PKCa Attenuates Jagged-1 Mediated Notch Signaling in ErbB ... · receptor modiﬁers (SERM) such as tamoxifen (6), aromatase inhibitors (7), or selective estrogen receptor disruptor

Biology of Human Tumors

PKCa Attenuates Jagged-1–Mediated NotchSignaling in ErbB-2–Positive Breast Cancer toReverse Trastuzumab ResistanceKinnari Pandya1, Debra Wyatt2, Brian Gallagher2, Deep Shah3, Andrew Baker4,Jeffrey Bloodworth1, Andrei Zlobin2, Antonio Pannuti5, Andrew Green6, Ian O. Ellis6,Aleksandra Filipovic7, Jason Sagert8, Ajay Rana3, Kathy S. Albain9, Lucio Miele5,Mitchell F. Denning2,10, and Clodia Osipo2,10,11

Abstract

Purpose: Breast cancer is the second leading cause of cancermortality among women worldwide. The major problem withcurrent treatments is tumor resistance, recurrence, and diseaseprogression. ErbB-2–positive breast tumors are aggressive andfrequently become resistant to trastuzumab or lapatinib. Weshowed previously that Notch-1 is required for trastuzumabresistance in ErbB-2–positive breast cancer.

Experimental Design: Here, we sought to elucidate mechan-isms by which ErbB-2 attenuates Notch signaling and how this isreversed by trastuzumab or lapatinib.

Results: The current study elucidates a novel Notch inhib-itory mechanism by which PKCa downstream of ErbB-2 (i)restricts the availability of Jagged-1 at the cell surface to

transactivate Notch, (ii) restricts the critical interactionbetween Jagged-1 and Mindbomb-1, an E3 ligase that isrequired for Jagged-1 ubiquitinylation and subsequent Notchactivation, (iii) reverses trastuzumab resistance in vivo, and (iv)predicts better outcome in women with ErbB-2–positive breastcancer.

Conclusions: The clinical impact of these studies is PKCa ispotentially a good prognostic marker for low Notch activityand increased trastuzumab sensitivity in ErbB-2–positive breastcancer. Moreover, women with ErbB-2–positive breast tumorsexpressing high Notch activation and low PKCa expression couldbe the best candidates for anti-Notch therapy. Clin Cancer Res; 22(1);175–86. �2015 AACR.

IntroductionBreast cancer is the second leading cause of cancer-related

deaths in women. Main hurdles for women are drug resistance,recurrence, and disease progression (1, 2). Breast cancer is a

heterogeneous disease comprising at least four major subtypes,that is, luminal A, luminal B, HER2, and basal/triple-negative (3).The majority of breast cancers express estrogen, progesteronereceptors (ER, PR), and/or the human epidermal growth factorreceptor-2 (HER2 or ErbB-2). The ErbB-2 proto-oncogene isamplified in 15% to 25% of breast cancers (4, 5). ER and/orErbB-2 are targets of therapies that include selective estrogenreceptor modifiers (SERM) such as tamoxifen (6), aromataseinhibitors (7), or selective estrogen receptor disruptor (SERD)fulvestrant (8) for ER/PR-positive breast cancer or trastuzumab(9) for ErbB-2–positive breast cancer.

Despite the efficacy and dramatic effects of trastuzumab onsurvival, 20% to 50% of women with ErbB-2–positive meta-static breast cancer exhibit intrinsic resistance (10). Further-more, 10% to 15% of the women treated with trastuzumab pluschemotherapy developed acquired resistance within the firstyear (11). Thus, understanding resistant mechanisms is criticalfor identifying novel targets to prevent and/or reverse resis-tance. Numerous mechanisms are implicated in resistance,including loss of PTEN, overexpression of IGF-1R, truncationof ErbB-2, hyperactivation of PI3K and mTOR signaling, amongothers (12).

PKCa is another mediator of the ErbB-2 pathway. ErbB-2activates phospholipase C g (PLCg), which cleaves phosphatidy-linositol 4,5-bisphosphate (PIP2) into diacylglycerol (DAG) andinositol 1,4,5-trisphosphate (IP3), triggering an elevation ofintracellular Caþ2 and activation of PKCa (13). PKCa overexpres-sion promotes tamoxifen resistance (14) and invasion (15).

1Molecular Biology Program, Louisiana State University, New Orleans,Louisiana. 2Oncology Research Institute, Louisiana State University,NewOrleans, Louisiana. 3DepartmentofMolecular PharmacologyandTherapeutics, Louisiana State University, New Orleans, Louisiana.4Integrated Cell Biology Program, Louisiana State University, NewOrleans, Louisiana. 5Louisiana State University, New Orleans, Louisi-ana. 6Department of Histopathology, University of Nottinghamand University Hospital NHS Trust, Nottingham, United Kingdom.7Imperial College of London, London, United Kingdom. 8CytomXTherapeutics, San Francisco, California. 9Department of Medicine/Hematology and Oncology, Cardinal Bernardin Cancer Center ofLoyola University Chicago: Health Sciences Division, Maywood,Illinois. 10Department of Pathology, Cardinal Bernardin Cancer Centerof Loyola University Chicago: Health Sciences Division, Maywood,Illinois. 11Department of Microbiology and Immunology, Cardinal Ber-nardin Cancer Center of Loyola University Chicago: Health SciencesDivision, Maywood, Illinois.

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

K. Pandya and D. Wyatt contributed equally to this article.

Corresponding Author: Clodia Osipo, Loyola University Chicago, 2160 SouthFirst Avenue Maywood, IL 60153. Phone: 708-327-2372; Fax: 708-327-2245;E-mail: [email protected]

doi: 10.1158/1078-0432.CCR-15-0179

�2015 American Association for Cancer Research.

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Recently, PKCa was shown to mediate breast cancer stem cellsurvival (16) and is a therapeutic target in triple-negative breastcancer (17). However, the role of PKCa in the ErbB-2–positivebreast cancer and its significance for anti-ErbB-2–targeted therapyremain unclear.

Notch ligands (Delta-like 1, 3, and 4 and Jagged-1 and -2) andreceptors (Notch-1, -2, -3, and -4) are implicated in breast cancerdevelopment and drug resistance (18). They require cell–cellcontact for engagement and subsequent cleavage of membrane-bound receptors to intracellular transcriptional activators. Theligand-induced Notch activation is regulated by E3 ubiquitinligases, Mindbomb-1 (18), and Neuralized (19). Co-overexpres-sion of Notch-1 and Jagged-1 predicts for the poorest overallsurvival (20). Notch-1, -3, and -4 are breast oncogenes and potentregulators of cell differentiation, proliferation, and apoptosis(21). High Notch-1 and -4 expression correlate to poor prognosis(22) and Notch-3 promotes ErbB-2–negative breast cancer cellproliferation (23). Notch receptor–ligand interactions are impor-tant for mammary stem cell differentiation (24) and tumor-initiating cells (25).

We showed that trastuzumab or a dual EGFR/ErbB-2 inhibitorincreased Notch-1 activity, and trastuzumab resistance wasreversed by Notch-1 knockdown or a g-secretase inhibitor (GSI;ref. 26). Subsequently, we demonstrated that dual targeting ofErbB-2 and Notch prevented recurrence and partially reversedresistance to trastuzumab (27). Here, we demonstrate a novelmechanism of action: PKCa attenuates Mib-1–mediated Jagged-1–Notch activation to predict sensitivity to trastuzumab. Further-more, breast cancers expressing lowPKCamight bemore sensitiveto anti-ErbB-2 agents. Importantly, anti-Jagged-1 therapy couldreverse resistance to trastuzumab by attenuating Jagged-1–medi-ated Notch activity.

Materials and MethodsCell culture and reagents

MDA-MB-453, BT474, and HCC1954 breast cancer cells werepurchased fromATCCwithin the last 6 years. BT474 trastuzumab-resistant (BT474 Resistant) cells were generated by treating paren-tal BT474 cells with increasing concentrations of trastuzumab for

6 months (26). All cell lines were authenticated using shorttandem repeat (STR) allelic profiling (DCC Medical).

Biotinylation assayMDA-MB-453 cells were seeded in 10-cm plates at a density of

40% to 50% confluency. After 48 hours, cells were treated witheithermouse IgG (20 mg/mL in PBS) or trastuzumab (20mg/mL inPBS) for 48hours. The cells werewashed 3 times inPBS, and a cell-impermeable biotinylation reagent EZ-Link Sulfo-NHS-BiotinReagent (Pierce Chemicals) as previously described (28) wasadded to cells to label cell surface proteins at 4�C under constantshaking. Cells were scraped, centrifuged at 1,000 rpm for 1minute, washed twice with PBS, and lysed in RIPA lysis buffer.The biotinylated cell surface proteins were precipitated from thetotal protein lysate using 30 mL of Immobilized NeutravidinProtein (Cat. 29200, Pierce Chemicals). The beads were washed4 timeswithPBS and theproteinwas elutedwith 4� SDSLaemmlibuffer and then heated at 95�C for denaturation.Western blottingwas performed to detect Jagged-1 and ErbB-2 as described in theSupplementary Section.

Confocal immunofluorescence microscopyMDA-MB-453, BT474HS, or BT474HR cells were either trans-

fected with control siRNA, PKCa siRNA, or ErbB-2 siRNA ortransfected with LRZS-linker alone or LRZS-PKCa and subse-quently plated into chamber slides or untransfected cells weretreated with PBS or 20 mg/mL trastuzumab directly in the well.Cells were then fixed in the chamber with 3.7% paraformalde-hyde, permeabilized with 0.1% saponin, and nonspecific inter-actions were blocked with 1% BSA. The staining protocol forspecific proteins of interest is provided in the SupplementarySection.

Co-immunoprecipitationJagged-1 (Jagged-1 2H28 rabbit monoclonal antibody, Cat.

2620, Cell Signaling) was immunoprecipitated under treatmentconditions as described in the figure legends. Lysates at a con-centration of 3 to 5 mg/mL were incubated with 6 mg of antibodyfor the specific protein of interest or rabbit isotype control IgG(Cat. SC-2027, Santa Cruz) overnight with gentle rocking at 4�C.Thirty microliters of protein A-plus beads (sc-2002, Santa Cruz)were added to the immune complexes for 2 hours, the beads werewashed with lysis buffer, and the proteins attached to the pelletedbeads were eluted with 20 mL of 2� Laemmli sample buffer plusb-mercaptoethanol and heated for 10 minutes at 95�C whilevigorously shaking. Immunoblots are described in Supplemen-tary Section.

Coculture assayMDA-MB-453 cells were plated at a density of 3� 106 cells in a

10-cm plate. Mouse fibroblast (LTK) cells expressing no ligand oroverexpressing Jagged-1 were added in equal parts (1:1) to pre-viously platedMDA-MB-453 cells. The cellswere then treatedwithPBS or 20 mg/mL trastuzumab for 18 hours. Cells were thenstained with 10 mL of phycoerythrin (PE)-conjugated humanErbB-2 antibody (Cat. 340552, Becton Dickinson). Stained cellswere then sorted for ErbB-2 expression at the cell surface by FACS.Total RNA was extracted from sorted cells, reverse transcribed tototal cDNA, and real-time PCR was performed as previouslydescribed. The PCRprimers thatwere used for detectionof specific

Translational Relevance

Breast cancer is the second leading cause of cancer mortalityamong women worldwide. The major problem with currenttreatments is tumor resistance, recurrence, and disease pro-gression. The current study elucidates a novel mechanism bywhich ErbB-2 via PKCa attenuates Jagged-1–mediated Notchsignaling in ErbB-2–positive breast cancer. The clinical impactof these studies is that PKCa is potentially a good prognosticmarker for low Notch activity and increased trastuzumabsensitivity in ErbB-2–positive breast cancer.Moreover, womenwith ErbB-2–positive breast tumors expressing high Notchactivation and low PKCa expression could be the bestcandidates for anti-Notch therapy. These studies provide apreclinical proof of concept for future clinical trials usingcombinations of trastuzumab/lapatinib plus an anti-Jagged-1–targeted therapy for trastuzumab-resistant, ErbB-2–positivebreast cancer expressing low levels of PKCa.

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Clin Cancer Res; 22(1) January 1, 2016 Clinical Cancer Research176




transcripts were Hes-1 and Deltex-1 with RPL13a as a loadingcontrol.

Trastuzumab-resistant xenograftsFive million BT474 trastuzumab-resistant cells were injected

bilaterally into mammary fat pads of ovariectomized FoxN1nu/nu athymic nude mice (Harlan Sprague-Dawley) followed byimplantation of a 17b-estradiol–containing silastic capsule of 0.3cm in length with a constant release providing 83 to 100 pg/mL asdescribed previously (29). The identity of each mouse and tumorwas tracked by an ear tag. Once tumors grew to a mean crosssectional area (CRA) of 0.30 to 0.50 cm2, mice were randomizedto four treatment groups: (i) vehicle control (PBS), (ii) trastuzu-mab (10 mg/kg in a total volume of 100 mL sterile PBS, intraper-itoneally once weekly), (iii) CTX-033 (5 mg/kg, intraperitoneallyonce weekly), or (iv) trastuzumab plus CTX-033. In case ofBT474HR cells retrovirally transduced with the LRZS-linker orLRZS-PKCa, these tumors were randomized to two treatmentgroups each: (i) PBS or (ii) trastuzumab. Tumor area (l � w) wasmeasured weekly using Vernier calipers and cross sectional area[(l � w)p)/4] was calculated and graphed. All animal studyprotocols were approved by Loyola University's InstitutionalAnimal Care and Use Committee.

Flow cytometryMDA-MB-453 cells were treated with indicated dose of lapa-

tinib/trastuzumab for 48 hours. DMSO and IgG were used asnegative controls. Cells were harvested using Cellstripper (Cat.25–056-CI Corning Cellgro) and were stained with fluoresceinisothiocyanate (FITC)–conjugated Jagged-1 antibody as per themanufacturer's protocol. The cells were then resuspended in FlowCytometry Staining Buffer (Cat. FC001 R&D Systems) and ana-lyzedwith BD FACSCanto II. Data acquisitionwas done using BDFACSDiva software anddata analysis was performed using FlowJosoftware.

Proliferation assayMDA-MB-453 cells were seeded into 10-cm dishes at 5 � 106

cells. HCC-1954 cells were seeded into 10-cm dishes at 3.5� 106

cells. The cells were transfected as described in the RNA interfer-ence of Methods. After completion of the siRNA transfection, thecells were seeded in triplicate into 6-well plates at a density of50,000 for MDA-MB-453 cells or HCC-1954 cells. BT474HSsensitive cells were seeded into 6-well plates at a density of60,000 cells. BT474HR resistant cells were seeded into 6-wellplates at a density of 60,000 cells. After plating into the 6-wellplates, cells were treated with PBS or 20mg/mL trastuzumab on adaily basis. Cells were counted at days 2, 4, 6, and 8 posttreatmentand cells were trypsinized into single-cell suspensions andcounted using the Countess Automated Cell Counter (Cat.C10310, Life Technologies). The live cell number was then usedto calculate the total number of live cells over the number of livecells plated.

Immunohistochemical staining of human ErbB-2–positivebreast tumors

Tissuemicroarray (TMA) sectionswereplaced in a 58�C to60�Coven overnight for tissue to adhere. The sections were deparaffi-nized in xylene, rehydrated through graded ethanol, and washedwith PBS before being treated with 1� Reveal in a Decloaking

Chamber (Biocare Medical) for antigen retrieval following themanufacturer's protocol. After rinsed in PBS for 15 minutes, thesectionswere soaked in 3%H2O2 in PBS for 20minutes to quenchendogenous peroxidase activity. Sections were incubated for 60minutes in 3% normal rabbit serum (Vector Laboratories) in PBSat room temperature to block nonspecific binding sites and thenprobed with primary antibodies (PKCa antibody was Santa CruzC20 at 1 mg/mL), with nonimmune IgG used as controls. Thedetails of the staining protocol are in the Supplementary Section.

Statistical analysisMost experiments were performed at least three times.Means�

SDs were calculated on at least n ¼ 3 experiments. A two-tailedStudent t test was performed on results with two comparisons.ANOVA was performed on results with multiple comparisons.Linear regression analysis was performed on tumor growth stud-ies, as eachmouse was tagged with a number and each tumor wasmeasured independently followed by ANOVA for multiple com-parisons. A Kaplan–Meier curve for recurrence-free survival oroverall survival of human patients was generated by the Kaplan–Meier Plotter software (30) or generated by GraphPad Prismsoftware and analyzed with a log-rank (Mantel—Cox) test.

Supplementary Materials and MethodsMore detailed methods are provided in the Supplementary

Section.

ResultsJagged-1 is required for trastuzumab-induced Notch activation

We demonstrated that inhibiting ErbB-2 increased NICD1expression, CBF-1–driven reporter activity, Deltex-1 and Hey-1transcripts, and thus most likely canonical Notch activation (26).Jagged-1 predicts poor outcome in women with breast cancer(31). Here, we sought to understand whether ErbB-2 attenuatesNotch signaling by regulating Jagged-1 expression and/or cellularlocalization in breast cancer cells. Figure 1A confirms that ErbB-2inhibits Notch transcriptional activity. Conversely, ErbB-2 inhi-bition using a kinase-dead mutant, ErbB-2 siRNA, or lapatinibincreased RNA transcripts of Notch gene targets, Hes-1, Deltex-1,and/or Hey-1 (Fig. 1A). Figure 1B shows that trastuzumab orlapatinib increased Jagged-1 protein expression on the cell surfaceby flow cytometry. Furthermore, this increase in Jagged-1 proteinexpression by trastuzumab treatment is restricted to the cellsurface as measured by a cell surface–specific biotinylation assay(Fig. 1C). These results indicate that ErbB-2 inhibition promotedthe accumulation of Jagged-1 at the cell surface.

To confirm the supporting cell surface biochemical assaysfrom Fig. 1B and C that ErbB-2 overexpression may restrict cellsurface expression of Jagged-1, we performed confocal fluores-cence microscopy to visualize Jagged-1 cellular localization. TheIgG control cells showed that a portion of Jagged-1 colocalizedwith early endosomal antigen-1 (EEA-1; Fig. 1D). In contrast,trastuzumab-treated cells showed that Jagged-1 was no longerretained in EEA-1–positive endosomes and primarily accumulat-ed at the cell surface (Fig. 1D). Furthermore, Notch-1 colocalizedwith Jagged-1 in control cells (Fig. 1D). However, trastuzumabtreatment induced accumulation of Notch-1 throughout the celland Jagged-1 at sites of cell–cell contacts (Fig. 1D). These resultsindicate that ErbB-2 overexpression possibly traps both Jagged-1and Notch-1 in subplasma membrane compartments, including

PKCa Regulates Notch to Prevent Trastuzumab Resistance

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early endosomes. However, anti-ErbB-2 treatment with trastuzu-mab disrupts the Jagged-1–Notch-1 colocalization.

If Jagged-1–mediated transactivation of Notch is attenuatedby ErbB-2, we predicted that supplying the Notch-expressingbreast cancer cells with stromal cells expressing abundantsurface Jagged-1 would restore Notch activation. We coculturedErbB-2–expressing MDA-MB-453 breast cancer cells withmouse fibroblasts expressing no ligand (LTK-P) or overexpres-sing Jagged-1 (LTK-JAG-1) and then treated with vehicle ortrastuzumab. Trastuzumab increased both Hes-1 (4-fold; Fig.1E right) and Deltex-1 mRNAs (7-fold; Fig. 1E, left) in ErbB-2þ

cells when cocultured with LTK-P cells. Providing MDA-MB-453 cells with abundant Jagged-1 in trans increased Hes-1mRNA by 3-fold (Fig. 1E, right) and Deltex-1 mRNA by morethan 2-fold (Fig. 1E, left) in the presence or absence of trastu-zumab. Interestingly, trastuzumab treatment in the presence ofLTK-JAG1 did not increase Deltex-1 levels compared with LTK-Pcells. It is possible that there are cis-inhibitory mechanismswithin the breast cancer cells that limited the activation of theNotch gene target. These results indicate that ErbB-2 most likelyrestricts Jagged-1 cell surface localization and limits transacti-vation of Notch.

Figure 1.ErbB2 inhibits Notch signaling and Jagged-1 cell surface expression. A, ErbB2 expression and activity inhibits Notch activation. Left, MDA-MB-453 cells werestably transfected with vector alone or kinase dead ErbB2 (ErbB2KD) in an expression plasmid. Middle, MDA-MB-453 cells were transfected with a scrambledcontrol siRNA (SCBi) or an ErbB2 siRNA. Right, MDA-MB-453 cells were treated with DMSO or lapatinib (LAP) for 6 days. Total protein from cell lysates weresubjected to SDS-PAGE followed by Western blotting to detect tyrosine phosphorylated ErbB2 (PY-ErbB2), total ErbB2, and actin proteins. Bottom, similarstudies were performed as described in A, and real-time PCR was performed to detect RNA transcript levels for Hes-1, Hey-1, and/or Deltex-1. Bar graphs, means�SDs of three independent experiments. � , statistical significance P < 0.05 using a nonpaired Student t test. B, trastuzumab (Trast) or lapatinib increases cellsurface Jagged-1 protein expression. MDA-MB-453 cells were treated with IgG control, DMSO, or increasing concentrations of trastuzumab or lapatinib for48 hours. Flow cytometry was performed on live cells to detect cell surface expression of Jagged-1 protein. The results are representative of at least threeindependent studies. C, inhibition of ErbB-2 increases cell surface Jagged-1 expression. MDA-MB-453 cells were treated with IgG or trastuzumab for 48 hours.Cell surface proteins were biotinylated as described in experimental procedures followed by streptavidin precipitation and Western blotting to detect thefollowing proteins: Jagged-1 (JAG1) and total ErbB-2 in both precipitates and total lysates. D, trastuzumab promotes Jagged-1 surface localization and Notch-1activation as shown by confocal immunofluorescence. Top, scale bars ¼ 20 mm; bottom, scale bars ¼ 10 mm. E, Jagged-1 mediated transactivation of Notch.MDA-MB-453 cellswere coculturedwithmouse fibroblasts expressing no ligand (LTK-Parental) or overexpressing Jagged-1 (LTK-JAG1) in a 1:1 ratio and then treatedwith PBS or trastuzumab for 18 hours. Cells were sorted by flow cytometry for ErbB-2–positive cells. RNAwas extracted and real-time PCRwas performed to detectrelative transcript levels: Hes-1 (right) or Deltex-1 (left). Western blotting was performed on LTK cells to detect levels of Jagged-1 protein (middle). � , P < 0.05between PBS and trastuzumab; ��, statistical significance between LTK-P and LTK-JAG1; and �� , statistical significance between LTK-P and LTK-JAG1 in response totrastuzumab using an ANOVA.

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To determine whether Jagged-1 is necessary for trastuzumab-induced Notch activation, we used a genetic approach to down-regulate Jagged-1 in two ErbB-2–positive breast cancer cell lines.Different Jagged-1 siRNAs (JAG1iA or JAG1iNew) decreased Jag-ged-1 protein expression (Figs. 2A, 2B; Supplementary Fig. S1A)and significantly inhibited the trastuzumab-induced increase ofNotch gene targets, Hes-1, Deltex-1, Hey-1, and/or Notch-4transcripts (Figs. 2A and 2B; Supplementary Fig. S1A). Theseresults show that Notch activationmediated by ErbB-2 inhibitionis most likely Jagged-1–dependent.

Dual blockade of ErbB-2 and Jagged-1 inhibits proliferation invitro and reverses resistance in vivo

We tested whether decreasing Jagged-1 by siRNA wouldenhance the effectiveness of trastuzumab in a panel of ErbB-2–positive breast cancer cells that are sensitive (MDA-MB-453 andBT474 sensitive) or resistant (BT474 resistant and HCC1954) totrastuzumab. Figure 3A shows that proliferation of MDA-MB-453cells was decreased by 50% upon trastuzumab treatment. Twodistinct Jagged-1 siRNAs (JAG1iA or JAG1iNew) decreased theproliferation similarly to trastuzumab, by almost 50% (Fig. 3A).However, the proliferation of MDA-MB-453 cells was decreasedby almost 75% when cells were treated with both trastuzumaband a Jagged-1 siRNA (Fig. 3A). Proliferation of BT474 (Sensitive)was slightly decreased upon Jagged-1 knockdown (Fig. 3B).Proliferation of trastuzumab resistant BT474 cells was significant-ly decreased upon Jagged-1 knockdown alone. In addition,resistance was reversed upon Jagged-1 knockdown (Fig. 3B). Inaddition, HCC1954 cells that are inherently resistant to trastu-zumab were partially inhibited by Jagged-1 knockdown and

trastuzumab treatment (Supplementary Fig. S1B). These resultsindicate that Jagged-1 is required for trastuzumab resistance.

Previously, Pandya and colleagues demonstrated partial resto-ration of trastuzumab sensitivity and prevention of ErbB-2–pos-itive breast tumor recurrence using a combination of trastuzumabplus a GSI(27). GSIs are pan-Notch inhibitors and might notspecifically target Notch-1. To determine whether Jagged-1 isrequired for trastuzumab resistance in vivo, we chose a novelformat of an anti-Jagged antibody, termed a Probody therapeutic(CTX-033; refs. 32, 33). The anti-Jagged Probody, CTX-033, hasdemonstrated antitumor activity in a mouse xenograft model ofpancreatic cancer (34). Figure 3D showed that xenograft breasttumors generated from trastuzumab-resistant cells were resistantto trastuzumab following 4 weeks of treatment. Treatment withthe CTX-033 inhibited tumor growth when used alone. However,trastuzumab plus CTX-033 significantly decreased tumor growthcompared with CTX-033 or trastuzumab alone (Fig. 3D). Thesedata suggest that using a combination of trastuzumab plus CTX-033 can partially reverse trastuzumab resistance in this model.

ErbB-2 limitsMib-1–mediatedubiquitinylationof Jagged-1 andsubsequent transactivation of Notch

Because ubiquitinylation of Jagged-1 by Mib-1 is required toactivate Notch (35, 36), we hypothesized that ErbB-2 attenuatesexpression ofMib-1 and/or its associationwith Jagged-1 to inhibitNotch activity. To test this hypothesis, Jagged-1 was immunopre-cipitated from MDA-MB-453 cells treated with trastuzumab orlapatinib, andWestern blotting was performed to detect Jagged-1and Mib-1 proteins. Co-immunoprecipitation (co-IP) demon-strated that trastuzumab or lapatinib increased the amount of

Figure 2.Jagged-1 is required to promotetrastuzumab-induced Notch activation.A, Jagged-1 is required fortrastuzumab-induced increase ofNotch gene targets. MDA-MB-453 cellswere transfected with control siRNA(SCBi) or two distinct Jagged-1 siRNAs(JAG1iA or JAG1i New) and then treatedwith PBS or trastuzumab. Proteinexpression of Jagged-1 or actin wasdetermined by Western blotting (top).Transcript levels of Notch gene targets,Deltex-1, Hey-1, and Notch-4 weremeasured by real-time PCR (bottomtwo). The results are means � SDs ofthree independent experiments. B,BT474 cells were transfected with SCBior JAG1iA siRNA and then treated withPBS or trastuzumab for 48 hours.Western blotting was performed todetect efficiency of Jagged-1knockdown (top) and real-time PCRwas performed to detect transcripts ofNotch gene targets: Hes-1 and Deltex-1(bottom). The results are means � SDsof three independent experiments.� and �� , Statistical significance ofP < 0.05 as determined by an ANOVA.






Mib-1 in a complexwith Jagged-1 (Fig. 4A). From these results, weconclude that ErbB-2 possibly limits the interaction betweenJagged-1 and Mib-1.

To investigate the role ofMib-1 onNotch-l activity, the effect ofa Mib-1 siRNA on the expression of Hes-1 protein levels wasmeasured. As measured by Western blot analysis, Mib-1 proteinlevels were decreased upon Mib-1 knock down (SupplementaryFig. S2). Either trastuzumab or lapatinib increased Hes-1 protein(Supplementary Fig. S2). Importantly,Mib-1 siRNAdecreased theinduction of Hes-1 (Supplementary Fig. S2), indicating that Mib-1 is necessary for Notch activation by ErbB-2 inhibitors.

Todeterminewhether ErbB-2or downstreammediators such asPI3K or MEK attenuate Jagged-1 ubiquitinylation, we performedan IP of Jagged-1 similar to Fig. 4A except we used the PI3Kinhibitor, LY294002, or theMek1/2 inhibitor, U0126, in additionto lapatinib. Lapatinib increased Jagged-1 ubiquitinylation at 150kDa and enhanced the recruitment of Mib-1 to a Jagged-1 com-plex (Fig. 4B). Neither LY294002 nor U0126 induced recruitment

of Mib-1 to Jagged-1 or Jagged-1 ubiquitinylation compared withlapatinib (Fig. 4B). Western blot analyses confirmed that thekinase inhibitors inhibited their targets (Supplementary Fig.S3A). These results suggest that ErbB-2 restricts the interactionof Mib-1 to Jagged-1 and its ubiquitinylation possibly through adifferent kinase downstream of ErbB-2.

ErbB-2 promotes an association between Jagged-1 and PKCaTo determine the mechanism by which ErbB-2 attenuates the

association between Jagged-1 andMib-1, the protein sequences ofJagged-1 andMib-1were scannedusing Prosite Expasy and severalhigh scoring putative PKC phosphorylation sites were identified.ErbB-2 activates PKCa in breast cancer cells via PLCg (13) or byupregulating c-Src (37). Co-IP demonstrated that Jagged-1 wasassociated with PKCa when ErbB-2 was active but failed to bindPKCa when cells were treated with lapatinib (Fig. 4C). Down-regulation of PKCa using an siRNA facilitated the association ofMib-1with Jagged-1 and subsequent ubiquitinylation of Jagged-1

Figure 3.Jagged-1 is necessary for trastuzumab resistance. A, MDA-MB-453 cells were transfected with SCBi or JAG1iA or JAG1i new siRNAs and then treated with PBS ortrastuzumab once daily for up 8 days. Proliferation was measured and calculated as total live cells/total live cell plated at the designated days. BT464 parentalcells (B) and trastuzumab-resistant BT474 cells (C) were transfected with SCBi or JAG1iA and treated as described in A. � , statistical significance P < 0.05;�� , P < 0.01 were calculated on the basis of means � SDs of three replicates using an ANOVA. D, trastuzumab-resistant breast tumors are inhibited by JaggedProbody CTX-033 plus trastuzumab. Trastuzumab-resistant BT474 cells were injected into mammary fat pads of forty 5- to 6-week-old female nude athymicmice. Tumorswere allowed to grow to amean tumor cross-sectional area of 40mm2 and then randomized to four treatment groups: (i) PBS, (ii) Trastuzumab (Trast),(iii) CTX-033, or (iv) CTX-033 plus trastuzumab for up to 9 weeks. Tumor area (L � W) was measured weekly using Vernier calipers. The results are means �SD of 5 to 10 tumors per group. Statistical significance was calculated using an ANOVA.

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similarly to lapatinib treatment (Fig. 4D). Control Western blotanalyses were performed to detect protein levels of active ErbB-2(PY), total ErbB-2, andPKCa to confirm the efficiencyof thePKCasiRNA and that it had little effect on the expression and activity ofErbB-2 (Fig. 4D).

Jagged-1 and PKCa colocalize in ErbB-2–positive breast cancercells and dissociate upon trastuzumab treatment

We performed confocal fluorescence microscopy to explorewhether Jagged-1 and PKCa colocalize in ErbB-2–overexpressingbreast cancer cells. Jagged-1 colocalizeswithPKCa (Fig. 4E, left) inPBS-treated cells. In contrast, Jagged-1 was no longer colocalized

with PKCa (Fig. 4E, left) in response to trastuzumab. Similar,results were observed in trastuzumab-sensitive BT474 cells (Fig.4E, right). Interestingly, trastuzumab-resistant BT474 cells expressless PKCa and Jagged-1 is primarily near the cell surface (Fig. 4E,right, top). PKCa knockdown in BT474 sensitive cells disrupts theJagged-1–PKCa colocalization similar to what we observed inresistant cells (Fig. 4E, right, bottom).

PKCa inhibits Jagged-1–mediated Notch activation andrestores trastuzumab sensitivity in vivo

To determine whether PKCa is sufficient to restrict Notchactivation, we tested whether a constitutive active PKCa

Figure 4.ErbB-2 or PKCa attenuates Mib1-mediated ubiquitinylation of Jagged-1. A, trastuzumab or lapatinib increases the interaction between Mib1 and Jagged-1.MDA-MB-453 cells were treated with controls (IgG or DMSO) or trastuzumab or lapatinib, respectively, for 30 minutes. A Jagged-1 antibody was used toimmunoprecipitate Jagged-1 from treated cell lysates. An IgG antibody was used as a control for nonspecific immunopreciptation. Western blotting wasperformed to detect co-immunoprecipitated proteins such as Mib-1 (Mib1). Jagged-1 (JAG1) protein was detected to determine specificity of theimmunoprecipitation. Western blotting was also performed on total lysates to detect levels of total Mib1 and JAG1. B, lapatinib-induced Jagged-1 ubiquitinylation isindependent of PI3K and MAPK signaling. Cells were treated with DMSO, lapatinib, LY294002, or U0126 and then lysed. Jagged-1 was immunoprecipitatedandWestern blotting was performed to detect ubiquitinylation (Ub), Mib1, and JAG1. C, recruitment of PKCa to Jagged-1 is inhibited by lapatinib. MDA-MB-453 cellswere transfected with the FLAG-tagged Ub for 48 hours and treated with DMSO or lapatinib for 30 minutes. JAG1 was immunoprecipitated from cell lysatesfollowed by Western blotting to detect Ub, Mib1, PKCa, and JAG1. D, PKCa inhibits recruitment of Mib1 and Jagged-1 ubiquitinylation. MDA-MB-453 cells weretransfected with SCBi or PKCai siRNA for 72 hours. Cells were then treated with DMSO or lapatinib for 30 minutes followed by Jagged-1 immunoprecipitationand Western blotting to detect Ub, Mib1, and JAG1. Western blotting was also performed on total lysates to detect PY-ErbB2, ErbB2, PKCa, and actin. E, Jagged-1and PKCa colocalize in ErbB-2–positive breast cancer cells. MDA-MB-453 cells were treated with PBS or trastuzumab for 48 hours. Trastuzumab-sensitive (Sens)and -resistant (Res) BT474 cells were transfected with SCBi or PKCai siRNA for 72 hours. Cells were fixed and stained for JAG1 (green) and PKCa (red) andvisualized using confocal immunofluorescence microscopy. The results are representative of three independent experiments. Left two, scale bare ¼ 10 mm;right, scale bars ¼ 20 mm. ImageJ software was used to measure densitometry of protein bands on Western blot analyses. Ratios of proteins are presentedbelow images.






(D22–28) would prevent the lapatinib-induced Mib-1 and Jag-ged-1 interaction, Jagged-1 ubiquitinylation, and increase inNotch gene targets. Figure 5A showed that lapatinib increasedrecruitment ofMib-1 to a Jagged-1 complex and increased Jagged-1 ubiquitinylation in cells transducedwith control LRZS-linker. Incontrast, lapatinib treatment of cells transduced with LRZS-PKCaD22–28, which increased PKC activation as shown in Fig.5A, right, failed to increase Mib-1 recruitment to Jagged-1 andubiquitinylation (Fig. 5A). Furthermore, Fig. 5C showed thatlapatinib significantly increased 3 of 5 Notch gene targets (Del-tex-1, Hey-1, and Notch-4) in cells transduced with LRZS-linkerbut not in cells expressing PKCaD22–28. In contrast, knockdownof PKCa increased expression of Hes-1 and Hey-1 proteins (Fig.

5B, left) and Deltex-1 transcripts (Fig. 5B, right), respectively.Taken together, these results indicate that ErbB-2 facilitates aPKCa–Jagged-1 association to limit Mib-1–mediated ubiquitiny-lation of Jagged-1 and activation of Notch.

To determine whether PKCa expression limits Notch-1 nuclearlocalization, we performed confocal immunofluorescence. Theresults showed that cellular localization of Notch-1 and Jagged-1was relatively unchanged in cells expressing LRZS-linker alone orLRZS-PKCa upon vehicle treatment (Fig. 5D, top). In contrast,trastuzumab treatment of cells transduced with the LRZS-linkerinduced nuclear localization of a proportion of the Notch-1protein, whereas Jagged-1 accumulated in the cytoplasm or nearthe cell surface (Fig. 5D, bottom left). PKCa overexpression

Figure 5.PKCa is necessary and sufficient to inhibit Notch activation. A, active PKCa prevents Mib1-mediated Jagged-1 ubiquitinylation. MDA-MB-453 cells were transducedwith a retrovirus LRZS-linker or LRZS-PKCaD22-28 for 48 hours and then treated with DMSO or lapatinib for 30 minutes. Jagged-1 was immunoprecipitatedfrom cell lysates followed byWestern blotting to detect Ub, Mib1, and JAG1 proteins. Total lysates were subjected with Western blotting to detect total PY-ErbB-2,ErbB-2, p-Ser-PKC substrate, Mib1, and actin. B, PKCa is necessary to inhibit Notch target gene expression. MDA-MB-453 cells were transfected with SCBior PKCai siRNA for 72 hours. Cell lysates were prepared and Western blotting performed to detect PKCa, Hes-1, Hey-1, and actin proteins. In addition, RNA wasextracted from cells and real-time PCR was performed to detect relative expression of Deltex-1 transcripts normalized to HPRT. The results from the PCRrepresent means � SD of three independent experiments. C, active PKCa attenuates lapatinib-induced Notch target gene expression. MDA-MB-453 cells weretransduced with a retrovirus LRZS-linker or LRZS-PKCaD22-28 for 48 hours and then treated with DMSO or lapatinib for 24 hours. RNA was extracted fromcells and real-time PCRwas performed to detect relative expression of Hes-1, Deltex-1, Hey-1, Notch-1, andNotch-4 transcripts. The results are shown asmeans� SDsof three independent experiments. � , statistical significance P < 0.05 was calculated using an ANOVA. D, PKCa is sufficient to prevent trastuzumab-inducedNotch-1 nuclear localization. Trastuzumab-sensitive BT474 cellswere transducedwith a retrovirus LRZS-linker or LRZS-PKCa for 48 hours and then treatedwith PBSor trastuzumab for 18 hours. Cells were fixed and stained for JAG1 (Green) and Notch-1 (Red) followed by visualization using confocal immunofluorescencemicroscopy. Scale bars, 20 mm.

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almost completely prevented Notch-1 nuclear localization (Fig.5D, bottom right). These results indicate that PKCa is sufficient toprevent Notch-1 nuclear localization and activation in ErbB-2–positive breast cancer cells.

On the basis of results shown so far, we hypothesized thattrastuzumab-resistant cells that have high Notch-1 activation andrequire Notch-1 for their survival (26) should have lower PKCaprotein levels or activity than their sensitive parental cells. Theresults show that trastuzumab-sensitive BT474 cells express higherPKCa protein than trastuzumab-resistant BT474 cells and thattrastuzumab treatment of both sensitive and resistant cellsdecreased levels of PKCa protein and kinase activity as measuredby detection of phosphorylated Serine-specific PKC substrates(Supplementary Fig. S3B). We hypothesized that overexpressionof PKCamight restore trastuzumab sensitivity. To address this, wetransduced the trastuzumab-resistant BT474breast cancer cell linewith LRZS-linker or LRZS-PKCa and injected these cells (Supple-mentary Fig. S3B) into female nude mice to determine theirsensitivity to trastuzumab treatment in vivo. The results demon-strated that trastuzumab-resistant BT474 cells expressing LRZS-linker are resistant to trastuzumab (Fig. 6A). However, PKCaoverexpression significantly decreased BT474 breast tumor

growth compared with LRZS-linker. This result is not surprising,as we have shown previously that Notch-1 knockdown alone intrastuzumab-resistant BT474 cells was necessary to inhibit cellproliferation in vitro in the absence or presence of trastuzumab(26). These current results would suggest that PKCa overexpres-sion could also be necessary to inhibit ErbB-2–positive breasttumor growth after acquired resistance to trastuzumabpossibly bysuppressing Notch signaling. PKCa overexpression partiallyrestored trastuzumab sensitivity, as the tumors almost completelyregressed upon trastuzumab treatment (Fig. 6A). These resultsdemonstrate that PKCa could limit Notch-1 activation and thuspossibly trastuzumab resistance in ErbB-2–positive breast cancercells.

PKCa and Hey-1 inversely predict ErbB-2–positive breastcancer recurrence and survival

These results suggest that ErbB-2–positive breast cancer patientswith high PKCa expression should have better outcomes, asPKCa can attenuate Notch signaling to promote sensitivity toanti-ErbB-2 therapy. To address this, we used survival analysis viathe Kaplan–Meier Plotter software, version 2014 (30) andsearched for expression of PRKCA and Hey-1 transcripts in a

Figure 6.PKCa overexpression reverses trastuzumab resistance and predicts better survival in women with ErbB-2þ human breast cancer. A, overexpression of PKCainhibits growth of trastuzumab-resistant BT474 tumor xenografts. Five million trastuzumab-resistant BT474 cells retrovirally transduced with LRZS-linkeror LRZS-PKCa were injected into mammary fat pads of 20 female nude athymic mice, respectively. Tumors were allowed to grow to a mean cross-sectionalarea of approximately 10 to 20 mm2 and then mice were randomized to PBS or trastuzumab, injected intraperitoneally once weekly for up to10 weeks. Statistical significance was calculated by a two-way ANOVA. B, low PKCa protein predicts poorer overall survival in women with ErbB-2–positivebreast cancer. The Nottingham cohort of 100 ErbB-2–positive breast tumor tissues was stained for PKCa protein and scored 0.00 for negative stainingor 1.00 for positive staining. Overall survival follow-up results were collected prospectively up to 400 months. Kaplan–Meier analysis for overallsurvival was performed on negative and positive expression of PKCa. Statistical significance was calculated using log-rank (Mantel–Cox) test.Immunohistochemistry was performed to detect PKCa protein in ErbB-2–positive breast cancer tissue. The three panels are representative of negativeexpression (top), high expression (middle), and moderate expression (lower). C, model summarizes conclusion of current study. ErbB-2 through PKCablocks the interaction of Mib1 with Jagged-1, subsequently limiting ubiquitinylation of Jagged-1 to restrict Notch activation. Jagged-1–mediated Notchactivation by trastuzumab or lapatinib is required for ErbB-2–positive breast cancer survival and resistance.






cohort of 208 patients with ErbB-2–positive breast cancer topredict recurrence-free survival (RFS) outcome. Results showedthat high PRKCA transcript expression based on an average of fourprobes predicted better RFS outcome compared with low with anHR of 0.64 (log-rank P¼ 0.037). In contrast, high expression of aNotch gene target,Hey-1, predicted poor RFS outcome (HR, 1.72;log-rank P ¼ 0.011) using an average of two Hey-1 probes(Supplementary Fig. S3). Interestingly, when we used the multi-gene analyzer and asked whether the same breast cancer samplesthat express high Hey-1 and low PRKCA predict worse RFS out-comes compared with the individual genes, the results showed aslightly worse outcome with HR ¼ 1.77 and log-rank P ¼ 0.0074(Supplementary Fig. S3).

We examined another cohort, the Nottingham primary breastcancer series (the Tenovus cohort) prepared as TMA. Outcomedata were collected on a prospective basis. These include breastcancer–specific survival (BCSS) defined as the time in monthsfrom the date of surgery to the breast cancer-related death(38, 39). One hundred nine ErbB-2–positive breast cancer tissueswere stained for PKCaprotein by immunohistochemistry, blindlyscored for staining intensity (0–3). Samples were classified intotwo groups: Group 1.0 with high PKCa (staining intensity, 2–3)and group 0.0with lowor negative PKCa (staining intensity, 0–1)and labeled as "0.00." The results demonstrated that high PKCaprotein expression predicted better BCSS compared with lowexpressing patients (log-rank P ¼ 0.019; Fig. 6B). Figure 6Bshowed a sample of the immunohistochemistry for PKCa proteinstained as negative (top), positive sample #1 (middle), andpositive sample #2 (bottom). Therefore, on the basis of the resultsof our current study, we propose a model where PKCa attenuatesthe association between Jagged-1 and Mib-1 to restrict Jagged-1ubiquitinylation and subsequent transactivation of Notch-1 toprevent Notch-1–driven resistance to ErbB-2–targeted therapies(summarized in Fig. 6C).

DiscussionErbB-2–positive breast cancer is currently treated with trastu-

zumab, lapatinib, pertuzumab, or trastuzumab emtansine. Manypatients will not initially respond to these drugs and, amongresponders, more than 25% develop resistance within the firstyear of therapy (40). Thus, resistance remains a clinical problemthat requires identification of new targets for future therapeuticstrategies andnovel prognostic biomarkers.Our results reveal thatPKCa downstreamof ErbB-2 limits an association betweenMib-1and Jagged-1 to attenuate Notch-1 activation. The PKCa-medi-ated attenuationofNotch-1 signaling is necessary and sufficient toenhance sensitivity to trastuzumab and possibly prevent resis-tance to potentially improve survival for women with ErbB-2–positive breast cancer.

Notch signaling promotes breast cancer progression (41, 42)and is critical for survival and self-renewal of breast cancer stemcells (24, 25). These cells are hypothesized to be the dormant orslowly self-replicating cell population resistant to standardtherapies. We showed that Notch-1 is required for trastuzumabresistance (26) and ErbB-2 breast tumor recurrence (27). Othershave shown that Notch cooperates with ErbB-2 to promote amore invasive phenotype in ductal carcinoma in situ (DCIS;ref. 43). Furthermore, Notch expression and activity have beenimplicated in ErbB-2–driven cancer stem cells (44). Recently,Abravanel and colleagues showed that Notch promotes recur-

rence of breast tumor cells after anti-ErbB-2 therapy (45). Thus,Notch could promote breast tumor development during earlystages of breast tumorigenesis, and at later a stage, its activityand function is attenuated as other pathways become domi-nant. From our studies, we conclude that ErbB-2 attenuatesNotch signaling via PKCa under conditions whereby ErbB-2 isthe primary growth driver. However, when ErbB-2 is inhibitedby trastuzumab or lapatinib, PKCa is inhibited and Jagged-1 ismade competent to transactivate Notch to promote a compen-satory survival signal.

The role of PKCa in breast cancer remains controversial, as itis not clear whether PKCa is a tumor promoter or a suppressor.PKCa is implicated in human breast cancer progression (46).However, there are conflicting data, as some have shown PKCais decreased in human breast tissue (47). Ectopic expression ofPKCa in cell lines promotes a more aggressive phenotype. Forexample, PKCa overexpression induces tamoxifen resistance inER-positive cell lines (14). Furthermore, PKCa is required forinvasion of breast cancer cells (48). In addition, PKCa activatesNotch-4 expression in ER-positive breast cancer cells to pro-mote tamoxifen resistance (49). PKCa is also a mediator ofbreast cancer stem cell expansion (16), regulates recycling ofErbB2 in breast cancer cells (50), and a therapeutic target intriple-negative breast cancer (17). One possible explanation forthe conflicting results could be that the activation status ofPKCa, its subcellular localization, and its immediate substratesin breast cancer remain unclear. As breast cancer is a hetero-geneous disease, it would not be surprising that PKCa couldhave pleiotropic functions in different subtypes of breast can-cer. For example, patients with ErbB-2–positive tumors expres-sing low PKCa have poorer RFS than high expressors. Thesedata included both ER-positive and -negative tumors. Ideally, ifwe could stratify and separate these data based on ER expres-sion, results could determine whether ER status changes theprediction. However, because of the low number of ErbB-2þ

patients from the Kaplan–Meier Plotter dataset and the Not-tingham cohort, this could not be done. Increasing the numberof retrospective samples and/or a prospective clinical trial willhelp increase power and answer some of these critical ques-tions. Results from clinical trials using PKC inhibitors havebeen disappointing. The ISIS 3521 (aprinocarsen or LY900003)trial failed to show a response and this could be due tomultifactorial roles of PKCs. More than 20 trials from phaseI to III have been conducted using various PKC inhibitors inmany solid tumors (51). However, results of trials have beendisappointing in women with metastatic breast cancer (52),suggesting that targeting PKCa might depend on the cellcontext.

The mechanism by which PKCa attenuates Jagged-1 vesiculartrafficking and recruitment of Mib-1 to Jagged-1 is under inves-tigation. Both Jagged-1 and Mib-1 contain putative PKC phos-phorylation sites. Therefore, Jagged-1 and/or Mib-1 could bedirect substrates of PKCa. Phosphorylation events regulaterecruitment of E3 ligases and thus PKCa could directly phos-phorylate Jagged-1 and/or Mib-1 to prevent their interaction.Alternatively, PKCa could regulate vesicular trafficking of Jag-ged-1 possibly sequestering Jagged-1 in endosomes targeted forrecycling. Classical PKCs such as a and bII regulate proteins thatare normally recycled by sequestering them in the pericentrion(53). Our results are consistent with this as we showed thatJagged-1 is localized to endosomes when ErbB-2 was hyperactive

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but accumulated at the cell surface upon ErbB-2 blockade. Thus,PKCa might limit the cell surface availability of Jagged-1 byfacilitating sequestration of Jagged-1 in recycling endosomes.

We describe a critical function for PKCa downstream ofErbB-2, which is to attenuate the competency of Jagged-1 toactivate Notch-1. Notch ligands are made competent by Mib-1and subsequent ubiquitinylation that drives endocytosis. Weshow that Jagged-1 is required for trastuzumab resistance inbreast cancer (Fig. 3) providing the first preclinical proof ofconcept for the use of an anti-Jagged-1, such as the ProbodyCTX-033, targeted therapy for prevention or reversal of resis-tance possibly in breast cancers expressing low PKCa levels(Fig. 6) and/or high Hey-1 levels (Supplementary Fig. S3). Ananti-Jagged Probody therapeutic may be well positioned forsuch a strategy given that it demonstrates less systemic toxicitycompared with other Notch targeting approaches because it isonly activated within the tumor microenvironment (34). Thehuman data from two cohorts of ErbB-2–positive breast tumorspredict that women with breast tumors expressing low PKCamRNA (Supplementary Fig. S4) or protein (Fig. 6B) havepoorer RFS or BCSS, respectively. Conversely, high Hey-1expression predicts poor RFS outcome in women with ErbB-2–positive breast cancer (Supplementary Fig. S4). To determinewhether the same tumors that express low PKCa also expresshigh Hey-1 would be better targets for anti-Notch therapy, aprospective clinical trial should be conducted using the Jagged-1 Probody or a Notch-specific inhibitor in combination withtrastuzumab. These data are consistent with the overall modelwhere the ErbB-2–PKCa axis attenuates Notch activation andthis subset of tumors could predict better sensitivity to anti-ErbB-2–targeted agents. Conversely, low PKCa tumors couldrequire concomitant Notch inhibition to prevent or reversetrastuzumab resistance. These results could have clinical impactfor the treatment of women with ErbB-2–positive breast cancer.Future clinical trials need to be designed to test whether PKCaprotein levels will predict for trastuzumab sensitivity.

Disclosure of Potential Conflicts of InterestJ. Sagert has ownership interest in CytoMx. L. Miele is a consultant/advisory

board member for CytoMx. No potential conflicts of interest were disclosed bythe other authors.

Authors' ContributionsConception and design: K. Pandya, D. Wyatt, D. Shah, A. Baker, A. Rana,M.F. Denning, C. OsipoDevelopment of methodology: K. Pandya, D. Wyatt, D. Shah, A. Baker,C. OsipoAcquisition of data (provided animals, acquired and managed patients,provided facilities, etc.): K. Pandya, D. Wyatt, B. Gallagher, D. Shah, J. Blood-worth, A. Green, I.O. EllisAnalysis and interpretation of data (e.g., statistical analysis, biostatistics,computational analysis): K. Pandya, D. Wyatt, D. Shah, A. Zlobin, A. Pannuti,A. Green, I.O. Ellis, A. Filipovic, A. Rana, L. Miele, M.F. DenningWriting, review, and/or revision of the manuscript: K. Pandya, D. Wyatt,D. Shah, A. Baker, A. Green, I.O. Ellis, J. Sagert, A. Rana, K.S. Albain, L. Miele,M.F. Denning, C. OsipoAdministrative, technical, or material support (i.e., reporting or organizingdata, constructing databases): K. Pandya, D. Wyatt, B. Gallagher, D. Shah,J. Bloodworth, A. RanaStudy supervision: C. OsipoOther (development of a molecule that was used in the study): J. Sagert

AcknowledgmentsThe authors thankDr. AdrianoMarchese for generously providing the FLAG-

tagged Ub construct and the ubiquitinylation protocols. They also thank Dr.Gerry Weinmaster for generously providing the LTK-parental and LTK-JAG1mouse fibroblasts. They are grateful to Dr. Maurizio Bocchetta for scientificdiscussions and advice on this project.

Grant SupportThis work was supported in part by an NIH grant R01 (CA160378) to

C. Osipo.The costs of publication of this articlewere defrayed inpart by the payment of

page charges. This article must therefore be hereby marked advertisement inaccordance with 18 U.S.C. Section 1734 solely to indicate this fact.

Received January 26, 2015; revised August 6, 2015; accepted August 25, 2015;published OnlineFirst September 8, 2015.

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Pandya et al.




2016;22:175-186. Published OnlineFirst September 8, 2015.Clin Cancer Res Kinnari Pandya, Debra Wyatt, Brian Gallagher, et al. Positive Breast Cancer to Reverse Trastuzumab Resistance

−Mediated Notch Signaling in ErbB-2− Attenuates Jagged-1αPKC

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