Targeting Nodal in Conjunction with Dacarbazine Induces ...mcr.aacrjournals.org/content/molcanres/13/4/670.full.pdf · Targeting Nodal in Conjunction with Dacarbazine Induces Synergistic

Cell Death and Survival

Targeting Nodal in Conjunction with DacarbazineInduces Synergistic Anticancer Effects inMetastatic MelanomaKatharine M. Hardy1, Luigi Strizzi1, Naira V. Margaryan1, Kanika Gupta1,2,George F. Murphy3, Richard A. Scolyer4, and Mary J.C. Hendrix1

Abstract

Metastatic melanoma is a highly aggressive skin cancer with apoor prognosis. Despite a complete response in fewer than 5% ofpatients, the chemotherapeutic agent dacarbazine (DTIC) remainsthe reference drug after almost 40 years. More recently, FDA-approved drugs have shown promise but patient outcome remainsmodest, predominantly due to drug resistance. As such, combina-torial targeting has received increased attention, and will advancewith the identification of new molecular targets. One attractivetarget for improvingmelanoma therapy is the growth factorNodal,whose normal expression is largely restricted to embryonic devel-opment, but is reactivated in metastatic melanoma. In this study,we sought to determine how Nodal-positive human melanomacells respond toDTIC treatment and to ascertain whether targetingNodal in combination with DTIC would be more effective thanmonotherapy. A single treatment with DTIC inhibited cell growth

but did not induce apoptosis. Rather than reducing Nodal expres-sion,DTIC increased the size of theNodal-positive subpopulation,an observation coincident with increased cellular invasion. Impor-tantly, clinical tissue specimens from patients with melanomasrefractory to DTIC therapy stained positive for Nodal expression,both in pre- and post-DTIC tumors, underscoring the value oftargeting Nodal. In vitro, anti-Nodal antibodies alone had someadverse effects on proliferation and apoptosis, but combiningDTIC treatment with anti-Nodal antibodies decreased cell growthand increased apoptosis synergistically, at concentrations incapa-ble of producing meaningful effects as monotherapy.

Implications: TargetingNodal in combinationwithDTIC therapyholds promise for the treatment of metastatic melanoma. MolCancer Res; 13(4); 670–80. �2015 AACR.

IntroductionMetastatic melanoma is the leading cause of skin cancer deaths

in the United States, with a median overall survival of only 6–9months (1). Despite advances in the field, the reference therapyfor patients diagnosed with metastatic melanoma is dacarbazine(DTIC),whichwasfirst approved for treatment in the 1970s.DTICtherapy is associated with poor patient outcome, with completeresponse occurring in less than 5% of cases (2). More recently,FDA approval has been granted for a limited number of alterna-

tive treatments, including vemurafenib (3) and dabrafenib (ref. 4;B-RAF inhibitors approved only for V600E B-RAF mutation–positive melanomas), trametinib (ref. 5; a MEK inhibitor alsolimited to B-RAF–mutant melanomas), as well as immunomo-dulating agents such as ipilimumab (ref. 6; an immunotherapeu-tic monoclonal antibody to CTLA-4), pembrolizumab (anti-PD1monoclonal antibody; ref. 7), IFNa-2b (8), and IL2 (9). However,these monotherapies are typically restricted in their efficacy andtend to provide only a small improvement in progression-freesurvival before resistance develops (10, 11). As such, combina-torial targeting with multiple agents to overcome drug resistancehas become an attractive potential strategy, but has so far hadlimited success (12–14). Thus, the identification of alternativetargets and development of new therapies are still needed.

Nodal is a growth factor of the TGFb superfamily. Predomi-nantly expressed during early embryonic development, Nodal isimportant in embryonic stem cell maintenance and body axisestablishment (15–17). Importantly, Nodal is not typicallyobserved inmost normal adult tissues but is reactivated in variousadvanced stage cancers such as metastatic melanoma, as well asbreast, prostate, and ovarian carcinomas (18–23). CanonicalNodal signaling occurs via Nodal binding to type I (ALK4/7) andtype II (ActRIIB) activin-like kinase receptors in a Cripto-depen-dent or -independent fashion. Subsequent phosphorylation ofSmad2/3/4 enables the complex to translocate to the nucleus andactivate a transcriptional program that typically includes Nodalitself and the Nodal antagonist, Lefty (15–17). In advancedcancers, Nodal expression is maintained by a positive feedbackloop (where signaling upregulates expression), but concurrent

1Program in Cancer Biology and Epigenomics, Stanley Manne Chil-dren's Research Institute at Ann and Robert H. Lurie Children's Hos-pital of Chicago, Robert H. Lurie Comprehensive Cancer Center,Northwestern University Feinberg School of Medicine, Chicago, Illi-nois. 2Howard Hughes Medical Institute NU Bioscientist Program,Weinberg College of Arts and Sciences, Northwestern University,Evanston, Illinois. 3Department of Pathology, HarvardMedical School,Brigham & Women's Hospital, Boston, Massachusetts. 4MelanomaInstitute Australia; Sydney Medical School, The University of Sydney;and Department of Tissue Pathology and Diagnostic Oncology, RoyalPrince Alfred Hospital, Sydney, New South Wales, Australia.

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

Corresponding Author: Mary J.C. Hendrix, Stanley Manne Children's ResearchInstitute, Northwestern University Feinberg School of Medicine, 225 East Chi-cago Avenue, Box 222, Chicago, IL 60611. Phone: 773-755-6528; Fax: 773-755-6534; E-mail: [email protected]

doi: 10.1158/1541-7786.MCR-14-0077

�2015 American Association for Cancer Research.

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Lefty upregulation is absent due to DNA methylation of thepromoter (19, 24), leading to unregulated Nodal expression andsignaling.

Efforts to target Nodal in metastatic melanoma cells haveshown inhibition of cell aggressiveness in vitro and reduced tumorgrowth in in vivo xenograft models (18, 19, 21, 25). It is notcurrently known how Nodal signaling is affected by standardtherapy for metastatic melanoma nor whether targeting Nodalsignaling offers any improvement over conventional monother-apy. In this study, we sought to identify the effects of DTIC onNodal-expressingmelanoma cell lines and evaluate the efficacy oftargeting Nodal in combination with DTIC.

Materials and MethodsCells

Melanoma cell lines utilized were: C8161 (University ofArizona, Tuscon, AZ; 1999); MV3 (a gift of Dr. van Muijen,University Hospital Nijmegen, Nijemegen, the Netherlands;2006); SK-MEL-28 (ATCC, 2010). Cell lines were authenticatedby short tandem repeat genotyping at Lurie Children's Hospitalof Chicago (Chicago, IL; 2009–2010). Lines were routinely testedformycoplasma contaminationwith a PCR ELISAKit (Roche). Allcell lines were maintained as previously described (21).

Chemicals and antibodiesDacarbazine (DTIC) was dissolved in serum-free medium at

stock concentrations before use (LKT Laboratories). Antibodiesused were mouse anti-actin (Calbiochem), mouse anti-BCL2(Santa Cruz Biotechnology), rabbit anti-phospho-HistoneH3(Ser10), rabbit anti-HistoneH3, and rabbit anti-PARP (Cell Sig-naling Technology), rabbit anti-Nodal (dialyzed to remove pre-servative contamination prior to cell culture treatments; SantaCruz Biotechnology), mouse anti-Nodal (Abcam, immunohis-tochemistry), donkey anti-rabbit AlexaFluor-488, and anti-mouseAlexaFluor-546 (Life Technologies).

Drug and antibody treatmentsFor DTIC experiments, working concentrations were prepared

immediately before application. Control conditions employedserum-free medium alone that was diluted equivalent to thehighest concentration of DTIC. Cells were typically evaluatedafter 72 hours. For immunofluorescence experiments, cells wereseeded on glass coverslips. For antibody experiments, cells wereantagonized with rabbit anti-Nodal antibodies or whole-mole-cule rabbit IgG (Jackson ImmunoResearch Laboratories) dilutedin medium, and were analyzed after 72 hours. In combinationexperiments, parallel cultures were incubated inDTIC diluted to 5mg/mL or equivalent volume of serum-free medium (control) for72hours, afterwhich cells werewashed andmediumwas replacedevery 24 hours. After 72 hours, anti-Nodal antibodies or rabbitIgG diluted to 3 mg/mL was added to cells and cultured for 72hours before analysis.

Flow cytometryCell viability assays were evaluated on a Guava Easycyte HT

Flow Cytometer using Guava viacount reagent according to themanufacturer's instructions (Millipore). Parameters were setusing untreated cells. Averages of triplicate samples were analyzedfor each data point.

Western blot analysisWhole-cell lysates were prepared as previously described (18),

and SDS-PAGE gel electrophoresis and Western blotting usedstandard techniques. Polyvinylidene difluoride membranes wereblocked in 5% nonfat milk, and antibodies diluted in either 5%milk or 5% bovine serum albumin overnight at 4�C, dependingon the manufacturer's recommendations. Signal was detectedusing West Pico Chemiluminescence Reagent (ThermoFisher).Membranes were stripped between antibodies using RestoreWestern blot stripping buffer (ThermoFisher). Specifically forNodal, relative protein levels were evaluated using ImageJ soft-ware with actin as reference.

ImmunofluorescenceFor phospho-HistoneH3 staining, cells on coverslipswerefixed

with 4% paraformaldehyde, and washed, blocked, and incubatedin antibodies according to the manufacturer's recommendedprotocol (Cell Signaling Technology). For Nodal staining, cellson coverslipswerefixedwith ice-coldmethanol, washed, blocked,and incubated in antibodies as previously described (23). Stain-ing was visualized on a Zeiss LSM510 Meta confocal microscopeunder a 25X LD.LCI.PlanApo multi-immersion objective andimages captured using ZEN 2009 software (Zeiss). Cells werecounted in at least 5 random fields in each of three independentexperiments. Mean and SD was presented graphically.

Reverse transcription and real-time PCRMessenger RNA was isolated from cells using the PerfectPure

Cell RNA Isolation Kit (5Prime). Reverse transcription and real-time PCR (RT-PCR)were performed as previously described usingTaqMan gene expression primer-probes (Applied Biosystems):Nodal (Hs00250630_s1) and RPLPO large ribosomal protein(4333761F). Experiments included cDNA samples generatedwithno reverse transcriptase to verify the absence of DNA contami-nation. Experiments were run in triplicate wells and were per-formed at least three times.

Matrigel invasion assaysInvasion assays were performed using BD biocoat growth

factor-reduced Matrigel invasion chambers in accordance withthe manufacturers' protocol (Corning). Briefly, after treatment,cells were plated in triplicate and invaded cells adhered to theunderside of the chamber were stained with 0.1% crystal violet.Dye was extracted using 10% acetic acid and optical density wasmeasured on a Bio-Rad plate reader at 570 nm. Raw data wereaveraged and converted to percentage of control for graphing. ForDTIC experiments, cells were treated with different DTIC con-centrations for 72 hours as described, the medium removed andcells washed every 24 hours for 72 hours before plating. Forcombination experiments, cells were treated with DTIC followedby anti-Nodal antibodies, rabbit IgG, or medium only control asdescribed before plating.

Patient samples and immunohistochemistryArchival formalin-fixed and paraffin-embedded melanoma

tissue sections from five patients that failed to respond to DTICtherapy were obtained from the Biospecimen Bank of the Mela-noma Institute Australia. Immunohistochemistry was performedon aMicromHMS710i autostainer usingmouse anti-Nodal (19).Color was developed with 3,30-diaminobenzidine and sections

Synergistic Effect of DTIC and Nodal Targeting in Melanoma

www.aacrjournals.org Mol Cancer Res; 13(4) April 2015 671




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were counterstained with Hematoxylin (Biocare Medical). Adja-cent serial sections were incubated with ChromPure mouse IgG(Jackson Immunoresearch Labs) as negative control. Sectionswere visualized and digital images captured and analyzed usinga Leica DM4000B microscope.

Multicellular tumor spheroid (MCTS) assaysSpheroids were generated essentially as previously described

(26). C8161 cellswere diluted in completemediumor inmediumcontaining DTIC (5 mg/mL) and either rabbit IgG or anti-Nodalantibodies (3mg/mL). Each conditionwas replicated in16wells ofa 96-well plate precoated with 1% (w/v) agar. Cells were forceaggregated by centrifugation at 1,000 � g. Plates were incubatedfor 72 hours before analysis. Spheroids were fixed in 4% para-formaldehyde overnight at 4�C, before being dehydrated andembedded in paraffin wax. Parallel 4 mm sections were depar-affinized and rehydrated before staining. Hematoxylin and eosinstaining used standard techniques. Immunofluorescence labelingof phospho-Histone H3 was performed using the recommendedprotocol except that antigen retrieval was performed in citratebuffer (pH 6.0) for 37 minutes at 97�C. Terminal deoxynucleo-tidyl transferase–mediated dUTP nick end labeling (TUNEL) wasperformed using a kit according to the manufacturer's protocol(Millipore). Sections were visualized and images captured asdescribed above, except that a 10� LD.LCI.PlanApomulti-immer-sion objective was utilized. For clarity, phospho-Histone H3fluorescence was pseudocolored red after image capture to dis-tinguish it from TUNEL staining.

ResultsEffects of DTIC on Nodal-expressing melanoma cells in culture

To address how exposure to DTIC affects Nodal-expressing,metastatic melanoma cells, we treated three cell lines (C8161, SK-MEL-28, and MV3) with increasing doses of DTIC for 72 hoursand evaluated cells by flow cytometry. Compared with controlcells that were exposed only to carrier, increasing doses of DTICresulted in a reduction in total viable cells in the population thatseemed to plateau at the highest concentrations tested (Fig. 1A).Importantly, the response to DTIC was somewhat dissimilaramong cell lines; C8161being themost sensitive cells,MV3havingan intermediate response, and SK-MEL-28 cells being least sensi-tive. Based upon this information, we identified the dose of DTICresponsible for a 50% reduction in cell population size (5 mg/mLfor C8161, 10 mg/mL for MV3, and 25 mg/mL for SK-MEL-28) andthe lowest dose capable of achieving an apparent maximal effect(25 mg/mL for C8161, 50 mg/mL for MV3, and 150 mg/mL forSK-MEL-28; Supplementary Fig. S1A–S1C). These identified con-centrations were then utilized in subsequent experiments.

To determine whether the response of these cell lines to DTICwas a consequence of effects on cellular growth, survival, or acombination of the two, cells treated with DTIC were analyzed byWestern blot analysis for markers of cell proliferation [phospho-

Histone H3 (Ser10)] and apoptosis (cleaved PARP; Fig. 1B). Incomparison with untreated controls, increasing doses of DTICcaused a dose-dependent reduction in Histone H3 phosphoryla-tion, whereas total Histone H3 levels remained consistent acrosstreatment groups. Conversely, PARP cleavage was not observed ineither C8161 or SK-MEL-28 cells at any drug concentration, whilein MV3 cells, some cleaved PARP was detected at the highestconcentration tested. To ascertain the response of these cells overtime, parallel treated wells were collected at 24-hour time points,and analyzed by flow cytometry for cell population size andprogrammed cell death. Comparedwith control cells that expand-ed in a fairly linear fashion and underwent 1 to 2 doublings in the72-hour time frame, DTIC treatment caused a dose-dependentreduction in the expansion rate (Fig. 1C). To determine whetherthis responsewas due to cell death aswell as growth inhibition,wealso analyzed the proportion of dead cells and found littledifference among the treatment groups (Fig. 1D), suggesting thatthe effect of DTIC is predominantly on restricting cellular growth.To evaluate this on a cell-by-cell basis, we performed immuno-fluorescence studies of phospho-Histone H3 on DTIC-treatedcells (Fig. 1E). Compared with control cells where numerousnuclei contained phosphorylated Histone H3, DTIC treatmentcaused a dose-dependent reduction of phospho-Histone H3–positive nuclei. Of note, positive cells were rarely detected at thehighest concentrations. In general, the difference in phospho-Histone H3–positive nuclei between control and treated cells wasstatistically significant (Supplementary Fig. S1D). Collectively,this suggests that DTIC treatment limits population growth pre-dominantly by reducing cell proliferation and not by inducingapoptosis.

Residual cells remaining after DTIC treatment maintainexpression of Nodal and are more invasive than parental cells

Nodal expression in cells treated with increasing doses of DTICwas evaluated at themRNA level by reverse transcriptase real-timePCR (RT-PCR) and at the protein level by Western blot analysis.Nodal mRNA levels were relatively unchanged among treatmentgroups (Fig. 2A) and Nodal protein levels were unchanged orslightly elevated relative to untreated cells (Fig. 2B). Given that wehave previously described Nodal protein to be limited to asubpopulation of cells in culture (21), we also analyzed Nodalprotein by immunofluorescence (Fig. 2C and Supplementary Fig.S2A and S2B). Surprisingly, comparedwith untreated control cellsthat displayed the typical proportion of Nodal-positive cells (Fig.2D; 22–31%), cultures exposed to increasing doses of DTICcontained a greater percentage of Nodal-positive cells that typi-cally increased proportionally with higher dosages of DTIC. In allcases, this was significantly different from controls (P < 0.05).Taken together, this suggests that residual cells persisting afterDTIC exposure retain Nodal expression.

To determine whether the residual cells surviving DTIC treat-ment differed from parental cells in their aggressiveness, we

Figure 1.DTIC affects proliferation but not cell death in metastatic melanoma cell lines. A, C8161, MV3, and SK-MEL-28 cells treated with increasing concentrations ofDTICwere analyzed by flowcytometry and viable cell population size determined as a proportion of untreated control cells. B, protein lysates fromDTIC-treated cellswere evaluated for Histone H3 phosphorylation at Ser10 (proliferation) and PARP cleavage (apoptosis) by Western blotting. Actin was the loading control.C and D, cell lines treated with DTIC for 72 hourswere analyzed at 24-hour time points for viable cell population size (calculated as a proportion of cells at plating; C)and percentage of cell death (D). E, histone H3 phosphorylation at Ser10 (green; white arrows) examined by confocal microscopy in DTIC-treated cells.Nuclei were counterstained with DAPI (blue). Scale bar, 50 mm.






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evaluated invasive behavior by exposing C8161 and SK-MEL-28cells toDTIC before plating inMatrigel invasion assays. Comparedwith controls, cells treated with DTIC typically exhibited a dose-dependent increase in the proportion of invaded cells (Fig. 2E).This increase was significantly greater than control cells at thehighest concentrations tested (P < 0.05), suggesting that DTIC-resistant residual cells exhibit more invasiveness, an observationcoincident with the increase in Nodal-positive cells.

DTIC therapy fails to eliminate Nodal-positive cells frompatient melanomas

To determine how Nodal expression is affected in humanmelanomas treated with DTIC, biopsy samples from a cohort offive relapsed patients whose melanomas were refractory to DTICtherapy were evaluated for Nodal. Tissue sections from tumorsbefore and after DTIC therapy were labeled with anti-Nodalantibodies and expressiondeterminedby immunohistochemistry(Fig. 3). In all patients, Nodal expressionwas observed both in thespecimens collected before exposure to a DTIC treatment regimeand in biopsy tissue taken following DTIC therapy. The observa-tion of Nodal protein in both DTIC-na€�ve and residual DTIC-resistantmelanomas offers proof-of-concept support for Nodal asa potential drug target in combination with chemotherapy.

Anti-Nodal antibodies adversely affect proliferation andapoptosis at high concentrations

Considering that Nodal expression is maintained in cellsexposed to DTIC, we reasoned that additional targeting of Nodalmight improve the response of cells to DTIC treatment. Anti-Nodal antibodies have previously been utilized to inhibit Nodalsignaling (23, 25), but have not been extensively evaluated inmelanoma cells. To first determine how cultured cells respond toNodal inhibition (independent of DTIC treatment), we exposedcells to increasing concentrations of anti-Nodal antibodies for 72hours, and evaluated cell growth and death by flow cytometry.Compared with untreated or rabbit IgG-treated control cells,cultures of C8161 and SK-MEL-28 cells treated with anti-Nodalantibodies at intermediate (3 mg/mL) and high (4–5 mg/mL)concentrations exhibited a reduced viable cell population size,that was significantly different from controls at the highest con-centrations (Fig. 4A and Supplementary Fig. S3A; P < 0.05).Furthermore, cells exposed to high concentrations of anti-Nodalantibodies (4–5 mg/mL) exhibited increased cell death that wassignificantly different from control cells (Fig. 4B and Supplemen-tary Fig. S3B; P < 0.05). In C8161 cells, the majority of thepopulation remained viable (54%), even at the highest antibodyconcentration evaluated, while, in SK-MEL-28 cells, death of themajority of cells (78%)was observed at the highest concentration.

To confirm the effects of anti-Nodal antibodies, Nodal proteinlevels, as well as markers of proliferation and apoptosis, wereevaluated in cell lysates (Fig. 4C). Importantly, Nodal expression

was downregulated in a concentration-dependent manner veri-fying that Nodal signaling was decreased by anti-Nodal antibo-dies (expected as Nodal expression is maintained by a positivefeedback loop). Similarly, a decrease in Histone H3 phosphory-lation was observed with increasing concentrations of anti-Nodalantibodies, while total Histone H3 levels remained constant.Surprisingly, when we evaluated apoptosis, we were unable todetect PARP cleavage, even at the highest concentrations of anti-bodies tested. Aswe had observed an increase in cell death by flowcytometry, we evaluated levels of an alternative apoptosis marker,B-cell lymphoma 2 (BCL2; an antiapoptotic protein). In contrastto lack of PARP cleavage, we did observe a decrease in BCL2,suggesting the likely early induction of apoptosis.

Targeting Nodal in addition to DTIC treatment is significantlymore effective than DTIC alone

To determine how cells would respond to anti-Nodal antibo-dies in addition to DTIC treatment, we pretreated cultures with

Figure 2.Effects of DTIC on Nodal expression in aggressive melanoma cell lines. A, Nodal mRNA levels were determined by real-time RT-PCR using RPLPO as endogenouscontrol (displayed as proportion of untreated control). B, protein lysates were evaluated for Nodal in C8161 cells by Western blotting. Densitometric analysisofNodal relative toActinwasperformedusing ImageJ software and calculated as a proportionof untreated controls. C,Nodal (green;white arrows)was examinedbyconfocal microscopy in DTIC-treated C8161 cells. Cell cytoplasm was labeled with an actin antibody (red) and nuclei were counterstained with DAPI (blue).Scale bar, 50mm. Inset shows corresponding area inwhite box.D, Nodal-positive cellswere counted inmultiplefields of view relative toDAPI-positive nuclei (averageis shown). E, cellular invasion was evaluated in DTIC-treated cells plated in Matrigel invasion assays. Average optical density is graphed as percentage ofuntreated control (� , P < 0.05).

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Figure 3.Nodal expression in DTIC-resistant melanoma. Images of Nodalimmunohistochemistry (brown) in tissue sections of DTIC na€�ve (pre-DTICtreatment; A, A0) and DTIC-resistant (post-DTIC treatment; B, B0) melanomafrom one representative patient. Mouse IgG at the same concentration asantibody was utilized as control (IgG control; C, C0). Sections werecounterstained with hematoxylin (blue).






DTIC, washed the cells, and then exposed cultures to anti-Nodalantibodies. At the lowest tested DTIC dose of 5 mg/mL, weobserved that further exposing cells to anti-Nodal antibodies atan intermediate concentration (3 mg/mL; only modestly effectiveby itself; Fig. 4 andSupplementary Fig. S3)was sufficient to inducea dramatic effect on cells. In contrast to all other treatment groups,cells that were pretreated with DTIC followed by anti-Nodalantibodies exhibited a striking decrease in the viable cell popu-lation (Fig. 5A and Supplementary Fig. S4A; P < 0.05). In contrast,cells exposed to DTIC and anti-Nodal antibodies displayed astriking increase in the proportion of programmed cell death thatwas significantly greater than untreated cells or cells exposed onlyto antibody (Fig. 5B and Supplementary Fig. S4B; P < 0.05).Importantly, the DTIC and anti-Nodal antibody combinationaffected the majority of the cell population (78% in C8161cells; Fig. 5B, 75% in SK-MEL-28 cells; Supplementary Fig.S4B). We observed a concurrent significant decrease in NodalmRNA levels in C8161 cells exposed to DTIC and anti-Nodalantibodies (P < 0.05; Fig. 5C), as well as a dramatic decrease inNodal protein, a complete loss of Histone H3 phosphorylation,and an increase in PARP cleavage, thatwas not observed under anyother conditions (Fig. 5D). Taken together, these data suggest thatcombining DTIC and anti-Nodal antibody treatments has asynergistic effect on apoptosis, far greater than either singletreatment at the corresponding concentrations.

To address how combined DTIC and anti-Nodal treatmentsmight affect cell behavior, invasiveness was evaluated in SK-MEL-28 cells treated with DTIC alone, or with DTIC followed by rabbitIgG or anti-Nodal antibodies. After treatment, cells were plated inMatrigel invasion assays where cultures treated with DTIC andanti-Nodal antibodies exhibited a significant reduction in cellularinvasion compared with DTIC control (Fig. 5E; P < 0.05). Thesedata suggest that targeting Nodal impairs the invasive ability ofDTIC-resistant melanoma cells.

Cells grown in multicellular tumor spheroid culture alsorespond to combinatorial DTIC and anti-Nodal antibodytreatment by undergoing apoptosis

The multicellular tumor spheroid (MCTS) assay more accu-rately represents in vivo tumors than monolayer cell culturesgiven its three-dimensional (3D) cellular arrangement (27). Toaddress the response of C8161 cells grown in 3D culture tocombined DTIC and anti-Nodal antibody exposure, MCTScultures were formed by centrifugation and grown eitheruntreated, with DTIC and rabbit IgG as a control, or with DTICand anti-Nodal antibodies (Fig. 6). Spheroids were fixed andserial sections were analyzed for proliferation and apoptosisusing phospho-Histone H3 staining and TUNEL assay, respec-tively. While some phospho-Histone H3–positive cells weredetected in MCTS sections from untreated and control cells,positive cells were rarely detected in sections of MCTS samplestreated with DTIC and anti-Nodal antibodies (compare Fig. 6C0

with Fig. 6A0 and B0). In contrast, TUNEL-positive, apoptoticcells were uncommon in untreated and control MCTS sections,but were abundant in MCTSs exposed to DTIC and anti-Nodalantibodies (compare Fig. 6C00 with Fig. 6A00 and B00). Thissuggests that combinatorial targeting also limits proliferationand induces apoptosis in a 3D assay.

Nodal expression is maintained and targetable in V600E B-RAFmutation-positive melanoma cells surviving vemurafenibtreatment

While DTIC is considered an older conventional therapy,targeted therapies, such as B-RAF inhibitors, are among newerstrategies aimed at improving patient response. Therefore, weperformed initial experiments to determine whether the persis-tence of Nodal is unique to cells surviving DTIC or is common tocells acquiring resistance to other drug treatments. SK-MEL-28cells (harboring the V600E B-RAF mutation) were treated with

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Figure 4.Response of C8161 cells to anti-Nodalantibody treatment. A and B, cellswere exposed to increasingconcentrations of anti-Nodalantibodies or rabbit IgG for 72 hours.Viable cell population size (relative tountreated control; A) and percentageof cell death (B) was determined byflow cytometry (� , P < 0.05). C, celllysates were analyzed by Westernblotting for Nodal, phospho-HistoneH3 (Ser10; proliferation), cleavedPARP (proapoptotic) and BCL2(antiapoptotic).

Hardy et al.





increasing doses of the B-RAF inhibitor RG7204 (vemurafenib)for 72 hours. Compared with DMSO-treated control cells,RG7204 treatment caused a dose-dependent reduction in thenumber of viable cells that plateaued at around 0.5 mmol/L,leaving approximately 50% of the cell population intact (Sup-plementary Fig. S5A). Importantly, in cell lysates, Nodal proteinwas detected at similar levels at all RG7204 concentrations tested(0.1–10 mmol/L; Supplementary Fig. S5B). To verify potency, ERKactivity was evaluated in lysates fromparallel cultures treatedwithRG7204 for 1 hour. A dose-dependent decrease in phosphory-lated ERK1/2was observed comparedwith total ERK1/2 (data notshown). In contrast, a Nodal-positive, B-RAF wild-type cell line(C8161) failed to respond to RG7204 at any concentration tested(0.1–10 mmol/L) and ERK phosphorylation was not altered (datanot shown).

To evaluate how treatment with anti-Nodal antibodiesaffects SK-MEL-28 cells surviving RG7204 treatment, cultures

were first treated with 0.5 mmol/L RG7204, then survivingcells exposed to anti-Nodal antibodies. Compared with cellspretreated with RG7204 (RG7204-resistant cells) or withRG7204-resistant cells exposed to rabbit IgG that doubledtwice over 72 hours, RG7204-resistant cultures exposed toanti-Nodal antibodies failed to expand (Supplementary Fig.S5C). Coincident with this, anti-Nodal antibody-treated cellsexhibited a marked decrease in viability (Supplementary Fig.S5D), and dramatic increase in cell death (Supplementary Fig.S5E) that was significantly different from control (P < 0.05).Taken altogether, these data indicate that Nodal expressionmaintained in B-RAF inhibitor–resistant cells is also targetablewith anti-Nodal antibodies. While this line of inquiry is notconsidered the primary focus of our study, the results arecomplementary and highly informative regarding the persis-tence of Nodal-positive subpopulations that are unaffected byB-RAF–targeted therapy.

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Figure 5.Combining DTIC and anti-Nodalantibody treatment induces dramaticcell death. C8161 cells were eitheruntreated or treated with 5 mg/mLDTIC, followed by no treatment, or3 mg/mL anti-Nodal antibodies orrabbit IgG. Endpoint viable cellnumber graphed as a percentage ofuntreated cells (A) and percent celldeath (B) were evaluated by flowcytometry. Nodal mRNA levels wereevaluated using real-time RT-PCR (C).Protein lysates were analyzed byWestern blotting forNodal, HistoneH3phosphorylation at Ser10(proliferation) and PARP cleavage(apoptosis). Actin was the loadingcontrol. � , significant difference fromuntreated cells and from cells treatedonly with anti-Nodal antibodies(P < 0.05). E, cellular invasion wasevaluated in SK-MEL-28 cellspretreated with DTIC, then exposed torabbit IgG or anti-Nodal or not furthertreated (no antibody) using Matrigelinvasion chambers. Invading cellswere quantified as percentage of noantibody control. #, P < 0.05.






DiscussionDespite recent advances in the field, metastatic melanoma is

still the leading cause of skin cancer deaths and remains highlyrefractory to current treatment strategies. In addition to thehistorically poor patient response to DTIC therapy, more recentlyFDA-approved treatments are unsuitable for many patients andtumors typically develop resistance to these agents over time(10, 11). The concept of combinatorial targeting is an attractivemeans of overcoming drug resistance in melanoma. Previouspreclinical and clinical studies have addressed possible ways tocircumvent the poor response to DTIC by including additionalchemotherapeutic agents (12) or by combining DTIC with tar-geted molecular inhibitors (28–33). In this study, we are first toestablish Nodal as a rational combinatorial target in melanomaand present a novel, synergistic combination of DTIC and anti-Nodal antibodies. The reexpression of Nodal in metastatic mel-anoma and other aggressive cancers is associated with advanceddisease progression, and contributes to the aggressive behavior ofcancer cells in vitro and in vivo (18, 19, 21, 23, 25). Hence, ourpreclinical study targeting Nodal in combination with an alreadystandardized treatment is a potentially exciting therapeutic strat-egy that warrants future indepth investigation.

In this study, in agreement with others, we show that DTICtreatment limits cell growth, but does not induce apoptosis in avariety of melanoma cell lines (29, 30). Of note, these cell linesdiffer in their B-RAF status: C8161 and MV3 cells are B-RAF wild-type, while SK-MEL-28 cells carry the V600E B-RAF mutation.While we observed SK-MEL-28 cells to be the most resistant to

DTIC treatment and C8161 cells to be the most sensitive (Fig. 1),the B-RAF status of these cells likely does not drastically impactresistance to DTIC as, in our hands, combining DTIC with theB-RAF inhibitor RG7204 has a minimal effect over either singleagent (K. Hardy and M. Hendrix, personal communication). Thethree metastatic melanoma cell lines (C8161, MV3, and SK-MEL-28) were primarily chosen for study because they have previ-ously been shown to express Nodal (18, 21) and not because oftheir B-RAF status, thus indicating that Nodal expression inmelanoma cells does not appear to correlate with B-RAF status.It is clear from our study that, regardless of the B-RAF status,Nodal continues to be expressed in these cells following DTICtreatment, even at high drug concentrations. Complementary tothis, Nodal was also observed to persist in tissue samples frommelanomas refractory to DTIC therapy. While the B-RAF status ofthese tumors is not known, as approximately 50% of all mel-anomas are thought to harbor a B-RAF mutation (34), it ispossible that at least one patient had B-RAF mutation-positivemelanoma. Also importantly, Nodal is still detected in B-RAFmutant SK-MEL-28 cells with acquired resistance to the B-RAFinhibitor, RG7204. Together, this suggests Nodal signaling as apossible broad combinatorial target to help circumvent drugresistance in both B-RAF wild-type and B-RAF mutation-carryingmelanomas.

Certainly in B-RAF mutation-positive melanomas, the MAPKpathway is a critical effector of tumor progression. In patientstreated with B-RAF andMEK inhibitors, resistance often developsvia the reactivation of MAPK signaling (10). Importantly, drugresistance still develops with recently FDA-approved combinedB-RAF/MEK inhibitor therapy (35, 36), likely via alternate MAPKpathway mutations (37). As targeting B-RAF mutations in mel-anoma does not eliminate MAPK dependency, the continuedsearch for alternative targetable MAPK-inducing mechanisms isjustified. In breast cancer, a noncanonical Nodal signaling path-way converges on MAPK signaling, and contributes to epithelial–mesenchymal transition, invasion, and tumor progression(38, 39).While it is not currently knownwhetherMAPK signalingis activated downstream of Nodal in melanoma, given our obser-vation that B-RAF inhibitor–resistant cells retain Nodal expres-sion and are sensitive to Nodal inhibition, it is exciting tospeculate that Nodal signaling may be an alternative mechanismcapable of inducing MAPK signaling in melanoma that could betargeted in combination with other MAPK-directed therapies.

Tumor cell heterogeneity is now a well-accepted phenomenonin vitro and in vivo (40, 41). Intracellular Nodal protein waspreviously detected in only a subpopulation of melanoma cellsin culture (21), highlighting its heterogeneous expression pattern.In agreement with this, we observe Nodal protein in 22%–31%ofuntreated cells (depending on the cell line) in the current study(Fig. 2). Interestingly though, in cell lines treated with DTIC, wedetect a dose-dependent increase in the proportion of Nodal-positive cells by immunofluorescence. One possible explanationfor this observation is that Nodal-expressing cells are somehowinsensitive to DTIC treatment. Another possibility is that Nodalexpression is upregulated by DTIC. The latter is unlikely as NodalmRNA levels remain relatively unchanged by all DTIC concentra-tions, suggesting no impact on Nodal gene transcription. Surpris-ingly, relative Nodal protein levels in cell lysates from DTIC-treated cells remain either unchanged or only slightly elevatedcompared with untreated control cells, suggesting no significantquantitative difference on a cell population basis, despite the

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Figure 6.C8161 multicellular tumor spheroids (MCTS) respond to combinatorial DTICand anti-Nodal antibody treatments. MCTS were formed from C8161cells and grown either untreated (A–A00), with DTIC plus 3 mg/mL rabbit IgG(B–B00) or with DTIC plus 3 mg/mL anti-Nodal antibodies (C–C00). Parallelsections from fixed and embeddedMCTSswere labeledwith hematoxylin andeosin (H&E; A–C), anti-phospho-Histone H3 (pHH3; red; indicated by whitearrowheads; A0–C0), or TUNEL reagent (green; indicated by white arrows;A00–C00) and counterstained with DAPI (blue). Scale bar, 100 mm.


Hardy et al.




observed expanded Nodal-positive subpopulation. The reasonsfor this discrepancy are unclear, but the expansion of the Nodal-positive subpopulation is a likely explanation for the dramaticeffect on cells in combinatorial DTIC and anti-Nodal antibodyexperiments. We show that combinatorial treatment renders cellssignificantly more sensitive to apoptosis than either single treat-ment, resulting in the death of close to 80%of cells in culture (Fig.5). Importantly, at the concentration utilized in combinatorialexperiments, DTIC treatment alone causes an expansion of theNodal-positive subpopulation to approximately 60%, whichcould not fully explain the increased sensitivity to anti-Nodalantibodies. Thus, we reason that the disproportionate increase incell death may be attributable to the "bystander effect", wherebysignals generated by one dying cell influence the survival ofneighboring cells in a paracrine manner (42).

Despite the acceptance that tumor cell populations are hetero-geneous with distinct subpopulations of cells that may retain theplasticity to transition between molecular phenotypes, the con-cept of a true stem cell population in cancer remains controversial(40, 41). Nodal signaling plays an important role in maintainingpluripotency in embryonic stem cells (43, 44), so it is conceivablethat Nodal may also contribute to a cancer stem cell phenotype.However, as Nodal is a secreted protein, it has been challenging tospecifically isolate and study the live Nodal-positive subpopula-tion to evaluate this possibility, as has been done with putativecancer stem cell surface markers (45, 46). Certainly, Nodal isimportant in tumor cell plasticity, as it is associated with vasculo-genic mimicry in melanoma (18, 25, 47); specifically, down-regulation of Nodal suppresses this endothelial phenotype andabrogates vasculogenic mimicry (18, 25). In addition, the het-erogeneous cancer cell phenotype within a tumor may be asso-ciated with drug resistance, whereby cells with specific propertiesenabling them to withstand drug treatment are preferentiallyselected (48). Whether Nodal is specifically associated or simplycoincident with a drug-resistant subpopulation is not entirelyclear from the current study. Interestingly, a recent study ofABCB5-expressing melanoma cells (an ABC transporter associat-ed with drug resistance) identified the selective survival of thissubpopulation in response toDTIC treatment (49). Given that weobserve a similar increase in the Nodal-positive subpopulation inthe current study, it is exciting to speculate that Nodal may beassociated with a drug-resistant subpopulation with plasticproperties.

We found a synergistic increase in cell killing utilizingDTIC andanti-Nodal antibody concentrations incapable of producingmeaningful effects alone, in cultured cells and tumor spheroids.This has potential significance for future in vivo studies where the3D architecture, host immune response, and other side effects

must be considered. DTIC at currently administered clinicaldosages causes numerous side effects (12). Our study suggeststhe possibility that, in combination with anti-Nodal antibodies,the effective concentration of DTIC could be decreased, poten-tially reducing side effects. The efficacy of anti-Nodal antibodieshas previously been tested in an experimental lung colonizationnudemousemodel using onemetastaticmelanoma cell line (25).This study showed both a decrease in lung melanoma coloniesand an increase in apoptotic cells, suggesting that anti-Nodalantibodies canbe effective in in vivomodels.Most noteworthy, theantibody concentration employed in the in vivo study was greaterthan that utilized in combinatorial experiments in the current invitro study. While we understand that concentrations utilized invitro arenot necessarily directly applicable in vivo, the current studyat least suggests the exciting possibility that combinatorial experi-ments may be effective. Thus, we conclude that the pursuit of invivo combinatorial studies with DTIC and Nodal as a target areboth warranted and should include the evaluation of agents atreduced concentrations that may be ineffective administeredindividually.

Disclosure of Potential Conflicts of InterestM.J.C. Hendrix holds a patent to target Nodal. No potential conflicts of

interest were disclosed by the other authors.

Authors' ContributionsConception and design: K.M. Hardy, L. Strizzi, M.J.C. HendrixDevelopment of methodology: K.M. Hardy, L. StrizziAcquisition of data (provided animals, acquired and managed patients,provided facilities, etc.): K.M. Hardy, N. Margaryan, K. Gupta, G.F. Murphy,R.A. ScolyerAnalysis and interpretation of data (e.g., statistical analysis, biostatistics,computational analysis): K.M. Hardy, L. StrizziWriting, review, and/or revision of the manuscript: K.M. Hardy, L. Strizzi,N. Margaryan, R.A. Scolyer, M.J.C. HendrixStudy supervision: M.J.C. Hendrix

Grant SupportThis work was supported by NIH grants U54CA143869, R37CA59702,

and R01CA121205 from the NCI (M.J.C. Hendrix) and the EisenbergResearch Scholar Fund (to L. Strizzi), as well as a kind gift from the RobertKris family.

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 February 7, 2014; revised December 2, 2014; accepted January 5,2015; published OnlineFirst March 12, 2015.

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2015;13:670-680. Published OnlineFirst March 12, 2015.Mol Cancer Res Katharine M. Hardy, Luigi Strizzi, Naira V. Margaryan, et al. Synergistic Anticancer Effects in Metastatic MelanomaTargeting Nodal in Conjunction with Dacarbazine Induces

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