Tissue Tranglutaminase Regulates Interactions between ...of the interaction between TG2 and FN also...

Tumor Biology and Immunology

Tissue Tranglutaminase Regulates Interactionsbetween Ovarian Cancer Stem Cells and theTumor NicheSalvatore Condello1, Livia Sima1, Cristina Ivan2,3, Horacio Cardenas1, Gary Schiltz4,Rama K. Mishra4, and Daniela Matei1,5,6

Abstract

Cancer progression and recurrence are linked to a rare popula-tion of cancer stem cells (CSC). Here, we hypothesized that inter-actions with the extracellular matrix drive CSC proliferation andtumor-initiating capacity and investigated the functions of scaffoldprotein tissue transglutaminase (TG2) in ovarian CSC. Complexesformed by TG2, fibronectin (FN), and integrin b1 were enriched inovarian CSC and detectable in tumors. A function-inhibiting anti-body against the TG2 FN-binding domain suppressed complexformation, CSC proliferation as spheroids, tumor-initiating capac-ity, and stemness-associated Wnt/b-catenin signaling. Disruptionof the interaction between TG2 and FN also blocked spheroidformation and the response to Wnt ligands. TG2 and the Wnt

receptor Frizzled 7 (Fzd7) form a complex in cancer cells andtumors, leading to Wnt pathway activation. Protein docking andpeptide inhibition demonstrate that the interaction between TG2and Fzd7 overlaps with the FN-binding domain of TG2. Theseresults support a new function of TG2 in ovarian CSC, linked tospheroid proliferation and tumor-initiating capacity andmediatedthroughdirect interactionswithFzd7.Wepropose this complex as anew stem cell target.

Significance: These findings reveal a newmechanism by whichovarian CSCs interact with the tumor microenvironment, pro-moting cell proliferation and tumor initiation. Cancer Res; 78(11);2990–3001. �2018 AACR.

IntroductionOvarian cancer (OC) is the most lethal gynecological cancer

(1), characterized by rapid growth, dissemination in the perito-neal space and universal development of chemoresistance, lead-ing to fatal tumor recurrence after primary treatment (2). Theaccumulation of malignant ascites in the peritoneal cavity pro-vides a favorable tumor microenvironment (TME) enriched insecreted inflammatory cytokines (3), growth factors (4), andextracellular macromolecules [collagen, fibronectin (FN), andlaminin; ref. 5], which regulates oncogenic signaling. In thismilieu, cancer cells form multicellular spheroids enriched instem/progenitor cells. The goal of this study was to mechanisti-cally dissect signaling pathways engaged by the extracellular

matrix (ECM), which regulate ovarian cancer stem cells (OCSC)proliferation in the peritoneal environment.

Tissue transglutaminase (TG2) is a multifunctional proteinwith protein crosslinking, GTPase and scaffold functions. Itsactivities are regulated by environmental factors and cellularlocalization. High intracellular GTP and low Ca2þ concentrationsinhibit TG2's enzymatic function, while in the extracellulardomain, in the presence of Ca2þ, the protein exerts transgluta-minase activity, crosslinking glutamine-rich ECM proteins. Reso-lution of the three-dimensional (3D) structure of TG2 providedimportant insight into the complex regulation of its functions (6).In addition to the catalytic triad, the protein has a FN-bindingdomain within its N terminus. Previous studies identified the bhairpin loop as the FN-binding region (amino acids 88–106;ref. 7). However,more recent analyses using hydrogen/deuteriumexchange andmass spectrometry point to residues K30, R116, andH134 in the N-terminus as being critical to forming a complexwith the 42-kDa gelatin-binding domain of FN (8).On the surfaceof cells, the complexbetweenTG2andFN is further supported andstabilized by direct interactions of both proteins with integrins,themajor receptors involved in cellular adhesion to the ECM.Dueto strong noncovalent association with both proteins, TG2 sig-nificantly enhances the interaction of cells with thematrix, servingas a bridge between integrins and FN (9). While little is knownabout specifics of the integrin–TG2 complexes, the complemen-tary TG2–FN binding sites have been delineated, and disruptionof this interaction appears a promising approach for interferingwith cell–ECMinteraction (9).Here,we study the functions of thiscomplex in regulation of OCSC signaling and functions.

TG2 was identified as an overexpressed transcript in OC cellsand tumors (10), and its aberrant expression was correlated withperitoneal dissemination (10, 11) and spheroid proliferation

1Department of Obstetrics and Gynecology, Feinberg School of Medicine,Northwestern University, Chicago, Illinois. 2Department of Experimental Ther-apeutics, The University of Texas MD Anderson Cancer Center, Houston, Texas.3Center for RNA Interference andNon-Coding RNAs, TheUniversity of TexasMDAnderson Cancer Center, Houston, Texas. 4Center for Molecular Innovation andDrug Discovery, Northwestern University, Evanston, Illinois. 5Robert H LurieComprehensive Cancer Center, Chicago, Illinois. 6Jesse Brown VA MedicalCenter, Chicago, Illinois.

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

Corresponding Authors: Daniela Matei, Northwestern University, 303 EastSuperior Street, Chicago, IL 60610. Phone: 312-503-4853; Fax: 312-472-4688;E-mail: [email protected]; and Salvatore Condello, Phone: 312-503-4933; Fax: 312-503-0095; E-mail: [email protected]

doi: 10.1158/0008-5472.CAN-17-2319

�2018 American Association for Cancer Research.

CancerResearch

Cancer Res; 78(11) June 1, 20182990

on May 30, 2021. © 2018 American Association for Cancer Research. cancerres.aacrjournals.org Downloaded from

Published OnlineFirst March 6, 2018; DOI: 10.1158/0008-5472.CAN-17-2319

http://crossmark.crossref.org/dialog/?doi=10.1158/0008-5472.CAN-17-2319&domain=pdf&date_stamp=2018-5-22http://cancerres.aacrjournals.org/

(12). TG2 expression was induced by TGFb in the TME via NFkBactivation and linked to the rare, but highly tumorigenic OCSCpopulation (12). The link between TG2 andCSCswas recorded inother solid tumors. For example, TG2 upregulation was alsorecorded in the highly tumorigenic subpopulation of CD44þ/CD24� breast CSCs characterized by self-renewal and mammo-sphere-forming capacity (13), in epidermal and glioblastomaCSCs, where it promoted spheroid proliferation and tumor for-mation (14, 15). The connection between TG2 and chemotherapyor radiation resistance in cancer cells (16) was attributed toactivation of the NFkB survival pathway (17, 18) and of "out-side-in" signaling (19), being consistent with the chemoresistantphenotype of CSCs. However, the mechanism by which TG2regulates the functions of CSCs remains unclear.

Here,we show that not only TG2, but also the othermembers ofthe ECM adhesion complex (FN and integrin b1) are enriched inOCSCs and contribute to activation of the stemness-associatedWntpathway.Disruptionof this complex by a function-inhibitingantibody disrupted sphere proliferation, stem cell characteristics,and tumor initiation capacity (TIC). We demonstrated that theanti-TG2 antibody or TG2 knockdown inhibited ovarian CSCsthrough blockade ofWnt signaling. This pathway was engaged byTG2 via direct interaction with the receptor Fzd7, which wasidentified here as a new TG2 partner. In all, our findings proposea newCSC target and point to a novel protein–protein interaction(PPI), which is critical to the maintenance of the CSC phenotype.

Materials and MethodsChemicals and reagents

Unless stated otherwise, chemicals and reagents were fromSigma-Aldrich. Monoclonal FN was from BD Biosciences; mono-clonal TG2 (clone 4G3), integrinb1 (MAB1959, cloneP5D2), andintegrin b1 (MAB2251, clone B3B11) were from EMD MilliporeCorporation, monoclonal TG2 (CUB 7402) and polyclonal TG2(Ab-4) were from Thermo Fisher Scientific; polyclonal Fzd1 andFzd7 antibodies were from Abcam; and GAPDH from BiodesignInternational. Secondary HRP-conjugated antibodies were fromAmersham Biosciences and Santa Cruz Biotechnology. TheALDEFLUORkit assaywas fromStemCell Technologies. Lentiviralparticles containing shRNA-targeting Fzd7 and scrambled shRNAwere purchased from Sigma-Aldrich. Recombinant humanWnt7A and 3A were purchased from R&D Systems. The TG2peptide was synthesized by Biosynthesis. Recombinant full-length andmutant TG2were expressed and purified, as previouslydescribed (20).

Cell linesHuman OC SKOV3 and HEY cell lines were from the ATCC.

OVCA432, COV362, and OVCAR5 cells were provided by Drs.Robert Bigsby and Kenneth Nephew, respectively (Indiana Uni-versity). All cell lines were authenticated by short tandem repeat(STR) analysis (IDEXX BioResearch, and tested to be Mycoplasmanegative by the Skin Tissue Engineering Core at NorthwesternUniversity. For the experimental procedures, OC cell lines wereused between 5 and 10 passages after thawing. For cell culturemethod specifications see Supplementary Material.

Primary human cellsDeidentified malignant ascites fluid specimens from OC

patients (n ¼ 4) were obtained at the Indiana University

Simon Cancer Center (IUSCC) under an IRB-approvedprotocol (IUCRO#505). The study was conducted in accor-dance with the International Ethical Guidelines for BiomedicalResearch Involving Human Subjects, and represented minimalrisk to subjects. All subjects had stage III or IV high-gradeserous OC or primary peritoneal carcinomatosis. For primarycells isolation from human specimens (OC ascites andprimary tumors) and culture method specifications see Sup-plementary Material.

Sphere formation assay, ALDEFLUOR assay, and fluorescence-activated cell sorting, coimmunoprecipitation (Co-IP), immuno-fluorescence, quantitative RT-PCR, Annexin V/7-AAD staining,IHC, transduction, gene reporter assay, and Western blot analysiswere performed as previously described (11, 12, 21, 22); fordetails, see Supplementary Material. The primers used are inSupplementary Table S1.

In vivo xenograft studiesAll animal experiments were conducted following protocols

approved by the IU Animal Care and Use Committee. An equalnumber of IgG control and 4G3-treated spheres were injectedsubcutaneously into the left and right flanks of 7- to 8-week-oldfemale athymic nude mice, respectively, with 6 mice randomlyassigned to each group. At the end of the study (e.g., when at leastone tumor reached 2,000 mm3), mice were euthanized, tumorswere harvested,measured, andweighed. For detailed informationsee Supplementary Material.

In situ proximity ligation assayInteraction between TG2 and integrin b1 or Fzd7 was mea-

sured in OC paraffin-embedded tissue sections by proximityligation assay (PLA; ref. 23) using Duolink reagents (Millipore-Sigma) and following the manufacturer recommendations.Deidentified human OC specimens and on a tissue microarraywere obtained from Pantomics Inc. (#OVC1021, n ¼ 93).Normal fallopian tube control was obtained from PantomicsInc. Details are included in Supplementary Material. Percentageof cells staining and intensity of the staining was graded from 0(no staining) to 3þ (strong staining). An H score was calculatedas the product between intensity and percentage of stained cellsand tumors were classified as positive or negative if H score was> or < median score, respectively.

Homology model of human Fzd7As the full-length crystal structure of Fzd7 has not yet been

solved, we built a robust homology model using the Prime 3.1software implemented in Schrodinger platform (24). For details,see Supplementary Material.

Statistical analysisThemRNAmicroarray (Agilent 244K Custom Gene Expression

G4502A-07 and Affymetrix Human Exon 1.0 ST Array) andRNASeqv2 level 3 data for The Cancer Genome Atlas (TCGA)ovarian cancer cohort were retrieved from Broad GDAC Firehosehttp://gdac.broadinstitute.org/. The clinical information associ-ated with these samples was obtained from cBioPortal (http://www.cbioportal.org). Survival analysis was performed in R(version 3.2.5; http:///www.r-project.org/) and the statisticalsignificance was defined as P < 0.05 (see Supplementary Mate-rial). One-way ANOVA and the Student t test were used forother comparisons between groups.

Tissue Transglutaminase in Ovarian Cancer Stem Cells

www.aacrjournals.org Cancer Res; 78(11) June 1, 2018 2991



http://gdac.broadinstitute.org/http://www.cbioportal.orghttp://www.cbioportal.orgwww.r-project.org/http://cancerres.aacrjournals.org/

ResultsOCSCs express high levels of TG2, FN1, and integrin b1

To determine TG2 expression in OCSCs, we used qRT-PCRand compared ALDHþ/CD133þ and ALDH�/CD133� cellsFACS sorted from OVCAR5 and COV362, two representativeHGSOC cell lines (25, 26). TG2 mRNA expression wassignificantly increased in OCSCs compared with non-CSC(ALDH�/CD133�Fig. 1A and B). Additionally, integrin b1and FN expression levels were increased in CSCs versusnon-CSCs (Fig. 1A and B). ALDHþ/CD133þ were also highlyenriched in CSC-specific transcription factors (Sox2, Nanog,

Oct-4) and ALDH1A1 expression (Supplementary Fig. S1Aand S1B).

Given that cells growing as spheroids under nondifferentiatingconditions are enriched in CSCs compared with cells growing asmonolayers (21, 27), TG2, integrin b1 (ITGB1), and FN expres-sion levels were also quantified in these models and found to besignificantly upregulated in OC spheroids versus monolayersacross several HGSOC cell lines and primary OC cells derivedfrom malignant ascites at the mRNA (Fig. 1C–D; SupplementaryFig. S1C–S1D) andprotein levels (Fig. 1E). Consistentwith a stemcell–enriched phenotype, expression of stemness-associated tran-scription factors (Oct-4, Nanog, and Sox2) and of ALDH1A1 was

Figure 1.

Ovarian CSCs express high levels ofTG2, integrin b1, and FN1. A and B, TG2,integrin b1 (ITGB1), and FN1 mRNAlevels measured by quantitative real-time PCR in ALDHþ/CD133þ versusALDH�/CD133� isolated from humanovarian cancer OVCAR5 and COV362cells (N � 3; � , P < 0.05; �� , P < 0.01).C and D, Expression of TG2, integrin b1(ITGB1), and FN1 in OVCAR5 andCOV362 cells grown as monolayersor spheroids under low adherenceconditions (N � 3; � , P < 0.05;�� , P < 0.01). E,Western blotting showsTG2, integrin b1, and FN expressionlevels in OVCAR5, COV362, andSKOV3 grown as monolayers (m) andspheroids (s). Densitometry quantifiesTG2, integrin b1, and FN expressionlevels normalized for GAPDH. F,Western blot measured TG2 expressionlevels in OVCA432 cells stablytransduced with nontargeting shRNAvector control (ShCtr) compared withpool of shRNA targeting TG2 (ShTG2).G, Flow cytometry measuresALDEFLUOR-FITCþ/CD133-APCþ cellsin OVCA432 cells transduced withShCtr empty vector comparedwith TG2 knockdown (Sh-TG2).DEAB/APC-Isotype–treated cells serveas negative controls. Measurementswere performed in three replicates.

Condello et al.

Cancer Res; 78(11) June 1, 2018 Cancer Research2992



http://cancerres.aacrjournals.org/

increased in OC spheroids versus monolayers (SupplementaryFig. S1E and S1F).

To further establish the significance of TG2 to the CSC pheno-type, we usedOC cells in which TG2was knocked down by stabletransduction of a pool of shRNA or of an antisense constructtargeting TG2 (11, 12). Western blotting confirmed decreasedTG2 protein levels in transduced OVCA432, HEY, and SKOV3cells (Fig. 1F; Supplementary Fig. S1G). The ALDHþ and theALDHþ/CD133þ cell populations were significantly decreasedin all three OC cell lines (Fig. 1G; Supplementary Fig. S1H),supporting the critical role of TG2 in the maintenance of ovarianCSCs.

TG2, FN, and integrin b1 form a complex in ovarian CSCsBy binding to integrin b1 and FN, TG2 stabilizes a ternary

complex at the plasmamembrane, which propagates "outside-in"signaling (9). TG2/integrin b1 colocalization by immunofluores-cence staining and quantified by Metamorph was increased inCSC-rich spheroids compared with monolayers (Fig. 2A and B,n � 5; P < 0.0001). Likewise, TG2 colocalized with FN in bothmonolayer and spheroid cultures (Fig. 2C) and significant enrich-ment of TG2–FN clusters was noted in CSC-rich 3D culturesystems compared with monolayers (Fig. 2D, n � 5; P < 0.01).Collectively, these data support the enrichment in TG2/FN/integ-rin b1 complexes in spheroids and suggest a functional role inovarian CSCs.

To determine whether this PPI is detectable in human tumors,we usedPLA, a technique capable of identifying proteins localizedwithin 40 nm distance in tissue. TG2 and integrin b1 expressionand colocalization were measured on a tissue multiarray includ-ing 93 ovarian tumors. Complex formation was detectable in 85of 93 ovarian malignant tumors, of which 43 displayed intensestaining, but not in normal surface ovarian or fallopian tubeepithelium (n¼ 7; Fig. 2E; Supplementary Table S2), supporting afunctional interaction between the two proteins in malignanttissue in vivo.

Further, we explored the TCGA ovarian cancer database, whichhouses gene expression data of 560 clinically annotated HGSOCtumors. TG2 expression was strongly correlated with ITGB1 (R ¼0.23, P < 0.0001; Fig. 2F) and with FN1 (R¼ 0.39, P < 0.0001; Fig.2G; Supplementary Fig. S2A and S2B). Furthermore, TG2 andITGB1 were independent markers of survival (not shown) andwere therefore included in a finalmultivariable analysis of overallsurvival in both Agilent 244K (Fig. 2H) and Affymetrix HumanExon 1.0 ST (Supplementary Fig. S2C), along with tumor stage.Patients with high TG2 and ITGB1 expression levels had anincreased estimated risk of death when compared with thosewith low TG2 and ITGB1 expression levels (Fig. 2H). The differ-ence in median survival time between the groups associated withhigh levels of ITGB1 and TG2 was significantly larger than thedifference between the groups classified into high and low basedon the expression of TG2 or ITGB1 alone. Similar results wereobtained by exploring Agilent 244K (Fig. 2F–H; SupplementaryTables S3–S6). These data support the significance of TG2 at theinterfacewith the ECM inhumanovarian tumors affecting clinicaloutcomes.

TG2–FN blockade suppresses sphere formation and thetumor-initiating capacity of ovarian CSCs

When secreted in the ECM, TG2 binds to the I6II1,2I7-9 modulesforming the 42-kDa gelatin-binding domain of FN with high

affinity (Kd � 8–10 nmol/L), regulating matrix assembly (28).Hence, we used an inhibitory anti-TG2 monoclonal antibody(mAb) 4G3 against the N-terminal domain of TG2 (aa 1–165;ref. 29), responsible for FNbinding (Fig. 3A). Co-IPwith anti-TG2and anti-FN antibodies demonstrated that 4G3 disrupts theTG2/FN interaction in ALDHþ/CD133þ cells grown as spheres(Fig. 3B–C). In addition, the FNprotein level was decreased in celllysates from 4G3-treated comparedwith control cells (Fig. 3B–C).

Next, we assessed the effects of disrupting the TG2/FN/integrinb1 complex in ovarian CSCs. The anti-TG2 antibody significantlysuppressed spheroids' proliferation compared with IgG isotypecontrol in COV362 (n¼ 8, P < 0.0001) and OVCAR5 cells (n¼ 8,P < 0.0001; Fig. 3D–E). By comparison, the function-blockingantibody P5D2 directed against integrin b1, which had beenreported to inhibit stemness in other models (30), also blockedsphere proliferation (n ¼ 8, P < 0.001), but less efficiently than4G3 (Fig. 3D–E, P < 0.05). Similar results were observed inprimary CSCs flow sorted from human OC ascites (Fig. 3F, n ¼4 specimens tested). Importantly, 4G3 did not induce apoptosisor necrosis in OVCAR5, SKOV3, and COV362 spheroids, asmeasured by Annexin V/7-AAD staining (Supplementary Fig.S3A). In addition, the TG2/FN and TG2/integrin b1 complexeswere disrupted by 4G3, as shown by immunofluorescence stain-ing of ovarian CSCs derived from ascites specimens (Supplemen-tary Fig. S3Band S3C). These data support that disruption of thiscomplex at the interfacewith the ECM inhibits CSCs proliferation.

The rare populationofovarianCSCs is responsible for initiatingtumor formation in vivowhen injected in small numbers in nu/numice (31). Ten thousand ALDHþ/CD133þ cells sorted fromOVCAR5 cells were cultured under stem cell conditions andtreated ex vivo with 4G3 or IgG isotype control for 6 days beforesubcutaneous inoculation into the flanks of female nu/nu mice.Tumor volumes (means � SEM) were reduced from 781.9 �246.2 mm3 (control) to 45.9 � 12.9 mm3 (4G3-treated ovarianCSCs; n ¼ 5, P < 0.01; Fig. 3G). Tumor weights (means � SEM)were also reduced from 0.148 � 0.032 g (control) to 0.013 �0.003 g (4G3-treated cells, n ¼ 5, P < 0.01; Fig. 3H). Tumorinitiation frequency was also significantly delayed and inhibitedby 4G3 (Fig. 3I). To verify whether the reduced TIC was due to adepletion of CSCs maintained in vivo, cells dissociated from 4G3and IgG pretreated tumors were cultured ex vivo in anchorage-independent conditions. Single cells derived from 4G3-treatedtumors were not able to form spheroids compared with cellsderived from control tumors (n¼ 3, P 2; Fig. 4A) and validated throughqRT-PCR in 4G3-treated cells compared with controls.ALDH1A1,Nanog, Oct-4, and Sox2 mRNA expression levels were significantlydecreased in 4G3-treated OVCAR5 and COV362 cells comparedwith controls (n� 3, P < 0.05, and P < 0.01, respectively; Fig. 4B–D; Supplementary Fig. S4A).Hedgehog andWnt signaling, two key






Figure 2.

TG2/FN/Integrin b1 form a complex in OC spheroids. A, Immunofluorescence staining for TG2 (Alexa Fluor 488, green) and integrin b1 (Alexa Fluor 568, red) in OCspheroids and monolayers. Protein colocalization is identified by yellow spectra on merged images. B, Quantification of colocalized proteins was calculated byvolume area of green over red spectra by using Metamorph software (N� 3; �� , P < 0.0001). C, Immunofluorescence staining for TG2 (Alexa Fluor 488, green) andFN (Alexa Fluor 568, red) in OCmonolayers and spheroids.D,Quantification of colocalized proteinswas calculated by volume area of green over red spectra (N� 3;�� , P < 0.01). E, TG2/integrin b1 colocalization detected by PLA in human ovarian tumors, normal surface ovarian, and fallopian tube epithelium, included on amultitissue array. Representative images are shown (magnification, �200). Bar, 10 mm. F and G, Correlation between TG2 and integrin b1 (ITGB1) mRNAexpression levels (R¼0.23; P

developmental pathways also linked to cancer stemness, wereamong the most significantly downregulated mechanisms afterTG2/FN complex disruption by 4G3 in OC spheroids. mRNAexpression levels of b-catenin and of target genes c-Myc and cyclinD1 were significantly decreased in 4G3-treated OVCAR5 andCOV362 cells compared with controls (n � 3, P < 0.01; Fig.4E–G; Supplementary Fig. S4B).

TG2–FN complex disruption inhibits Wnt signalingHaving previously demonstrated that TG2 activates b-catenin

in OC cells by altering cell adhesion to the matrix (32) and the

critical role ofWnt/b-catenin in themaintenance ofOC spheroidsby direct transcriptional regulation of ALDH1A1 (21), we furtherexplored the mechanism by which the TG2–FN–integrin b1complex regulates Wnt/b-catenin and cancer stemness. By usingaqRT-PCR array focused onWnt/b-catenin signaling,we observedanoverall global downregulation of genes in this pathway in 4G3-treated spheroids compared with the IgG control group. Specif-ically, 32 Wnt-related transcripts, of which 24 are known positiveregulators of the pathway, were downregulated >2.0-fold (P <0.05) in 4G3-treated cells compared with controls (Fig. 5A).Reversely, Wnt inhibitors were upregulated, suggesting a role of

Figure 3.

TG2/FN/Integrin b1 complex regulates spheroids proliferation and tumor-initiating capacity. A, Graphical representation of the epitope targeted by the4G3 mAb overlapping with the FN-binding domain of TG2 (amino acids 1–165). B, Co-IP with anti-TG2 and anti-FN mAbs of cell lysates from OVCAR5spheroids treated with 4G3 (10 mg/mL) for 6 days. Western blotting was performed by using anti-TG2 and FN monoclonal antibodies. C, Densitometricanalysis results are shown as means � SEM (N ¼ 3; �, P < 0.05; �� , P < 0.01). D–F, CCK-8 assay quantifies proliferation of spheroids derived from OC cell linesand primary cells treated with inhibitory mAbs directed against the FN-binding domain of TG2 (4G3), and integrin b1 (clone P5D2) (N ¼ 8; � , P < 0.05; �� , P < 0.01;�� , P < 0.0001). G–H, Tumor weights and volumes derived from ALDHþ/CD133þ sorted from OVCAR5 cells and treated with 4G3 or IgG control andinjected subcutaneously in nudemice, as described (N¼ 5; �� ,P

the TG2/FN complex in fine-tuning Wnt signaling. Among theWnt pathway elements affected by disruption of the TG2/FNcomplex, the Frizzled (Fzd) receptors 1 and 7 were significantlydownregulated (>4.0-fold), along with b-catenin, its cotranscrip-tional regulator LEF1, and the target gene c-Myc (>2.0 foldchange). qRT-PCR confirmed Fzd1 and Fzd7 mRNA expressionlevel downregulation in 4G3-treated ovarian CSCs fromOVCAR5and COV362 cells compared with controls (n � 3, P < 0.05 andP < 0.01, respectively; Fig. 5B-C; Supplementary Fig. S5A). 4G3-mediated disruption of TG2–FN complex significantly reducedb-catenin/TCF transcriptional activity as measured by TCF/LEF1reporter assay in OVCAR5 and COV362 cells (n � 3, P <0.01; Fig. 5D; Supplementary Fig. S5B). This inhibition wasmaintained in vivo as determined by decreased b-catenin stainingin xenografts derived from ovarian CSCs pretreated with 4G3versus IgG (Supplementary Fig. S5C).

To investigate the mechanism by which the TG2/FN complexalters Wnt signaling, we tested the possibility that components ofthe complex directly interact with the Fzd receptors. Fzd7, but notFzd1, was detected in endogenous protein lysates from CSCspulled down with an anti-TG2 antibody (Fig. 5E). The TG2–Fzd7complex was disrupted by 4G3, suggesting that the potential areaof interaction between TG2 and Fzd7 is located in the N-terminal

domain of TG2 (amino acids 1–165), which is targeted by 4G3(Fig. 5E–F). In contrast, no interaction between FN and Fzd7 orbetween FN and Fzd1 was observed, as measured by co-IP withan anti-FN antibody. In addition, immunoblotting of totallysates showed a downregulation of Fzd7 expression levels in4G3-treated ovarian CSCs compared with control-treated cells,consistent with mRNA results.

Immunofluorescence and confocal microscopy confirmedcolocalization of TG2 and Fzd7 at the plasma membrane (Spear-man r ¼ 0.5, Pearson r ¼ 0.43; Fig. 5G). 4G3 induced a 10%reduction in the overlap between TG2 and Fzd7 compared withIgG (Spearman r ¼ 0.4, Pearson r¼ 0.3). In addition, integrin b1colocalized with Fzd7 (Spearman r ¼ 0.42, Pearson r ¼ 0.57;Supplementary Fig. S5D) and FN colocalized with Fzd7 (Spear-man r ¼ 0.58 and Pearson r ¼ 0.3; Supplementary Fig. S5E). Theanti-TG2 antibody 4G3decreased both integrin b1/Fzd7 (�60%–30%) and FN/Fzd7 colocalization (50%–25%). PLA demonstrat-ed TG2/Fzd7 colocalization in ovarian tumors, but not in normalfallopian tube epithelium (Fig. 5H). However, the signal was lessintense compared with the colocalization signal for TG2–integrinb1 and was detectable in only 22 of 93 tumors stained, presum-ably due to the restricted expression of Fzd7 in ovarian tumors.These data suggest that the TG2/FN complex through its interac-tions with Fzd7 may act as a direct Wnt/b-catenin coactivator.

TG2 forms a complex with FZD7To further define the interaction between TG2 and Fzd7, we

used full-length recombinant TG2, integrin b1, and Fzd7 proteins.Co-IP with anti-TG2 antibody demonstrates direct interactionbetween TG2 and Fzd7 (Fig. 6A). In contrast, recombinant integ-rin b1 and Fzd7 did not co-IP when an anti-integrin b1 antibodywas used (Fig. 6B). In the ECM, TG2 can act as an active enzyme oras a scaffold protein noncovalently interacting with other ECMcomponents (9, 33). To determine whether the TG2/Fzd7interaction depends on its enzymatic function, we used recom-binant TG2 carrying a C277A inactivating mutation (34). Co-IPwith anti-TG2 antibody demonstrated that Fzd7 was found ina complex with C277A-TG2, suggesting independence of theprotein's catalytic function (Fig. 6C). To further delineate theTG2/Fzd7 interaction we used the 81DAVEEGDWTATVVDQQ-DCTLSLQLTTPANA110 peptide overlapping with part of theFN-binding domain (7). The peptide disrupted partially theTG2–Fzd7 complex in a dose dependent manner, as shown byco-IP experiments with anti-TG2 antibody (Fig. 6D), suggestingthat the TG2 region binding Fzd7 may be located near theFN-interacting N-terminal domain.

Protein–protein dockingTo further study the interaction between TG2 and FZD7, we

considered the crystal structure of TG2 (2Q3Z.pdb) available inprotein database and themodel structure of Fzd7 obtained earlierthrough homologymodeling. The protein–protein docking panelavailable in Schrodinger suite (35), which has an interface to thePiper program (36), was used, considering TG2 as the receptorand Fzd7 as the ligand. The ligand was rotated 70,000 timesvarying every 5� in the space of Euler angles and each of theorientations of the ligand were translated to find the best dockingscorewith respect to the receptor. Based on the docking scores andthe energetics of the interaction, we selected 32 distinct potentialdocking poses of the two proteins. Out of the 32 poses, twoillustrated that themost probable region of interactionswith Fzd7

Figure 4.

TG2/FN/Integrin b1 complex regulates b-catenin activation. A,Expression levels of stemness-associated genes in 4G3 compared withcontrol (IgG)-treated OVCAR5 cells grown as spheroids were quantified byRT2 Profiler PCR Array. Pie chart analysis illustrates the fold changes (�2.0) ofdownregulated genes (% of total) for each represented pathway in controlversus treated spheres.B–D,QuantitativeRT-PCR forALDH1A1, Sox2, andNanogmRNA expression levels in 4G3 compared with control (IgG)-treatedOVCAR5 spheroids. E–G, Quantitative RT-PCR for b-catenin, c-Myc, andcyclin D1 in 4G3 comparedwith control (IgG)-treatedOVCAR5 spheroids (N� 6;� , P < 0.05; �� , P < 0.01).

Condello et al.





Figure 5.

TG2 and Fzd7 form a complex in OC cells. A, List of Wnt/b-catenin pathway genes downregulated in OVCAR5 spheroids by 4G3 treatment comparedwith IgG control (�2.0-fold change). B–C, Quantitative RT-PCR for Fzd1 and Fzd7 in OVCAR5 spheroids (N � 6; � , P < 0.05; �� , P < 0.01). D, OVCAR5 cellswere cotransfected with TCF/LEF1 luciferase reporter and Renilla control plasmid prior to treatment with 4G3 or IgG (control) and plated as spheroids. Luciferasesignal relative to Renilla activity is expressed as fold increase (N � 6; �� , P < 0.01). E, Co-IP with anti-TG2 and anti-FN mAbs of cell lysates from OVCAR5spheroids treatedwith 4G3.Western blotting was performed by using anti-Fzd7, Fzd1, and GAPDH antibodies. F,Densitometric analysis results are shown asmeans� SEM (N ¼ 3; �� , P < 0.0001). G, Immunofluorescence staining for TG2 (red) and Fzd7 (green) in control and 4G3-treated OC cell lines (magnification, �400).Quantification of colocalized proteins was calculated by volume area of green over red spectra in IgG control (N � 3; Spearman rank correlation ¼ 0.5) versus4G3-treated cells (N � 3; Spearman rank correlation ¼ 0.40). H, TG2/Fzd7 colocalization detected by PLA in human ovarian tumors included on amultitissue array and in normal fallopian tube epithelium. Representative images are shown (magnification, �200). Bar, 10 mm.






overlaps with W88-T106 residues of TG2. We performed energyminimization and compared the energetics of the two poses andfound the PPI pose shown in Fig. 6E as the most likely interactingposition. By analyzing the proposed PPIs, we found that the TG2residue Gln96 can form at least two potential strong hydrogenbonds with the protonated Lys368 of Fzd7. Furthermore, Gln103of TG2 can form a hydrogen bondwith the side chain of Ser438 ofFzd7 and Asp97 of TG2 with Trp369 of Fzd7. In addition to thesepotential hydrogen bonds, a few electrostatic and Van der Waalsinteractions between the two proteins are shown in Fig. 6F.

TG2/Fzd7 interaction regulates Wnt signaling and ovarianspheroid proliferation

Next, we tested how TG2 and Fzd7 affect Wnt signaling inovarian CSCs. TG2 inhibition by 4G3 blocked OVCAR5,COV362, SKOV3, and primary OC derived CSCs' proliferationas spheres in basal conditions and after treatment with Wnt3A(Fig. 7A–D) or Wnt 7A (Supplementary Fig. S6A–S6D). To deter-mine the function of Fzd7 in this pathway, the receptor was stablyknocked down by using two shRNA sequences in OVCAR5(Fig. 7E) and SKOV3 cells (Supplementary Fig. S7A). Fzd7 knock-down blocked OVCAR5- and SKOV3-derived CSCs' proliferationas spheres in basal conditions andafter treatmentwithWnt3A and

Wnt7A (Fig. 7F; Supplementary Fig. S7B–S7C) and expression oftarget genes in response to Wnt3A and Wnt7A (Fig. 7G–H;Supplementary Fig. SD-I).

Interestingly, TG2 expression was also strongly correlated withFzd7 (R ¼ 0.12, P ¼ 0.0038) in the ovarian TCGA's Agilent 244K(Fig. 7I) and Affymetrix Human Exon 1.0 ST platforms (Supple-mentary Fig. S7J). Collectively, these results strongly supportthe role of TG2-promoting CSC signaling and proliferation bydirect interaction with Fzd7 and activation of Wnt/b-cateninsignaling (Fig. 7J).

DiscussionOur results demonstrate that TG2 and its partner proteins, FN1

and integrin b1, are upregulated and forma functional complex inOCSCs coexpressing ALDHþ/CD133þ (22, 37) compared withnon-CSCs (ALDH�/CD133�). This complex is enriched in cancercells grown as spheroids in ultra-low adherence conditions com-pared with cells grown under differentiating conditions as mono-layers. We show that targeting the TG2/FN1/integrin b1 complexby using a blocking antibody disrupts the stem cell phenotype byinhibiting Wnt/b-catenin signaling. Importantly, we found Fzd7,aWnt ligand receptor, as a new TG2-interacting protein in OCSCs

Figure 6.

TG2 forms a complex with Fzd7 in OC spheroids. A, Co-IP with anti-TG2 mAb and Western blotting for TG2 and Fzd7 using full-length recombinant TG2and Fzd7. B, Co-IP with anti-integrin b1 mAb and Western blotting for integrin b1 and Fzd7 using recombinant integrin b1 and Fzd7. C, Co-IP withanti-TG2 mAb and Western blotting for TG2 and Fzd7 using recombinant mutant TG2 (C277A) and Fzd7. D, Co-IP with anti-TG2 mAb and Western blottingfor TG2 andFzd7using recombinant TG2andFzd7 in thepresenceof different concentrations of a synthetic peptide 81DAVEEGDWTATVVDQQDCTLSLQLTTPANA110.E, Putative poses for protein–protein interaction were identified by using the crystal structure of TG2 (2Q3Z.pdb) available in the protein database andthe virtual structure of Fzd7 obtained through homology modeling. F, Proposed interacting amino acids residues for TG2 and Fzd7.

Condello et al.





required for transduction ofWnt ligand signals. Our findings haveseveral implications.

First, we reaffirm the significance of TG2 to the CSC phenotypeand point to a new mechanism by which the protein promotesstem cell survival and tumor-initiation capacity. The observationsthat TG2 expression is enriched in ovarian CSCs are consistentwith our previous findings (12) as well as with recent reports inother solid tumors (13–15). Importantly, here, we show that TG2directly modulates the interaction between OCSCs and the ECMby forming a complex with integrin b1. The ternary complexformed and stabilized by TG2 with integrin b1 and FN has beendescribed innormalfibroblasts (7).Here,wedemonstrate that theTG2/integrin b1 and TG2/FN complexes are significantly enrichedin ALDHþ/CD133þ OCSCs grown as spheroids compared withnon-CSCs and cells grown as monolayers. The involvement ofintegrins in cancer stemness has been recognized in other contexts(38, 39), yet our results provide a new understanding of themechanisms by which integrins anchoring CSCs in the matrixengage stemness pathways. The TG2/integrin b1 complexes werealso detectable in humanovarian tumors by PLA, supporting their

functional role in vivo. Furthermore, the expression of each of theproteins in this complex, individually, as well as together, corre-lated with clinical outcomes, establishing clinical relevance ofour findings.

To understand the role of TG2/FN/integrin b1 complexespromoting the OCSC phenotype and spheroids proliferation, weused an inhibitory antibody for the TG2/FN interaction. The anti-TG2 antibody (29) was more effective than anti-integrin b1 mAbdecreasing ALDHþ/CD133þ survival and spheroids proliferation.Anti-TG2 mAbs have been shown to inhibit the migration ofmonocytic cells in inflamed tissue rich in FN matrices (29).Possible explanations of this phenomenon include the specificand high-affinity binding of TG2 to the 42-kDa gelatin-bindingdomain of FN (28), and with the critical importance of TG2/FNorganization inmodulatingb integrins expression and function ina model of tumor growth and dissemination (10). Additionally,TG2 knockdown diminished the OCSC population.

One of the hallmarks of CSCs is to recapitulate hierarchicallyorganized human tumors when injected in immunodeficientmice (31). Here, we show that 4G3-mediated TG2–FN blockade

Figure 7.

TG2/Fzd7 direct interaction regulates spheroid proliferation. A–D, Proliferation assay measured the number of cells growing as spheroids derived fromOC cell lines (OVCAR5, COV362, and SKOV3) and from primary OC cells derived from malignant ascites after treatment with 4G3 and/or Wnt3A for 6 dayscomparedwith IgG controls (N� 3; � ,P

inhibited the tumor-initiating capacity of ALDHþ/CD133þ cellsin nude mice and cells isolated from 4G3-treated tumors andcultured ex vivo were unable to form spheroids. Unbiased expres-sion profiling analysis demonstrated that 4G3-mediated TG2/FNcomplex disruption downregulated genes related to CSC-main-tenance, self-renewal, and proliferation, linking TG2-mediatedECM components assembly with the expression of ALDH1A1,Nanog, Oct-4, and Sox2, well-known CSC markers.

Second, our results point to the Wnt/b-catenin pathway as themain regulator of stemness disrupted by inhibition of TG2/FN/integrin b1 complexes. b-Catenin activation has been correlatedwith progression of human cancers (40) and the functions ofCSCs in lung adenocarcinoma (41) and colorectal cancer (42).Wepreviously showed that b-catenin directly regulates the expressionof the stem cell marker ALDH1A1 in OC, thus being involved inmaintenance of the OCSC phenotype (21). We have previouslydemonstrated that the TG2/FN complex regulates b-catenin acti-vation through a c-Src–dependent mechanism (32). ExtracellularTG2 was shown to activate canonical b-catenin signaling invascular smooth muscle cells by directly binding to LRP5/6(43, 44). Here, we identify the Fzd7 receptor as a new TG2substrate, linking the adhesion complex mediated by TG2 tostemness-associated b-catenin pathway. Although interactionsbetween TG2 and all Wnt receptors were not fully explored, theinteraction between TG2 and Fzd7 appears to be specific anddirect. Additionally, the other components of the complex (integ-rin b1 or FN) did not directly interact with Fzd7. To betterdelineate this PPI, we used both chemical and virtual dockingstrategies. Both the full length and theC277Amutant TG2 formeda complex with Fzd7, excluding the possibility that the enzymaticfunction of TG2 plays a role in this interaction. However, asynthetic peptide, comprising the amino acid residues corre-sponding to the FN binding sequence of TG2, blocked complexformation in a dose-dependent manner, suggesting that theinteracting domain is near the TG2's N terminus. Virtual dockingusing the available TG2 structure and a reconstituted Fzd7 struc-ture predicted that the interaction sites were in the FN-bindingdomain of TG2 (W88-T106) and involved amino acids(>358–379) and (>445–470) of Fzd7.

The functions of Fzd7 in cancer are incompletely elucidated.Fzd7 was found to be upregulated in OC compared with normalsurface ovarian epithelium (45) and was linked to the canonicalWnt pathway in colorectal cancer (46), breast (47), and hepato-cellular carcinoma (48). Our data demonstrate a role for thereceptor in a complex with TG2, transducing Wnt signals andpromoting OCSCs' proliferation as spheres. Colocalization of thereceptor with TG2 was demonstrated in ovarian tumors for thisfirst time here, supporting future investigation of its functions inthis context.

Finally, we propose a positive feedback loop mechanism toexplain the overall decrease in Wnt-related genes after 4G3 treat-ment in OCSCs. TG2/FN complex formation acting as Fzd7coactivator increases Wnt/b-catenin signal transduction that inturn fuels a positive feedback loop, mediating the transcriptionalregulation and membrane distribution of frizzled receptors andactivation of other positive Wnt/b-catenin key regulatory genes.Several Wnt/b-catenin signaling-related genes such as LEF1 andFzd7 contain putative TCF/LEF1-binding sites within their pro-moter regions, suggesting they may be self-regulated throughWnt/b-catenin canonical signaling.

In conclusion, our results provide strong evidence supporting akey function of TG2/FN/integrin b1 complexes in OCSCs. Bydemonstrating that TG2 directly binds the Fzd7 receptor, weidentify anovel functionof TG2andanewmechanismpromotingCSCs' proliferation and tumorigenicity. These results point toTG2/FN/integrin clusters or the newly discovered coreceptorcomplex TG2/Fzd7 as potential new therapeutic CSC targets.

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

Authors' ContributionsConception and design: S. Condello, G.E. Schiltz, D. MateiDevelopment of methodology: S. Condello, L.E. Sima, D. MateiAcquisition of data (provided animals, acquired and managed patients,provided facilities, etc.): S. Condello, H. Cardenas, D. MateiAnalysis and interpretation of data (e.g., statistical analysis, biostatistics,computational analysis): S. Condello, L.E. Sima, C. Ivan, G.E. Schiltz,R.K. Mishra, D. MateiWriting, review, and/or revision of the manuscript: S. Condello, L.E. Sima,R.K. Mishra, D. MateiAdministrative, technical, or material support (i.e., reporting or organizingdata, constructing databases): D. MateiStudy supervision: S. Condello, D. MateiOther (Carried out protein–protein interaction studies and wrote the in silicopart of the manuscript): R.K. Mishra

AcknowledgmentsThis work was made possible by funding from the U.S. Department of

Veterans Affairs (I01 BX000792-06), the Diana Princess of Wales endowedProfessorship from the Lurie Cancer Center (to D. Matei), and Friends ofPrentice award (to S. Condello). We acknowledge the technical support pro-vided by the Imaging Core Facility and Flow Cytometry Core supported by theLurie Cancer Center and the NCI-P30 (CA 060553) award.

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 July 31, 2017; revised December 15, 2017; accepted March 2, 2018;published first March 6, 2018.

References1. Siegel R, Ma J, Zou Z, Jemal A. Cancer statistics, 2014. CA Cancer J Clin

2014;64:9–29.2. Lengyel E. Ovarian cancer development and metastasis. Am J Pathol

2010;177:1053–64.3. Matte I, Lane D, Laplante C, Rancourt C, Piche A. Profiling of cytokines in

human epithelial ovarian cancer ascites. Am J Cancer Res 2012;2:566–80.4. Mills GB, May C, Hill M, Campbell S, Shaw P, Marks A. Ascitic fluid from

human ovarian cancer patients contains growth factors necessary forintraperitoneal growth of human ovarian adenocarcinoma cells. J ClinInvest 1990;86:851–5.

5. Burleson KM, Casey RC, Skubitz KM, Pambuccian SE, Oegema TR Jr.,Skubitz AP. Ovarian carcinoma ascites spheroids adhere to extracellularmatrix components and mesothelial cell monolayers. Gynecol Oncol2004;93:170–81.

6. PinkasDM, Strop P, Brunger AT, Khosla C. Transglutaminase 2 undergoes alarge conformational change upon activation. PLoS Biol 2007;5:e327.

7. Hang J, Zemskov EA, Lorand L, Belkin AM. Identification of a novelrecognition sequence for fibronectin within the NH2-terminal beta-sandwich domain of tissue transglutaminase. J Biol Chem 2005;280:23675–83.

Condello et al.





8. Cardoso I, Osterlund EC, Stamnaes J, Iversen R, Andersen JT, Jorgensen TJ,et al. Dissecting the interaction between transglutaminase 2 and fibronec-tin. Amino Acids 2017;49:489–500.

9. Akimov SS, KrylovD, Fleischman LF, Belkin AM. Tissue transglutaminase isan integrin-binding adhesion coreceptor for fibronectin. J Cell Biol2000;148:825–38.

10. Satpathy M, Cao L, Pincheira R, Emerson R, Bigsby R, Nakshatri H, et al.Enhanced peritoneal ovarian tumor dissemination by tissue transgluta-minase. Cancer Res 2007;67:7194–202.

11. Shao M, Cao L, Shen C, Satpathy M, Chelladurai B, Bigsby RM, et al.Epithelial-to-mesenchymal transition and ovarian tumor progressioninduced by tissue transglutaminase. Cancer Res 2009;69:9192–201.

12. Cao L, Shao M, Schilder J, Guise T, Mohammad KS, Matei D. Tissuetransglutaminase links TGF-beta, epithelial to mesenchymal transitionand a stem cell phenotype in ovarian cancer. Oncogene 2012;31:2521–34.

13. Kumar A, Gao H, Xu J, Reuben J, Yu D, Mehta K. Evidence that aberrantexpression of tissue transglutaminase promotes stem cell characteristics inmammary epithelial cells. PLoS One 2011;6:e20701.

14. Kerr C, Szmacinski H, Fisher ML, Nance B, Lakowicz JR, Akbar A, et al.Transamidase site-targeted agents alter the conformation of the transglu-taminase cancer stem cell survival protein to reduce GTP binding activityand cancer stem cell survival. Oncogene 2017;36:2981–90.

15. SullivanKE,Rojas K,CerioneRA,Nakano I,WilsonKF. The stemcell/cancerstem cell marker ALDH1A3 regulates the expression of the survival factortissue transglutaminase, in mesenchymal glioma stem cells. Oncotarget2017;8:22325–43.

16. Mehta K, Fok J, Miller FR, Koul D, Sahin AA. Prognostic significance oftissue transglutaminase in drug resistant andmetastatic breast cancer. ClinCancer Res 2004;10:8068–76.

17. Mann AP, Verma A, Sethi G, Manavathi B, Wang H, Fok JY, et al. Over-expression of tissue transglutaminase leads to constitutive activation ofnuclear factor-kappaB in cancer cells: delineation of a novel pathway.Cancer Res 2006;66:8788–95.

18. Cao L, Petrusca DN, Satpathy M, Nakshatri H, Petrache I, Matei D. Tissuetransglutaminase protects epithelial ovarian cancer cells from cisplatin-induced apoptosis by promoting cell survival signaling. Carcinogenesis2008;29:1893–900.

19. Verma A,WangH,Manavathi B, Fok JY,MannAP, Kumar R, et al. Increasedexpression of tissue transglutaminase in pancreatic ductal adenocarcinomaand its implications in drug resistance and metastasis. Cancer Res2006;66:10525–33.

20. Yakubov B, Chen L, Belkin AM, Zhang S, Chelladurai B, Zhang ZY, et al.Small molecule inhibitors target the tissue transglutaminase and fibronec-tin interaction. PLoS One 2014;9:e89285.

21. Condello S, Morgan CA, Nagdas S, Cao L, Turek J, Hurley TD, et al. beta-Catenin-regulated ALDH1A1 is a target in ovarian cancer spheroids.Oncogene 2015;34:2297–308.

22. Li J, Condello S, Thomes-Pepin J, Ma X, Xia Y, Hurley TD, et al. Lipiddesaturation is ametabolic marker and therapeutic target of ovarian cancerstem cells. Cell Stem Cell 2017;20:303–14 e5.

23. Soderberg O, Gullberg M, JarviusM, Ridderstrale K, Leuchowius KJ, JarviusJ, et al. Direct observation of individual endogenous protein complexes insitu by proximity ligation. Nat Methods 2006;3:995–1000.

24. Jacobson MP, Pincus DL, Rapp CS, Day TJ, Honig B, Shaw DE, et al.A hierarchical approach to all-atom protein loop prediction. Proteins2004;55:351–67.

25. Mitra AK, Davis DA, Tomar S, Roy L, Gurler H, Xie J, et al. In vivo tumorgrowth of high-grade serous ovarian cancer cell lines. Gynecol Oncol2015;138:372–7.

26. Domcke S, Sinha R, LevineDA, Sander C, Schultz N. Evaluating cell lines astumour models by comparison of genomic profiles. Nat Commun2013;4:2126.

27. Lawrenson K, Notaridou M, Lee N, Benjamin E, Jacobs IJ, Jones C, et al. Invitro three-dimensional modeling of fallopian tube secretory epithelialcells. BMC Cell Biol 2013;14:43.

28. Lorand L, Dailey JE, Turner PM. Fibronectin as a carrier for the transglu-taminase from human erythrocytes. Proc Natl Acad Sci U S A 1988;85:1057–9.

29. Akimov SS, Belkin AM. Cell surface tissue transglutaminase is involved inadhesion and migration of monocytic cells on fibronectin. Blood2001;98:1567–76.

30. Lu Z, Roohani-Esfahani SI, Li J, Zreiqat H. Synergistic effect of nano-materials and BMP-2 signalling in inducing osteogenic differentiationof adipose tissue-derived mesenchymal stem cells. Nanomedicine 2015;11:219–28.

31. Zhang S, Balch C, Chan MW, Lai HC, Matei D, Schilder JM, et al. Iden-tification and characterization of ovarian cancer-initiating cells from pri-mary human tumors. Cancer Res 2008;68:4311–20.

32. Condello S, Cao L, Matei D. Tissue transglutaminase regulates beta-catenin signaling through a c-Src-dependent mechanism. FASEB J2013;27:3100–12.

33. Telci D, Wang Z, Li X, Verderio EA, Humphries MJ, Baccarini M, et al.Fibronectin-tissue transglutaminase matrix rescues RGD-impaired celladhesion through syndecan-4 and beta1 integrin co-signaling. J Biol Chem2008;283:20937–47.

34. Liu S, Cerione RA, Clardy J. Structural basis for the guanine nucleotide-binding activity of tissue transglutaminase and its regulation of transami-dation activity. Proc Natl Acad Sci U S A 2002;99:2743–7.

35. Schroedinger. Small-molecule drug discovery Suite 2017-1: LLC, NewYork; 2017.

36. Chuang GY, Kozakov D, Brenke R, Comeau SR, Vajda S. DARS (Decoys Asthe Reference State) potentials for protein–protein docking. Biophys J2008;95:4217–27.

37. Silva IA, Bai S, McLean K, Yang K, Griffith K, Thomas D, et al. Aldehydedehydrogenase in combination with CD133 defines angiogenic ovariancancer stem cells that portend poor patient survival. Cancer Res 2011;71:3991–4001.

38. GoelHL,Grits T, Purcell B, ChangC, Shultz LD,GreinerDL, et al. Regulatedsplicing of the alpha6 integrin cytoplasmic domain determines the fate ofbreast cancer stem cells. Cell Rep 2014;7:747–61.

39. Li Q, Eades G, Yao Y, Zhang Y, Zhou Q. Characterization of a stem-likesubpopulation in basal-like ductal carcinoma in situ (DCIS) lesions. J BiolChem 2014;289:1303–12.

40. Reya T, Clevers H. Wnt signalling in stem cells and cancer. Nature2005;434:843–50.

41. Tammela T, Sanchez-Rivera FJ, Cetinbas NM, Wu K, Joshi NS, Helenius K,et al. AWnt-producing niche drives proliferative potential and progressionin lung adenocarcinoma. Nature 2017;545:355–9.

42. Li J, Yu B, Deng P, Cheng Y, Yu Y, Kevork K, et al. KDM3 epigeneticallycontrols tumorigenic potentials of human colorectal cancer stemcells through Wnt/beta-catenin signalling. Nat Commun 2017;8:15146.

43. Faverman L, Mikhaylova L, Malmquist J, Nurminskaya M. Extracellulartransglutaminase 2 activates beta-catenin signaling in calcifying vascularsmooth muscle cells. FEBS Lett 2008;582:1552–7.

44. Deasey S, Nurminsky D, Shanmugasundaram S, Lima F, Nurminskaya M.Transglutaminase 2 as a novel activator of LRP6/beta-catenin signaling.Cell Signal 2013;25:2646–51.

45. Matei D, Graeber TG, Baldwin RL, Karlan BY, Rao J, Chang DD.Gene expression in epithelial ovarian carcinoma. Oncogene 2002;21:6289–98.

46. Ueno K, Hiura M, Suehiro Y, Hazama S, Hirata H, Oka M, et al. Frizzled-7as a potential therapeutic target in colorectal cancer. Neoplasia 2008;10:697–705.

47. Yang L,WuX,Wang Y, ZhangK,Wu J, YuanYC, et al. FZD7has a critical rolein cell proliferation in triple negative breast cancer. Oncogene 2011;30:4437–46.

48. Merle P, Kim M, Herrmann M, Gupte A, Lefrancois L, Califano S, et al.Oncogenic role of the frizzled-7/beta-catenin pathway in hepatocellularcarcinoma. J Hepatol 2005;43:854–62.






2018;78:2990-3001. Published OnlineFirst March 6, 2018.Cancer Res Salvatore Condello, Livia Sima, Cristina Ivan, et al. Cancer Stem Cells and the Tumor NicheTissue Tranglutaminase Regulates Interactions between Ovarian

Updated version

10.1158/0008-5472.CAN-17-2319doi:

Access the most recent version of this article at:

Material

Supplementary

http://cancerres.aacrjournals.org/content/suppl/2018/03/06/0008-5472.CAN-17-2319.DC1

Access the most recent supplemental material at:

Cited articles

http://cancerres.aacrjournals.org/content/78/11/2990.full#ref-list-1

This article cites 47 articles, 14 of which you can access for free at:

Citing articles

http://cancerres.aacrjournals.org/content/78/11/2990.full#related-urls

This article has been cited by 9 HighWire-hosted articles. Access the articles at:

E-mail alerts related to this article or journal.Sign up to receive free email-alerts

Subscriptions

Reprints and

[email protected]

To order reprints of this article or to subscribe to the journal, contact the AACR Publications Department at

Permissions

Rightslink site. Click on "Request Permissions" which will take you to the Copyright Clearance Center's (CCC)

.http://cancerres.aacrjournals.org/content/78/11/2990To request permission to re-use all or part of this article, use this link



http://cancerres.aacrjournals.org/lookup/doi/10.1158/0008-5472.CAN-17-2319http://cancerres.aacrjournals.org/content/suppl/2018/03/06/0008-5472.CAN-17-2319.DC1http://cancerres.aacrjournals.org/content/78/11/2990.full#ref-list-1http://cancerres.aacrjournals.org/content/78/11/2990.full#related-urlshttp://cancerres.aacrjournals.org/cgi/alertsmailto:[email protected]://cancerres.aacrjournals.org/content/78/11/2990http://cancerres.aacrjournals.org/

/ColorImageDict > /JPEG2000ColorACSImageDict > /JPEG2000ColorImageDict > /AntiAliasGrayImages false /CropGrayImages false /GrayImageMinResolution 200 /GrayImageMinResolutionPolicy /Warning /DownsampleGrayImages true /GrayImageDownsampleType /Bicubic /GrayImageResolution 300 /GrayImageDepth -1 /GrayImageMinDownsampleDepth 2 /GrayImageDownsampleThreshold 1.50000 /EncodeGrayImages true /GrayImageFilter /DCTEncode /AutoFilterGrayImages true /GrayImageAutoFilterStrategy /JPEG /GrayACSImageDict > /GrayImageDict > /JPEG2000GrayACSImageDict > /JPEG2000GrayImageDict > /AntiAliasMonoImages false /CropMonoImages false /MonoImageMinResolution 600 /MonoImageMinResolutionPolicy /Warning /DownsampleMonoImages true /MonoImageDownsampleType /Bicubic /MonoImageResolution 900 /MonoImageDepth -1 /MonoImageDownsampleThreshold 1.50000 /EncodeMonoImages true /MonoImageFilter /CCITTFaxEncode /MonoImageDict > /AllowPSXObjects false /CheckCompliance [ /None ] /PDFX1aCheck false /PDFX3Check false /PDFXCompliantPDFOnly false /PDFXNoTrimBoxError true /PDFXTrimBoxToMediaBoxOffset [ 0.00000 0.00000 0.00000 0.00000 ] /PDFXSetBleedBoxToMediaBox true /PDFXBleedBoxToTrimBoxOffset [ 0.00000 0.00000 0.00000 0.00000 ] /PDFXOutputIntentProfile (None) /PDFXOutputConditionIdentifier () /PDFXOutputCondition () /PDFXRegistryName () /PDFXTrapped /False

/CreateJDFFile false /Description > /Namespace [ (Adobe) (Common) (1.0) ] /OtherNamespaces [ > /FormElements false /GenerateStructure false /IncludeBookmarks false /IncludeHyperlinks false /IncludeInteractive false /IncludeLayers false /IncludeProfiles false /MarksOffset 18 /MarksWeight 0.250000 /MultimediaHandling /UseObjectSettings /Namespace [ (Adobe) (CreativeSuite) (2.0) ] /PDFXOutputIntentProfileSelector /NA /PageMarksFile /RomanDefault /PreserveEditing true /UntaggedCMYKHandling /LeaveUntagged /UntaggedRGBHandling /LeaveUntagged /UseDocumentBleed false >> > ]>> setdistillerparams> setpagedevice

Tissue Tranglutaminase Regulates Interactions between ...of the interaction between TG2 and FN also...

Documents

Transcript of Tissue Tranglutaminase Regulates Interactions between ...of the interaction between TG2 and FN also...