RON Receptor Tyrosine Kinase as a Therapeutic Target for ...RON Receptor Tyrosine Kinase as a Therapeutic Target for Eradication of Triple-Negative Breast Cancer: Efﬁcacy of Anti-RON

Large Molecule Therapeutics

RON Receptor Tyrosine Kinase as a TherapeuticTarget for Eradication of Triple-Negative BreastCancer: Efficacy of Anti-RON ADC Zt/g4-MMAESreedhar Reddy Suthe1,2,3,4, Hang-Ping Yao1,2, Tian-Hao Weng1,2, Chen-Yu Hu1,2,Liang Feng3,4, Zhi-Gang Wu5, and Ming-Hai Wang1,3,4,5

Abstract

Triple-negative breast cancer (TNBC) is a highly diversegroup of malignant neoplasia with poor outcome. Currently,the lack of effective therapy has fostered a major effort todiscover new targets to treat this malignant cancer. Here weidentified the RON receptor tyrosine kinase as a therapeutictarget for potential TNBC treatment.We analyzedRONexpres-sion in 168 primary TNBC samples via tissuemicroarray usinganti-RON IHC staining and demonstrated that RON waswidely expressed in 76.8%TNBC samples with overexpressionin 76 cases (45.2%). These results provide the molecular basisto target RON for TNBC therapy. To this end, anti-RONmonoclonal antibody Zt/g4-drug monomethyl auristatin Econjugate (Zt/g4-MMAE) was developed with a drug toantibody ratio of 3.29 and tested in a panel of TNBC cell lineswith different phenotypes. In vitro, Zt/g4-MMAE rapidly

induced RON internalization, resulted in cell-cycle arrestfollowed by massive cell death. The calculated IC50 valuesranged from 0.06 to 3.46 mg/mL dependent on individualTNBC cell lines tested. Zt/g4-MMAE also effectively killedTNBC stem-like cells with RONþ/CD44þ/CD24� phenotypesand RON-negative TNBC cells through the bystander effect.In vivo, Zt/g4-MMAE at 10 mg/kg in a Q12 � 2 regimencompletely eradicated TNBC xenografts without theregrowth of xenograft tumors. In conclusion, increased RONexpression is a pathogenic feature in primary TNBC samples.Zt/g4-MMAE is highly effective in eradicating TNBCxenograftsin preclinical models. These findings lay the foundation forusing anti-RON Zt/g4-MMAE in clinical trials as a novelstrategy for TNBC treatment. Mol Cancer Ther; 17(12); 2654–64.�2018 AACR.

IntroductionTriple-negative breast cancer (TNBC), defined by the absence

of the estrogen receptor (ER), progesterone receptor PR), andHER2, belongs to a highly malignant subgroup of breast cancerwith poor prognosis and high mortality (1). Genetic andcellular profiling has revealed that TNBC is heterogeneous withdifferent phenotypes including two basal-like, one immuno-modulatory, one mesenchymal, one mesenchymal stem-like,

and one luminal androgen receptor subtype (1, 2). Thesephenotypes constitute the molecular basis for various TNBCmalignant behaviors, which are manifested through alterationsat genetic and cellular levels (1–2).

A major clinical challenge in managing TNBC is the lack ofeffective therapies (1–3). Currently, chemotherapy is the pri-mary established treatment for patients with TNBC in bothearly and advanced-stages of the disease (1–3). However, theefficacy of chemotherapy is very limited, which leads to poorsurvival outcomes (1, 2). Targeted therapeutic agents haverecently been applied to treat patients with advanced TNBC.The identified targets include androgen receptor, phosphoino-sitide 3-kinase/protein kinase B (PI3K/AKT), trophoblast anti-gen-2, programmed cell death-1, programmed cell deathligand-1 receptors, and others (3, 4). In addition, inhibitionof PARP is appealing in TNBC patients with a BRCA mutantphenotype (5, 6). Nevertheless, initial observations from dif-ferent stages of clinical trials indicate that these targeted ther-apies exert only moderate efficacy against TNBC (1–6). In lightof these observations, it is clear that additional exploration isnecessary to understand the TNBC biology and to identify newdrug targets for effective TNBC therapy.

The RON receptor tyrosine kinase, amember of theMET proto-oncogene family (7), has been implicated in breast cancer malig-nancy and chemoresistance (8–14). Studies in murine modelshave revealed that aberrant RON expression is critical for breastepithelial cell transformation, tumorigenesis, and distancemetas-tasis (8). Clinically, increased RON expression has been found inmore than 50% of primary breast cancer samples (15–18). RONoverexpression also is an independent predictor for breast cancer

1State Key Laboratory for Diagnosis and Treatment of Infectious Diseases, FirstAffiliated Hospital, Zhejiang University School of Medicine, Hangzhou, China.2Collaborative Innovation Center for Diagnosis and Treatment of InfectiousDiseases, First Affiliated Hospital, Zhejiang University School of Medicine,Hangzhou, China. 3Cancer Biology Research Center. 4Department of Pharma-ceutical Sciences, School of Pharmacy, Texas Tech University Health SciencesCenter, Amarillo, Texas. 5Zhejiang Provincial Key Laboratory for PrecisionDiagnosis and Treatment of Hepatobiliary and Pancreatic Cancers, FirstAffiliated Hospital, Zhejiang University School of Medicine, Hangzhou, China.

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

S.R. Suthe and H.-P. Yao contribute equally to this article.

CorrespondingAuthors:Ming-HaiWang, Texas TechUniversity Health SciencesCenter, School of Pharmacy, 1406 Coulter Street, Amarillo, TX 79106; E-mail:[email protected] or Hang-Ping Yao, First Affiliated Hospital, ZhejiangUniversity School of Medicine, 79 Qingchun Road, Hangzhou, Zhejiang 310003,P.R. China. E-mail: [email protected]

doi: 10.1158/1535-7163.MCT-18-0252

�2018 American Association for Cancer Research.

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distant relapse and disease-free survival (16). Molecularly, aber-rant RON expression results in constitutive kinase phosphoryla-tion, which activates various intracellular signaling cascadesincluding the b-catenin pathway. This leads to increased breastcancer cell proliferation, migration, invasion, stemness, and drugresistance (8–14). In addition, aberrant RON expression has beenvalidated as a therapeutic target for breast cancer therapy (18–22).Various therapeutics targeting RON including small moleculekinase inhibitors BMS777607, PHA665752, and therapeutic anti-bodies such as Zt/g4, 29B06, and nanatumab have been devel-oped and undergoing preclinical and clinical evaluation (23, 24).Preliminary results indicate that suppressing RON signaling ortargeting RON for drug delivery has the therapeutic potential forbreast cancer treatment (18). Recently, a novel anti-RON anti-body–drug conjugates (ADC) with significantly improved thera-peutic index has been developed and used in preclinical studies(21, 25, 26), which open a new avenue for RON-targeted targetedtherapy of breast cancer.

This study was to determine RON expression in primaryTNBC samples and to evaluate the efficacy of a novel anti-RON ADC for targeted TNBC therapy. The objective was toestablish proof-of-concept that anti-RON ADC has the thera-peutic value for TNBC therapy. As described above, the path-ogenesis of RON in breast cancer has been extensively studied.However, the role of RON in TNBC tumorigenesis and therapyhas not been investigated. Currently, knowledge about aber-rant RON expression and signaling in TNBC are unknown.Moreover, inhibition of RON signaling as a therapeutic poten-tial for TNBC has not been thoroughly investigated. Consid-ering these facts, we reason that understanding RON expres-sion in TNBS could help dissect the molecular mechanismsunderlying TNBC malignancy and find a new strategy forTNBC treatment.

Materials and MethodsTNBC microarrays and IHC staining

TNBC tissue microarrays containing 168 samples with certainclinical information were from US Biomax, Inc. All samples werenegative for ER, PR, and HER2 expression (www.biomax.us). TheIHC staining of RON was performed using a mouse anti-RONmAb Zt/f2 followed by DAKO EnVision System as previouslydescribed (17). Pathologic digital evaluation was used for imageanalysis as previously described (27). The intensity of RON-staining was determined as negative, weak, moderate, or strongpositive. The area reactivity of RON-stainingwas set as 0�1%,1¼2%–30%, 2¼ 31%–70%, and 3¼71%–100%) in reference to theFDA-approved criterion for assessing IHC staining of EGFR (28).Two pathologists blinded to clinical information independentlyscored RON positivity in each sample. The combined scores thatdefine RON expression were as: 0, negative; 1–2, low; 3–4,moderate; and 5–6, high, corresponding to RON 0, RON 1þ,RON 2þ, and RON 3þ, respectively.

Cell lines and reagentsBreast cancer T-47D cell line and TNBC cell lines BT-20,

DU4475, HCC1806, HCC1937, HCC2185, MDA-MB231,MDA-MB468, and SUM52PE were from ATCC. All cell lineswere authenticated in 2015 with cytogenesis analysis per-formed by ATCC. Individual cell lines were cultured withsupplementation of 10% FBS. Mouse anti-RON mAb Zt/g4,

Zt/f2, and rabbit anti-RON or phosphorylated RON polyclonalIgG antibodies were used as previously described (17, 18).Rabbit IgG antibodies specific to cyclin B1 and PARP fragmentswere from Cell Signaling Technology. Goat anti-mouse IgGlabeled with fluorescein isothiocyanate (FITC) or rhodaminewas from Jackson ImmunoResearch.

Generation and analysis of anti-RON ADC Zt/g4-MMAEConjugation of synthetic maleimidocaproyl-valine-citrulline-

p-aminobenzoyl-oxycarbonyl-monomethyl auristatin E [MC-VC-PAB-MMAE to Zt/g4 was performed under conditions toachieve a drug to antibody ratio (DAR) of 4:1 according tothe manufacture's instruction (www.concortis.com)]. Briefly,MC-VC-PAB-MMAE was coupled to cysteine residues of Zt/g4after partial reduction of the interchain disulfide bonds to formZt/g4-MMAE with an average DAR of 3.29:1. Zt/g4 conjugatedwith maytansinoid (Zt/g4-DM1) was used as previouslydescribed (26). Isotype-matched mouse IgG was conjugatedwith MC-VC-PAB-MMAE (CmIgG-MMAE) as the control. Allconjugates were purified using a PC10 Sephadex G25 column,sterilized through a 0.22 mmol/L filter, and stored at 4�C. Theconjugation was verified by hydrophobic interaction chroma-tography (HIC) using a Varian Prostar 210 Quaternary HPLCsystem coupled with a TSK butyl-NPR 4.6� 3.5 column (TosohBiosciences) as previously described (26, 29). Stability of Zt/g4-MMAE in storage conditions in PBS was analyzed by incubatingZt/g4-MMAE at 10 mg/mL with PBS at 37�C for various times.Changes of DARs at different time points were determined bythe HIC analysis.

Assays for Zt/g4-MMAE-mediated cell surface RONinternalization

Zt/g4-MMAE-mediated RON internalization by TNBC cellswas determined as previously described (26). Residual RONremained on cell surface after Zt/g4 treatment was detected bythe immunofluorescence assay using anti-RONmAb Zt/f2 (26).A time required to achieve 50% reduction of cell surface RON(internalization efficacy, IE50) was calculated for individual celllines. Detection of intracellular RON was performed usingimmunofluorescence staining as previously described (26).Nuclear DNAs were stained with 40,6-diamidino-2-phenylin-dole (DAPI). Lysosomal-associated membrane protein(LAMP)-1 was detected by goat anti-mouse IgG coupled withAlexa Fluor-647. Cellular immunofluorescence was observedunder an Olympus microscope equipped with DUS/fluorescentapparatus.

Analysis for Zt/g4-MMAE-mediated changes in cell cycle,viability, death, and colony formation

Individual TNBC cell lines (1 � 106 cells per dish) weretreated with different amounts of Zt/g4-MMAE for varioustimes. Measurement of cell-cycle changes was performed bylabeling cells with propidium iodide and analyzed by measur-ing DNA contents as previously described (21). Cell viability 96hours after Zt/g4-MMAE treatment was determined by the MTSassay (26). Levels of dead cells were determined by using thetrypan blue exclusion assay (26). TNBC cell colony formationand Zt/g4-MMAE treatment was performed as previouslydescribed (20).

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Western blot detection of RON proteins and cellular apoptoticmarkers

TNBC cellular proteins (20 mg per sample) were separated in a12% SDS-PAGE under reduced conditions. RON and its phos-phorylated proteins were detected using rabbit anti-RON IgGantibody or phospho anti-RON antibody as previously described(26). Cyclin B1 and PARP fragment were detected in Westernblotting using specific antibodies, respectively. Membranes werereprobed with anti-actin antibody to ensure equal sampleloading.

Assay of cellular caspase-3 and -7 activities for cellularapoptosis

TNBC HCC1937, HCC2185, MDA-MB-468, and SUM52PEcell lines (10,000 cells per well in a 96-well plate in triplicate)were treated with 5 mg/mL Zt/g4-MMAE for 48 hours. Cellstreated with control ADC CmIgG MMAE served as the control.Activities of caspase-3 and -7 were measured using the Cas-pase-Glo 3/7 assay system according to the manufacture'sinstruction (www.promega.com). Results were expressed asfold induction of control (caspase-3/7 activities from controlcells were set as 0).

Assays for Zt/g4-MMAE-mediated bystander killing of TNBCcells

HCC1806 cells were labeled with CellTrace CFSE (ThermoFisher Scientific) according to manufacturer's instructions.Labeled cells were then mixed at 1:1 ratio with RON-positiveHCC1937, MDA-MB231, or SUM52PE cells and incubated at 1�105 cells per well in a six-well plate in duplicate. After treatmentwith 1 mg/mL CmIgG-MMAE or Zt/g4-MMAE for 96 hours, cellswere collected and labeled with 7-AAD (Thermo Fisher Scientific)to identify dead cells. Fluorescence intensities were determinedusing the BD C6 Accuri flow cytometer. The presence of CFSE-labeledHCC1806 cells facilitate the detection and gating betweenthe RON-negative HCC1806 cells and the RON-positive unla-beled TNBC cells. Dead cells were identified by gating cellsaccording to 7-AAD staining. The percentage of viable cells wascalculated on the basis of the total cell population. Viable cellsplus dead cells are set as 100%.

Isolation of breast stem-like CD44þ/CD24� TNBC cells forcytotoxic study

Existence of CD44þ/CD24� cells in seven TNBC cell lines wereevaluated by flow cytometric analysis using FITC-conjugated anti-CD44 and PE-conjugated anti-CD24 mAbs (BD Biosciences),respectively. MDA-MB231, MDA-MB468, and SUM52PE cellswith a relatively high population of CD44þ/CD24� cells wereselected for isolation TNBC stem-like cells using a magnetic cell-sorting method as previously described (30). Briefly, TNBC cells(5� 106 cells/mL) were incubated with anti-CD24mAb followedby anti-mouse IgG dynal magnetic beads (Thermo Fisher Scien-tific) to eliminate CD24þ cells in the cell population. Cells werethen incubated with anti-CD44 mAb and goat anti-mouse IgGdynal beads to obtain CD44þ cells. Cells with RONþ/CD44þ/CD24� phenotypes (designated as TNBCSLC) were confirmed byflow cytometric analysis.

TNBC xenograft in mouse models and Zt/g4-MMAE treatmentAll experiments on mice were approved by the institutional

animal care committee. Female athymic nude mice at 6 weeks of

age (Taconic) were injected with 5 � 106 cancer cells in thesubcutaneous space of the right flank and randomized intodifferent groups (five mice per group). Treatment began whentumors reaches a mean tumor volume of �100 to 150 mm3. Zt/g4-MMAEwas injected through the tail vein at 10mg/kg in a Q12� 2 schedule. Tumor volumes were measured every 4 days andcalculated as previously described (26). Mice were monitored fortumor growth up to 68 days. Animals were euthanized whentumor volumes exceeded2,000mm3or if tumors becamenecroticor ulcerated through the skin.

Statistical analysisGraphPad Prism 7 software was used for statistical analysis.

Results are shown as mean � SD. The data between control andtreatment groups were compared using Student t test. Statisticaldifferences at P < 0.05 were considered significant.

ResultsRON expression profile in human primary TNBC samples

Clinical features of 168 TNBC samples were summarized asfollows: The age of patients ranged from30 to 79 years oldwith anaverage of 51.37 � 10.96 years old. One hundred fifty-one caseswere confirmed as invasive ductal carcinoma. Ten cases wereinvasive lobular carcinoma. The remaining seven cases weremedullary carcinoma. Majority of the patients (134 cases,79.76%) had clinical stage II tumors. Twenty-six cases(15.48%) were at stage III and eight cases at stage I. Tumor sizedistributions were: T1, 8 cases; T2, 115 cases; T3, 21 cases; and T4,24 cases. Tumor differentiation status included: grade 1, 7 cases;grade 2, 97 cases; and grade 3, 38 cases. The grade for remaining 26caseswasunknown. Lymphnode involvementwasobserved in44cases (26.19%). Distance metastasis was confirmed in only onepatient (0.59%).

Immunoreactivity of RON by Zt/f2 IHC staining with dif-ferent intensities is shown in Fig. 1A. The patterns of RONexpression from individual TNBC samples were differentwith heterogeneous and homogeneous staining appearances(Fig. 1B), which are similar to the IHC staining patterns of RONin other epithelial cancers (16, 17). In addition, analysis of IHCstaining identified RON residing in different cellular compart-ments including: predominant membrane localization, pre-dominant cytoplasmic localization, and mixed membrane andcytoplasmic localization (Fig. 1B). Such appearances probablyindicate the maturation process of RON moving from intracel-lular compartment to cell surface. Particularly, it is the reflec-tion of differential RON expression with intrinsic heteroge-neous natures of individual TNBC cells.

Information about RON expression in TNBC samples aresummarized in Supplementary Table S1. A total of 129 samplesexpressed RON (76.79%) with variable intensities and patterns.The remaining 39 cases were RON negative (23.21%). AmongRON positive samples examined, 38 cases showed low levelexpression (combined score: 1–2, 22.62%); 15 cases moderateexpression (combined scores: 3–4, 8.93%); and 76 cases over-expression (combined scores: 5–6, 45.24%). Because of incom-plete pathologic and clinical information, RON expression incorrelation with pathological parameters was not performed.Nevertheless, the IHC staining confirmed that RON was broadlyexpressed in primary TNBC samples. The fact that RON wasoverexpressed in �45% cases suggests that aberrant RON

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expression is a TNBC pathogenic feature, which renders it asuitable therapeutic target.

Generation and characterization of Zt/g4-MMAETo target RON for TNBC treatment, we conjugated Zt/g4 with

MMAE through cathepsin B-sensitive dipeptide linker to generateZt/g4-MMAE (Fig. 2). The conjugation achieved the average DARof 3.29 as confirmed by HIC analysis. The percentages of con-jugates with different DARs from the integrated areas of theconjugates are shown in Supplementary Fig. S1A. Major peaksincluded peak 2 (33.02%)with aDARof 2:1 andpeak 4 (46.16%)with a DAR of 4:1. Zt/g4-MMAE with DARs at 2:1, 4:1, and 6:1accounted for 87.07% of the total conjugates. DARs for CmIgG-MMAE were 3.54.

We determined the stability of Zt/g4-MMAE in PBS, whichreflects the storage condition. Zt/g4-MMAE appeared to be stableat 37�C in PBS as evident by HIC analysis (Supplementary Fig.S1B).Onday 28, Zt/g4-MMAEhad an averageDARof 3.06,whichrepresented only �7.0% reduction from DAR of 3.29 on day 0.Themajor changes appeared to be peak 4 and 6. Fromday 0 to day28, peak 4 increased from 46.16% to 55.32% and P6 decreasedfrom 7.89% to 0.00%. These results suggest that Zt/g4-MMAE isstable in storage condition in PBS.

Induction by Zt/g4-MMAE of cell surface RON internalizationPathologic features of several TNBC cell lines are summa-

rized in Supplementary Table S2. Levels of RON expressionwere determined by flow cytometric analysis using Zt/g4 as theprimary antibody (Fig. 3A). T-47D cells expressing 15,756 �314 RON molecules per cell were used as the control. Specificbinding was not observed in BT-20 or HCC1806 cells (<50RON molecules per cell), which served as the negative control.Zt/g4-MMAE treatment caused a progressive reduction of cellsurface RON in a time-dependent manner in RON-positiveTNBC cell lines tested (Fig. 3B). Less than 30% of RONremained on the cell surface after a 36-hour treatment. Thecalculated IE50 values were 10.21 hours for HCC1937, 10.25hours for HCC2185, 10.88 hours for MDA-MB231, 22.86 hoursfor MDA-MB468, and 19.86 hours SUM52PE cells, respectively.Cellular immunofluorescence analysis confirmed Zt/g4-MMAE-induced RON internalization in three TNBC cell lines tested(Fig. 3C). RON was detected on the cell surface at 4�C. Inter-nalization occurred at 37�C after Zt/g4-MMAE treatment. More-over, cytoplasmic RON was colocalized with LAMP-1 in TNBCcells. Thus, results presented in Fig. 3 demonstrate that Zt/g4-MMAE is highly effective in induction of RON internalizationby RON-expressing TNBC cells.

Figure 1.

Expression of RON in primary triple-negative breast cancer samples. A, Various intensity of RON staining. Semiquantitative IHC staining was performed on 168primary TNBC samples using mouse anti-RON mAb Zt/f2. Representative images showing negative staining and different levels of RON immunoreactivity areshown. The combined score �5 to 6, defined as RON 3þ, was judged as overexpression. B, Different expression patterns of RON in TNBC samples. Imagesshowing cell membrane (MEM), cytoplasm (CYT), and mixture staining of RON expression are presented with individual enlarged images. Scale bars for A and Brepresent 50 mmol/L.






Effect of Zt/g4-MMAE in vitro on TNBC cell cycle, growth, death,and colony formation

We first determined sensitivities of individual TNBC celllines to free MMAE using the MTS assay. TNBC cells weresensitive to free MMAE regardless levels of RON expression(Supplementary Table S2). We then studied the effect of Zt/g4-MMAE on cell cycles (Fig. 4A). Changes in cell cycle wereobserved as early as 6 hours after addition of Zt/g4-MMAE inRON-positive TNBC cell lines with a significant reduction inG0–G1 phase, a decrease in S phase, and a dramatic increasein G2–M phase. RON-negative HCC1806 cells did not respondto Zt/g4-MMAE. These results indicate that Zt/g4-targeteddelivery of MMAE has a profound effect on cell cycles ofRON-expressing TNBC cells.

The effect of Zt/g4-MMAE on TNBC cell viability is shownin Fig. 4B. A significant reduction in cell viability was observedin a Zt/g4-MMAE dose-dependent manner in all RON-positiveTNBC cell lines tested. The IC50 values were 1.42 mg/mL forHCC1937, 0.06 mg/mL for HCC2185, 3.46 mg/mL for MDA-MB231, 0.98 mg/mL for MDA-MB468, and 1.26 mg/mL forSUM52PE cells, respectively. BT-20 and HCC1806 cells wereinsensitive to Zt/g4-MMAE. The kinetic effect of Zt/g4-MMAEon cell viability of HCC1937, HCC2185, MDA-MB468, andSUM52PE cells are shown in Supplementary Fig. S2A. Zt/g4-MMAE caused a time-dependent reduction in cell viabilityamong TNBC cell lines tested. Thus, Zt/g4-MMAE reduces cellviability in both dose- and time-dependent manners in RON-positive TNBC cells.

Observation of cell morphology under a microscope indi-cated massive cell death after Zt/g4-MMAE treatment (Supple-mentary Fig. S2B). Dose-dependent cell death was documentedamong TNBC cell lines tested after a 96-hour treatment (Fig.4C). The calculated IC50 values were 1.81 mg/mL for MDA-MB468, 3.92 mg/mL for HCC1937, 0.15 mg/mL for HCC2185,and 3.64 mg/mL for SUM52PE cells, respectively. Flow cyto-metric analysis confirmed increased apoptotic cell death inRON-expressing TNBC cell lines tested (Fig. 4D). Moreover,we performed Western blot analysis to determine Zt/g4-MMAE-

caused cyclin B1 expression and proteolytic activation of PARP(Fig. 4E and F), both served as indicators for cellular apoptosis(31). Western blot analysis indicated spontaneous RON acti-vation as evident by the detection of phosphorylated RON insix cell lines tested (Supplementary Fig. S3). Increased cyclin B1expression and fragments of activated PARP were evident afterZt/g4-MMAE treatment. Increased caspase-3 and -7 activities inZt/g4-MMAE-treated TNBC cell lines also were documented(Fig. 4G). These observations provide the evidence of apoptoticcell death caused by Zt/g4-MMAE. Finally, we observed theinhibitory effect of Zt/g4-MMAE on TNBC cell colony forma-tion (Fig. 4H). Zt/g4-MMAE at 1 mg/mL dramatically inhibitedthe colony formation by three RON-expressing TNBC cell lines.Thus, results shown in Fig. 4 demonstrate that Zt/g4-MMAE iscapable of arresting cell cycles, decreasing cell viability, reduc-ing viable cell numbers, inhibiting clonogenic growth, andinducing massive apoptotic cell death.

Cytotoxic effect of Zt/g4-MMAE on bystander TNBC cells andstem-like TNBC cells

To explore the possibility of Zt/g4-MMAE in killing RON-negative bystander TNBC cells, we cocultured RON-negativeHCC1806 cells with one of the following RON-positive TNBCcells, HCC1937 (RON3þ),MDA-MB231 (RON1þ), or SUM52PEcells (RON 2þ) in the presence of CmIgG-MMAE, Zt/g4-DM1, orZt/g4-MMAE. The reason we include Zt/g4-DM1 formed withnoncleavable linker was to determine if Zt/g4-MMAE with thecleavable linker was superior in killing bystander cancer cells. Asshown in Fig. 5A, viable HCC1806 cells remained at high levels inthe presence of CmIgG-MMAE but decrease significantly inresponse to Zt/g4-MMAE. In contrast, high levels of viableHCC1806 cells were observed in the presence of Zt/g4-DM1.Similar results were observed when HCC1806 cells were co-incubated with SUM52PE and HCC1937 cells. We further con-firmed by using the MTS assay that HCC1806 cells was highlysensitive to Zt/g4-MMAE cytotoxicity but resistant to Zt/g4-DM1action when they were cocultured with MDA-MB231, SUM52PE,or HCC1937 (Fig. 5B). In these cases, levels of HCC1806 cell

Figure 2.

Schematic representation of Zt/g4-MMAE structure: Zt/g4, mouse mAbspecific to the RON Sema domain, wasconjugated with MMAE through thevaline-citruline dipeptide linker toreach a DAR of 3.29 according to themanufacturer's instruction (www.concortis.com).

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survival were significantly reduced in the presence of Zt/g4-MMAEbut not Zt/g4-DM1. These results indicate that Zt/g4-MMAEformed with the cleavable linker is superior to Zt/g4-DM1 withnoncleavable linker due to the reduction of viability of bystanderRON-negative TNBC cells.

Another interesting finding was that the survival levels ofHCC1806 cells were significantly lower in the presence of TNBCcells expressed high levels of RON (SUM52PE or HCC1937 cells)than those in the presence of TNBC cells expressed low levels ofRON (MDA-MB231, Fig. 5A and B). The killing of bystander cells

Figure 3.

Expression of RON and itsinternalization by TNBC cells:A, Individual TNBC cell lines(1 � 106 cells/mL) in 1 mL PBS induplicate were incubated at 4�Cwith 5.0 mg Zt/g4 (red line) for60 minutes. Isotope-matchedmouse IgG (black line) was use asthe control. Cell surface RON wasquantitatively determined byimmunofluorescence analysis usingQIFKIT (DAKO) as previouslydescribed (17). B, Zt/g4-inducedinternalization of cell surface RONwas determined using anti-RONmAb Zt/c1 immunofluorescenceanalysis as previously described (21).Immunofluorescence from cellstreated with Zt/c1 at 4�C was set as100%. Among six RON-positive celllines tested, the percentages of RONremaining on the cell surface at24 and 36 hours were significantlylower than those at 0 hour(P < 0.05). C, Detection ofinternalized RON using theimmunofluorescence stainingmethod was performed aspreviously described (21). NuclearDNAs were stained with DAPI.LAMP-1 was used as a marker forprotein cytoplasmic localization.Englarged images were from theareas indicated with yellow arrows.Scale bar: 10 mmol/L.






was more prominent in the presence of TNBC cells overexpressedRON after treatment of Zt/g4-MMAE. These data suggest that Zt/g4-MMAE is capable of killing the RON-negative bystander TNBCcells and its effectiveness is associated with levels of RONexpressed by TNBC cells.

Next, we studied the effect of Zt/g4-MMAE in reducingviability of TNBCSLC cells with CD44þ/CD22� phenotypes.

Breast cancer with CD44þ/CD22� phenotypes are known tobe tumor-initiating stem-like cells (32, 33). We isolated RONþ/CD44þ/CD24� TNBCSLC cells from MDA-MB231, MDA-MB468, and SUM52PE cell lines expressed different levels ofRON. The flow cytometric analysis confirmed RON coexpres-sion with CD44þ/CD24� TNBCSLC cells (SupplementaryFig. S4). All three TNBCSLC populations displayed an increase

Figure 4.

Effect of Zt/g4-MMAE on TNBC cell-cycle change, viability, death, and colony formation. A, Cell-cycle changes caused by Zt/g4-MMAE were conducted by flowcytometric analysis after labeling with propidium iodide as previously described (21). RON-negative HCC1806 cells were used as the control. Changes in cell cycleswere marked with arrows. B, Cell viability was determined by the MTS assay as previously described (20). CmIgG-MMAE served as the control. The individual IC50

values were calculated by using GraphPad Prism 7 software. All IC50 values derived from six RON-positive cell lines treated with Zt/g4-MMAE were significantlylower than those from control cells treated with CmIgG-MMAE (P < 0.05). C,Death of TNBC cells were determined by treating cells with different amounts of Zt/g4-MMAE for 96 hours. Percentages of cell deathwere determined by the trypan blue exclusionmethod. All IC50 values derived from four RON-positive cell lines treatedwith Zt/g4-MMAE were significantly lower than those from control cells treated with CmIgG-MMAE (P < 0.05). Data shown here are derived from one of threeexperiments with similar results. D, Treatment of cells with Zt/g4-MMAE was performed as described in C. Cell apoptotic death was also determined by flowcytometric analysis after labeling cells with markers as detailed in the Materials and Methods. Percentages of cell death obtained from HCC1937 and SUM52PE cellstreated with Zt/g4-MMAE were significantly higher than those from control cells treated with CmIgG-MMAE (P < 0.05). E and F, Western blot analysis of Zt/g4-MMAE-induced cyclin B1 expression (E) and fragmentationof PARP (F)wasperformedusing specific antibodies as previously described (25). Samemembraneswereused to detect both proteins with actin as the loading control. G, Zt/g4-MMAE-mediated activation of caspase-3 and -7 in four TNBC cell lines were determinedusing the caspase-Glo 3/7 assay system as detailed in the Materials and Methods. Cells were treated with Zt/g4-DM1 or Zt/g4-MMAE. Caspase-3/7 activities fromcontrol TNBC cells treated with CmIgG-MMAE served as the baseline (zero-fold). Increased caspase-3/7 activities expressed as the fold induction of controlwere statistically significant in comparison with those from controls. H, The assays to determine the effect of Zt/g4-MMAE on TNBC cell colony formation wasperformed as previously described (20). After incubation of cells with Zt/g4-MMAE for 20 days, cells were fixed and stained. Colonies consisting of at least50 cells were counted using a stereo-microscope as previously described (20). The number of colonies in controls wad set as 100%. Percentages of colony inADC-treated cells were calculated as: [(colonies from ADC treated plate � 100%)/colonies from control plate]. , Student t test with P < 0.05.

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in chemoresistance in response to a panel of chemoagentswhen compared with their corresponding parental cell lines(Supplementary Table S3). Zt/g4-MMAE significantly decreasedcell viability in a dose-dependent manner among three

TNBCSLC populations tested. The observed IC50 values for cellviability were in the range of 0.9 to 2.0 mg/mL, similar to thosefrom their parental cells (Fig. 5C). These results suggestthat the Zt/g4-MMAE reduces the viability of CD44þ/CD24�

Figure 5.

Cytotoxic effect of Zt/g4-MMAEonRON-negative TNBC cells and RON-positive stem-like TNBC cells.A, The assay to determine the bystander killing effect of Zt/g4-MMAE on a panel of TNBC cell lines was detailed in the Materials and Methods. Both Zt/g4-MMAE and Zt/g4-DM1 were used for comparison. CmIgG-MMAEwas used as the control. B, Experimental conditions were similar to those in (A). Cell survival was determined by flow cytometric analysis as detailed in the Materialsand Methods. The Co-Culture set is a measurement of HCC1806 cells. C, The viability of TNBC stem-like cells after Zt/g4-MMAE treatment was determined bytheMTS assay. Parental TNBC cells were used for comparison. The IC50 values derived from three paired TNBC cell lines showed no statistical significance (P >0.05).Data shown here are from one of three experiments with similar results.






tumor-initiating TNBC cells regardless their chemoresistantphenotype.

Therapeutic activity of Zt/g4-MMAE in TNBC xenograft tumormodel

Among eight TNBC cell lines tested, HCC1806, HCC1937, andMDA-MB468 cells were able to grow in athymic nude mice andselected for therapy studies. It is noticed that some TNBC cell linessuch as SUM52PE and HCC2185 were known to cause tumorgrowth (34). However, we were unable to reproduce these results.To test the therapeutic activity, Zt/g4-MMAE at 10 mg/kg with aQ12 � 2 schedule was used for in vivo studies. The rationale touse this treatment regimen was based on the terminal half-life(t1/2: 6.01 days) of anti-RON ADCs in mice (25). As shownin Fig. 6A, HCC1806 cell-initiated TNBC xenografts grew rapidlyand were insensitive to Zt/g4-MMAE treatment. No statisticaldifferences were observed between control and Zt/g4-MMAEtreated mice. In contrast, Zt/g4-MMAE was highly effective inblocking tumor growth caused by HCC1937 and MDA-MB468cell lines. It is worth to note that the growth rates of xenograftsinitiated by HCC1937 and MDA-MB468 cells were different.

Nevertheless, Zt/g4-MMAE was able to completely inhibit tumorgrowth initiated by both cell lines. The effect of Zt/g4-MMAEin vivo was long lasting as measured by average tumor volume.After the second ADC injection at day 24, tumor growth wascompletely blocked for both HCC1937 and MDA-MB468 cell-derived TNBC xenografts. In both cases, the regrowth of tumorswas not observed.

A comparisonof average tumorweight between the control andZt-g4-MMAE treated mice from the end of experiment are shownin Supplementary Fig. S5A and S5B. Consistent with those shownin Fig. 6A, Zt/g4-MMAE did not exhibit an inhibitory effect on theHCC1806-mediated xenograft. An exciting finding was the com-plete eradication by Zt/g4-MMAE of both HCC1937 and MDA-MB468-mediated TNBC xenografts. Detailed examination in thecell injection site did not find any trances of tumor residues. Thus,Zt/g4-MMAE as a single agent at 10mg/kg in theQ12� 2 regimenis highly effective not only in inhibition but also in eradication ofTNBC xenografts.

To exclude any possibilities that reduction of tumorwas relatedto in vivo general toxicity of Zt/g4-MMAE, we monitored animalbehavior, food consumption, daily activity, and body weight in

Figure 6.

Therapeutic efficacy of Zt/g4-MMAE in TNBC xenograft tumor models. A, Athymic nude mice (five mice per group) were subcutaneously inoculated with 5 � 106

TNBC cells. Zt/g4-MMAE at 10 mg/kg was injected through tail vein in the Q12� 2 regimen after tumor volumes reached 100–150 mm3. Mice injected with CmIgG-MMAEat 10mg/kgwere used as the control. Xenografts initiated byRON-negative HCC1806 cellswere used as the control. Arrows indicate the time ofADC injection.B, The effect of Zt/g4-MMAE on mouse body weight and survival as described in (A) was monitored every day up to day 68. Average body weight reduction andnumbers of dead mice were calculated for each group.

Suthe et al.





both control and Zt/g4-MMAE-treated mice. All mice behavednormallywith regular activity and food consumption. The averagebody weight from mice injected with HCC1806, HCC1937, andMDA-MB468 cells were not affected by Zt/g4-MMAE treatment(Fig. 6B). Also, Zt/g4-MMAE treatment did not halt mousegrowth. The average body weight of Zt/g4-MMAE treated groupswas comparable to that of control mice with no statistical differ-ences. Thus, Zt/g4-MMAE at 10 mg/kg in the Q12 � 2 schedulehad no negative impact on mouse bodyweight, implying thattumor reduction was not related to the general toxicity of Zt/g4-MMAE on mice.

DiscussionThe study presented here is our attempt to determine the

feasibility of anti-RON ADCs for TNBC treatment. Anti-RONIHC staining confirmed RON overexpression in �45% TNBCsamples, which provide the rationale to target RON for TNBCtreatment. ADCs conjugated with maytansinoid such as Zt/g4-DM1 have been previously used for RON-targeted cancertherapy (21, 25, 26). However, the efficacy of Zt/g4-DM1 invivo is relatively weak, which lacks the tumor-eradicatingactivities (21, 25, 26). Thus, we produced Zt/g4-MMAE withgoals to improve its stability and to increase its therapeuticindex. Both in vitro and in vivo studies demonstrated that Zt/g4-MMAE effectively induced RON internalization by TNBCcells, which leads to massive apoptotic cell death. Moreover,we show that Zt/g4-MMAE was capable of killing RON-neg-ative or low-expressive TNBC cells through the bystander effectand the RON-positive TNBC stem-like cells. Results fromTNBC xenograft studies in mice further confirmed that Zt/g4-MMAE at 10 mg/kg in the Q12 � 2 regimen not onlyinhibited and but also eradicated TNBC xenografts with thelong-lasting effect. This thoroughly shows that the RON recep-tor is a suitable target for TNBC therapy using anti-RON ADCZt/g4-MMAE.

The IHC staining of RON expression in primary breast cancersamples has been studied with increased RON expression in therange of 30% to 80% (15–17). However, the detailed analysis ofRON expression in TNBC samples has not been performed. Thisstudy was our first attempt to profile RON expression in TNBCsamples, which shows that RON was expressed in �75% ofprimary TNBC samples with overexpression in �45% cases. Thisexpression pattern is comparable to those from previous studiesshowing increased RON expression in breast cancer samples.Therefore, aberrant RON expression can be characterized as apathogenic feature in this particular subtype of breast cancer.Moreover, increased RON expression renders it a suitable targetfor therapeutic application.

Identification of RON as the target opens a new avenue forTNBC treatment. The following evidences support this notion.First, Zt/g4 was effective in induction of RON internalization byTNBC cells. we observed that the kinetics of Zt/g4-induced RONinternalization among five TNBC cell lines tested are differentwith IE50 values varied from 10 to 23 hours regardless their levelsof RONexpression.However, Zt/g4-induced RON internalizationwas highly effective with more than 70% of cell surface RONinternalized within 36 hours. In the case of HCC2185 cells withcell surface RON molecules per cell at �30,000, it was estimated21,000 cell surface RON receptors internalized within 36 hours.This is equivalent to �67,200 MMAE molecules delivered into a

single cell, sufficient to cause cell-cycle arrest. Thus, Zt/g4-inducedRON internalization facilitated intracellular delivery of MMAE inTNBC cells overexpressing RON.

Second, Zt/g4-MMAE had a profoundly cytotoxic impact onTNBC cells. We showed by flow cytometric analysis that Zt/g4-MMAE caused cell-cycle arrest inG2–Mphase, which is a feature ofMMAE that impairs microtubule dynamics. The effect wasobserved as early as 6 hours after the addition of Zt/g4-MMAEand characterized by progressive reduction of the G1 phase andthe accumulation of cells at the G2–Mphase. Moreover, we foundthat Zt/g4-MMAEprogressively decreased cell viability.More than80% reduction in cell viability was observed 96 hours aftertreatment among TNBC cell lines tested. Decreased viability wasaccompanied with massive cell death in a Zt/g4-MMAE dose-dependent manner. Analysis of cellular apoptotic markers con-firmed Zt/g4-MMAE induced cyclin B1 expression and generationof PARP fragments. Finally, we confirmed that Zt/g4-MMAE killedRON-negative TNBC cells through the bystander effect and stem-like TNBC cells. These activities indicate that Zt/g4-MMAE ishighly effective in elimination of TNBC cells.

Third, the therapeutic efficacy of Zt/g4-MMAE is confirmed inmouse TNBC xenograft models as evident in the 10 mg/kg inthe Q12 � 2 schedule. This dosing-schedule regimen wasdesigned to determine the capability of Zt/g4-MMAE at10 mg/kg in a two terminal half-life cycle to inhibit TNBCxenograft growth. We show that Zt/g4-MMAE effectively inhib-ited TNBC xenograft growth mediated by HCC1937 and MDA-MB468 cells but had no effect on HCC1806 cell-mediatedtumors. By monitoring Zt/g4-MMAE concentrations based onits terminal half-life, we confirmed that Zt/g4-MMAE displayedthe long-lasting tumor-inhibitory activity up to 6 weeks with-out signs of tumor regrowth (from day 24 to day 68, Fig. 6A).Significantly, we demonstrated that Zt/g4-MMAE not onlyinhibited but also eradicated TNBC xenografts in both TNBCmodels tested. This effect is important since anticancer biother-apeutics often exert their activities by inhibition not by erad-ication. Finally, we observed that Zt/g4-MMAE at the therapeu-tic dose and regimen in vivo had no major toxic activities inanimals, evident by the increase in average body weight in Zt/g4-MMAE treated mice. Considering these facts, it is suggestedthat Zt/g4-MMAE is effective and safe in the targeted treatmentof TNBC xenograft tumors. Furthermore, these findings lay thefoundation for transition of Zt/g4-MMAE into clinical trials inthe future.

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

Authors' ContributionsConception and design: S.R. Suthe, H.-P. Yao, M.-H. WangDevelopment of methodology: S.R. Suthe, H.-P. Yao, T.-H. Weng, L. Feng,Z.-G. Wu, M.-H. WangAcquisition of data (provided animals, acquired and managed patients,provided facilities, etc.): S.R. Suthe, H.-P. Yao, T.-H. Weng, C.-Y. Hu,Z.-G. Wu, M.-H. WangAnalysis and interpretation of data (e.g., statistical analysis, biostatistics,computational analysis): S.R. Suthe, H.-P. Yao, C.-Y. Hu, L. Feng, M.-H. WangWriting, review, and/or revision of the manuscript: S.R. Suthe, H.-P. Yao,M.-H. WangAdministrative, technical, or material support (i.e., reporting or organizingdata, constructing databases): M.-H. WangStudy supervision: H.-P. Yao, M.-H. Wang






AcknowledgmentsWe appreciate Ms. Rachel Hudson from Texas Tech University Health

Sciences Center in editing the manuscript. This work was supported in part bythe Amarillo Research Support #242041 from the Amarillo Area Foundation toMing-Hai Wang. The work was also supported by National Natural ScienceFoundation of China grant #81872883 and Zhejiang Major Medical Health &Sciences Technology Foundation Projects #WKJ-ZJ-13 and #2014C33204 toH.-P. Yao.

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 March 12, 2018; revised May 21, 2018; accepted September 26,2018; published first October 1, 2018.

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Suthe et al.




2018;17:2654-2664. Published OnlineFirst October 1, 2018.Mol Cancer Ther Sreedhar Reddy Suthe, Hang-Ping Yao, Tian-Hao Weng, et al. ADC Zt/g4-MMAEEradication of Triple-Negative Breast Cancer: Efficacy of Anti-RON RON Receptor Tyrosine Kinase as a Therapeutic Target for

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