cellmigration (34–36), mechanisms for how PAI1 promotes cancercell migration and tumor progression remain incompletelydefined. Here we make the unique discovery that PAI1 activatesERK, connecting PAI1 signaling to glycolytic metabolism andcytoskeletal reorganization.

Building on our discovery that highly migratory TNBC cellsexpress high levels of PAI1, this study established that PAI1 drivescell migration by promoting cytoskeletal reorganization and

Figure 3.Stable expression of PAI1 promotes actin cytoskeletal reorganization and amore invasive phenotype in MDA-MB-231 TNBC cells. A, Representative overlaidimages of immunofluorescence staining of phalloidin (red) and nuclear DAPI (blue) ofWT cells; WT cells treated with rPAI1 (40 nmol/L); PAI1 cells; and PAI1 cellstreated with tiplaxtinin (5 mmol/L; left). Arrows point to actin-rich migratory structures. Right, Frequency distribution of number of actin-rich migratorystructures per cell for each group (n¼ 50 cells). B, Box plot and whiskers for the aspect ratio (length/width) of cells in each group demonstrates more elongatedmorphology of PAI1 cells (n¼ 50 cells). � , P < 0.05; ��, P < 0.01; �� , P < 0.0001. C, Representative images of spheres formed from single cells of MDA-MB-231 WTand PAI1 cells that express LifeAct-GFP. D, Representative images of spheroids composed of MDA-MB-231 WT or PAI1 cells that express LifeAct-GFP (green) andhumanmammary fibroblasts (HMFs, red; i). Arrows in zoomed picture point to actin-rich migratory structures. Example images that define the perimeter ofcancer cells in a spheroid (green line), the center of the spheroid (red dot), and the spheroid core (red circle) as defined in Fig. 2 (ii). Frequency distribution of thedistance of points on the perimeter of the spheroid from the spheroid core (n� 9 spheroids; iii). Graph showing the mean number of points on the perimeterþSD that fall outside of the spheroid core (n� 9 spheroids; iv; � , P < 0.05).






Figure 4.

PAI1 activates ERK signaling and promotes glycolytic metabolism in TNBC cells. A, Representative fluorescence images of ERK KTR (yellow) and nuclear H2B-mCherry (red) in SUM159 and MDA-MB-231 WT cells at the initial timepoint (0 minutes) and 20minutes after addition of rPAI1 (40 nmol/L) showing translocationof yellow fluorescence out of the nucleus (darker nucleus, ERK activation; left). Right, frequency distribution of cytoplasmic-to-nuclear ratio (CNR) of SUM159 (n> 350 cells) and MDA-MB-231 (n > 670 cells) WT cells at the initial timepoint (0minutes) and 20minutes after addition of rPAI1 (n¼ 2). A shift to the rightdemonstrates activation of ERK signaling. B, Frequency distributions for NADH lifetime and representative FLIM images of SUM159 and MDA-MB-231 WT andPAI1 cells in 2D (B, SUM159: n� 12, and MDA-MB-231: n� 9) and in 3D fibrin gels (C, SUM159: n� 39, and MDA-MB-231: n� 36). Both SUM159 and MDA-MB-231PAI1 cells shift toward glycolytic metabolism as defined by a shorter NADH lifetime in 2D and 3D environments.

Humphries et al.





glycolytic metabolism. Recent studies show migrating cancercells utilize energy generated through glycolysis rather thanOXPHOS (7, 8), suggesting that elevated glycolysis mediated byPAI1drives cellmigration.Mechanistically, weobserved that PAI1activates several processes that potentially link cell migration andglycolytic metabolism. Cells with high expression of PAI1 (i)activate ERK; (ii) exhibit fragmented mitochondria; and (iii)utilize glycolysis to a greater extent than control breast cancercells in environments from 2D cultures to metastases in mice.Overall, our data suggest anovelmetabolic regulatory function forPAI1 in migration and tumor progression.

Dysregulation of cell migration during cancer progressionshapes the metastatic capacity of cancer cells. Cell migration isthe result of amulti-step process driven by dynamic changes in the

actin cytoskeleton. Consistent with other studies (37, 38), wefound that PAI1 expression reduces stress fiber density whileincreasing the formation of actin-rich migratory structures instandard cell culture. We also found that cells expressing PAI1displayed reduced focal adhesions as determined by paxillinstaining. Paxillin is a key component of integrin signaling, andphosphorylation of paxillin is required for integrin-mediatedreorganization of the actin cytoskeleton. Previous work demon-strated that phosphorylation of paxillin by ERK promotes trans-location of paxillin from focal adhesions to the cytosol, resultingin disassembly of focal adhesions (39, 40). Therefore, one expla-nation for reduced focal adhesions in cells expressing PAI1maybethrough activation of ERK. Although cells displayed a moremigratory phenotype in culture conditions, breast cancer cells

Figure 5.

PAI1 reduces orthotopic tumorgrowth and promotes collagenalignment. A, Area-under-the-curvephoton flux for orthotopic tumorgrowth of SUM159 and MDA-MB-231WT and PAI1 cells. Graphs showmeanþ SEM (n� 6 tumors;� , P < 0.05; �� , P < 0.01).B, Representative overlaid imagesof tumors (green) and collagen(gray) from SUM159 or MDA-MB-231WT or PAI1 cells that expressLifeAct-GFP. Tumors from PAI1expressing cells demonstrate morealigned collagen fibers. C,Graphsdemonstrate reduced cell density intumors from PAI1 cells relative toWT (SUM159: n > 30 z-axis sections� 20 mm in depth, and MDA-MB-231: n¼ 36 z-axis sections� 20 mmin depth). Graphs showmeanþSEM. � , P < 0.05; �� , P < 0.0001.D, Frequency distribution ofcollagen alignment angle revealsgreater alignment of collagen fibersperpendicular to margins of tumorsfrom PAI1 cells. Graphs show theorientation of the collagen fibersrelative to the tumor (n� 5tumors).






with elevated expression of PAI1 exhibited greater adhesion toHMFs. Apparent discordance between greater migration andadhesion may arise from measuring these processes in 2D and

3D environments, respectively. Furthermore in orthotopictumors, we found that PAI1 promotes alignment of tumor-asso-ciated collagen perpendicular to the margin of a tumor, a

Figure 6.

PAI1 promotes glycolytic metabolism in both orthotopic tumor andmetastatic sites. Left, Frequency distributions of NADH lifetime for SUM159 (A) or MDA-MB-231 (B) WT control and PAI1 cells in an orthotopic tumor (n� 5 tumors) and MDA-MB-231 WT and PAI1 cells in lung metastatic sites (C; n� 3 lungs, n� 15metastatic sites per group). Decreased lifetime of NADH fluorescence in PAI1 cells indicates increased glycolysis in both the orthotopic tumor and lungmetastases. Right, Representative fluorescence (red) and FLIM images of cells in orthotopic tumor and lung metastatic sites.

Humphries et al.





signature associated with tumor invasion and poor progno-sis (32). These data suggest that PAI1 enhances migration notonly through effects on cancer cells, but also on the surroundingtumor microenvironment.

Reprogramming of cellular metabolism represents a hallmarkfeature of cancer with malignant cells frequently shifting toglycolytic metabolism (41). Breast cancers upregulate glycolyticenzymes relative to normal breast tissue and benign breastlesions, and glycolytic metabolism correlates with worse out-comes (42, 43). Mechanisms underlying a switch from oxidativeto glycolytic metabolism in cancer cells remain incompletelydefined. Here, we discovered a previously unknown function ofPAI1 to promote glycolysis in TNBC cells, a metabolic switchlinked to cell migration (7). Consistent with a previous studyshowing mitochondrial effects of PAI1 (12), we found that PAI1promotes mitochondrial fission in TNBC cells. Mitochondrialfission promotes a shift to glycolytic metabolism in both normaland malignant settings (44–46), linking changes in mitochon-drialmorphology to effects of PAI1 onmetabolic reprogramming.ERK signaling drives mitochondrial fission (28, 29), suggestingactivation of this kinase as a mechanism through which PAI1promotes mitochondrial fission and glycolysis. In addition, wefound that PAI1 cells retain their enhanced glycolytic phenotypefollowing colonization to lung. This contrasts with other studiesdemonstrating that breast cancer cells engage OXPHOS to metas-tasize to the lung (47, 48). Differences among studies may be dueto the use of human breast cancer cells in our current study versusmurine breast cancer cells in prior publications. In addition, wequantified metabolism in single cells in situ using FLIM of endog-enous NADH (49, 50), while studies showing OXPHOS in lungmetastases relied on profiles of gene expression and cell cultureassays with populations of cancer cells selected for enhanced lungmetastasis. Our data establish that PAI1 drives glycolysis in cell-based assays, orthotopic xenografts, and lungmetastases, suggest-ing that PAI1 glycolytic reprogramming as an integral function ofPAI1 in breast cancer.

In summary, this study not only provides the first evidence thatPAI1 promotes glycolytic metabolism but also suggests an under-lying mechanism mediated by ERK and known effects of this

kinase on mitochondrial morphology. Our data support recentresearch proposing that highly migratory cells rely on energyobtained primarily through enhanced glycolysis for migrationand chemotaxis (7, 8). Data from our study provide a strongrationale for targeting cancer cell metabolism as a potentialtherapeutic approach not only for aggressive andhighlymigratorycancers, such as TNBC, but also for patients with elevated levels ofPAI1 in primary tumors and/or circulating tumor cells.

Disclosure of Potential Conflicts of InterestG.D. Luker is a consultant/advisory board member for Polyphor. No poten-

tial conflicts of interest were disclosed by the other authors.

Authors' ContributionsConception and design: B.A. Humphries, E. Yoon, K.E. Luker, G.D. LukerDevelopment of methodology: B.A. Humphries, J.M. Buschhaus, Y.-C. Chen,B. Chiang, Y.-H. Cheng, E. Yoon, K.E. Luker, G.D. LukerAcquisition of data (provided animals, acquired and managed patients,provided facilities, etc.): B.A. Humphries, J.M. Buschhaus, Y.-C. Chen,H.R. Haley, T. Qyli, B. Chiang, N. Shen, A. Cutter, K.E. Luker, G.D. LukerAnalysis and interpretation of data (e.g., statistical analysis, biostatistics,computational analysis): B.A. Humphries, J.M. Buschhaus, Y.-C. Chen,H.R. Haley, T. Qyli, B. Chiang, S. Rajendran, A. Cutter, J. Cong, P.C. Spinosa,K.E. Luker, G.D. LukerWriting, review, and/or revision of the manuscript: B.A. Humphries,J.M. Buschhaus, Y.-T. Chen, K.E. Luker, G.D. LukerAdministrative, technical, or material support (i.e., reporting or organizingdata, constructing databases): Y.-T. Chen, E. Yoon, K.E. Luker, G.D. LukerStudy supervision: E. Yoon, K.E. Luker, G.D. Luker

AcknowledgmentsThe authors acknowledge funding from NIH grants R01CA196018,

R01CA195655, U01CA210152, and R37CA222563. This study was supportedby NIH grants R01CA196018, R01CA195655, U01CA210152, andR37CA222563.

The costs of publication of this articlewere defrayed inpart by the payment ofpage charges. This article must therefore be hereby marked advertisement inaccordance with 18 U.S.C. Section 1734 solely to indicate this fact.

Received August 7, 2018; revised October 22, 2018; accepted January 29,2019; published first February 4, 2019.

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Humphries et al.




2019;17:1142-1154. Published OnlineFirst February 4, 2019.Mol Cancer Res Brock A. Humphries, Johanna M. Buschhaus, Yu-Chih Chen, et al. Triple-Negative Breast CancerCytoskeleton Reorganization and Glycolytic Metabolism in Plasminogen Activator Inhibitor 1 (PAI1) Promotes Actin

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PlasminogenActivatorInhibitor1(PAI1)Promotes ... · PAI1 enhanced cytoskeletal features associated with migra-tion, actin-rich migratory structures, and reduced actin stress ﬁbers