Constitutive NOTCH3 Signaling Promotes the …Therapeutics, Targets, and Chemical Biology Constitutive NOTCH3 Signaling Promotes the Growth of Basal Breast Cancers Lisa Choy1,Thijs

Therapeutics, Targets, and Chemical Biology

Constitutive NOTCH3 Signaling Promotes theGrowth of Basal Breast CancersLisa Choy1, Thijs J. Hagenbeek1, Margaret Solon2, Dorothy French2, David Finkle3,Amy Shelton1, Rayna Venook3, Matthew J. Brauer4, and Christian W. Siebel1

Abstract

Notch ligands signal through one of four receptors on neigh-boring cells to mediate cell–cell communication and control cellfate, proliferation, and survival. Although aberrant Notch activa-tion has been implicated in numerous malignancies, includingbreast cancer, the importance of individual receptors in distinctbreast cancer subtypes and the mechanisms of receptor activationremain unclear. Using a novel antibody to detect active NOTCH3,we report here that NOTCH3 signals constitutively in a panel ofbasal breast cancer cell lines and in more than one third of basaltumors. Selective inhibition of individual ligands revealed that thissignal does not require canonical ligand induction. A NOTCH3

antagonist antibody inhibited growth of basal lines, whereas aNOTCH3 agonist antibody enhanced the transformed phenotypein vitro and in tumor xenografts. Transcriptomic analyses generateda Notch gene signature that includedNotch pathway components,the oncogene c-Myc, and the mammary stem cell regulator Id4.This signature drove clustering of breast cancer cell lines andtumors into the common subtypes and correlated with the basalclassification. Our results highlight an unexpected ligand-indepen-dent inductionmechanism and suggest that constitutive NOTCH3signaling can drive an oncogenic program in a subset of basalbreast cancers. Cancer Res; 77(6); 1439–52. �2017 AACR.

IntroductionBreast cancer is the second most common cancer among

women in the United States and is the second leading cause ofcancer death. Threemain subtypes have beendistinguished on thebasis of the expression of progesterone and estrogen receptors andthe HER2 receptor tyrosine kinase: luminal (hormone receptor–positive; �50%–60%), HER2-enriched (�10%), and basal (tri-ple-negative;�10%–20%; ref. 1). The basal subtype lacks targetedtherapies and suffers from the poorest prognosis. Illuminatingthe molecular mechanisms supporting maintenance of this sub-type thus holds therapeutic promise.

Notch ligands and receptors comprise a conserved family oftransmembrane proteins that conduct cell–cell communicationto regulate cell fate and stem/progenitor cell proliferation anddifferentiation. Current understanding holds thatNotch receptorsremain quiescent until ligand binding triggers a conformationalchange that enables ADAM-protease cleavage (at site S2) withinthe juxtamembrane negative regulatory region (NRR; refs. 2, 3).The g-secretase complex subsequently cleaves within the mem-

brane (site S3), liberating the Notch intracellular domain(NICD�) to translocate to the nucleus and direct the Notchtranscriptional program (4). Mammals express 4 Notch receptors(NOTCH1–4) and 4 canonical ligands, includingmembers of theJagged (Jag)-Serrate family, JAG1 and JAG2, and the Delta-likefamily, DLL1 and DLL4.

Widely expressed and functioning broadly in tissue homeosta-sis, the Notch pathway has been linked to disease, includingcancer. The strongest case supporting Notch as an oncogenicdriver in humans stems from T-cell acute lymphoblastic leukemia(T-ALL), where NOTCH1 signal–stimulating mutations, includ-ing missense changes that destabilize the NRR and enable ligand-independent signaling, are found inmore thanhalf of the patients(5). Hyperactive Notch signaling has been implicated in numer-ous other leukemias and solid tumors, although the rationale forNotch as a cancer driver in these other indications generally lackssuch "smoking gun" data of frequentmutations (6). These links tocancer have compelled therapeutic targeting, beginning withsmall-molecule g-secretase inhibitors (GSI). However, GSIs targetall Notch signaling plus other g-secretase–dependent signals andare not well-tolerated.

Genetic and expression studies have linked Notch signalingto breast cancer. A subset of breast cancers display JAG1 andNOTCH1 overexpression, correlating with a poor prognosis(7, 8), and genetic translocations leading to constitutive Notch1andNotch2 signaling occur in 2.6% of basal breast tumors (9). Inmice, NOTCH3 expression marks a self-renewing populationof luminal progenitor cells in the normal gland (10), whereasconstitutive NOTCH3 signaling generates tumors and revealsNOTCH3 oncogenic potential (11). In the human disease, basalbreast cancers show NOTCH3 amplification and overexpression(12, 13), and Notch3 knockdown reduces proliferation of breastcancer cell lines (14). Jag1 or Jag2 knockdown reduced Notchactivity in these lines, suggesting that these ligands were required

1Department of Discovery Oncology, Genentech, Inc., South San Francisco,California. 2Department of Pathology, Genentech, Inc., South San Francisco,California. 3Department of Translational Oncology, Genentech, Inc., South SanFrancisco, California. 4Department of Bioinformatics & Computational Biology,Genentech, Inc., South San Francisco, California.

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

CorrespondingAuthor:ChristianW. Siebel, Department ofDiscoveryOncology,Genentech, 1 DNA Way, South San Francisco, CA 94080. Phone: 650-225-2751;Fax: 650-467-7571; E-mail: [email protected]

doi: 10.1158/0008-5472.CAN-16-1022

�2017 American Association for Cancer Research.

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Published OnlineFirst January 20, 2017; DOI: 10.1158/0008-5472.CAN-16-1022

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to induce the NOTCH3 signal (14). While these studies provide afoundation, the mechanism and prevalence of NOTCH3 activa-tion in breast cancer have not been rigorously characterized.

To address these questions, (i) we developed a new reagentantibody that selectively detects activated NOTCH3 and (ii) weexploited therapeutic antibodies that selectively agonize orantagonize individual Notch ligands and receptors, movingbeyond GSIs to precisely manipulate Notch function and min-imize toxicity. We discovered that NOTCH3 signals constitu-tively in breast cancer cell lines, independent of canonical ligandinduction, as well as in human breast cancers, particularly thoseof the basal/triple-negative subtype. Selective NOTCH3 inhibi-tion antagonizes breast cancer cell line and tumor growth,whereas NOTCH3 agonism promotes a malignant phenotypeand increases proliferation. Transcriptomic analyses establish aNotch signature that includes increased expression of the c-Myconcogene as well as the mammary stem cell regulator Id4. Thissignature is sufficient to cluster breast cancer cell lines andpatient tumors into the major subtypes and positively correlateswith basal samples, indicating that NOTCH3 activity may beclinically relevant in this subclass.

Materials and MethodsAntibody characterization, immunohistochemistry

Hybridoma supernatants (rabbits immunized with humanNOTCH3 peptide 1662–1675) were screened by immunoblot-ting of MDA-MB-468 nuclear lysates, after using N-[N-(3,5-difluorophenylacetyl)-l-alanyl]-S-phenylglycine tert-butyl ester(DAPT) or 10 mmol/L EDTA to inhibit or stimulate NOTCH3,respectively. This concentration of EDTA chelates calcium ionsneeded for proper folding of the LNR modules of the NRR anddisrupts receptor quiescence without inhibiting ADAM proteasecleavage (15–17). Tissues from 4- to 8-week-old knockout mice(B6;129S1-NOTCH3tm1Grid/J, Jackson Labs) and age-matchedcontrols (B6129SF1/J) were fixed in formalin and embedded inparaffin. After rehydration, slides were pretreated with TargetRetrieval solution (Dako) for 20 minutes at 99�C, rinsed, treatedwith KPL (Kierkegaard and Perry Laboratories) and avidin/biotinblocking solution (Vector Labs), TNB buffer (Perkin Elmer), andprimary antibody (2.5 or 10 mg/mL) for 1 hour at room temper-ature. Slides were rinsed and incubated with donkey anti-rabbitbiotinylated secondary (Jackson Immunoresearch) followed bytyramide signal amplification (Perkin Elmer). Visualization usedmetal-enhanced DAB staining (Thermo Scientific), counterstain-ing, and dehydration. Isotype control antibody controlled forstaining specificity. Tissue arrays were from Biomax.

Cell linesShort tandem repeat (STR) profiles using Promega Power-

Plex16 were compared with external profiles to authenticate celllines (ATCC).

Notch expression, activationCell lines were cultured in: RPMI/10% FBS/1% GlutaMAX

(Life Technologies; breast cancer), DMEM/20% FBS/1% Glu-taMAX (C2C12 myoblasts), and DMEM with 10% FBS/1%GlutaMAX/1% nonessential amino acids (Life Technologies;U87 cells). Cells grown to about 80% to 90% confluencewere treated with 5 mmol/L DAPT (EMD), DMSO (0.02% finalconcentration), or antibodies. Isotype controls were a-gD

(hIgG1) or a-ragweed (mIgG2a). Antibody concentrationswere kept constant between samples by adding control anti-body as needed. For treatments lasting more than 24 hours,DAPT was re-added 3 hours before harvest. MG132 (EMD;20 mmol/L) was added at this time, as needed. For Notchinduction, cells were washed with PBS and incubated for20 minutes at 37�C in 10 mmol/L EDTA. JAG1-induced sig-naling was triggered for 3 hours with rat recombinant JAG1-Fc(R&D) immobilized to anti-Fc–coated paramagnetic particles(Bangs Labs).

siRNA transfectionExtracts were prepared in RIPA buffer after 72-hour transfec-

tions with 50 nmol/L OTP-modified siRNA pools (Dharmacon)or nontargeting siRNA. For IHC, cells were treated with 5 mmol/LDAPT after 48-hour transfections, incubated for 24 hour, washed,and incubated 20 for minutes with 10mmol/L EDTA� 5 mmol/LDAPT.

Protein analysesNuclear and cytoplasmic/membrane fractions were prepared

after lysis (RIPA, Pierce; ref. 18), separated using 4%–12%NuPAGE gels/MOPS (Life Technologies), transferred to polyvi-nylidene difluoride (PVDF; TransBlot Turbo, BioRad), blockedwith 5%milk, and probed for 12 to 16 hours with: 1 mg/mL anti-NICD3� (V1662), 0.2 mg/mL anti-NICD2� (V1697), 1:1,000 CellSignaling antibodies (anti-NICD1� D3B8, anti-NOTCH3 2889,anti-JAG2C83A8, anti-CREB48H2), 1:500 anti-JAG1 (SantaCruzH-114), or 1:5,000 anti-tubulin (Sigma DM1a). Secondary anti-bodies (1:15000) were from BioRad. Chemiluminescent detec-tion used Super Signal West Pico or Femto reagents (Thermo).

Growth assaysSoft agar. Cells (n ¼ 1,500) in 40 mL 0.29% low-melting tem-perature agarose (Life Technologies)with 1�RPMI/10%FBSwereplated in 96-well plates prefilled with 40 mL 0.6% agarose in thesame medium and then overlaid with 40 mL medium plus a 3�concentration of test articles: 15 mmol/L DAPT/0.06% DMSO or60mg/mL antibody. Cells were fed twiceweekly over 12 to 19 dayswith 40 mL medium plus 1� test articles. Plates were scannedusing Gelcount (Oxford Optronix Ltd.).

Hanging drop. Cells (n¼ 1,000) in 40 mL drops (test articles as insoft agar) were prepared in GravityPLUS plates using 10 mL PBS/0.1% Triton X-100 in the humidifier pad (InSphero). Mediumand test articles were changed twice weekly over 8 days beforecentrifuging to 96-well plates, lysing cells (30 minutes, 100 mL),and processing (CellTiter-Glo, Promega).

Colony formation. A total of 1.7� 104 cells were plated in 1.5 mLper well in a 6-well plate in medium plus test articles, which wererefreshed twice weekly over 11 to 18 days before counting cells.

XenograftsAnimal studies were conducted in accordance with the Guide

for the Care and Use of Laboratory Animals, National AcademyPress (2006) and approved by the Institutional Animal Careand Use Committee (IACUC, Genentech). C.B-17 SCID.bg mice(Charles River Labs)were inoculatedwith5million cells in 0.2mLHBSS/Matrigel into the 2/3mammary fat pad.When tumors grew

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to 80 to 150 mm3 [length and width measured with UltraCal-IVcalipers 54-10-111, Fred V. Fowler Company; tumor volume ¼(length � width2) � 0.5], mice were randomly assigned totreatment groups (10/group) and injected intraperitoneally twiceweekly with 30 mg/kg mouse IgG2a antibodies: a-ragweed, anti-N3.A4, or anti-N3.A13. In the HCC1143 study, anti-N3.A13 wasdosed once a week for 2 doses. For the MDA-MB-468 geneexpression studies, the anti-N3.A13 dose amount was loweredto 20 mg/kg after the initial dose and supplemented with anti-ragweed to keep total IgG at 30 mg/kg. Animal weights weremonitored; none of the antibody regimens caused weight loss.Tumors were fixed, embedded, sectioned, and stainedwith hema-toxylin or anti-V1662.

Gene expressionFor the NOTCH3 antagonism experiment, 2.5 � 106 MDA-

MB-468 cells/10-cm plate were treated after 40 hours with10 mg/mL antibody (anti-gD or anti-N3.A4) þ 0.05% DMSO� 5 mmol/L DAPT and harvested 24 hours later. For NOTCH3agonism, 6.25 � 106 cells/10-cm plates were treated with0.05% DMSO (baseline) or 5 mmol/L DAPT/0.05%DMSO (Notch inhibited) for 16 hours and then cells werewashed and incubated for 2 hours in medium alone. Afterwashout, cells were treated with DMSO, 5 mmol/L DAPT/DMSO, 20 mg/mL control IgG, 20 mg/mL anti-N3.A13, JAG1-Fc–coated beads, or Fc-coated beads. DAPT or DMSO wasrefreshed in their corresponding plates 22 hours later. Cellswere harvested after 24 hours. RNA was extracted (QiagenRNeasy Plus kit) and yields analyzed (Agilent Bioanalyzer).Microarray methods used Affymetrix HGU133plus2 expressionmicroarrays and GeneChip protocols, and data were processedusing Affymetrix Microarray Analysis Suite software version 5 toyield signal data; results were deposited to GEO (GSE82298).Three arrays were performed per condition using biologicreplicates. RNASeq reads were mapped to the hg19 humanreference genome using "GSNAP" (19) and normalized for eachgene by transcript length and total number of mapped reads toyield RPKM. Differential expression was estimated usingDESeq2 (Bioconductor; ref. 20).

ResultsDetection of active NOTCH3

Antibodies recognizing the active form (S3-cleaved) of Notchreceptors, rather than just receptor expression, have provenvaluable for measuring NOTCH1 and NOTCH2 activity(21, 22), and recent work has described a similar antibody forstudying NOTCH3 signaling in T-ALL (23). We characterized anew tool for assessing NOTCH3 activity, with a focus onNOTCH3 signaling in basal breast cancer, starting with thegeneration of a monoclonal antibody directed at the humanNOTCH3 S3 site at amino acid V1662 (Supplementary Fig. S1).We tested whether the V1662 antibody recognized activeNICD3 (NICD3�) using the basal breast cancer cell lineMDA-MB-468 because this line could be manipulated to createa range of NICD3� levels. V1662 recognized a polyprotein ofthe molecular mass expected for NICD3� (Fig. 1A, Supplemen-tary Fig. S1). The band intensity correlated with signal strength,increasing or decreasing after treating the cells with a NOTCH3agonist (anti-N3.A13) or antagonist antibody (anti-N3.A4),respectively (Fig. 1A and B); similarly, treatment with EDTAto destabilize the NRR and induce signaling (15–17) or with a

GSI (DAPT) to inhibit signaling affected band intensity in thepredicted manner (Fig. 1A). Targeting NOTCH3 with siRNAsefficiently silenced NOTCH3 expression (Fig. 1C) and elimi-nated the V1662 signal, confirming antibody specificity. Fur-thermore, V1662 recognized a band of the expected size, andonly after Notch activation, in the mouse cell line C2C12(Fig. 1D, left), yet did not reveal any signal at the expectedpositions for either NICD1� or NICD2� (Fig. 1D, left), despitethe fact that these NICDs were clearly detected by anti-NOTCH1 V1774 in 3T3-N1 cells and by anti-NOTCH2V1697 in U87 cells, respectively (Fig. 1D, right). These datademonstrate that anti-NOTCH3 V1662 specifically detectshuman and mouse NICD3� but does not cross-react withNICD1� or NICD2�.

To enable assessment of activated NOTCH3 in clinical sam-ples, we tested V1662 for IHC applications in formalin-fixed,paraffin-embedded (FFPE) material. EDTA activation yielded astrong V1662 signal that was significantly reduced followingDAPT or siRNA inhibition (Fig. 1E, top). To rigorously teststaining specificity, we compared wild-type (WT) or NOTCH3knockout (KO) mice, focusing on NOTCH3 in brain vascula-ture and muscle satellite cells. Whereas WT tissues showed aclear nuclear signal of V1662 staining, KO tissues did not(Fig. 1E, bottom), supporting the assertion that V1662 specif-ically detects active NICD3�.

NOTCH3 activation is constitutive and ligand-independentOur detection of constitutively active NOTCH3 signaling

seemed surprising, as Notch signaling typically requires induc-tion with a ligand expressed on neighboring cells. Mutationaldestabilization of the NRR, as found in certain cancers, providesone hypothesis to explain this result. However, DNA sequencingof cell lines with constitutively active NOTCH3 signaling didnot identify any alterations in the Notch3 NRR (data notshown). Consistent with previous results (23), our sequencingdid reveal that MDA-MB-468 cells express both a WT and amutant Notch3 allele with a truncated PEST domain (data notshown), expected to prolong but not induce signaling. More-over, such truncations are irrelevant with regards to the full-length, WT NOTCH3 protein on which we focus. We hypoth-esized that signaling might be induced by endogenouslyexpressed ligand. Immunoblots revealed expression of JAG1 inMDA-MB-468 and JAG1 plus JAG2 in HCC1143 cells, a secondbasal line with active NICD3� (Fig. 2A and B). However, potentantibody inhibitors of these JAG ligands (24) failed to block theconstitutive NOTCH3 signal, in contrast to the inhibition froma GSI or anti-N3.A4 (Fig. 2B and C) and as observed inadditional basal lines (data not shown). Likewise, anti-DLL1(25) and anti-DLL4 (26) blocking antibodies, used alone or incombination with high concentrations of all 4 anti-DLL/JAGinhibitors, did not affect NOTCH3 signaling (Fig. 2D). To testwhether ligand-induced signaling could originate in an intra-cellular compartment inaccessible to antibodies, we used siRNAto silence Jag1, the most abundantly expressed canonical ligandin MDA-MB-468 cells (Fig. 2A). While knockdown of Notch3significantly inhibited the NOTCH3 signal, evidenced by thereduction in NICD3� and JAG1 protein (NOTCH3 signalinginduces Jag1 expression; see below), knockdown of Jag1 hadlittle or no effect on the NOTCH3 signal (Fig. 2E). Inhibitionusing the NOTCH3 antagonist antibody further argues against

Constitutive NOTCH3 Signaling in Basal Breast Cancer

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this hypothesis of intracellular signal induction. Our resultsusing multiple methods thus demonstrate (i) constitutiveNOTCH3 signaling that occurs (ii) independent of canonicalNotch ligands.

Constitutive NOTCH3 signaling is prevalent in basal cell linesand tumors

To determine the frequency at which constitutive NOTCH3signaling occurs in breast cancer cell lines, we expandedour screento include 17 basal plus 5 hormone receptor–positive lines.

NICD3� levels roughly correlated with expression of totalNOTCH3, with clearly detectable NOTCH3 signaling in 13 of17 basal lines and weaker but detectable signaling in 3 of 5hormone receptor-positive lines (Fig. 3A). Although the majorityof lines expressed JAG ligands, JAG levels did not correlate withNICD3� levels (Fig. 3A), consistent with NOTCH3 occurringindependent of canonical ligands. DLL1 andDLL4were expressedweakly, if at all, across the entire cell line panel (data not shown).

We exploited our NICD3� IHC method to determine theprevalence of NOTCH3 signaling in human breast cancers. We

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Immunoblots demonstratingconstitutive and ligand-independentNOTCH3 signaling in basal lines. A,Untreated MDA-MB-468 and HCC1143basal lines. � , n.s., nonspecific band. Band C, JAG antagonist antibodies donot inhibit NOTCH3 signaling,suggesting ligand-independentsignaling. Anti-J1 and anti-J2, antibodyantagonists of JAG1 and JAG2,respectively, at 10 or 50 mg/mL;anti-N3.A4 at 20 mg/mL; DAPT at5 mmol/L. D, Antibody inhibition ofligands, blocked alone or incombination, in MDA-MB-468 cells. E,Jag1 siRNA silencing does not inhibitNOTCH3 activation in MDA-MB-468cells. Note, the decrease in JAG1 afterNotch3 silencing is consistent with Jag1being a NOTCH3 transcriptional target(see main text).

Figure 1.Antibody V1662 selectively detects active NOTCH3. A, Immunoblots (IB) of MDA-MB-468 lysates probed with V1662, anti-NOTCH3 (total), or anti-tubulin. Treatments: 5 mmol/L DAPT; DMSO (control); 20 mmol/L MG132 (stabilizes NICD3�); 10 mmol/L EDTA (Notch activation); 15 mg/mL anti-gD (isotype-control); 15 mg/mL anti-N3.A4, NOTCH3 antagonist. Numbers, molecular mass markers. B, Nuclear lysates probed with V1662 or anti-CREB (control).Treatments included immobilized JAG1 (iJAG1) and 15 mg/mL anti-N3.A13 agonist. C, siRNA knockdown of Notch3. Lysates were probed using V1662(detects S3 cleavage) or anti-NOTCH3 (detects full-length and S1 cleavage), after knockdown using nontargeting (NT) or Notch3 (siN3) siRNAs. D, V1662selectively recognizes human and mouse NICD3� . Nuclear lysates � EDTA activation were probed using V1662, V1744 (NICD1�), or V1697 (NICD2�).3T3-N1 and U87 cells served as controls for NICD1� and NICD2� , respectively. � , Nonspecific band detected by V1697. E, V1662 detects NICD3� by IHC.MDA-MB-468 cells were treated with (i) EDTA, (ii) DAPT, (iii) EDTA plus NT control siRNA, or (iv) EDTA plus Notch3 siRNAs. v–viii, Brain or gastrocnemiusmuscle samples from Notch3 WT or knockout mice. Black arrows, positive-stained nuclei.






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1 83 35 (42%) ND 83 35 (42%)

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NOTCH3 activity is prevalent in basallines and tumors. A, Immunoblots oflysates (nuclear for NICD3� and CREB;cytoplasmic for all others) from a panelof breast cancer lines. B, V1662 IHCexamples of basal tumors positive fornuclear NICD3� . Solid arrows, NOTCH3-ICD–positive nuclei. Open arrows,stromal cells that stained negative.NICD3� was evident in some stromalcells and is expected in macrophagesand smooth muscle vascular. C,Summary of the V1662 IHC screen forNOTCH3 activity in patient breasttumors in three tumor tissue arrays. ND,not done.

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observed a range of NICD3� signals, from strong nuclear stainingin themajority of tumor cells to weak staining in a fraction of cells(Fig. 3B), in 73 of 371 samples (20%; Fig. 3C). Triple-negativebreast cancer (TNBC) sampleswere enriched forNICD3� staining,observed in 52 of 153 cases (34%; Fig. 3C). Consistent withpublished results (27), we detectedNICD1� in tumor cells in only4%of total samples (Fig. 3C). Thus, NOTCH3 signaling is evidentin a significant percentage of TNBCs and is foundmore frequentlythan is NOTCH1 signaling.

NOTCH3 signaling promotes breast cancer cell line growthTo assess whether NOTCH3 signaling fostered basal tumor cell

growth, we exploited NOTCH3 function–modulating antibodiesin assays that involve cell–cell contact, a hallmark of Notchsignaling. These therapeutic antibodies selectively target thehuman NOTCH3 NRR, either antagonizing (anti-N3.A4) or ago-nizing (anti-N3.A13) signaling (28). We first examined anchor-age-independent growth in soft agar and found that selectiveNOTCH3 blockade using anti-N3.A4 significantly inhibited col-ony growth, to an extent similar to that seen using the pan-Notchinhibitor DAPT (Fig. 4A and B). Selective inhibition of NOTCH1,NOTCH2, or JAG1 had little or no effect (Fig. 4B and Supple-mentary Fig. S2A). In contrast, NOTCH3 agonism using anti-N3.A13 modestly stimulated colony growth, an effect that wasreversed when anti-N3.A4 was included (Fig. 4A and B andSupplementary Fig. S2A). We observed similar results with avariety of basal lines and growth assays (Fig. 4C and D, Supple-mentary Fig. S2B, data not shown). Colony formation on platesrevealed the most striking effects, with NOTCH3 blockade dra-matically inhibiting growth (Fig. 4E and F). Our results withmultiple cell lines and assays, using selective and general Notchinhibitors, indicate that NOTCH3 signaling promotes growth orsurvival in a subset of basal lines and is sufficient to account for theobserved growth effects.

The colony formation assay also provided a means to investi-gate cell morphology. Control colonies displayed a mix of 2 cellmorphologies, with some cells appearing in a cobblestone pat-tern, typical of epithelial cells, and other cells appearing rounded,consistent with a transformed phenotype (Fig. 4G, middle).NOTCH3 antagonism shifted the colony phenotype to onealmost exclusively comprised of a cobblestone epithelial layer(Fig. 4G, left), whereas NOTCH3 agonism shifted the phenotypeto one primarily composed of rounded cells (Fig. 4G, right). SuchNOTCH3-controlled shifts in phenotype evoke classic views ofNotch regulating binary cell fate decisions and are consistent withNOTCH3 signaling promoting a transformed state.

NOTCH3 activation drives tumor growth in vivoTo examine whether NOTCH3 signaling affected tumor

growth in vivo, we grew basal lines as xenografts in the mam-mary fat pads of immunocompromised mice, allowed tumorsto establish, and then treated with our NOTCH3 antibodies.Tumors grown from HCC1143 cells, which display high levelsof active NOTCH3 signaling (Fig. 3A), dramatically respondedto NOTCH3 agonism, doubling in volume approximately twiceas rapidly in the presence of anti-N3.A13 compared withcontrol (Fig. 5A). NOTCH3 antagonism using anti-N3.A4 slo-wed tumor growth, although the effect was modest relative tothe growth stimulation induced by agonism (Fig. 5A). Tumorsfrom other basal lines responded similarly, although the mag-nitude of the responses varied, with MDA-MB-468 cells show-

ing the most aggressive growth acceleration following NOTCH3stimulation (Fig. 5B, Supplementary Fig. S3). Immunoblottingand IHC using V1662 showed high levels of active NOTCH3signaling in the vast majority of tumor cells following anti-N3.A13 treatment, indicating that tumor growth rates correlatedwith levels of nuclear NICD3� (Fig. 5C and D). NOTCH3-agonized tumor cells appeared more aggressive than did theircontrol counterparts, with increased numbers of active mitoses(Fig. 5D). Given that NOTCH3 antagonism significantly inhib-ited colony formation in vitro (Fig. 4), the modest growthinhibition observed in vivo suggested that differences in thegrowth settings impacted the responses to NOTCH3 inhibition.Indeed, MDA-MB-468 cells expressed lower levels of bothtotal and active NOTCH3 in vivo compared with in vitro(Fig. 5C, note the near absence of NICD3� in the controlanti-RW tumors and compare with Fig. 3), consistent with slowtumor growth and only a modest further slowing afterNOTCH3 inhibition (Fig. 5B).

NOTCH3 drives an oncogenic expression signature sufficientto cluster breast cancer subtypes and correlating with basalphenotype

We pharmacologically toggled NOTCH3 signaling up ordown from its baseline state in basal lines (i) to compareNOTCH3-specific versus pan-NOTCH manipulation and (ii) todefine the downstream gene signature (Fig. 6A). Gene expres-sion changes following 24-hour treatment with DAPT or anti-N3.A4 appeared very similar (Fig. 6B, Supplementary Table S1).To ascertain whether the expression changes in the baseline statediffered from those driven by a full "on" signal after NOTCH3agonism, we performed a wash-out experiment (29). FollowingDAPT incubation to inhibit Notch signaling, cells were replatedwithout DAPT but with (i) anti-gD isotype control antibody, toallow the cells to re-establish baseline signaling (the de-repressed state) or (ii) anti-N3.A13, to agonize NOTCH3 sig-naling (the stimulated state). Expression changes from the sameset of genes were observed under both conditions, although themagnitude of the changes was greater following NOTCH3agonism, as expected (Fig. 6C, Supplementary Table S2). Wealso replated in the presence of (i) immobilized JAG1, toprovide pan-Notch stimulation or (ii) anti-N3.A13, to provideNOTCH3-specific agonism and found that both inducers causedsimilar expression changes (Fig. 6D, Supplementary Table S3).These studies support the conclusion that NOTCH3 signaling isconstitutively "on" and that this baseline signal can be bothinhibited and stimulated. Moreover, these results indicate thatNOTCH3 signaling is sufficient to account for the Notch effectsin these cells, without invoking contributions from other Notchreceptors.

To elucidate a Notch basal transcriptional program, we definedaNotch gene signature inMDA-MB-468 cells by comparingDAPTand anti-N3.A13 treatments. This comparison identified 83genes for which expression varied significantly (adj. P < 0.01,Benjamini–Hochberg correction), with at least a 2-fold change(Fig. 6D, Supplementary Table S4). NOTCH3positively regulateda number of oncogenes and cell-cycle genes as well as Notchtranscriptional targets identified in other cell types (Fig. 6E,Supplementary Table S4). For example, 2 well-established Notchtargets, Nrarp (30) and Olfm4 (31), increased expression follow-ing NOTCH3 agonism, underscoring the validity the signature.The downstreamoncogenic program includes the oncogene c-Myc






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as well as stem cell markers/oncogenes Id4 and Kit. Consistentwith its driving cell-cycle progression and proliferation, NOTCH3activity also increased expression ofCCND1 andCdk6. TheNotchligand Jag1 itself appeared as downstream target, suggesting a

positive feedback loop. Intriguingly, NOTCH3 signaling down-regulated expression of Dkk1, a negative regulator of Wnt signal-ing, and gene set enrichment analysis revealed a positive corre-lation of Notch and Wnt signaling (data not shown), suggesting

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Figure 5.

NOTCH3 activation acceleratestumor growth in vivo. A and B,NOTCH3 agonism stimulates tumorgrowth, whereas NOTCH3antagonism slows tumor growth invivo. Mice carrying establishedxenograft tumors from the indicatedcell lineswere treated twiceperweekwith 30 mg/kg of NOTCH3antagonist (anti-N3.A4) or agonist(anti-N3.A13). C, Immunoblotanalysis of MDA-MB-468 tumorsshows that NOTCH3 signalingcorrelates with tumor growth rates.D, Sample tumors from B stainedwith hematoxylin and eosin (H&E)and V1662 IHC. NOTCH3 signaling isactive in the majority of tumor cellsfollowing systemic agonism.

Figure 4.NOTCH3 signaling promotes a transformed phenotype in basal lines. A, Phase-contrast micrograph of MDA-MB-468 colonies in soft agar, with treatmentsas in Fig. 1. B, Quantification of total colony area. Anti-N1 and anti-N2, anti-NRR inhibitory antibodies targeting NOTCH1 and NOTCH2, respectively. C,HCC70 colony growth also depends on NOTCH3 signaling. D, Growth in hanging drops depends on NOTCH3 but not NOTCH1, NOTCH2, or JAG1.Viability measured using CellTiter-Glo after 8 days. RLU, relative luminescence units. E and F, Pan-Notch and selective NOTCH3 inhibition inhibit colonyformation to a similar extent. MDA-MB-468 (E) or HCC70 (F) cells plated at low density were treated as indicated and counted after 11 to 17 days. Mean � SD,n ¼ 6 (B–D) or 3 (E, F). G, Toggling NOTCH3 signaling off/on alters cell morphology, consistent with NOTCH3 controlling a binary state. Phase-contrastexamples from cells treated as in E.






that NOTCH3 may act upstream to stimulate the Wnt pathway.Supporting the notion that Notchmay favor a basal-over-luminallineage decision in breast cancer, NOTCH3 signaling correlatedwith higher levels of keratins 5, 6B, and 14—markers of the breastmyeoepithelial lineage and basal cancers—and with lower levelsof FOXA1 and keratins 7, 8, and 18—markers of the luminallineage (32, 33).

We next sought to determine whether this Notch genesignature, defined in the MDA-MB-468 cell line, could demar-cate triple-negative samples and, more importantly, revealNotch activity in patient tumor samples. We first performednonsupervised hierarchical clustering on a RNA sequencingdata set derived from a panel of 70 breast cancer cell lines.

Our Notch basal signature was sufficient to cluster the lines into2 main groups, one enriched for basal and the other for luminaland Her2 overexpression (Fig. 7A). Notably, a similar analysisusing of a large set of patient tumors (34) showed that theNotch signature sufficed to tightly cluster the patient samplesinto the main subclasses of breast tumors (Fig. 7B).

We calculated the average correlation (Pearson coefficient) ofsignature gene expression within each cancer subtype to oursignature (Fig. 6E). The basal tumors displayed the highest cor-relation (basal correlation ¼ 0.26), indicating that the NOTCH3-controlled transcriptional network is active in this subtype ofcancer and supporting the notion that a Notch signal may beclinically relevant in basal tumors. In contrast, the other groups

Figure 6.

NOTCH3 provides the primary Notchsignal andgenerates agene signature inbasal cells. A, Strategy to assessbaseline and activated Notch signaling.Treatments for 24 hours. B, Scatterplotof log fold expression changes (logFC)in response to NOTCH3-specific or pan-Notch inhibition. C, As in B, comparingderepression (control anti-gDantibody) with NOTCH3 activation(anti-N3.A13) after DAPT washout. D,As in C, comparing JAG1 induction withNOTCH3 agonism.E,As inC, comparingpan-Notch inhibition (DAPT) withNOTCH3 activation. The gene list fromE defined the Notch signature.

Choy et al.





Figure 7.

The Notch gene signature separates the basal subclass from other breast cancer subtypes. A, Eighty-three genes comprising the Notch signature (Fig. 6E) wereused in an unsupervised hierarchical clustering of RNA sequencing data from a panel of breast cancer lines. Subclass (basal/triple-negative, Her2þ, luminal, ornone/normal) is depicted along the top. B, Clustering of RNA sequencing data from The Cancer Gene Atlas set of breast cancer patient tumors.






showed a negative correlation (Her2, Luminal A, and Luminal Bcorrelations¼�0.17,�0.08, and�0.17, respectively), indicatingthat the state of the signature in these subtypes is inconsistentwithNOTCH3 activation.

DiscussionNotch receptor activation appears oncogenic in certain cel-

lular contexts. Pan- Notch inhibition using GSIs supports thisconclusion (13, 35), although use of GSIs fails to distinguishthe particular Notch receptor driving growth. In human cancers,the clearest cases supporting Notch as a tumor driver derivefrom genomic studies revealing Notch1 mutations in the(i) NRR, which activate signaling in a ligand-independentmanner, and (ii) PEST domain, which prolong signaling. Suchmutations were reported over a decade ago in T-ALL (5), andthis leukemia remains the best-studied example of oncogenicNotch signaling in humans. However, the subsequent wealth oftumor genomic data revealed that such mutations are rare inmost tumor types, including breast (13). Arguments for Notchas a cancer driver largely turned to receptor and ligand over-expression, including in breast cancers (7, 9, 12), with Notch1highlighted in the basal subtype. The discovery of transloca-tions that activate NOTCH1 or NOTCH2 (9), as well as PESTdomain mutations in multiple Notch receptors (13), althoughrare, lend genetic weight to the argument that Notch is impor-tant in basal cancers. Notch3 gene amplifications (12, 13) aswell as mouse models and in vitro studies (7, 14) reinforced thepossible relevance of NOTCH3 in this cancer subtype.

In the absence of the "smoking gun" of frequent activatingmutations, we argue that it is imperative not to focus on receptorexpression but rather on activity. Notch target gene expression(36, 37) can provide a surrogate for activity, but Notch target geneprofiles vary between cell types and context. Thus, we havefocused on the presence of g-secretase–cleaved NICD�. Indeed,an antibody that specifically detects NICD1� has proven valuablefor assessingNOTCH1activity, andwe recently exploited a similarantibody for NOTCH2 (21). However, such an antibody has onlyrecently been described for NOTCH3 (23), and use of thisantibody centered on Notch in T-ALL. The fact that NICD1� wasdetected in only 3 of 78 breast tumor samples examined (27)highlights the importance of examining activity of other Notchreceptors, including NOTCH3, in breast cancer. Our characteri-zation of anti-NICD3� V1662, including the use ofNotch3 knock-out tissues, shows that this antibody specifically detects NICD3�

from human and mouse by both immunoblotting and IHC. Wenote that the ability to detect the NICD3� neo-epitope generatedby g-secretase cleavage demonstrates that NOTCH3 activity inthese samples does not originate from the type of NOTCH3fusions reported in glomus tumors (38)—an important consid-eration for thedeployment of therapeutic antibodies because suchfusions delete the amino terminus ofNICD3 and, thus, would notbe targetable by antibodies.

We first detected active NOTCH3 signaling in several basal celllines. Surprisingly, we discovered that this signaling appears to beligand-independent (although it remains possible that signalingis induced by a noncanonical ligand not targeted in our antibodypanel). While Drosophila studies provide precedence for ligand-independent Notch signaling (39), the examples of ligand-inde-pendent signaling in mammals have largely centered on NRRdestabilizing mutations, such as those found in T-ALL. However,

studies of NRR3 dynamics point to a less stable autoinhibitedstate for NRR3 compared with other Notch receptor NRRs, and arecent structural analysis of NRR3 bolsters this assertion, furtherrevealing elevated basal activity in reporter assays of NOTCH3compared with NOTCH1 or NOTCH2 (40).

Our findings suggest that this less stable autoinhibited state isrelevant in cancer. Specifically, we discovered that endogenouslyexpressed NOTCH3 signals at a "de- repressed" baseline level inthe absence of ligand induction. Given the elevated baselineactivity ofNOTCH3, onemight viewNOTCH3as aWT equivalentof themutationally activatedNOTCH1 found in T-ALL, providingamoderate Notch signal in the absence of any receptor mutation.Indeed, we detected constitutively active NOTCH3 in a broadpanel of breast cancer cell lines and in patient samples. Prolifer-ation and viability studies, performed using a suite of assays invitro and in vivo, suggested that this activity is physiologicallymeaningful, revealing that the level ofNOTCH3 signaling, but notexpression levels of NOTCH1, NOTCH2, or JAG1, correlatedwithgrowth.

We exploited our ability to toggle the level of NOTCH3signaling with NOTCH3 agonist or antagonist antibodies todefine a Notch gene signature in basal cancer and to compareNOTCH3-specific versus pan-Notch effects. NOTCH3 appearedsufficient to explain the downstream Notch transcriptionalresponse, following either inhibition or agonism, without theneed to invoke signaling from other Notch receptors. The genesignature provocatively suggests mechanisms that may functiondownstream of NOTCH3 signaling to drive an oncogenicphenotype. JAG1 and NOTCH3 are downstream targets, andNOTCH3 agonism correlated with NOTCH3 increases—whichour data now indicate are sufficient for increased activity—highlighting a positive feedback loop. Our observation thatc-Myc expression clearly depends on NOTCH3 activity isintriguing, given that c-Myc is an established NOTCH1 targetand a key node in the oncogenic Notch network driving T-ALL(41). Two other oncogenes, Id4 and Kit, are also downstreamtargets of NOTCH3. Both genes are stem cell markers, prompt-ing speculation that NOTCH3 may function in basal breastcancer to maintain an undifferentiated state. ID4 has also beendescribed as a master regulator of the basal lineage (42),suggesting that NOTCH3, through control of a downstreamtranscriptional network that includes ID4, may regulate thesame cell fate. Extending this rationale to the malignant state, itis tempting to speculate that NOTCH3 may help maintain abasal tumor subtype and oppose establishment of luminal orother subtypes. Our data provide preliminary support for thisidea, given that NOTCH3 induces keratin markers of the basalsubtype, downregulates markers of the luminal fate, and con-trols a cell morphologic switch consistent with a fate switch.

Our gene signature was sufficient to cluster not just cell lines,but also a large panel of patient tumors, into the well-establishedsubtypes. Most importantly, the Notch "on" signature correlatedwith the basal phenotype, consistent with our IHCmeasurementsindicating that NOTCH3 is active in more than one third of basaltumors. This finding suggests that therapeutic targeting of Notch3could provide therapeutic benefit without the known toxicitiesassociated with pan-Notch inhibition. As a whole, our studieshighlight constitutive NOTCH3 signaling in a subset of TNBCsand underscore the importance of additional studies aimed atthoroughly ascertaining how strongly these tumors depend onNotch signaling for growth and survival.

Choy et al.





Disclosure of Potential Conflicts of InterestL. Choy reports receiving other commercial research support from Genen-

tech. No potential conflicts of interest were disclosed by the other authors.

Authors' ContributionsConception and design: L. Choy, C.W. SiebelDevelopment of methodology: L. Choy, M. Solon, D.M. French, A. SheltonAcquisition of data (provided animals, acquired and managed patients, pro-vided facilities, etc.): L. Choy, T. Hagenbeek, D. Finkle, A. Shelton, R. VenookAnalysis and interpretation of data (e.g., statistical analysis, biostatistics,computational analysis): L. Choy, T. Hagenbeek, A. Shelton, M.J. Brauer,C.W. SiebelWriting, review, and/or revision of the manuscript: L. Choy, C.W. Siebel

Administrative, technical, or material support (i.e., reporting or organizingdata, constructing databases): T. Hagenbeek, A. Shelton, R. Venook,C.W. SiebelStudy supervision: C.W. Siebel

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

Received June 2, 2016; revised December 1, 2016; accepted December 19,2016; published OnlineFirst January 20, 2017.

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2017;77:1439-1452. Published OnlineFirst January 20, 2017.Cancer Res Lisa Choy, Thijs J. Hagenbeek, Margaret Solon, et al. Breast CancersConstitutive NOTCH3 Signaling Promotes the Growth of Basal

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