activation of notch signaling by tenascin-c promotes ......tumor and stem cell biology activation of...

Tumor and Stem Cell Biology

Activation of NOTCH Signaling by Tenascin-CPromotes Growth of Human BrainTumor-Initiating CellsSusobhan Sarkar1,2, Reza Mirzaei1,2, Franz J. Zemp3,4,Wu Wei3,4,Donna L. Senger3,4, Stephen M. Robbins3,4, and V.Wee Yong1,2

Abstract

Oncogenic signaling by NOTCH is elevated in braintumor-initiating cells (BTIC) in malignant glioma, but themechanism of its activation is unknown. Here we provideevidence that tenascin-C (TNC), an extracellular matrix pro-tein prominent in malignant glioma, increases NOTCHactivity in BTIC to promote their growth. We demonstratethe proximal localization of TNC and BTIC in human glio-blastoma specimens and in orthotopic murine xenograftsof human BTIC implanted intracranially. In tissue culture,TNC was superior amongst several extracellular matrix pro-teins in enhancing the sphere-forming capacity of gliomapatient-derived BTIC. Exogenously applied or autocrine TNCincreased BTIC growth through an a2b1 integrin-mediated

mechanism that elevated NOTCH ligand Jagged1 (JAG1).Microarray analyses and confirmatory PCR and Westernanalyses in BTIC determined that NOTCH signaling compo-nents including JAG1, ADAMTS15, and NICD1/2 were ele-vated in BITC after TNC exposure. Inhibition of g-secretaseand metalloproteinase proteolysis in the NOTCH pathway,or silencing of a2b1 integrin or JAG1, reduced the prolifer-ative effect of TNC on BTIC. Collectively, our findingsidentified TNC as a pivotal initiator of elevated NOTCHsignaling in BTIC and define the establishment of a TN-a2b1-JAG1-NOTCH signaling axis as a candidate therapeutictarget in glioma patients. Cancer Res; 77(12); 3231–43. �2017AACR.

IntroductionMalignant gliomas are thought to be maintained by a rare

population of transformed stem-like cells referred to as gliomastem cells or brain tumor-initiating cells (BTIC; refs. 1, 2). BTICsexhibit increased resistance to radiation (3, 4) and chemotherapy(5, 6) as compared with their more differentiated transformedprogeny (henceforth referred to as differentiated glioma cells),and they account for glioma recurrence following efficient che-motherapy in mice (7). Thus, the identification of mechanismsthat maintain the growth of BTICs will be important for thera-peutic purposes.

NOTCH proteins (NOTCH 1-4 in mammals) are transmem-brane proteins activated by ligands such as delta 1-4 andserrate/jagged 1 (JAG1) and JAG2. The interaction of NOTCH

with its ligand results in the proteolytic cleavage of the NOTCHreceptor and release of the intracellular domain (NOTCH-intracellular domain, NICD) that translocates to the nucleusto affect NOTCH-dependent transcription of genes (8, 9).Proteases implicated in the generation of NICD include met-zincin metalloproteinases [e.g., matrix metalloproteinases(MMP), a disintegrin and metalloproteinases (ADAM), andADAMs with thrombospondin motifs (ADAMTS)] at the prox-imal extracellular loop of NOTCH, and the g-secretase complexat the intracellular juxtamembrane (10).

The NOTCH signaling pathway maintains normal stem cellturnover in the brain. Excessive or aberrant NOTCH signaling,however, is a feature of tumorigenesis (8). The BTICs present inmalignant glioma exhibit elevated NOTCH activity (11–13)that has recently been implicated in BTIC growth and self-renewal, and their resistance to radiation and chemotherapy(11, 14, 15). However, the cause of the elevated NOTCHactivation in BTICs is unknown.

The microenvironment of tumors includes the surroundingextracellular matrix (ECM). For most tumor types, the availabilityof ECM proteins is a critical factor for tumorigenesis (16–18) asECM components activate integrins on the cell surface to triggersurvival and proliferative signaling, and because the ECM seques-ters a rich source of growth factors. The ECM inmalignant gliomais unique and includes vitronectin, collagen-I and -IV, osteopon-tin, and tenascin-C (TNC; refs. 19, 20).Of these, arguably themostimportant component is TNC as its amount in situ is correlatedproportionally with increased glioma grade (21–25) and prolif-erative capacity (21, 23, 26–29). Furthermore, antibodies thatblock TNC interactions reduce the motility of differentiated

1Department of Clinical Neurosciences, University of Calgary, Calgary, Alberta,Canada. 2Hotchkiss Brain Institute, University of Calgary, Calgary, Alberta,Canada. 3Department of Oncology, University of Calgary, Calgary, Alberta,Canada. 4Arnie Charbonneau Cancer Institute, University of Calgary, Calgary,Alberta, Canada.

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

R. Mirzaei and F.J. Zemp share second authorship of this article.

Corresponding Author: V. Wee Yong, University of Calgary, 3330 HospitalDrive, Calgary, Alberta T2N4N1, Canada. Phone: 403-220-3544; Fax: 403-210-8840; E-mail: [email protected]

doi: 10.1158/0008-5472.CAN-16-2171

�2017 American Association for Cancer Research.

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glioma cells in culture and suppress the growth of gliomas inmice(30). These preclinical results have evolved to clinical trials ofglioma patients using locally introduced small interfering RNA(siRNA) to TNC (31), or 131I-labeled anti-TNC antibody (32),where the survival results have been encouraging.

TNC also plays an important role in promoting tumor stem-ness. For instance, Wnt/b-catenin signaling has an important rolein regulating the balance between differentiation and stemness ina number of adult stem cell niches (33), and TNCmodulatesWnt/b-catenin signaling in glioma cells (27). In addition, TNC hasbeen shown to elevate components of stem cell signaling, such asmusashi homolog 1, a positive regulator of NOTCH signaling inbreast cancer cells, thus linking TNC to NOTCH signaling path-way (34). Whether or not TNC can promote epithelial-to-mes-enchymal transition in glioma stem-like cells is likely an impor-tant future area of research.

We have reported that TNC facilitates the invasiveness ofdifferentiated glioma cells in culture, through regulatingMMP-12 (35) and protein kinase C (36); moreover, we foundthat TNC promotes BTIC invasion through ADAM-9 and the c-Jun NH2-terminal kinase pathway (37). However, the role ofTNC on BTIC biology has not been investigated in detail. Arecent study has identified TNC as a novel marker for BTICs(38). Herein, we sought to test the hypothesis that the ele-vated NOTCH signaling that regulates BTIC growth is medi-ated by TNC. Our results implicate TNC in the maintenance ofglioma stem cells in the cancer stem cell niche, by modulatingNOTCH activity.

Materials and MethodsBTICs generated from human glioma patients

BTICs were generated and stable lines were formed fromresected specimens of patients with malignant glioma asdescribed before (39, 40). We used two BTIC lines for most ofthe tissue culture experiments in the present study designatedBT025 and BT048 with divergent genetic background; these lineshave been referred to previously as 25EF and 48EF, respectively(39).Other patient-derived BTIC lines used in culture or xenograftexperiments included BT012, BT53M, BT067, BT069, BT075,BT134, BT143, BT147, BT157, and BT161 (Supplementary Figs.S1–S4; ref. 41). All of these lines were cultured chronologically,maintained, and authenticated to the present time within theUniversity of Calgary BTIC Core directed by Dr. Sam Weiss andDr. Greg Cairncross. Their stemness features have been previous-ly described in our publications (39, 41) and our citationstherein. Lines were initiated in culture from resected specimensin year 2005 (BT012 and BT025), 2007 (BT048, BT53M, BT067,BT069, and BT075), 2009 (BT134, BT143, and BT147), and 2010(BT157 and BT161).

Neurosphere assayTo study the growth of BTICs, dissociated cells from BTIC

spheres were plated at a density of 10,000 cells/100 mL inserum-free BTICmedium(39). BTICswere treatedwithorwithoutTNC or other ECM proteins (10–50 mg/mL; Millipore), inhibitorsor antibodies.Weused soluble TNC in themajority of our study asa matter of convenience as our previous studies found that thisECM molecule exerts its effects on differentiated glioma cellseither in the soluble form or when embedded in a 3-dimensionalmatrix of collagen gel (35, 37). Although TNC purified from the

U251 human glioma line (Millipore, catalog # CC065, molecularweight 280–300, and glycosylated) was used in the majority ofexperiments unless otherwise stated, we also tested TNC (recom-binant human, R&D Systems, catalog #3358-TC) from a differentcommercial source.

Cultures were maintained at 37�C in 5% CO2 incubator.After 72 hours, four random fields per well were photographedunder a 10� objective in a phase contrast microscope, and thenumber of spheres over 60 mm in diameter was tabulated. Insome experiments, cells were treated with metalloproteinaseinhibitors [BB94 (500 nmol/L, British Biotech) or GM6001(10 mmol/L, Calbiochem)], or a NOTCH/g-secretase inhibitor(DAPT, with a final concentration of 2 or 5 mmol/L). TNC wasadded 1 hour after the addition of inhibitors. In selectedexperiments, cells were treated with neutralizing antibodies toa2b1, a9b1, and aVb6 integrins or isotype antibodies (Abcam)followed by TNC after 1 hour.

Western blot analysis and immunoprecipitationGBM tumor samples were used for Western blot analyses. The

samples were probed with rabbit human TNC antibody asdescribed before (37).

For immunoprecipitation, 500 mg of cell lysates was incubatedfor 3 hours at 4�C with 4 mg of the a2b1 antibody (Abcam) and30 mL of protein A/G-agarose beads (Santa Cruz Biotechnology).To control for nonspecific binding, an isotype antibody replacedthe primary antibody. The immunoprecipitates were subjectedto Western blot analysis as described before (42).

Cell cycle analysisBTIC cells were treated with or without TNC for 48 hours, and

cell-cycle analysis was performed with propidium iodide using astandard flow cytometry protocol (39).

Microarray and data analysisTo determine the effect of TNCon BTIC gene expression, BT025

cells were treated with TNC for 6 hours and subjected to micro-array analysis as described elsewhere (39). All microarray datahave been submitted to the US National Institute of Health GEOdatabase under accession number GSE94640.

Quantitative real-time PCR for JAG1BTICs were lysed in 1 mL of TRIzol reagent (Invitrogen) by

leaving plates at room temperature for 5 minutes before thecontent of the well was harvested and stored at �80�C prior touse. Following extraction, RNA was reverse transcribed and theresulting cDNA was used as a template for the Bio-Rad iCyclerMyiQ detection system and 2� SYBR green mastermix (Qia-gen). Primers (10� QuantiTect Primer Assay) were purchasedfrom Qiagen. Expression of gene transcripts was normalizedagainst at least two housekeeping genes, that is, GAPDH andb-actin. Relative expression levels for genes of interest weredetermined using the formula 2�DCT, where DCT ¼ CT (gene ofinterest) � CT (housekeeping gene).

NOTCH activityBTICs were plated in 12-well plates (100,000 cells/well) and

treated with or without TNC either in the presence or absence ofinhibitors or integrin antibodies. After 6 hours, cell lysate wasprepared and analyzed for the detection of cleaved NOTCHproduct, NICD1 (Rabbit anti-cleaved NOTCH1 (1:1,000, Cell

Sarkar et al.

Cancer Res; 77(12) June 15, 2017 Cancer Research3232




Signaling Technology) or NICD2 (rabbit anti-NOTCH2, cleaved;Millipore), by Western blot analysis following protein separationby gradient gel or 10% SDS-PAGE.

Small interfering RNA to TNC, JAG1, and a2b1 integrinA predesigned small interfering RNA (siRNA, Santa Cruz Bio-

technology), designated JAG1a siRNA, was used to targethuman JAG1. A second siRNA designated JAG1b siRNA was usedto confirm the first siRNA results. Similarly, two predesignedsiRNAs were used to target human TNC. For a2b1 integrinknockdown, two sets of predesigned silencer select siRNAs wereused (Ambion, Life Technologies); each set comprised two siRNAsthat targeted the respective a2 and b1 genes. A matrix-assistedlaser desorption/ionization-time of flight mass spectrometer wasused to identify the correct mass of the single-stranded RNAoligonucleotides. The annealed siRNAs were analyzed by non-denaturing PAGE. A negative control siRNA, composed of a 19-bpscrambled sequence with three deoxythymidine overhangs, wasused; the sequences have no significant homology to any knowngene sequences frommouse, rat, or human. For transfection withsiRNA, BTICs were plated in 12-well plates and were incubatedwith 30 nmol/L siRNA and Lipofectamine (Invitrogen). After 24hours, cells were harvested for neurosphere assays, Western blot,and cell-cycle analysis.

Lentiviral-mediated TNC knockdownLentivirus-mediated transfection was performed to stably

knockdown TNC. Stable knockdown of TNC was carried outusing shRNA constructs in BT025 and BT048 using SigmaMission TNC shRNAs or Scrambled controls in pLKO.1-purovector as described elsewhere. Briefly, the shRNA vector wascotransfected using Lipofectamine 2000 (Invitrogen) into HEK-293 cells with pMD2.G (VSV.G env) and pCMV-DR8.91. After a12-hour transfection in DMEM, viral containing media werecollected over 2 days in BTIC media. Collected media were thensyringe-filtered with a 0.22-mm filter, and then ultracentrifugedat 26,000 RPM for 90 minutes at 4�C. Viral pellets wereresuspended in BTIC media and added to cultures overnight.One mg/mL of puromycin (Invitrogen) was added to cultures 3days after infection.

Detection of TNC by immunofluorescence orimmunohistochemistry in human glioblastoma specimens

Paraffin sectionswere deparaffinized and subjected to antibodystaining and confocalmicroscopy as described elsewhere (39).Weevaluated paraffin sections from 3 autopsied glioblastomapatients (obtained from the University of Calgary Neuropathol-ogy archive) for the expression of TNC (using rabbit anti-TNCantibody, 1:500, Novus Biologicals) in proximity to presumed

Figure 1.

Staining of human glioblastoma and xenografts shows BTICs to be in the vicinity of TNC. A, TNC expression in a resected human glioblastoma specimenshowing elevated level of TNC protein (brown) compared with normal human brain tissue (original magnification,�100). B, At higher �200 magnification,TNC in GBM specimen (patient 1) is in the same area as BTICs; the latter were identified by nestin and CD133 immunoreactivity. These sections werecounterstained with hematoxylin (for nestin) or nuclear fast red (for TNC and CD133). C, Sections from two other autopsied GBM specimens show TNC(red) in proximity to nestin-positive cells (green); nuclei were labeled with DAPI in blue. D, TNC immunoreactivity (brown) was observed in the brain ofmice implanted with human BTICs (BT048, BT53M, and BT147). Implanted BTICs were identified in adjacent sections by nestin staining and by thepresence of human nucleolin (see Supplementary Fig. S1).

Tenascin-C Regulation of BTIC Growth

www.aacrjournals.org Cancer Res; 77(12) June 15, 2017 3233




BTICs labeled for nestin (mouse anti-Nestin, 1:500,Chemicon)orCD133 (mouse anti-CD133, 1:500, Milteny Biotech). A fluores-cence-conjugated secondary antibody was then applied to revealthe antigen. We also used a biotinylated secondary antibody,ABC reagent (Vectastain ABC kit, Vector Laboratories) and dia-minobenzidine to detect antigens in glioblastoma specimens forcorroboration.

Mice and BTIC implantationBTIC spheres were dissociated into single cells using

Accumax solution, and 10,000 viable cells in 2 mL of salinewere stereotactically implanted into the right striatum ofeach 6–8 week-old female SCID mice (Charles River) as de-scribed elsewhere (37, 39). Animals were returned to theircages and allowed free access to food and water. Mice wereweighed every other day and observed for symptoms of neu-rologic deficits; they were sacrificed 7 weeks after implantationwhile still asymptomatic. The whole brain was removed, cutinto blocks, fixed in 10% buffered formalin, and embedded inparaffin. Sections of 6 mm were taken every 120 mm apart,

through the entire brain. The sections were deparaffinized,rehydrated, and stained with hematoxylin and eosin (H&E)for general histology. Sections were also labeled for TNC(rabbit anti-TNC antibody, 1:500, Novus Biologicals), nestin(1:500, Chemicon), human implanted cells (mouse anti-human nucleolin, 1:500, R&D; ref. 38), or for Jagged1 (mouseanti-Jagged1, 1:100, Santa Cruz Biotechnology). Following abiotinylated secondary antibody, ABC reagent (Vectastain ABCkit, Vector Laboratories), and diaminobenzidine reaction, theslides were lightly counterstained with hematoxylin or nu-clear fast red, dehydrated and mounted. All protocols wereapproved by the Animal Care Committee at the University ofCalgary in accordance with research guidelines from the Cana-dian Council for Animal Care.

Statistical analysesFor analyses of differences in sphere formation in culture, the

one-way ANOVA with post hoc Tukey comparisons was used formultiple groups, while the t test was used for comparisons of twogroups.

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Of several ECM proteins, TNC most efficiently increases the sphere-forming capacity of BTICs. A and B, TNC stimulates sphere-forming capacity of the BT025and BT048 lines in comparison to other ECM proteins or control (no ECM); 3 days treatment, � , P < 0.05; �� , P < 0.01; �� , P < 0.001 compared with control (ANOVAwith Tukey's post hoc comparisons). The TNC enhancement of sphere growth is pictorially depicted in C for the BT025 cell line. D, TNC also stimulatedgrowth of BTICwhen anchored in a 3-dimensional (3D)matrix of type I collagen (CL). The effect of soluble TNConBTIC growthwas supported byAlamar blue assays(E), cell counts (F), and PI flow cytometry (G). �� , P < 0.01; �� , P < 0.001 compared with controls (t test, D–G). Error bars, SEM of 3–4 analyses.

Sarkar et al.





ResultsBTICs are found in TNC-enriched areas in human glioblastomaspecimens

TNC has been documented to be markedly elevated in malig-nant glioma compared with surrounding normal brain tissue(22), and we have demonstrated by Western blot analyses thesubstantial increase in TNC content in glioblastoma comparedwith nontransformed humanbrain specimens (37).However, thelocalization of TNC in relation to BTICs in human glioblastomaspecimens has not been characterized. Figure 1A and B shows thatby immunohistochemistry, the high expression of TNCwithin theglioblastoma of patient 1, where presumed BTICs, detectedthrough CD133þ and nestinþ staining, was present. We corrob-oratedby immunofluorescencemicroscopy thehigh expressionofTNC in glioblastoma specimen from 2 other patients, in prox-imity to nestin-positive cells (Fig. 1C).

To further emphasize that TNC and BTICs are in proximityfor potential interactions to occur, we determined the expres-sion of TNC in SCID mice implanted with human BTIC cells(BT048, BT53M, BT134, BT143, BT147, BT157, and BT161) inthe right striatum. We sacrificed asymptomatic mice 7 weeksafter implantation and found tumor mass in mice. TNCexpression was detected in brain areas where nestinþ andhuman nucleolinþ cells were found (Fig. 1D and Supplemen-tary Fig. S1). Elevated level of TNC expression was observed inthe tumor mass and at the invasive front of the tumor (Sup-plementary Figs. S2 and S3).

We addressed the localization of TNC in relation to cells orthe ECM. As TNC is an ECM component, it would be reason-able to find some staining outside of cells (e.g., the diffuse TNCstaining in Fig. 1A). However, during its production and sub-sequent export out of cells, some signal for TNC immunore-activity should be inside cells. This appears to be the case in Fig.1C (patient 2) where there are yellow profiles that are overlapof nestin-positive BTICs (green) and TNC (red). The latter resultalso suggests that BTICs in glioblastomas have the potential toproduce TNC.

TNC promotes growth of glioma patient-derived BTICsAlthough others have reported on the capacity of TNC to

promote the invasiveness and proliferation of differentiatedglioma cells, we have not found reports of the potential of TNCto promote BTIC growth. Several BTIC lines isolated fromindividual glioma patients with diverse genetic mutation pro-files were used to address whether their responses were uniformto TNC. These lines were assessed to be BTICs rather thandifferentiated glioma cells based on their expression of nestin,Sox2 and Musashi-1; they exhibited self-renewal propertyand multilineage capacity by differentiation into GFAPþ andbIII tubulinþ cells; and they formed invasive tumors whenimplanted orthotopically as xenografts into mice (39, 40). TheBTICs grew as progressively larger spheres in suspension cul-tures and we mechanically dissociated the enlarging spheresinto single cells periodically, so that these could regrow asspheres to allow the lines to be maintained indefinitely.

To determine the role of ECM proteins on BTIC growth, thepatient-derived spheres were plated as dissociated cells (10,000cells/100 mL) and exposed to 10 mg/mL TNC, type III collagen (CLIII), type IV collagen (CL IV), fibronectin (FN), vitronectin (VN),or chondroitin sulfate proteoglycans (CSPG). Using a sphere size

cut off of 60 mm for ease of quantitation (39), we determined thatboth TNC and CL IV stimulated sphere formation of the BT025and BT048 lines (Fig. 2A–F), with TNC having a greater effect.Indeed, a sphere after 3 days of exposure to TNC (Fig. 2C)regularly contains�1,500 cells. TNC from a different commercialsource also promoted sphere formation of BTICs (SupplementaryFig. S4, D). CSPGpromoted sphere formation in one of Two lines,whereas cells grew as an adherent monolayer on VN. Moreover,TNC increased the number of viable cells (Fig. 2E), and the totalnumber of cells in the S and G2–Mphases of the cell cycle (Fig. 2Fand G). Importantly, TNC-enhanced sphere formation wasfound across all human-derived BTIC lines tested (Supplement-ary Fig. S4). Although TNC increased the number of cells in theS-phase of the cell cycle supporting a proliferative effect, anadditional effect of TNC on cell survival is not ruled out.

The above experiments involved the provision of soluble TNCto cells to simulate the release of TNC from its cellular source toaffect autocrine or paracrine interactions. To represent TNCanchored to the ECM, we encased TNC along with BTICs withina 3-dimensional matrix of collagen gel as described previously(35). In this environment, the increase in cell numbers in responseto TNC was still evident (Fig. 2D).

Integrin-mediated TNC-induced autocrine growth of BTICsWeexamined the receptors responsible for TNC-mediated BTIC

growth. TNC binds to integrin receptors on cells, including a2b1,a9b1, and aVb6 (43). BTICs were treated with neutralizingantibodies to these integrins in the presence of exogenously addedTNC, and the number of sphereswas quantified. Cells treatedwitha2b1-neutralizing antibody in the presence of exogenously TNCshowed significant reduction of BTIC growth compared withcontrol (Fig. 3A and B), whereas a9b1- and aVb6-neutralizingantibodies had no effect. Thus, the data suggest that exogenouslyadded TNC acted through a2b1 integrin on BTICs for growth.Unexpectedly, the a2b1 integrin antibody added to BTICs in theabsence of exogenously applied TNC also diminished sphereformation (Fig. 3A and B), suggesting that TNC was producedby BTICs to regulate autocrine growth. Consistent with thisobservation, we previously reported that TNC was detected inthe conditioned media of BTICs (37). As noted above, there isoverlap of nestin and TNC immunoreactivity in a glioblastomaspecimen (Fig. 1C).

We further corroborated the role of a2b1 integrin in TNC-stimulated BTIC growth using RNAi approaches. Indeed, theknockdown of a2b1 integrin attenuated the stimulatory effect ofTNC on BT025 and BT048 growth, both in presence and absenceof TNC (Fig. 3C–F).

We evaluated the TNC–a2b1 integrin interaction further usinglenti-viral-mediated knockdown of TNC. In TNC-silenced shTNCBTICs, the addition of the function blocking antibody to a2b1integrin in both BT025 and BT048 lines no longer reduced BTICgrowth below the level of control, supporting the above conten-tion that TNCwasproducedbyBTICs to regulate autocrine growth(Supplementary Fig. S5). Furthermore, exogenous TNC that pro-moted BTIC growth in TNC-silenced cells was blocked of thisactivity in the presence of the a2b1 integrin antibody (Supple-mentary Fig. S5). These results support a critical role for a2b1integrin in mediating autocrine or exogenously applied TNC instimulating BTIC growth.

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TNC is produced by BTICs for autocrine growth. A and B, Basal (no TNC added) or TNC-promoted BTIC growth (sphere-forming capacity or cell counts) of boththe BT025 and BT048 lines is abrogated by neutralizing a2b1, but not a9b1 or aVb6 integrin. �� , P < 0.001 compared with control. C and D, Knockdown bysiRNA of a2b1 integrin in BT025 and BT048 cells, as determined by FACS analysis (red, background staining; blue, positive population). E and F, When a2b1integrin knockdown cells were subjected to neurosphere assay either in presence or absence of TNC, both sphere growth and total cell numbers werereduced. ��, P < 0.001 compared with control siRNA. G and H, TNC knockdown with siRNA as evaluated by Western blot and cell-cycle progression of thetransfected cells. I, Control siRNA– or TNC siRNA–treated cells were subjected to neurosphere assay and evaluated after 72 hours. TNC siRNA–treated cells showedsignificant reduction of sphere formation compared with control, and this could be overcome by exogenous TNC. Control þ TNC was used as a positivecontrol for TNC stimulated BTIC growth. �� , P < 0.001 compared with their respective siRNA control. J, Immunofluorescence staining showing focal adhesionkinase (FAK) expression 6 hours after TNC stimulation of freshly dissociated cells compared with control (a representative image from four cultures each). Allerror bars represent SEM of 3–4 analyses, with statistical evaluation through ANOVA with Tukey multiple comparisons.

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had lower number of cells in the S-phase and correspondingmorein G1 of the cell cycle, and some death was evident after 3 days,indicating that cell cycle block and eventual death occurred.Notably, a2b1 integrin seems to be predominantly expressed inBTICs (BT025: 99.4% of cells; BT048: 65.5%) compared withother TNC binding integrins (Fig. 3C and D and SupplementaryFig. S5). TNC is also known to bind the aVb3 integrin; very lowexpression of aVb3 was detected in BTIC lines (SupplementaryFig. S5), and a neutralizing antibody toaVb3 integrin did not alterBTIC growth upon TNC stimulation.

We evaluated TNC-mediated autocrine signaling further byreducing TNC expression in BTICs using siRNAs (Fig. 3G). Con-sequently, cells were reduced in cell-cycle progression (Fig. 3H)and they formed spheres less readily; the exogenous applicationofTNC to TNC siRNA-treated BTICs resumed their capacity forsphere formation (Fig. 3I).

Finally, to begin to provide data (see also results below) thatsoluble TNC interacting with integrin promotes signaling inBTICs, we evaluated BTICs for focal adhesion kinase, a knowndownstream target of this interaction. We noted by immunoflu-orescence the pronounced focal adhesion kinase expression inTNC-treated BTICs (Fig. 3J).

TNC upregulates NOTCH ligand expression in BTICWe subjected BTICs to global gene expression analysis to

provide insights into the mechanisms by which TNC stimulatesBTIC growth. After 6 hours of TNC treatment, 917 genes (P<0.05)were differentially expressed compared with controls (Fig. 4A;GEO accession number GSE94640), with 178 genes altered by afold change of at least 1.5 (Supplementary Tables S1 and S2);these included previously known genes that regulate gliomagrowth, such as SMAD7, ID2, TGFb, ADAMTS15, and theNOTCH

Figure 4.

A, Microarray analysis of BTICs 6 hours after exposure to TNC. Hierarchical clustering upon 6 hours of TNC treatment (GEO accession number GSE94640). B,Upregulated genes (fold change �1.5) were classified according to their involvement in different pathways via Panther database. Numbers show the percentageof genes involved in the respective pathway. C, Volcano plot of all present genes. The most significantly upregulated genes are shown in blue. Amongthem, four genes are involved in different signaling pathways. The results were obtained from three experiments performed at different times, where eachexperiment involved a control and a TNC-treated culture in order to capture consistent changes.D, PCR validation of JAG1 gene in BT025 cells (GADPH normalized);�� , P < 0.001 compared with control (t test); error bars, SEM. E, The JAG1 expression was increased as well with TNC treatment at the protein level (BT025and BT048 lines). F, Metacore analysis of microarray data, showing interactome pathway in TNC-treated BT025 cells. G, Brain samples from 6 GBM patients;all had evidence of JAG1, NICD1, and NICD2 expression.






ligand, JAG1. Pathway analysis using Panther database demon-strated several signaling pathways were upregulated (P < 0.05)with TNC treatment, including the Notch signaling pathway (Fig.4B). When analyzing the data using a volcano plot of fold change(log2) of genes against their statistical significance, twoof themosthighly upregulated genes, JAG1 and ADAMTS15, are key compo-nents ofNOTCHsignalingpathways, andother upregulated genesare SKIL and ELK3 implicated for TGFb and interleukin signalingpathways, respectively (Fig. 4C and Supplementary Table S1)reported to play a critical role in glioma stem cell proliferationand self-renewal (12, 44). The increase in expression of JAG1 wasconfirmed by PCR transcript and protein analysis (Fig. 4D and E).

Metacore analysis of the microarray data was used to probeinteractive pathways or interactome (Fig. 4F). Mining of the

microarray data publicly available from the Oncomine (Com-pendia Biosciences, http://www.oncomine.org/) databaserevealed that glioma patients with higher TNC expression havea significantly poorer survival advantage. The mining data alsoshowed high JAG1 and NOTCH1 expression in glioma patients(Supplementary Fig. S6), and improved survival for gliomapatients with low expression of JAG1 or NOTCH1 based on otheravailable data sets from Rembrandt (Currently available fromG-Doc plus database) or The Cancer Genome Atlas (TCGA; usingAgilentg Platform; Supplementary Fig. S7). Our microarray dataalso showed increased expression of a2 integrin (ITGA2) withTNC treatment (Supplementary Table S1). Notably, TCGA dataanalysis showed significant correlation between TNC andNOTCH1, JAG1, or a2b1 integrin in GBM samples that strongly

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The TNC-stimulated NOTCH activation or growth of BTICs is reduced by blocking g-secretase or metalloproteinases, or in a2b1 integrin knockdown cells. A, TNCstimulates NOTCH activity in BTICs as evidenced by elevation of cleaved NOTCH receptors, NICD1 and NICD2, after 6 hours of TNC treatment and evaluatedthrough Western blot analysis. B and C, Addition of the NOTCH inhibitor, DAPT (2 or 5 mmol/L), or the metalloproteinase inhibitors, BB94 (500 nmol/L) orGM6001 (10 mmol/L), reduced total cell numbers under basal and TNC-stimulated condition. ��, P < 0.001 compared with TNC and compared with control (inabsence of TNC experiment) for all the bars enveloped by the drawn line (ANOVA with Tukey's multiple comparisons). Error bars represent SEM of fouranalyses, and the results were reproduced in two other experiments.D, TNC-stimulated NOTCH activity was abrogated by a blocking antibody to a2b1 integrin after6 hours of exposure to TNC in both the BT025 and BT048 lines (Supplementary Fig. S9). In contrast, isotype antibody treatment had no effect. Actin was used asloading control. Results were reproduced in another set of experiment. E, The TNC-stimulated JAG1 expression was abrogated by blocking g-secretase (DAPT),metalloproteinase (GM6001), ora2b1 integrin. F, TNC-stimulated NOTCH activity was abrogated by siRNA knockdown ofa2b1 integrin in BT025 cells. The result wasreproduced twoother times.G,Whena2b1 integrin knockdowncellswere treatedwith TNC,BTICgrowthwas significantly reduced; however, CLIVor LNhadnoeffectin a2b1 knockdown cells for BTIC growth. H, Immunoprecipitation demonstrates the association of a2b1 integrin with TNC in both the BT025 and BT048 lines.

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http://www.oncomine.org/


support our findings of these interacting components (Supple-mentary Fig. S8). To validate these findings, we analyzed tumorsamples from 6 GBM patients and readily observed JAG1 exp-ression. Moreover, 4 of these patients showed elevated levelsof active or cleaved forms of NOTCH (NICD1 and NICD2expression; Fig. 4G).

Interference of NOTCH activation reduces TNC-enhanced BTIC growth

Binding of ligand to the NOTCH receptor results in thestimulation of its cleavage by metalloproteinases and g-secre-tase. This results in the generation of the NICD that cantranslocate to the nucleus to modulate gene expression. Weassayed NOTCH activation bymeasuring NICD1 and NICD2 inBTICs exposed to TNC for 6 hours and found elevated levels ofthis cleavage fragment (Fig. 5A). Moreover, an inhibitor of theg-secretase complex, DAPT (N-[N-(3,5-Diflurophenaacetyl-L-alanyl)]-S-phenylglycine t-Butyl Ester), decreased BTIC sphereformation (Fig. 5B and Supplementary Fig. S9) with a concor-dant reduction in the total number of cells (Fig. 5C) in the

presence of TNC. Addition of the metzincin metalloproteinaseinhibitors BB94 and GM6001 also reduced TNC-stimulatedBTIC growth in BT025 and BT048 lines (Fig. 5C and Supple-mentary Fig. S9). Collectively, these data suggest that NOTCHsignaling is engaged following TNC treatment, and involved inBTIC growth.

a2b1 integrin and NOTCH activityFrom the observations that a function blocking antibody to

a2b1 integrin, or the siRNA knockdown of a2b1 integrin,blocked the effect of TNC on BTIC growth, and that NOTCHactivity was enhanced by TNC treatment, we evaluated whetherblocking a2b1 integrin would alter NOTCH activity. Figure 5Dshows elevated level of NOTCH activity (NICD1) following6 hours of TNC treatment and this was abolished by theneutralizing antibody to a2b1 integrin. In contrast, a neutral-izing antibody to a9b1 integrin did not alter TNC-stimulatedNOTCH activity (Supplementary Fig. S9). The neutralizingantibody to a2b1 integrin attenuated TNC-stimulated JAG1expression (Fig. 5E and Supplementary Fig. S9), which was

Figure 6.

The knockdown of JAG1 reduces TNC-stimulated BTIC growth. A, The reduction of TNC expression in BTIC lines (BT025 and BT048) by lentiviraltransfection of small hairpin loop RNA (shRNA) to TNC also exhibited reduced JAG1 expression, as determined using cell lysates by Western blots. Theclones generated using shRNA construct is designated as shRNA(1) to shRNA(4). shRNA(1) and shRNA(3) were found to express the least amount of TNCand corresponding JAG1 compared with control shRNA-generated cells. This evaluation was reproduced with another set of analysis. B, In a separateexperiment, JAG1 gene was transiently knocked down with two different siRNAs for JAG1 (designated JAG1a and JAG1b) in BT025 and BT048 celllines. BTICs treated with TNC for 6 hours after siRNA-mediated knockdown of JAG1 have reduced S-phase cell-cycle kinetics (C) and sphere formingcapacity (D). �� , P < 0.001 compared with TNC þ control siRNA. E, JAG1 and corresponding TNC immunoreactivity (brown) in the tumor-containing area ofthe brain of 5 mice at 7 weeks following implantation with BT025 cells.






also decreased in the presence of g-secretase (DAPT) andmetalloproteinase (GM6001) inhibitors. We could not obtaina consistent basal (in absence of TNC) expression of JAG1 toaddress whether the low level of JAG1 in unstimulated cellswould also be reduced by these inhibitors.

We determined that BTICs deficient of a2b1 integrin throughsiRNA knockdown had reduced NOTCH activity (NICD1)compared with control siRNA-treated cells in response to TNCstimulation (Fig. 5F), or in absence of TNC (Supplementary Fig.S9, E). Unexpectedly, unlike the function blocking antibody toa2b1 integrin that ameliorated TNC-stimulated JAG1 expres-sion (Fig. 5E and Supplementary Fig. S9), we did not findreduction of JAG1 expression in TNC-stimulated a2b1 knock-down cells; the turnover of JAG1 in siRNA transfected orantibody treated cells may be markedly different to accountfor this discrepancy.

a2b1 integrin also binds to CLIV and laminin (LN), butthese ECM proteins unlike TNC did not alter BTIC growth ina2b1 integrin knockdown cells (Fig. 5G). It is possible thatCLIV and LN promote BTIC growth through a a2b1-indepen-dent mechanism that compensates for lack of interactionbetween endogenous TNC and a2b1. Finally, the presence ofTNC bound to the a2b1 integrin was demonstrated by coim-munoprecipitation experiments (Fig. 5H) although it is stillconceivable that there is an intermediary molecule that anchorsTNC to the a2b1 integrin. Overall, these results support theresult that a2b1 integrin mediates the TNC-induced NOTCHactivation in BTICs.

TNC as a regulator of NOTCH signaling for BTIC growthBecause TNC stimulates NOTCH activity and elevates the

expression of the NOTCH1 ligand JAG1 in BTICs, we sought todetermine the functional implication of TNC–JAG1–NOTCH1link. We sought to stably knockdown TNC in BT025 andBT048 cells using lentiviral transduction. We used 4 differentTNC shRNAs to reduce TNC (Supplementary Fig. S10) in bothlines, and shTNC (1) and shTNC (3) were effective (Fig. 6A).Remarkably, JAG1 expression was reduced in the TNC knockeddown clones but not in BTIC clones where TNC was notsuccessfully lowered. This observation further links the func-tional association between TNC and NOTCH1 ligand JAG1expression.

Next, we knocked down JAG1 using two siRNAs in BT025and BT048 lines (Fig. 6B). The resultant cells were no longerresponsive to TNC for BTIC growth, suggesting that JAG1 isintegral to TNC's effect (Fig. 6C and D). This is supported byJAG1 immunoreactivity in TNC-expressing areas in brain sec-tions of asymptomatic mice at 7 weeks after implantation ofBT25 BTICs (Fig. 6E).

Overall, these results strongly suggest that NOTCH sig-naling is critically involved in TNC stimulated BTIC growth.Figure 7 shows an overview of TNC–NOTCH signalingpathway, which is presumed to be operative in BTICs inglioblastoma.

DiscussionWithin the CNS, glioma cells show preference to infiltrate

along the periphery of blood vessel walls, the subpial glialsurface (glial limitans externa) or white matter tracts (19).Besides the occurrence of glial cells or their processes, these

structures are enriched in ECM proteins. For most tumor types,including gliomas, the availability of ECM proteins is consid-ered a critical step in cell invasion and tumor growth as theyactivate integrins on the cell surface to trigger signaling andbecause the ECM is a rich source of growth factors. Amongmany glioma ECM proteins, TNC is arguably the most prom-inent component (45), and plays important roles in gliomaangiogenesis, growth, and invasion. However, there is dearth ofsuch information for the influence of TNC on BTICs in contrastto differentiated glioma cell lines.

Here, we demonstrate the potential of interactions betweenBTICs and TNC by displaying their proximity in human glioblas-toma specimens and in mouse brains implanted with humanBTICs (Fig. 1). We emphasize the capacity of exogenous TNC topromote BTIC growth in culture (Fig. 2) and we have uncoveredan autocrine pathway of TNC-mediated cellular proliferation thatengages the a2b1 integrin (Fig. 3); an association between TNCand the a2b1 integrin was also found by coimmunprecipitationand is corroborated by the TCGA mining data (Fig. 5 and Sup-plementary Fig. S8). Moreover, failure of exogenous TNC topromote BTIC growth in a2b1 integrin blocked–TNC-silencedBTIC cells further emphasize the critical requirement of functionala2b1 integrin heterodimers for TNC-promoted BTIC growth(Supplementary Fig. S5). We note that while several TNC-inter-acting integrins other than a2b1 were not prominently expressedon BTICs, and that their function blocking antibodies did notabrogate TNC-promoted sphere formation by BTICs, we have notexhausted the examination of integrins with the capacity to bindTNC, such as a5b1 (46).

Importantly, our results link TNC/a2b1 integrin to theNOTCH elevation that is noted in gliomas. This was supportedby microarray analyses and confirmatory PCR and Westernblots that TNC-elevated components of the NOTCH signalingpathway in BTICs, including JAG1, ADAMTS15, and NICD1/2(Figs. 4 and 5); and by perturbation experiments in cultureinvolving knockdown of JAG1 or pharmacologic inhibitors tog-secretase and metalloproteinases that abrogate TNC-promot-ed BTIC growth (Figs. 5 and 6).

Although we have posited TNC as an important factor inactivating NOTCH signaling in BTICs, it is likely that otherfactors in the tumor microenvironment can also activateNOTCH. For instance, TGFb signaling is elevated in TNC-stimulated BTICs as indicated by the microarray data (Fig. 4),and whether this is relevant to the enhanced BTIC growththough activating NOTCH remains to be explored in futureexperiments. In addition, other cell types may also activateNOTCH in BTICs; one example is endothelial cells thatexpress NOTCH ligands (47) and that may regulate braintumor stem cell niches via NOTCH signaling (48). Anotherarea for investigation in future studies is whether the TNC–JAG1–NOTCH signaling pathway plays a vital role in theproliferation of transformed cells differentiated from BTICs,or whether this cascade is a property of stemness withinglioblastomas.

In our study, we suggest the presence of autocrine TNCsignaling for BTICs as TNC and nestin immunoreactivity inBTICs in glioblastoma specimens may overlap (Fig. 1C), andbecause blocking the a2b1 integrin in unstimulated BTICs(Fig. 3) result in reduced basal sphere growth. Exogenouslyapplied TNC further stimulates growth, raising the question ofinteractions of TNC from autocrine and paracrine sources.

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Indeed, there appears to be some interaction between endog-enous and exogenous TNC because in the TNC siRNA-treatedcells without endogenous TNC for autocrine signaling, theaddition of exogenous TNC does not return sphere growth tothe extent seen with control þ TNC BTICs (Fig. 3I).

In reviewing the literature, we note that NOTCH signaling ingliomas has been found to upregulate TNC levels (49); how-ever, the converse that TNC accounts for the elevated NOTCHsignaling in gliomas, particularly in BTICs, has not been pre-viously reported. Moreover, we are unaware of publicationsthat addressed whether TNC is a growth-promoting factorfor BTICs. One publication reported that TNC-expressingBTICs were better at sphere formation in limiting dilutionassays than BTICs that were not expressing TNC (38), but thedirect role of TNC in promoting proliferation was notaddressed. Nonetheless, that manuscript supports our presentanalysis that BTICs produce TNC, and that the TNC in turn canregulate BTIC growth.

From the literature, it is evident that TNC is present in neuralstem cell niches in the normal central nervous system (50).Whether TNC maintains the stemness of neural stem cellsthrough a2b1 integrin–NOTCH signaling remains to be deter-mined. Whether TNCmaintains the tumor stemness phenotypethrough a2b1 integrin–NOTCH signaling in other cancer typesshould also be of interest. In breast cancer, tumor cells thatmetastasize to the lungs support their metastatic capacity byexpressing TNC that enhances the expression of a stem cellsignaling component, musashi homolog 1, a positive regulatorof NOTCH signaling (34).

In summary, and to the best of our knowledge, this study is thefirst to identify TNC for the elevated NOTCH signaling in BTICs;moreover, we demonstrate that the TNC-activated NOTCH sig-naling within BTICs regulates their proliferation and sphere-forming capacity. The TNC/NOTCH/JAG1 axis has pathologic

relevance as mining of the Oncomine, Rembrandt or TCGAdatabases shows each component to have survival disadvantagefor patients with gliomas; moreover, TCGA data analysis showedsignificant correlation between TNC and NOTCH1, JAG1 and a2integrin in GBM specimens. As the relative resistance of BTICs tochemo- or radiotherapy helps account for the intractability ofhuman gliomas, our findings of TNC-triggered activation ofNOTCH signaling could provide new insights and therapeuticsto target the components of this axis, such as by inhibiting theproteases or translocation of NICD involved in the activation ofNOTCH, in order to control the growth of BTICs and improve theprognosis of those with glioblastomas.

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

Authors' ContributionsConception and design: S. Sarkar, R. Mirzaei, V.W. YongDevelopment of methodology: S. Sarkar, R. Mirzaei, V.W. YongAcquisition of data (provided animals, acquired and managed patients,provided facilities, etc.): S. Sarkar, R. Mirzaei, F.J. Zemp, D.L. Senger,S.M. Robbins, V.W. YongAnalysis and interpretation of data (e.g., statistical analysis, biostatistics,computational analysis): S. Sarkar, R. Mirzaei, W. Wu, V.W. YongWriting, review, and/or revision of the manuscript: S. Sarkar, R. Mirzaei,F.J. Zemp, D.L. Senger, S.M. Robbins, V.W. YongAdministrative, technical, or material support (i.e., reporting or organizingdata, constructing databases): D.L. Senger, V.W. YongStudy supervision: V.W. Yong

AcknowledgmentsWe acknowledge the technical help of Claudia Silva, Yan Fan, Xiuling Wang,

and Fiona Yong. We thank the University of Calgary BTIC Core headed by Drs.Sam Weiss and Greg Cairncross for isolating BTIC lines from patient-resectedspecimens.

Figure 7.

Postulated mechanism of TNC-stimulated BTIC growth. The diagram depicts that TNC elevates level of JAG1 expression in BTICs upon binding to a2b1 integrin onthe same or proximal cell. The interaction of JAG1 and its receptor NOTCH1 then results in the proteolytic cleavage of the NOTCH receptor and release of theintracellular domain (NICD) that translocates to the nucleus to affect NOTCH-dependent transcription of genes. The generation of NICD is promoted bymetzincin metalloproteinases (denoted as S1) at the proximal extracellular loop of NOTCH and the g-secretase complex (denoted as S2) at the intracellularjuxtamembrane. TNC is either from the extracellular matrix or produced by BTICs for autocrine growth.






Grant SupportWe acknowledge grant support from Alberta Cancer Foundation/

Alberta Innovates – Health Solutions to V.W. Yong, and the CanadianInstitutes of Health Research to S.M. Robbins and V.W. Yong. R. Mirzaeiis supported by a University of Calgary Eyes High postdoctoralfellowship.

The costs of publication of this article were defrayed in part by the paymentof page charges. This article must therefore be hereby marked advertisementin accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

Received August 11, 2016; revised February 10, 2017; accepted April 10, 2017;published OnlineFirst April 17, 2017.

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2017;77:3231-3243. Published OnlineFirst April 17, 2017.Cancer Res Susobhan Sarkar, Reza Mirzaei, Franz J. Zemp, et al. Human Brain Tumor-Initiating CellsActivation of NOTCH Signaling by Tenascin-C Promotes Growth of

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