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Myeloma CellsRelease of NKG2D MIC Ligands in MultipleSenescence-Associated ADAM10-Dependent Genotoxic Stress Induces

SantoniCerboni, Rossella Paolini, Marco Cippitelli and AngelaMaria Giulia Amendola, Joannis Mytilineos, Cristina Teresa Petrucci, Maria Rosaria Ricciardi, Daniel Fuerst,Cinzia Fionda, Rosa Molfetta, Alessandra Soriani, Maria Alessandra Zingoni, Francesca Cecere, Elisabetta Vulpis,

ol.1402643http://www.jimmunol.org/content/early/2015/06/12/jimmun

published online 12 June 2015J Immunol 

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The Journal of Immunology

Genotoxic Stress Induces Senescence-AssociatedADAM10-Dependent Release of NKG2D MIC Ligands inMultiple Myeloma Cells

Alessandra Zingoni,*,1 Francesca Cecere,*,1 Elisabetta Vulpis,* Cinzia Fionda,*

Rosa Molfetta,* Alessandra Soriani,* Maria Teresa Petrucci,† Maria Rosaria Ricciardi,†

Daniel Fuerst,‡,x Maria Giulia Amendola,* Joannis Mytilineos,‡,x Cristina Cerboni,*

Rossella Paolini,* Marco Cippitelli,* and Angela Santoni*,{

Genotoxic stress can promote antitumor NK cell responses by upregulating the surface expression of activating ligands on cancer

cells. Moreover, a number of studies suggested a role for soluble NK group 2D ligands in the impairment of NK cell tumor rec-

ognition and killing. We investigated whether genotoxic stress could promote the release of NK group 2D ligands (MHC class I–

related chain [MIC]A and MICB), as well as the molecular mechanisms underlying this event in human multiple myeloma (MM)

cells. Our results show that genotoxic agents used in the therapy of MM (i.e., doxorubicin and melphalan) selectively affect the

shedding of MIC molecules that are sensitive to proteolytic cleavage, whereas the release of the short MICA*008 allele, which is

frequent in the white population, is not perturbed. In addition, we found that a disintegrin and metalloproteinase 10 expression is

upregulated upon chemotherapeutic treatment both in patient-derived CD138+/CD38+ plasma cells and in several MM cell lines,

and we demonstrate a crucial role for this sheddase in the proteolytic cleavage of MIC by means of silencing and pharmacological

inhibition. Interestingly, the drug-induced upregulation of a disintegrin and metalloproteinase 10 on MM cells is associated with

a senescent phenotype and requires generation of reactive oxygen species. Moreover, the combined use of chemotherapeutic drugs

and metalloproteinase inhibitors enhances NK cell–mediated recognition of MM cells, preserving MIC molecules on the cell

surface and suggesting that targeting of metalloproteinases in conjunction with chemotherapy could be exploited for NK cell–

based immunotherapeutic approaches, thus contributing to avoid the escape of malignant cells from stress-elicited immune

responses. The Journal of Immunology, 2015, 195: 000–000.

Increasing evidence supports a pivotal role for innate immuneeffector cells, such as NK cells, in tumor surveillance. NKcells’ rapid response is related primarily to their ability to

release cytolytic mediators, such as perforin and granzymes, or to

express ligands able to trigger death receptors on target cells. NKcell activation is tightly regulated by a delicate balance betweenactivating and inhibitory signals, with the latter being primarilytransduced by receptors for MHC class I molecules (KIRs, CD94/NKG2A). Recognition of abnormal self on tumor cells triggersa number of non-MHC class I–restricted activating receptors,such as NK group 2D (NKG2D), DNAX accessory molecule-1(CD226), and the natural cytotoxicity receptors (1).NKG2D, a C-type lectin-like receptor expressed on NK cells,

gd T cells, CD8+ T cells, and some autoreactive or immunosup-pressive CD4+ T cells (2), represents a major recognition receptorfor the detection and elimination of transformed cells (3, 4). Inhumans, NKG2D binds to MHC class I–related chain (MIC)A,MICB, and UL16-binding proteins, whose expression is either nullor low on normal tissues; however, it is induced by different typesof stress, including tumoral transformation, viral and bacterialinfections, and autoimmune diseases (5, 6).NKG2D ligand (NKG2DL) expression is finely regulated by

transcriptional, posttranscriptional, and posttranslational mech-anisms, with the latter comprising shedding through the action ofmetalloproteinases or exosome release. Thus, in addition to be-ing displayed on the cell surface, NKG2DLs can be shed or se-creted from different cell types (7–9), and they appear as solubleforms in the sera of most cancer patients (10, 11). The releaseof soluble (s)MIC was suggested to be a major mechanism oftumor cell evasion from NKG2D-mediated immune surveillance.Indeed, sMICA/B can cause systemic downregulation of NKG2Dsurface expression, thereby impairing NKG2D-dependent lysis oftumor cells (11, 12). Concomitantly, the reduction in NKG2DL

*Department of Molecular Medicine, “Sapienza” University of Rome, Rome 00161,Italy; †Department of Cellular Biotechnology and Hematology, “Sapienza” Univer-sity of Rome, 00161 Rome, Italy; ‡Institute for Clinical Transfusion Medicine andImmunogenetics Ulm of the German Red Cross Blood Transfusion Service, BadenWuerttemberg-Hessen, 89081 Ulm, Germany; xInstitute of Transfusion Medicine,University of Ulm, 89081 Ulm, Germany; and {Institute Pasteur-Fondazione CenciBolognetti, “Sapienza” University of Rome, 00161 Rome, Italy

1A.Z. and F.C. contributed equally to this work.

Received for publication October 17, 2014. Accepted for publication May 6, 2015.

This work was supported by grants from the Italian Association for Cancer Re-search (AIRC Investigator Grant and AIRC 5x1000), the Italian Ministry of Uni-versity and Research (Projects of National Interest and Basic Research InvestmentFund PRIN/2010NECHBX_004/MC), the “Sapienza” University of Rome, theCenter of Excellence for Biology and Molecular Medicine, and the Italian Instituteof Technology.

Address correspondence and reprint requests to Alessandra Zingoni and Angela San-toni, Department of Molecular Medicine, “Sapienza” University of Rome, Viale ReginaElena 291, Rome 00161, Italy. E-mail addresses: alessandra.zingoni@uniroma1.it(A.Z.) and angela.santoni@uniroma1.it (A.S.)

The online version of this article contains supplemental material.

Abbreviations used in this article: ADAM, a disintegrin and metalloproteinase; Ct,threshold cycle; DOX, doxorubicin; bGal, b-galactosidase; GAM, goat anti-mouse;GPI, glycosylphosphatidylinositol; MEL, melphalan; MIC, MHC class I–relatedchain; MM, multiple myeloma; MMP, matrix metalloproteinase; NAC, N-acetyl cys-teine; NKG2D, NK group 2D; NKG2DL, NKG2D ligand; PC, plasma cell; PI, propi-dium iodide; ROS, reactive oxygen species; s, soluble; SASP, senescence-associatedsecretory phenotype; shRNA, short hairpin RNA.

Copyright� 2015 by The American Association of Immunologists, Inc. 0022-1767/15/$25.00

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density on the tumor cell surface likely triggers less efficientkilling.Shedding of MIC molecules primarily depends on a proteolytic

process involving multiple enzymes, including a disintegrin andmetalloproteinase (ADAM)9, ADAM10, ADAM17, some mem-bers of the matrix metalloproteinases (MMP) family (MMP9 andMMP14), and the disulfide isomerase Erp5 (13). Another level ofcomplexity is reflected by the fact that MICA displays a highdegree of polymorphism leading to an allele-dependent shedding,as demonstrated for the allelic variant MICA*008 released inassociation with exosomes (14).Multiple myeloma (MM) is a clonal B cell malignancy

characterized by expansion of plasma cells (PCs) in the bonemarrow. It is an incurable disease with a median survival ofa few years, and its prognosis has been improved by the use ofautologous hematopoietic stem cell transplantation and newimmunochemotherapeutic approaches (15).Among immune cells controlling MM progression, NK cells have

long been considered key players, because they are expanded andactivated during the early stages of disease and can recognize andkill the tumor cells (16). NKG2D was reported to play an importantrole in the NK cell–mediated killing of MM cells (17–19), andhigh levels of sMICA in sera of MM patients correlate with thedisease stage, strongly suggesting sMICA as an independent prog-nostic factor in newly diagnosed MM patients (20, 21).A novel strategy to improve NK cell–mediated antitumor ac-

tivity may involve the upregulation of activating ligands on tu-mor cells. Enhancement of NK cell–mediated recognition ofMM cells was reported by us and other investigators, showingincreased surface expression of NKG2DLs on tumor cells fol-lowing treatment with genotoxic agents (22) or with GSK3,HSP-90, and histone deacetylase inhibitors (23–25). Importantly,drug-induced expression of these ligands on MM cells was al-ways associated with their ability to trigger increased NK celldegranulation in an NKG2D-dependent manner. Our previousfindings indicate that upregulation of NKG2DL expression onMM cells in response to genotoxic drugs depends on DNAdamage response activation and reactive oxygen species (ROS)signaling and is associated with the induction of a drug-inducedsenescent phenotype (22, 26).In the current study, we investigated the mechanisms regulating

the shedding of different allelic variants of MICA and MICBfollowing treatment of MM cells with sublethal doses of genotoxicdrugs: doxorubicin (DOX) and melphalan (MEL). To our knowl-edge, we first demonstrate that drug treatment can stimulate theshedding of MICA long alleles and MICB by a mechanism that isprimarily dependent on ADAM10, whereas the release of theMICA*008 short allele is not perturbed. We also provide novelevidence that genotoxic agents result in upregulation of ADAM10expression that is associated with a senescent phenotype and re-quires ROS generation. Finally, our data show that the combineduse of chemotherapeutic drugs and metalloproteinase inhibitorsstrongly increases NK cell–mediated recognition of MM cells,preserving MIC molecules on the cell surface.

Materials and MethodsAbs and reagents

Anti-MICA (clone 159227, IgG2b), anti-MICB (clone 236511; IgG2b),anti-ADAM10 (clone 163003; IgG2b), and anti-ADAM17 (clone 111633;IgG1) were from R&D Systems (Minneapolis, MN). Anti-PDIA6 (Erp5)Ab (ab11432) was purchased from Abcam (Cambridge, U.K.); anti-Erp5(sc-365260; IgG2b) was from Santa Cruz Biotechnology (Santa Cruz,CA). Control mouse IgG1 (clone MOPC-21) was from BioLegend (SanDiego, CA); F(ab)2 fragments of allophycocyanin-conjugated goat anti-

mouse ([GAM]-allophycocyanin or GAM-PE) IgG were from JacksonImmunoResearch Laboratories (West Grove, PA). Other Abs used wereanti-MICA (clone AMO1; IgG1; BamOmaB, T€ubingen, Germany);anti–b-actin (clone AC-74; IgG2a; Sigma-Aldrich, St Louis, MO); anti–CD3/allophycocyanin-H7 (clone SK7), anti-CD56/PE (clone NCAM16.2),anti–CD16/PerCP-CY5.5 (clone 3G8), anti-CD107a/allophycocyanin (cloneH4A3), anti-CD38/allophycocyanin (clone HIT2), anti-CD138/FITC,CD138/PerCP (clone MI15) (all from BD Biosciences); and AlexaFluor 488 GAM IgG Ab and Alexa Fluor 594 cholera toxin subunit Bconjugate (C22842) (Life Technologies, Gaithersburg, MD). ELISA kitsfor sMICA, sMICB, and syndecan (CD138) were from R&D Systems. TheADAM10 inhibitor GI254023X was kindly provided by Dr. A. Ludwig(Institute for Biochemistry, Christian-Albrechts-University Kiel, Kiel,Germany). Other reagents used were MMP inhibitor marimastat, para-formaldehyde, propidium iodide (PI), trypan blue, Polybrene (8 mg/ml)(hexadimethrine bromide), N-acetyl cysteine (NAC), and PI-PLC (all fromSigma-Aldrich).

Cell lines and clinical samples

The human MM cell lines SKO-007(J3), U266, RPMI 8226, ARP, ARK,OPM-2, and LP1 were provided by P. Trivedi (“Sapienza” University ofRome). The cell lines were maintained at 37˚C and 5% CO2 in RPMI1640 (Life Technologies, Gaithersburg, MD) supplemented with 10%FCS. All cell lines were mycoplasma free (EZ-PCR Mycoplasma test kit;Biological Industries). Bone marrow samples from patients with MMwere managed at the Division of Hematology (“Sapienza” University ofRome). Informed consent, in accordance with the Declaration of Hel-sinki, was obtained from all patients, and approval was obtained from theEthics Committee of “Sapienza” University of Rome. The bone marrowaspirates were processed, as previously described (22). In some experi-ments, myeloma cells were purified using anti-CD138 magnetic beads(Miltenyi Biotec, Auburn, CA). More than 95% of the purified cellsexpressed CD138 and CD38.

RNA isolation, RT-PCR, and real-time PCR

One microgram of total RNA, isolated by TRIzol reagent (Life Tech-nologies, Grand Island, NY), was used for cDNA first-strand synthesis ina 25-ml reaction volume, according to the manufacturer’s protocol forMoloney murine leukemia virus reverse transcriptase (Promega, Madison,WI). Real-time PCR was performed using the ABI Prism 7900 SequenceDetection system (Applied Biosystems, Foster City, CA). cDNAs wereamplified in triplicate with primers for ADAM10 (Hs00153853_m1),ADAM17 (Hs01041915_m1), and b-actin (Hs99999903_m1) conjugatedwith fluorochrome FAM (Applied Biosystems). The level of ADAMexpression was measured using threshold cycle (Ct), which was obtainedby subtracting the Ct value of the gene of interest from that of thehousekeeping gene (b-actin). In the current study, we used the Ct of theuntreated sample as the calibrator. The fold change was calculated as22DDCt, where DDCt is the difference between the Ct of the sample andthe Ct of the calibrator (according to the formula, the value of the cali-brator in each run is 1).

Plasmids

The pMSCV retroviral vector (Clontech Laboratories, Mountain View, CA)was used to clone the cDNA encoding the MICA*008, MICB, andMICA*019 sequences. MICA*008 and MICB cDNA were amplified fromthe SKO-007(J3) cell line, whereas MICA*019 cDNA was provided byLewis L. Lanier (University of California, San Francisco). For knocking downADAM10 and ADAM17, we used pLKO.1-sh-ADAM10 (TRCN0000006672)and pLKO.1-sh-ADAM17 (TRCN0000052168) lentivirus vectors and thecontrol vector pLKO nontarget short hairpin RNA (shRNA) (all from Sigma-Aldrich).

Virus production and in vitro transduction

For retrovirus production, the Phoenix retrovirus packaging cell lineHEK293 was transfected with viral DNA (pMSCV, pMSCV/MICA*008,pMSCV/MICB, and pMSCV/MICA*019) and the packaging vectors(pCMVgag-pol and pMD2.G) using Lipofectamine Plus (Invitrogen, SanDiego, CA). With regard to lentivirus production, Phoenix cells weretransfected with pLKO-sh-ADAM10, pLKO-sh-ADAM17, or the controlvector pLKO plus psPAX2 and pMD2.G packaging plasmids. After 48 h,virus-containing supernatants were harvested, filtered, and used immedi-ately for infection as follows: 2 ml viral supernatant containing Polybrene(8 mg/ml) was used to infect 1 3 106 SKO-007(J3) or LP1 cells for 2 h.Two infection cycles were performed.

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Drug treatment

Cells were cultured in six-well tissue culture plates for different times(24–72 h) at a concentration of 3 3 105 cells/ml with different drugs. Insome experiments, marimastat (10 mM) or GI254023X (10 mM) wasadded to the cell culture. A dose-response curve was constructed for bothmetalloproteinase inhibitors to identify the doses that did not affect cellviability. DOX and MEL doses used to treat the different cell lines weredetermined as previously described (22) and corresponded to sublethaldoses 10 times lower than the IC50 values: DOX: ARK (0.06 mM), LP-1(0.06 mM), OPM-2 (0.08 mM), RPMI-8226 (0.05 mM), SKO-007(J3)(0.05 mM), and U266 (0.1 mM); MEL: ARK (7 mM), LP-1 (21.5 mM),OPM-2 (1.6 mM), RPMI-8226 (1.5 mM), SKO-007(J3) (22 mM), andU266 (15 mM). In some experiments, cells were incubated with 10 mMNAC for 1 h before the drug treatment, as previously described (26).Patient-derived PCs were incubated with MEL (10 mM) or DOX (0.05mM) for 48–72 h at 37˚C and 5% CO2.

Immunofluorescence, FACS, and microscopic analysis

The expression of the NKG2DLs and ADAM10 and ADAM17 on MM cellswas analyzed by immunofluorescence staining using unconjugated mAbs,followed by secondary GAM-allophycocyanin or GAM-PE Abs. In someexperiments, cells were stained with PI (1 mg/ml) to assess cell viability. Theanalysis of ligand and ADAM expression on patient-derived PCs was per-formed by gating on the CD38+CD138+ PC population. Samples were ana-lyzed using a FACSCanto (BD Biosciences, San Jose, CA). For microscopyexperiments, samples were prepared and visualized using a fluorescencemicroscope equipped with the ApoTome system, as previously described (27).

Degranulation assay

As the source of effector cells, we used PBMCs isolated from healthy donorsby Lymphoprep (Nycomed, Oslo, Norway) gradient centrifugation andthen cocultured for 10 d with the irradiated (30 Gy) EBV-transformedB cell line RPMI 8866 at 37˚C in a humidified 5% CO2 atmosphere,as previously described (28). On day 10, the cell population was rou-tinely .95% CD56+CD16+CD3+, as assessed by immunofluorescenceand flow cytometry analysis. NK cells were activated overnight with200 U/ml human rIL-2 (R&D Systems). Drug-treated SKO-007(J3) cellswere incubated with activated NK cells in a U-bottom 96-well tissueculture plate in complete medium at 37˚C and 5% CO2 for 2 h. There-after, cells were washed with PBS/2% FCS and incubated with the ly-sosomal marker CD107a/allophycocyanin for 45 min at 4˚C. In someexperiments, before the assay, target cells were incubated with anti-MICA (clone 159227; IgG2b; R&D Systems) and anti-MICB (BamO-maB clone AUMO1; mouse IgG2a) at 2 mg/106 cells for 20 min at roomtemperature. Cells were washed with complete medium and used astarget in the degranulation assay. Blocking of the NKG2D receptor wasperformed as previously described (29).

When patient-derived PCswere used as targets, as the source of effector cells,we used autologous bone marrow purified NK cells or autologous bonemarrow CD1382 cells cultured for 2 d in complete medium supplementedwith 200 U/ml IL-2. Drug-treated patient-derived PCs were incubated witheffector cells in complete medium at 37˚C and 5% CO2 for 2 h. Thereafter,cells were washed with PBS/2% FCS and stained with CD107a/allophycocyanin and anti–CD3/allophycocyanin-H7, anti-CD56/PE, andanti-CD16/PerCP to gate the CD32CD56+CD16+ NK population. Analy-ses were performed using a FACSCanto (BD Biosciences).

Western blot analysis

ForWestern blot analyses, SKO-007(J3) or LP1 cells were pelleted, washedonce with cold PBS, resuspended in lysis buffer (1% Triton X-100 [v/v], 50mM Tris HCl [pH 7.6], 1 mM EGTA, 150 mMNaCl, 10 mMNaF, completeprotease inhibitor mixture [Sigma-Aldrich]), and incubated for 20 min onice. The lysate was centrifuged at 14,000 3 g for 15 min at 4˚C, and thesupernatant was collected as whole-cell extract. Protein concentration wasdetermined with the Bio-Rad Protein Assay. A total of 30–50 mg cellextract was run on 8% denaturing SDS-polyacrylamide gels. Proteinswere electroblotted onto nitrocellulose membranes (Schleicher & Schuell,Keene, NJ) and blocked in 5% milk in TBST buffer. Immunoreactivebands were visualized on nitrocellulose membranes, using HRP-coupledgoat anti-rabbit or GAM Igs and an ECL detection system (GE HealthcareAmersham), following the manufacturer’s instructions.

ADAM10 catalytic activity

ADAM10 catalytic activity was evaluated using a SensoLyte 520 ADAM10Activity Assay Kit *Fluorimetric* (cat. #72226; AnaSpec). SKO-007(J3)

cells were treated with DOX for 72 h. Cells were harvested, and 100 mgtotal cell lysate, prepared according to the manufacturer’s instructions, wasincubated with 5-FAM/QXL 520 ADAM10 substrate for 30 or 60 min.Fluorescence signal was measured at 490/520 nm. Fluorescence readingsare expressed in relative fluorescence units.

MICA gene typing

For the genotyping ofMICA, genomic DNA, derived from patients’ PBMCsor from different MM cell lines, was isolated from 1 3 106 cells using theGenomic DNA purification kit, according to the manufacturer’s instruc-tions (Bioline, London, U.K.). Sequence-based typing of MICA was per-formed as described (30).

Analysis and isolation of senescent cells

We performed a senescence-associated b-galactosidase (bGal) assay, us-ing the fluorogenic substrate C12FDG (Invitrogen), to measure bGal ac-tivity by flow cytometry, as previously described (26). Surface staining ofADAM10 was performed on SKO-007(J3) cells or on ex vivo primary PCs,as previously described, and the C12-fluorescein signal was measured onthe FL-1 detector. In some experiments, 72-h DOX-treated SKO-007(J3)cells were incubated with C12FDG, and bGalhigh and bGallow MM cellswere sorted using a FACSAria (BD Biosciences).

Statistics

Statistical analysis was performed with the Student paired test. Error barsrepresent SD or, where indicated, SEM.

ResultsDrug treatment of MM cells selectively affects the release ofMIC molecules sensitive to proteolytic cleavage

To investigate whether genotoxic stress results in MICA and MICBrelease from MM cells, we initially determined the expressionlevels and the release of sMICB and sMICA together with MICAgenotype in different MM cell lines (Table I). Notably, all MM celllines expressed the MICA*008 short allele that is present morefrequently in nearly all populations. Despite the fact that MICAwas detected on the cell surface (data not shown) and in celllysates (as evaluated by FACS and ELISA, respectively), it wasnot detectable in the supernatants of SKO-007(J3), U266, ARP-1,and RPMI cells. Instead, MICB was released from SKO-007(J3)and U266 cell lines (Table I).We then examined whether the release of MICB in the super-

natants of SKO-007(J3) and U266 cell lines could be modulated bytreatment with DOX and MEL at doses not affecting cell viability(22). Interestingly, drug treatment strongly promoted the release ofsMICB at 48 h, which increased further at 72 h (Fig. 1A, 1B, leftpanels). Because these treatments block cell proliferation, data alsowere normalized taking into account cell number; we noted aneven greater difference between untreated and drug-treated cells(Fig. 1A, 1B, right panels). When the relative shedding of MICBwas analyzed, a significant increase was observed in drug-treatedcells compared with untreated cells, indicating that increased MICBlevels in the supernatant were attributable to drug-induced MICBexpression (data not shown) (22) and were dependent on stimulationof the shedding process (Fig. 1C, 1D). In contrast, we did not detectsMICA after drug treatment (data not shown), despite the en-hancement of its cell surface expression (22) (data not shown).The notion that different MIC alleles exhibit distinct molecular

structures and different shedding modalities (14, 31) prompted usto examine whether the chemotherapeutic agents could differen-tially affect the release of distinct MICA alleles. To this end, theMM cell line LP1, which does not express endogenous MICA andMICB, was stably transduced with cDNA encoding MICA*008(carrying a truncated cytoplasmic tail and released by exosomes orthrough exocytosis), MICA*019 (which represents the prototypeof the long form of MICA alleles), or MICB and exposed to DOX

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treatment. Transfection resulted in ligand expression, as evaluatedby immunofluorescence, FACS analysis, fluorescence microscopy,and Western blot, and, importantly, transduced MIC molecules werereleased in the supernatants (Supplemental Fig. 1). Interestingly, DOXtreatment upregulated only the shedding of the long MICA*019 allele,whereas it did not perturb the release of MICA*008 (Fig. 1E). Asexpected, MICB shedding also was strongly stimulated, confirming ourobservations for SKO-007(J3) and U266 cells.Altogether, these data demonstrate that drug treatment of MM

cells stimulates the shedding of MICA and MICB and selectively

affects the release of MIC molecules that are sensitive to metal-loproteinase cleavage (i.e., MICA*019 and MICB).

Inhibition of MICB shedding on drug-treated cells by treatmentwith metalloproteinase inhibitors increases their susceptibilityto NK cell–mediated killing

To investigate whether drug-induced MICB release involves me-talloproteinase activity (32, 33), we evaluated MICB levels inthe supernatants of DOX- and MEL-treated SKO-007(J3) cells inthe presence of the metalloproteinase inhibitor marimastat. As

Table I. Expression levels and shedding profiles of endogenous MICA and MICB determined in different MM cell lines

MM Cell Lines

MICA MICB

Geonotype Cell Lysate (ng/ml) Supernatant (ng/ml) Cell Lysate (ng/ml) Supernatant (ng/ml)

SKO-007(J3) 008:01/008:01 1.108 ,0.0625 0.899 3.533U266 008:01/008:01 0.442 ,0.0625 0.223 0.525RPMI 008:02/008:01 0.299 ,0.0625 ,0.156 ,0.156ARP-1 004/008:01 0.829 ,0.0625 ,0.156 ,0.156

MICA gene typing was performed as described in Materials and Methods. Cells were plated at 0.3 3 106/ml and incubated for 72 h. The amount of MICA and MICB in celllysates and in the supernatants was evaluated using specific ELISA. Detection limits of MICA and MICB ELISA are indicated and correspond to 0.0625 and 0.156 ng/ml,respectively. A representative experiment of five is shown.

FIGURE 1. Drug treatment of MM cells selectively affects the shedding process of MIC molecules sensitive to proteolytic cleavage. SKO-007(J3) (A)

and U266 (B) cells were plated, at 3 3 105 cells/ml, in the presence of suboptimal doses of DOX and MEL. After 24, 48, and 72 h of incubation, cells were

harvested, and supernatants were collected and analyzed for the presence of sMICB using specific ELISA. The mean of three independent experiments is

shown. Relative shedding of MICB in SKO-007(J3) (C) and U266 (D) cells. Relative shedding, evaluated after 72 h, represents the ratio of the amount of

MICB (ng/ml) detected in conditioned medium/cellular lysates (ng/ml). The mean of four independent experiments is shown. (E) LP1 MM cells transduced

with MICA*008, MICA*019, or MICB were treated as described in (A). After 48 h, cells were washed and plated at 0.53 106 cells/ml in fresh medium and

incubated for an additional 18 h. Supernatants were collected and analyzed for the presence of sMICA or sMICB by specific ELISA. Mean of four in-

dependent experiments 6SEM is shown. *p , 0.05, **p , 0.01. ns, not significant.

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shown in Fig. 2A, marimastat inhibited the amount of sMICB, ina dose-dependent manner, on both untreated and drug-treatedcells, and this inhibition was accompanied by a concomitant in-crease in cell surface MICB (Fig. 2B, 2C). In accordance with theevidence that the MICA*008 allele is resistant to proteolyticcleavage (14), marimastat treatment did not result in a significantupregulation of cell surface MICA expression (Fig. 2B, 2C). We

explored whether the differential behavior of MICA*008 andMICB could be affected by the association mode to the plasmamembrane. Because one of the modifications acquired by manyNKG2DLs is the replacement of a short transmembrane regionwith a glycosylphosphatidylinositol (GPI) moiety (13), we in-vestigated whether MIC molecules could be sensitive to PI-PLC,an enzyme that specifically cleaves the GPI anchor. We found that

FIGURE 2. Inhibition of MICB shedding on drug-treated cells increases their susceptibility to NK cell–mediated killing. (A) SKO-007(J3) cells were plated

at 3 3 105 cells/ml and treated with DOX and MEL, as described in Fig. 1. After 48 h, cells were washed and plated at 0.5 3 106 cells/ml in fresh medium.

Different doses of the broad metalloproteinases inhibitor marimastat were added and left for an additional 18 h. Supernatants were collected and analyzed for

the presence of sMICB by specific ELISA. A representative experiment of three is shown. (B) SKO-007(J3) cells were treated as described above in the

presence of 10 mM of marimastat. Cell surface expression of MICA and MICB was evaluated by immunofluorescence and cytofluorimetric analysis by gating

on PI2 cells. Shaded line graphs represent the isotype-control Ig. A representative experiment of four is shown. (C) Cells were treated as described in (B). The

mean of four independent experiments is shown. Values represent the mean fluorescence intensity (MFI) of MICA or MICB subtracted from the MFI value of

the isotype-control Ig. (D) SKO-007(J3) cells, treated as described in (B), were used as targets, and long-term cultured NK cells were used as effectors in

a standard CD107a degranulation assay using an E:T ratio of 2.5:1. NK cells were distinguished from target cells by gating on forward scatter and side scatter.

Numbers indicate the percentage of CD107a+ cells. A representative experiment of four is shown. (E) Data are expressed as fold increase in CD107a values

(%) obtained on NK cells cocultured with drugs and inhibitor-treated MM cells divided by CD107a values (%) of NK cells cocultured with untreated MM

cells. The mean of four independent experiments is shown. (F) Degranulation assay was performed as described in (D). Before the assay, target cells were

incubated with cIg, anti-MICB, or anti-MICA plus anti-MICB Abs, as described in Materials and Methods. Anti-NKG2D mAb was used instead to block

NKG2D receptor on effector cells. The mean of four independent experiments 6SEM is shown. *p , 0.05, **p , 0.01, ***p , 0.001. ns, not significant.

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PI-PLC treatment of SKO-007(J3) cells affected MICA*008, butnot MICB, cell surface expression, thus indicating that MICA*008molecules can be anchored to the cell membrane through GPI(Supplemental Fig. 2). To investigate the functional consequencesof marimastat-induced expression of MICB on drug-treated SKO-007(J3) MM cells, we evaluated their ability to trigger NK celldegranulation. Upregulation of NKG2DLs on MM cells by thecombination of the metalloproteinase inhibitor and genotoxicagents was always verified before degranulation assays (data notshown). As shown in Fig. 2D and 2E, in accordance with themarimastat-induced enhancement of cell surface levels of MICB,but not MICA (Fig. 2B), NK cells contacting drug-treated MMcells exhibited higher expression of CD107a, a lysosomal markerthat correlates with NK cell cytotoxicity; this expression wasfurther enhanced upon marimastat target treatment. To directlyprove the contribution of MICB, we performed degranulationexperiments using a neutralizing Ab against MICB, alone or incombination with anti-MICA. Our results show that anti-MICBalone moderately, but significantly, reduced NK cell degranulationin untreated and drug-treated target cells, indicating that stabili-zation of this ligand on the cell surface renders tumor cells moresusceptible to NK cell lysis. In addition, the combined usage ofanti-MICA and anti-MICB Abs further reduced NK cell degran-ulation, indicating that the NKG2D-dependent lysis of the SKO-007(J3) cell line is mediated primarily by these ligands (Fig. 2F).In line with these observations, UL16-binding protein molecules,expressed at low levels on SKO-007(J3) cells, are not upregulatedby drug treatment, thus excluding their contribution in theNKG2D-dependent killing of drug-treated MM cells (22) (datanot shown). Collectively, these data show that chemotherapeutictreatment of SKO-007(J3) cells, while enhancing the surface ex-pression of both NKG2DLs, promotes MICB, but not MICA*008,shedding by enhancing metalloproteinase activity. Inhibition ofMICB shedding by marimastat in drug-treated cells results inincreased susceptibility of MM cells to NK cell lysis by preservingcell surface MICB expression.

The combined use of genotoxic drugs and metalloproteinaseinhibitors on patient-derived malignant PCs enhancesautologous NK cell degranulation

We next extended the analysis of the effects on NK cell degran-ulation by the combined use of metalloproteinase inhibitors andMEL in patient-derived PCs that express both MICA long allelesand MICB (Fig. 3A, 3B). To this end, highly purified patient-derived PCs were treated with MEL and marimastat for 48 hand then allowed to interact with IL-2–treated autologous bonemarrow–derived NK cells to assess their ability to trigger NK celldegranulation. The combined treatment with MEL and marimastatenhanced the expression of MICA long alleles and MICB onpatient PCs (Fig. 3A). We found that IL-2–treated bone marrow–derived autologous NK cells contacting CD138+ drug-treated cellsexhibited higher expression of CD107a, and this expression wasfurther enhanced upon marimastat target treatment (Fig. 3B, 3C).Similar results were obtained using CD1382 bone marrow cells (Fig.3B) or highly purified CD32CD16+CD56+ NK cells (Fig. 3C).Overall, our findings indicate that the increased expression of MIC

molecular species sensitive to proteolytic cleavage on MM cells re-sults in enhanced NK cell recognition and killing and further poten-tiates the immunostimulatory effects exerted by genotoxic agents.

ADAM10 is involved in drug-induced MICB release from MMcells

The relative roles for ADAM10 and ADAM17 metalloproteinaseson MIC shedding were primarily investigated in steady-state

conditions in a variety of tumor cell lines or transfected cells (9,32–34). To identify the ADAM enzymes responsible for the drug-induced MICB release, we examined whether the inhibition ofADAM10 and ADAM17 expression on drug-treated MM cellsinterfered with MICB shedding. To this end, ADAM10 andADAM17 were knocked down in SKO-007(J3) cells using lenti-viral vectors containing ADAM10 or ADAM17 shRNA. As shownin Fig. 4A–C, ADAM10 and ADAM17 expression were clearlysuppressed and ∼70% reduction was observed at the mRNA andprotein levels using real-time PCR, Western blot, immunofluo-rescence, and FACS analysis. Interestingly, ADAM10, but notADAM17, knockdown led to a dramatic inhibition of both con-stitutive and drug-induced MICB shedding, as observed in un-treated and DOX-treated MM cells (Fig. 4D). Of note, CD138,which is highly expressed and released as a soluble molecule fromMM cells, including SKO-007(J3) cells (35), was reduced inADAM17-silenced cells, making evident the specific effect ofADAM10-silencing on MICB shedding (Fig. 4E). The involve-ment of ADAM10 was further confirmed using the specificADAM10 inhibitor GI254023X (36), which affected MICB re-lease in a dose-dependent manner in both untreated and drug-treated MM cells (Fig. 4F). In addition, GI254023X inhibitedMICA*019 release from LP1-transduced cells in response to drugtreatment, revealing a role for this sheddase in drug-inducedMICA shedding as well (data not shown).Because efficient MICB proteolysis depends on ligand recruit-

ment to detergent-resistant microdomains (32), we also investi-gated the localization of MICB, ADAM10, and Erp5 within themembrane rafts in response to chemotherapeutic drugs. Ourfindings show that a high proportion of MICB and ADAM10colocalize in the membrane rafts, and their distribution is not al-tered by drug treatment. In contrast, Erp5 does not localize withinraft microdomains, suggesting that Erp5 and MIC form transitorymixed disulfide complexes on the cell membrane outside of theraft microdomains (Supplemental Fig. 3).Altogether, these data demonstrate that ADAM10 plays a predom-

inant role on drug-inducedMICB release from humanMMcells, withthe proteolytic process primarily occurring in the membrane rafts.

ADAM10 is upregulated in MM cell lines and inpatient-derived malignant PCs in response to genotoxic stress

Because we defined an important role for ADAM10 in the regu-lation of MIC shedding by genotoxic agents, we asked whether thiscould be attributable to the drug-induced modulation of ADAM10expression and activity. Fig. 5A and 5B show that ADAM10increases sharply at 48 h after DOX or MEL treatment, as eval-uated by immunofluorescence and FACS analysis, and this in-crease persists at 72 h. Similar results were obtained by Westernblot analysis, which did not reveal any change in the expression ofother enzymes involved in MIC proteolysis: ADAM17 and Erp5(Fig. 5C). In addition, increased levels of ADAM10 mRNA wereobserved in drug-treated cells (Fig. 5D). Accordingly, increasedADAM10 expression resulted in enhanced enzymatic activity inDOX-treated cells (Fig. 5E).To exclude that the drug-induced enhancement of ADAM10

expression was unique to the SKO-007(J3) MM cell line, weextended our analysis to a large panel of MM cell lines and ma-lignant PCs from MM patients by gating on CD138+/CD38+ PCs(Supplemental Fig. 4A). Drug treatment induced a significantupregulation of ADAM10 cell surface expression, from its basallevel of expression, in all MM cell lines tested independently(Fig. 5F). With regard to ADAM10 cell surface expression onmalignant PCs, we found that patient-derived PCs displayeddifferent levels of ADAM10 independent of the clinical stage

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(Supplemental Fig. 4B, 4C). In addition, consistent with the dataobtained in MM cell lines, drug treatment of ex vivo PCs resultedin higher levels of surface ADAM10 (Fig. 5G, 5H, SupplementalFig. 4C), and this increase was accompanied by a concomitantincrease in ADAM10 mRNA (Fig. 5I). We did not observe anychange in the expression of ADAM10 on CD1382CD382 cellsupon drug treatment (data not shown).These results show that ADAM10 expression is upregulated in

MM cell lines and in primary malignant PCs in response to gen-otoxic agents.

Drug-induced ADAM10 upregulation is dependent on ROSgeneration and is associated with a senescent phenotype

A large body of evidence describes an increased expression ofseveral metalloproteinases in cells undergoing senescence and

developing a senescence-associated secretory phenotype (SASP)(37). In addition, our recent data show that drug-induced NKG2DLsare preferentially expressed on senescent MM cells (22), and theirexpression requires DNA damage response activation and ROS sig-naling (26).Thus, we asked whether MICB secretion and ADAM10 ex-

pression were upregulated in MM drug-induced senescent cells.To this end, SKO-007(J3) MM cells were treated with DOX for72 h and then incubated with the fluorogenic substrate C12FDG tomeasure bGal activity, which is a marker of senescence; subse-quently, bGalhigh and bGallow cells were sorted, as previouslydescribed (26). Generally, the percentage of drug-induced sen-escent SKO-007(J3) cells was ∼60% (22, 26) (data not shown).We found that ADAM10 expression was more prominent onbGalhigh cells at the mRNA and protein levels (Fig. 6A, 6B).

FIGURE 3. The combined use of che-

motherapeutic drugs and metalloproteinase

inhibitors on patient-derived malignant PCs

enhances autologous NK cell degranula-

tion. (A) Total cells isolated from the bone

marrow of MM patients were purified with

CD138+ immunomagnetic beads and cul-

tured in the presence of IL-3 (20 ng/ml),

IL-6 (2 ng/ml), MEL (10 mM), marimastat

(10 mM), or MEL+marimastat for 48 h,

and expression of MICA and MICB on PCs

was evaluated by immunofluorescence and

FACS analysis. Results relative to two

patients (P7 and P13) are shown. Values

represent the mean fluorescence intensity

(MFI) of MICA and MICB subtracted from

the MFI value of the isotype-control Ig. (B)

Highly purified PCs were treated as de-

scribed in (A) and used as targets in

a standard degranulation assay. IL-2–treated

bone marrow CD1382 cells were used as

effectors. The assay was performed at an

E:T ratio of 2.5:1. After 2 h at 37˚C, cells

were stained with anti-CD56, anti-CD3,

anti-CD16, and anti-CD107a mAbs. Cell

surface expression of CD107a was ana-

lyzed on CD56+CD16+CD32 NK cells. (C)

Highly purified PCs were treated as de-

scribed in (A) and used as targets in

a standard degranulation assay. As effec-

tors, autologous IL-2–treated highly puri-

fied bone marrow–derived NK cells, as

evaluated by CD45+CD56+CD16+CD32

expression, were used. The assay was

performed at an E:T ratio of 2.5:1. After

2 h at 37˚C, cells were stained with anti-

CD56, anti-CD16, and anti-CD107a mAbs.

Cell surface expression of CD107a was

analyzed on CD56+CD16+ NK cells.

MICA gene typing of patients P39, P38,

and P42 was *006/009:01, *008:1/018:01,

and *007:01/008:01, respectively. Patient

characteristics are shown in Supplemental

Fig. 4C.

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FIGURE 4. ADAM10 strongly contributes to drug-induced MICB release. (A–C) SKO-007(J3) cells were infected with the pLKO lentiviral vector

containing shRNA for silencing ADAM10 (shADAM10) or ADAM17 (shADAM17) or with a scrambled sequence. After 72 h, real-time PCR, immu-

nofluorescence, FACS analysis, and Western blot were performed to evaluate the efficiency of the ADAM10 and ADAM17 silencing. One representative

experiment of three is shown. (D and E) pLKO/shADAM10, pLKO/shADAM17, and pLKO/scramble transduced cells were treated as described in Fig. 1.

After 48 h, supernatants were collected and analyzed for the presence of sMICB or CD138. The mean of three independent experiments is shown. (F) SKO-

007(J3) cells were treated as described in Fig. 1. After 48 h, cells were washed and plated at 0.5 3 106 cells/ml in fresh medium. Different doses of the

specific ADAM10 inhibitor GI254023X were added and left for an additional 18 h. Supernatants were collected and analyzed for the presence of sMICB. A

representative experiment of three is shown. *p , 0.05.

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FIGURE 5. ADAM10 is upregulated in response to genotoxic stress in several MM cell lines and in ex vivo malignant CD138+/CD38+ PCs. SKO-007

(J3) cells were plated at 33 105 cells/ml and treated with DOX and MEL, as described in Fig. 1. (A and B) After 24, 48, and 72 h, cell surface expression of

ADAM10 was evaluated by performing immunofluorescence and cytofluorimetric analysis by gating on PI2 cells. Values reported represent the mean

fluorescent intensity (MFI) of ADAM10 subtracted from the MFI value of the isotype-control Ig. One representative experiment (A) and the mean of three

independent experiments (B) are shown. (C) After 48 h of drug treatment, Western blot analysis was performed on total cell lysates using anti-ADAM10–,

anti-ADAM17–, or anti-ERp5–specific Abs. Protein loading was normalized using b-actin. One representative experiment of three is shown. (D) Real-time

PCR analysis of total mRNA obtained from MM cells, unstimulated or treated with drugs, as described above. Data, expressed as fold change units, were

normalized with b-actin and compared with the untreated cells, which were considered calibrators. Data represent the mean of at least four independent

experiments. (E) Cells treated with drugs for 72 h were harvested, and ADAM10 catalytic activity was evaluated on total cellular lysates in the presence

of 5-FAM/QXL 520 ADAM10 substrate after 30 and 60 min, as described in Materials and Methods. The mean of three (Figure legend continues)

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Accordingly, MICB release was dramatically increased onbGalhigh senescent cells (Fig. 6C). To further investigate whetherdrug-induced ADAM10 upregulation was associated with a sen-escent phenotype in primary PCs, we assayed its expression onbGalhigh and bGallow drug-treated PCs. By simultaneously eval-uating cell cycle and bGal activity, we found that the ability ofprimary PCs to undergo senescence was related to the percentage

that was able to divide and to consequently arrest following che-motherapeutic treatment (data not shown). Interestingly, and similarto the data obtained in the SKO(J3)-007 cell line, our results indi-cate that ADAM10 expression was significantly augmented onbGalhigh cells (Fig. 6D, 6E).It is noteworthy that the inhibition of oxidative stress by the

antioxidant agent NAC, a glutathione precursor acting as a ROS

FIGURE 6. Drug-induced ADAM10 upregulation is associated with a senescent phenotype. (A) A senescence-associated bGal assay was performed

using the fluorogenic substrate C12FDG to measure bGal activity by flow cytometry (26). Cells were treated with DOX for 72 h, incubated with C12FDG,

and analyzed by immunofluorescence. bGalhigh and bGallow cells were selected by a FACSAria cell sorter through FL-1 green fluorescence emission.

mRNA levels of ADAM10 were tested by RT-PCR. b-Actin was used to normalize. (B) ADAM10 expression was evaluated on bGalhigh- and bGallow-

sorted cells by immunofluorescence and FACS analysis. (C) bGalhigh- and bGallow-sorted cells were incubated for an additional 24 h in fresh medium, and

the presence of sMICB in the supernatants was evaluated by ELISA. Results are representative of one of three independent experiments. (D and E) Total

cells isolated from the bone marrow of MM patients were cultured in the presence of IL-3 (20 ng/ml), IL-6 (2 ng/ml), and DOX (10 mM) for 72 h.

A senescence-associated bGal assay was performed as described in (A) and then cells were stained with anti-ADAM10/PE, anti-CD38/PerCP, and CD138/

allophycocyanin. In (D), SA-bGal activity evaluated by gating on CD38+/CD138+ cells is shown. In (E), ADAM10 expression was analyzed on CD38+/

CD138+ bGalhigh and bGallow cells. Values reported represent the mean fluorescence intensity (MFI) of ADAM10 subtracted from the MFI value of the

isotype-control Ig. Four representative patients (P42, P39, P43, and P44) are shown.

independent experiments is shown. (F) Different MM cell lines were plated at 33 105 cells/ml and treated with low doses of DOX and MEL, as indicated in

Materials and Methods, for 48 h. Cell surface expression of ADAM10 was evaluated as described in (A). Values reported represent the MFI of ADAM10

subtracted from the MFI value of the isotype-control Ig. Mean of at least three independent experiments is shown. (G) Total cells isolated from the bone

marrow of MM patients were cultured in the presence of IL-3 (20 ng/ml), IL-6 (2 ng/ml), and DOX (0.05 mM) or MEL (10 mM) for 48 h. Cells were stained

with anti-ADAM10 mAb plus GAM-PE and with anti-CD38/FITC and CD138/allophycocyanin. ADAM10 expression was analyzed on CD38+/CD138+

cells. Three representative patients (P1, P5, and P9) are shown. (H) MFI values of ADAM10 subtracted from the MFI value of the isotype-control Ig on

CD138+/CD38+ cells from nine patients (P1–P9). Each patient is represented by an individual symbol. (I) CD138+ cells were purified using immuno-

magnetic beads and treated as described in (A). After 48 h, cells were harvested, RNAwas extracted, and real-time PCR was performed. Data, expressed as

fold change units, were normalized with b-actin and compared with the untreated cells, which were considered calibrators. Values reported represent the

mean of four independent experiments relative to patients P5, P9, P11, and P12. *p , 0.05, **p , 0.01. ns, not significant.

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scavenger, resulted in a marked reduction in ADAM10 on drug-treated SKO-007(J3) MM cells but did not substantially affect itsbasal expression (Fig. 7A, 7B), suggesting that ROS generation is in-volved in the upregulation of ADAM10 on senescent cells. In addi-tion, we validated these data using ex vivo primary malignant PCsderived from three patients. As shown in Fig. 7C, MEL treatment ofmalignant PCs in the presence of NAC reduced ADAM10 expression.Collectively, our results show that ADAM10-dependent MICB

release in response to genotoxic stress is associated with a sen-escent phenotype and requires ROS generation.

DiscussionThe release of NKG2DLs from the cell surface in their soluble formwas suggested to play an important role in tumor cell escape fromNKG2D-dependent NK cell–mediated immunosurveillance; thus,its inhibition could be exploited as a promising strategy to en-hance antitumor immunity.Indeed, soluble forms of NKG2DLs are present in the serum of

cancer patients, and their levels correlate with tumor stage andmetastasis (12, 20, 21, 38–41), as well as with reduced expression ofNKG2D on NK cells and other cytotoxic lymphocytes (11, 21, 42).In this study, we showed that treatment of MM cells with

chemotherapeutic drugs that induce genotoxic stress stimulates therelease of MICA andMICBmolecules through a mechanism that isprimarily dependent on ADAM10 protease, as well as that thecombined use of metalloproteinase inhibitors and chemothera-peutic agents strengthens NK cell–mediated killing of MM cells.We provide novel evidence that genotoxic stress selectively affects

the shedding process of MIC molecules that are sensitive to met-alloproteinases cleavage (i.e., MICA*019 or MICB), whereas therelease of the most frequent allelic variant in the white population,MICA*008 short allele, is not modulated. Our observations are inline with findings by Ashiru et al. (31), who demonstrated that

MICA*008 can be anchored to the plasma membrane via GPI andreleased in association with exosomes or secreted through a mech-anism that is dependent on exocytosis. Thus, effective blockade ofsMICA release and accumulation in patient sera might requiredifferent strategies, depending on the patient’s MICA genotype.We also provide further insight into the roles played by

ADAM10 and ADAM17 in the shedding of endogenous MIC fromMM cells and demonstrate, by means of silencing and usage ofpharmacological inhibitors, that ADAM10 plays a predominantrole in steady-state conditions, as well as in drug-induced MICrelease from MM cells. Several studies, primarily performed indifferent types of tumor cell lines or transfected cells in steady-stateconditions, indicate the direct involvement of both ADAM10 andADAM17 enzymes in MICA and MICB release, but the relativecontribution of these enzymes is still controversial. Boutet et al.(32) found that ADAM17 mediates MICB proteolytic cleavage inCV1 epithelial transfectants; however, recent evidence highlightsa cell type–specific role for these metalloproteinases (33). Ourresults also reveal high levels of ADAM10 expression in severalMM cell lines and in primary malignant PCs, with enzyme ex-pression not correlating with the disease stage. Previous studiesshowed that ADAM10 is required for normal PC function (43),and its expression increases during B cell differentiation, reach-ing higher levels at the PC stage (44). However, no evidence isavailable for the malignant cell counterpart, although a role forADAM10 in hematologic malignancies was recently reported (45,46). Importantly, we provide evidence that ADAM10 expression isenhanced in MM cell lines and in primary PCs in response togenotoxic agents, and its upregulation requires ROS generation andis associated with the acquisition of a senescent phenotype. Similarto our observations, etoposide, a topoisomerase II inhibitor able toactivate the response to DNA damage, was demonstrated to enhanceADAM10 expression (47). However, at variance with our results,

FIGURE 7. Drug-induced ADAM10 upregulation

is dependent on oxidative stress. (A) SKO-007(J3)

were pretreated with 10 mM of NAC for 1 h at 37˚C

and then treated with DOX or MEL for 72 h.

ADAM10 expression was evaluated by immunofluo-

rescence and FACS analysis. (B) Values reported

represent the mean fluorescence intensity (MFI) of

ADAM10 subtracted from the MFI value of the iso-

type-control Ig. Mean of three independent experi-

ments is shown. (C) Total cells isolated from the bone

marrow of MM patients were cultured in the presence

of IL-3 (20 ng/ml), IL-6 (2 ng/ml), and MEL (10 mM)

for 48 h. Cells were stained with anti-ADAM10/PE

and with anti-CD38/FITC and CD138/allophycocyanin.

ADAM10 expression was analyzed on CD38+/CD138+

cells. Three representative patients (P38, P40, and P41)

are shown. Values reported represent the MFI of

ADAM10 subtracted from the MFI value of the

isotype-control Ig.

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Kohga et al. (48) observed that MICA shedding upon epirubicintreatment in hepatocarcinoma cells was accompanied by reducedADAM10 expression. These discrepancies may depend on thedifferent cellular systems and/or experimental conditions used. Ourevidence for ROS involvement in ADAM10 upregulation andMICB shedding are in line with the notion that conditions causingcellular stress, including ionizing radiation, chemotherapeuticagents, or ROS, can lead to increased metalloproteinase-mediatedrelease of cell surface molecules (49–51) and suggest that stressstimuli promote the proteolytic process by upregulating metal-loproteinase expression and activity. We also are the first to de-scribe, to our knowledge, that genotoxic stress-induced upregulationof ADAM10 expression and sMICB secretion are primarily asso-ciated with senescent cells. Accordingly, senescence-inducedgeneration of sIL-6R was mediated by ADAM10-dependent ecto-domain shedding (52). Increased expression of several MMPs, in-cluding MMP2 and MMP3, was reported previously in cellsundergoing senescence that develop a SASP, but it primarily wasassociated with promotion of cell invasion (53). The role of theSASP in tumor progression remains unclear and can be beneficial ordeleterious, because senescent cells within a tumor can producesecreted factors with both tumor-promoting and tumor-suppressingactivities (54). We suggest that release of sNKG2DLs is a compo-nent of tumor cell SASP and contributes to the creation of a mi-croenvironment that is suitable for tumor escape. Because MICAand MICB expression on primary PCs was found in a substantialnumber of MM patients (70 and 50% of patients analyzed, re-spectively) (data not shown), strategies aimed at stabilizing MICligands on the cell surface of tumor cells look promising to increaseNK cell recognition mediated by NKG2D. Indeed, our findingsshow that the combined use of MEL and marimastat enhances NKcell degranulation triggered by autologous malignant PCs. Simi-larly, the combination of valproate, which is known to upregulatecell surface MICA/B (25, 55), and metalloproteinase inhibitorssubstantially stabilized cell surface MICA/B on ovarian carcinomacells and enhanced the efficacy of immune cell therapy in vivo (56).In addition, the use of ADAM10 and ADAM17 inhibitors wasshown to ameliorate the response to chemotherapy treatments indifferent in vivo models of cancer (57, 58), and ADAM inhibitorswere used in clinical trials in breast cancer patients (59). Of interest,combined treatment using chemotherapy and metalloproteinaseinhibitors recently was proposed as a therapeutic regimen in MM,because drug-induced ADAM-mediated CD138 release was shownto promote tumor growth (60). Overall, our findings suggest thattargeting of metalloproteinases in conjunction with chemotherapycould be exploited for NK cell–based immunotherapeutic ap-proaches, thus contributing to avoid the escape of malignant cellsfrom stress-elicited immune responses.

AcknowledgmentsWe thank A. Ludwig for kindly providing the ADAM10 inhibitor

GI254023X, Lewis L. Lanier for MICA*019 cDNA, and Dina Milana

for valuable technical assistance.

DisclosuresThe authors have no financial conflicts of interest.

References1. Lanier, L. L. 2005. NK cell recognition. Annu. Rev. Immunol. 23: 225–274.2. Dai, Z., C. J. Turtle, G. C. Booth, S. R. Riddell, T. A. Gooley, A. M. Stevens,

T. Spies, and V. Groh. 2009. Normally occurring NKG2D+CD4+ T cells areimmunosuppressive and inversely correlated with disease activity in juvenile-onset lupus. J. Exp. Med. 206: 793–805.

3. Bryceson, Y. T., and H. G. Ljunggren. 2008. Tumor cell recognition by the NKcell activating receptor NKG2D. Eur. J. Immunol. 38: 2957–2961.

4. Guerra, N., Y. X. Tan, N. T. Joncker, A. Choy, F. Gallardo, N. Xiong,S. Knoblaugh, D. Cado, N. M. Greenberg, and D. H. Raulet. 2008. NKG2D-deficient mice are defective in tumor surveillance in models of spontaneousmalignancy. Immunity 28: 571–580.

5. Raulet, D. H., S. Gasser, B. G. Gowen, W. Deng, and H. Jung. 2013. Regulationof ligands for the NKG2D activating receptor. Annu. Rev. Immunol. 31: 413–441.

6. Champsaur, M., and L. L. Lanier. 2010. Effect of NKG2D ligand expression onhost immune responses. Immunol. Rev. 235: 267–285.

7. Cerboni, C., M. Ardolino, A. Santoni, and A. Zingoni. 2009. Detuning CD8+T lymphocytes by down-regulation of the activating receptor NKG2D: role ofNKG2D ligands released by activated T cells. Blood 113: 2955–2964.

8. Mincheva-Nilsson, L., O. Nagaeva, T. Chen, U. Stendahl, J. Antsiferova,I. Mogren, J. Hernestal, and V. Baranov. 2006. Placenta-derived soluble MHCclass I chain-related molecules down-regulate NKG2D receptor on peripheralblood mononuclear cells during human pregnancy: a possible novel immuneescape mechanism for fetal survival. J. Immunol. 176: 3585–3592.

9. Salih, H. R., H. G. Rammensee, and A. Steinle. 2002. Cutting edge: down-regulation of MICA on human tumors by proteolytic shedding. J. Immunol.169: 4098–4102.

10. Salih, H. R., H. Antropius, F. Gieseke, S. Z. Lutz, L. Kanz, H. G. Rammensee,and A. Steinle. 2003. Functional expression and release of ligands for the acti-vating immunoreceptor NKG2D in leukemia. Blood 102: 1389–1396.

11. Groh, V., J. Wu, C. Yee, and T. Spies. 2002. Tumour-derived soluble MICligands impair expression of NKG2D and T-cell activation. Nature 419: 734–738.

12. Doubrovina, E. S., M. M. Doubrovin, E. Vider, R. B. Sisson, R. J. O’Reilly,B. Dupont, and Y. M. Vyas. 2003. Evasion from NK cell immunity by MHCclass I chain-related molecules expressing colon adenocarcinoma. J. Immunol.171: 6891–6899.

13. Chitadze, G., J. Bhat, M. Lettau, O. Janssen, and D. Kabelitz. 2013. Generationof soluble NKG2D ligands: proteolytic cleavage, exosome secretion and func-tional implications. Scand. J. Immunol. 78: 120–129.

14. Ashiru, O., P. Boutet, L. Fernandez-Messina, S. Ag€uera-Gonzalez, J. N. Skepper,M. Vales-Gomez, and H. T. Reyburn. 2010. Natural killer cell cytotoxicity issuppressed by exposure to the human NKG2D ligand MICA*008 that is shed bytumor cells in exosomes. Cancer Res. 70: 481–489.

15. Mahindra, A., T. Hideshima, and K. C. Anderson. 2010. Multiple myeloma:biology of the disease. Blood Rev. 24(Suppl. 1): S5–S11.

16. Godfrey, J., and D. M. Benson, Jr. 2012. The role of natural killer cells in im-munity against multiple myeloma. Leuk. Lymphoma 53: 1666–1676.

17. Carbone, E., P. Neri, M. Mesuraca, M. T. Fulciniti, T. Otsuki, D. Pende, V. Groh,T. Spies, G. Pollio, D. Cosman, et al. 2005. HLA class I, NKG2D, and naturalcytotoxicity receptors regulate multiple myeloma cell recognition by naturalkiller cells. Blood 105: 251–258.

18. El-Sherbiny, Y. M., J. L. Meade, T. D. Holmes, D. McGonagle, S. L. Mackie,A. W. Morgan, G. Cook, S. Feyler, S. J. Richards, F. E. Davies, et al. 2007. Therequirement for DNAM-1, NKG2D, and NKp46 in the natural killer cell-mediated killing of myeloma cells. Cancer Res. 67: 8444–8449.

19. Lu, X., A. Zhu, X. Cai, Z. Jia, W. Han, L. Ma, M. Zhou, K. Qian, L. Cen, andB. Chen. 2012. Role of NKG2D in cytokine-induced killer cells against multiplemyeloma cells. Cancer Biol. Ther. 13: 623–629.

20. Rebmann, V., P. Sch€utt, D. Brandhorst, B. Opalka, T. Moritz, M. R. Nowrousian,and H. Grosse-Wilde. 2007. Soluble MICA as an independent prognostic factorfor the overall survival and progression-free survival of multiple myelomapatients. Clin. Immunol. 123: 114–120.

21. Jinushi, M., M. Vanneman, N. C. Munshi, Y. T. Tai, R. H. Prabhala, J. Ritz,D. Neuberg, K. C. Anderson, D. R. Carrasco, and G. Dranoff. 2008. MHC class Ichain-related protein A antibodies and shedding are associated with the pro-gression of multiple myeloma. Proc. Natl. Acad. Sci. USA 105: 1285–1290.

22. Soriani, A., A. Zingoni, C. Cerboni, M. L. Iannitto, M. R. Ricciardi, V. DiGialleonardo, M. Cippitelli, C. Fionda, M. T. Petrucci, A. Guarini, et al. 2009.ATM-ATR-dependent up-regulation of DNAM-1 and NKG2D ligands on mul-tiple myeloma cells by therapeutic agents results in enhanced NK-cell suscep-tibility and is associated with a senescent phenotype. Blood 113: 3503–3511.

23. Fionda, C., A. Soriani, G. Malgarini, M. L. Iannitto, A. Santoni, andM. Cippitelli. 2009. Heat shock protein-90 inhibitors increase MHC class I-related chain A and B ligand expression on multiple myeloma cells and theirability to trigger NK cell degranulation. J. Immunol. 183: 4385–4394.

24. Fionda, C., G. Malgarini, A. Soriani, A. Zingoni, F. Cecere, M. L. Iannitto,M. R. Ricciardi, V. Federico, M. T. Petrucci, A. Santoni, and M. Cippitelli. 2013.Inhibition of glycogen synthase kinase-3 increases NKG2D ligand MICA ex-pression and sensitivity to NK cell-mediated cytotoxicity in multiple myelomacells: role of STAT3. J. Immunol. 190: 6662–6672.

25. Wu, X., Y. Tao, J. Hou, X. Meng, and J. Shi. 2012. Valproic acid upregulatesNKG2D ligand expression through an ERK-dependent mechanism and poten-tially enhances NK cell-mediated lysis of myeloma. Neoplasia 14: 1178–1189.

26. Soriani, A., M. L. Iannitto, B. Ricci, C. Fionda, G. Malgarini, S. Morrone,G. Peruzzi, M. R. Ricciardi, M. T. Petrucci, M. Cippitelli, and A. Santoni. 2014.Reactive oxygen species- and DNA damage response-dependent NK cell acti-vating ligand upregulation occurs at transcriptional levels and requires thetranscriptional factor E2F1. J. Immunol. 193: 950–960.

27. Molfetta, R., L. Quatrini, C. Capuano, F. Gasparrini, B. Zitti, A. Zingoni,R. Galandrini, A. Santoni, and R. Paolini. 2014. c-Cbl regulates MICA- but notULBP2-induced NKG2D down-modulation in human NK cells. Eur. J. Immunol.44: 2761–2770.

28. Zingoni, A., G. Palmieri, S. Morrone, M. Carretero, M. Lopez-Botel, M. Piccoli,L. Frati, and A. Santoni. 2000. CD69-triggered ERK activation and functions are

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by guest on April 12, 2018

http://ww

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negatively regulated by CD94/NKG2-A inhibitory receptor. Eur. J. Immunol. 30:644–651.

29. Cerboni, C., A. Zingoni, M. Cippitelli, M. Piccoli, L. Frati, and A. Santoni. 2007.Antigen-activated human T lymphocytes express cell-surface NKG2D ligandsvia an ATM/ATR-dependent mechanism and become susceptible to autologousNK- cell lysis. Blood 110: 606–615.

30. F€urst, D., G. Solgi, K. Recker, D. Mytilineos, H. Schrezenmeier, andJ. Mytilineos. 2011. Sequence-based typing of major histocompatibility complexclass I chain-related gene A alleles by use of exons 2-5 information. TissueAntigens 77: 201–205.

31. Ashiru, O., S. Lopez-Cobo, L. Fernandez-Messina, S. Pontes-Quero, R. Pandolfi,H. T. Reyburn, and M. Vales-Gomez. 2013. A GPI anchor explains the uniquebiological features of the common NKG2D-ligand allele MICA*008. Biochem.J. 454: 295–302.

32. Boutet, P., S. Ag€uera-Gonzalez, S. Atkinson, C. J. Pennington, D. R. Edwards,G. Murphy, H. T. Reyburn, and M. Vales-Gomez. 2009. Cutting edge: themetalloproteinase ADAM17/TNF-alpha-converting enzyme regulates proteo-lytic shedding of the MHC class I-related chain B protein. J. Immunol. 182: 49–53.

33. Chitadze, G., M. Lettau, J. Bhat, D. Wesch, A. Steinle, D. F€urst, J. Mytilineos,H. Kalthoff, O. Janssen, H. H. Oberg, and D. Kabelitz. 2013. Shedding of en-dogenous MHC class I-related chain molecules A and B from different humantumor entities: heterogeneous involvement of the “a disintegrin and metal-loproteases” 10 and 17. Int. J. Cancer 133: 1557–1566.

34. Waldhauer, I., D. Goehlsdorf, F. Gieseke, T. Weinschenk, M. Wittenbrink,A. Ludwig, S. Stevanovic, H. G. Rammensee, and A. Steinle. 2008. Tumor-associated MICA is shed by ADAM proteases. Cancer Res. 68: 6368–6376.

35. Ramani, V. C., A. Purushothaman, M. D. Stewart, C. A. Thompson,I. Vlodavsky, J. L. Au, and R. D. Sanderson. 2013. The heparanase/syndecan-1axis in cancer: mechanisms and therapies. FEBS J. 280: 2294–2306.

36. Hoettecke, N., A. Ludwig, S. Foro, and B. Schmidt. 2010. Improved synthesis ofADAM10 inhibitor GI254023X. Neurodegener. Dis. 7: 232–238.

37. Rodier, F., and J. Campisi. 2011. Four faces of cellular senescence. J. Cell Biol.192: 547–556.

38. Wu, J. D., L. M. Higgins, A. Steinle, D. Cosman, K. Haugk, and S. R. Plymate.2004. Prevalent expression of the immunostimulatory MHC class I chain-relatedmolecule is counteracted by shedding in prostate cancer. J. Clin. Invest. 114:560–568.

39. Holdenrieder, S., P. Stieber, A. Peterfi, D. Nagel, A. Steinle, and H. R. Salih. 2006.Soluble MICB in malignant diseases: analysis of diagnostic significance and cor-relation with soluble MICA. Cancer Immunol. Immunother. 55: 1584–1589.

40. Holdenrieder, S., P. Stieber, A. Peterfi, D. Nagel, A. Steinle, and H. R. Salih.2006. Soluble MICA in malignant diseases. Int. J. Cancer 118: 684–687.

41. Baragano Raneros, A., B. Suarez-Alvarez, and C. Lopez-Larrea. 2014. Secretorypathways generating immunosuppressive NKG2D ligands: New targets fortherapeutic intervention. OncoImmunology 3: e28497.

42. Arreygue-Garcia, N. A., A. Daneri-Navarro, A. del Toro-Arreola, A. Cid-Arregui, O. Gonzalez-Ramella, L. F. Jave-Suarez, A. Aguilar-Lemarroy,R. Troyo-Sanroman, A. Bravo-Cuellar, V. Delgado-Rizo, et al. 2008. Augmentedserum level of major histocompatibility complex class I-related chain A (MICA)protein and reduced NKG2D expression on NK and T cells in patients withcervical cancer and precursor lesions. BMC Cancer 8: 16.

43. Chaimowitz, N. S., D. J. Kang, L. M. Dean, and D. H. Conrad. 2012. ADAM10regulates transcription factor expression required for plasma cell function. PLoSONE 7: e42694.

44. Bret, C., D. Hose, T. Reme, A. Kassambara, A. Seckinger, T. Meissner,J. F. Schved, T. Kanouni, H. Goldschmidt, and B. Klein. 2011. Gene expressionprofile of ADAMs and ADAMTSs metalloproteinases in normal and malignantplasma cells and in the bone marrow environment. Exp. Hematol. 39: 546–557.e8.

45. Armanious, H., P. Gelebart, M. Anand, A. Belch, and R. Lai. 2011. Constitutiveactivation of metalloproteinase ADAM10 in mantle cell lymphoma promotescell growth and activates the TNFa/NFkB pathway. Blood 117: 6237–6246.

46. Zocchi, M. R., S. Catellani, P. Canevali, S. Tavella, A. Garuti, B. Villaggio,A. Zunino, M. Gobbi, G. Fraternali-Orcioni, A. Kunkl, et al. 2012. High ERp5/ADAM10 expression in lymph node microenvironment and impaired NKG2Dligands recognition in Hodgkin lymphomas. Blood 119: 1479–1489.

47. Lizama, C., D. Rojas-Benitez, M. Antonelli, A. Ludwig, and R. D. Moreno.2012. Involvement of TACE/ADAM17 and ADAM10 in etoposide-induced ap-optosis of germ cells in rat spermatogenesis. J. Cell. Physiol. 227: 829–838.

48. Kohga, K., T. Takehara, T. Tatsumi, T. Miyagi, H. Ishida, K. Ohkawa, T. Kanto,N. Hiramatsu, and N. Hayashi. 2009. Anticancer chemotherapy inhibits MHCclass I-related chain a ectodomain shedding by downregulating ADAM10 ex-pression in hepatocellular carcinoma. Cancer Res. 69: 8050–8057.

49. Fischer, O. M., S. Hart, A. Gschwind, N. Prenzel, and A. Ullrich. 2004. Oxi-dative and osmotic stress signaling in tumor cells is mediated by ADAM pro-teases and heparin-binding epidermal growth factor. Mol. Cell. Biol. 24: 5172–5183.

50. Vahdat, A. M., K. S. Reiners, V. L. Simhadri, D. A. Eichenauer, B. Boll,A. Chalaris, V. R. Simhadri, K. Wiegmann, H. W. Krell, S. Rose-John, et al.2010. TNF-alpha-converting enzyme (TACE/ADAM17)-dependent loss ofCD30 induced by proteasome inhibition through reactive oxygen species. Leu-kemia 24: 51–57.

51. Kyula, J. N., S. Van Schaeybroeck, J. Doherty, C. S. Fenning, D. B. Longley, andP. G. Johnston. 2010. Chemotherapy-induced activation of ADAM-17: a novelmechanism of drug resistance in colorectal cancer. Clin. Cancer Res. 16: 3378–3389.

52. Garbers, C., F. Kuck, S. Aparicio-Siegmund, K. Konzak, M. Kessenbrock,A. Sommerfeld, D. Haussinger, P. A. Lang, D. Brenner, T. W. Mak, et al. 2013.Cellular senescence or EGFR signaling induces Interleukin 6 (IL-6) receptorexpression controlled by mammalian target of rapamycin (mTOR). Cell Cycle12: 3421–3432.

53. Coppe, J. P., P. Y. Desprez, A. Krtolica, and J. Campisi. 2010. The senescence-associated secretory phenotype: the dark side of tumor suppression. Annu. Rev.Pathol. 5: 99–118.

54. Kuilman, T., and D. S. Peeper. 2009. Senescence-messaging secretome: SMS-ingcellular stress. Nat. Rev. Cancer 9: 81–94.

55. Armeanu, S., M. Bitzer, U. M. Lauer, S. Venturelli, A. Pathil, M. Krusch,S. Kaiser, J. Jobst, I. Smirnow, A. Wagner, et al. 2005. Natural killer cell-mediated lysis of hepatoma cells via specific induction of NKG2D ligands bythe histone deacetylase inhibitor sodium valproate. Cancer Res. 65: 6321–6329.

56. Huang, B., R. Sikorski, P. Sampath, and S. H. Thorne. 2011. Modulation ofNKG2D-ligand cell surface expression enhances immune cell therapy of cancer.J. Immunother. 34: 289–296.

57. Fridman, J. S., E. Caulder, M. Hansbury, X. Liu, G. Yang, Q. Wang, Y. Lo,B. B. Zhou, M. Pan, S. M. Thomas, et al. 2007. Selective inhibition of ADAMmetalloproteases as a novel approach for modulating ErbB pathways in cancer.Clin. Cancer Res. 13: 1892–1902.

58. Zhou, B. B., M. Peyton, B. He, C. Liu, L. Girard, E. Caudler, Y. Lo, F. Baribaud,I. Mikami, N. Reguart, et al. 2006. Targeting ADAM-mediated ligand cleavageto inhibit HER3 and EGFR pathways in non-small cell lung cancer. Cancer Cell10: 39–50.

59. Duffy, M. J., M. Mullooly, N. O’Donovan, S. Sukor, J. Crown, A. Pierce, andP. M. McGowan. 2011. The ADAMs family of proteases: new biomarkers andtherapeutic targets for cancer? Clin. Proteomics 8: 9.

60. Ramani, V. C., and R. D. Sanderson. 2014. Chemotherapy stimulates syndecan-1shedding: a potentially negative effect of treatment that may promote tumorrelapse. Matrix Biol. 35: 215–222.

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