HER2/HER3 Signaling Regulates NK Cell-Mediated Cytotoxicity via MHC Class I Chain-Related Molecule A and B Expression in Human Breast Cell Lines This information is current as of September 25, 2021. Riki Okita, Dimitrios Mougiakakos, Takashi Ando, Yumeng Mao, Dhifaf Sarhan, Erik Wennerberg, Barbara Seliger, Andreas Lundqvist, Kousaku Mimura and Rolf Kiessling J Immunol published online 1 February 2012 http://www.jimmunol.org/content/early/2012/02/01/jimmun Downloaded from ol.1102237

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The Journal of Immunology is published twice each month by The American Association of Immunologists, Inc., 1451 Rockville Pike, Suite 650, Rockville, MD 20852 Copyright © 2012 by The American Association of Immunologists, Inc. All rights reserved. Print ISSN: 0022-1767 Online ISSN: 1550-6606. Published February 1, 2012, doi:10.4049/jimmunol.1102237 The Journal of Immunology

HER2/HER3 Signaling Regulates NK Cell-Mediated Cytotoxicity via MHC Class I Chain-Related Molecule A and B Expression in Human Breast Cancer Cell Lines

Riki Okita,* Dimitrios Mougiakakos,*,† Takashi Ando,‡ Yumeng Mao,* Dhifaf Sarhan,* Erik Wennerberg,* Barbara Seliger,x Andreas Lundqvist,* Kousaku Mimura,{ and Rolf Kiessling*

Overexpression of the receptor tyrosine kinases HER2 and HER3 is associated with a poor prognosis in several types of cancer. Presently, HER2- as well as HER3-targeted therapies are in clinical practice or evaluated within clinical trials, including treatment with mAbs mediating growth inhibition and/or activation of Ab-induced innate or adaptive cellular immunity. A better understand- ing of how HER2/HER3 signaling in tumors influences cellular immune mechanisms is therefore warranted. In this study, we dem- Downloaded from onstrate that HER2/HER3 signaling regulates the expression of MHC class I-related chain A and B (MICA and MICB) in breast cancer cell lines. The MICA and MICB (MICA/B) molecules act as key ligands for the activating receptor NK group 2, member D (NKG2D) and promote NK cell-mediated recognition and cytolysis. Genetic silencing of HER3 but not HER2 downregulated the expression of MICA/B, and HER3 overexpression significantly enhanced MICA expression. Among the major pathways activated by HER2/HER3 signaling, the PI3K/AKT pathway was shown to predominantly regulate MICA/B expression. Treatment with the HER3-specific ligand neuregulin 1b promoted the expression in a process that was antagonized by pharmacological and genetic http://www.jimmunol.org/ interference with HER3 but not by the ataxia-telangiectasia–mutated (ATM) and ATM and Rad3-related kinases inhibitor caffeine. These observations further emphasize that HER2/HER3 signaling directly, and not via genotoxic stress, regulates MICA/ B expression. As anticipated, stimulating HER2/HER3 enhanced the NKG2D-MICA/B–dependent NK cell-mediated cytotoxicity. Taken together, we conclude that signaling via the HER2/HER3 pathway in breast carcinoma cell lines may lead to enhanced NKG2D-MICA/B recognition by NK cells and T cells. The Journal of Immunology, 2012, 188: 000–000.

atural killer cell activity is promoted via NK group 2, cells and at low levels also by other nonmalignant cell types. member D (NKG2D) on NK cells, CD8+ T cells, and gd Primary tumor cells and tumor cell lines frequently express by guest on September 25, 2021 N T cells (1, 2). The engagement between NKG2D and its MICA/B molecules, but the mechanisms responsible for the ligands enhances cell-mediated cytotoxicity and pro- induction of MICA/B and other NKG2D ligands duction against transformed cells (3). An important NKG2D li- (NKG2DLs) during oncogenesis are poorly understood. They are gand is the MHC class I chain A and B (MICA and MICB). Both described as stress-related proteins that can be induced by heat MICA and MICB (MICA/B) are expressed by intestinal epithelial shock and that are regulated by heat shock transcription elements activated during cell transformation (4). Also, there is evidence that the DNA damage pathway, induced by ionizing radiation and *Immune and Therapy Laboratory, Department of Oncology and Pathology, inhibitors of DNA replication, can induce NKG2DLs (5). A direct Cancer Center Karolinska, Karolinska Institute, 171 76 Stockholm, Sweden; control of MICA by the BCR/ABL in chronic mye- †Department of Internal Medicine 5, Hematology and Oncology, University of Erlan- gen–Nuremberg, D-91054 Erlangen, Germany; ‡Department of Orthopedic Surgery, logenous has also been described (6), but the regulation x University of Yamanashi, Yamanashi 409-3898, Japan; Institute of Medical Immu- of MICA/B expression by is incompletely understood. nology, Martin Luther University Halle–Wittenberg, 06112 Halle, Germany; and {First Department of Surgery, University of Yamanashi, Yamanashi 409-3898, Japan A better knowledge of this would seem important, because it is well established that NKG2DLs play an important role in the Received for publication August 2, 2011. Accepted for publication December 30, 2011. recognition of tumor cells by NK cells. Additionally, tumor cells This work was supported by grants from the Cancer Society of Stockholm, the expressing NKG2DLs can become susceptible to NK cell killing Karolinska Institutet, the Stockholm City Council, the Swedish Cancer Society, even if they have normal MHC class I expression (1, 7). Thus, the and the Swedish Research Council. balance between NKG2DLs and MHC class I expression on Address correspondence and reprint requests to Prof. Rolf Kiessling, Immune and transformed cells is important for their survival under the immune Gene Therapy Laboratory, Department of Oncology and Pathology, Cancer Center Karolinska, R8:01, Karolinska Hospital, 171 76 Stockholm, Sweden. E-mail address: surveillance of their host (8). [email protected] The epidermal growth factor (EGF) family consists of four The online version of this article contains supplemental material. closely related transmembrane receptors (EGFR, Abbreviations used in this article: ATM, ataxia-telangiectasia–mutated protein also known as HER1, 2, 3, and 4). On binding small peptide ligand kinase; ATR, ataxia-telangiectasia–mutated and Rad3-related ; EGF, molecules, HER receptors undergo homo- or heterodimerization epidermal growth factor; MFI, mean fluorescence intensity; DMFI, increase in mean fluorescence intensity; MICA, MHC class I chain A; MICA/B, MHC class I chains A and autophosphorylate, which activates downstream signaling and B; MICB, MHC class I chain B; NKG2D, NK group 2, member D; NKG2DL, pathways resulting in enhanced cell proliferation, migration, in- NK group 2, member D ligand; NRG, neuregulin; rMFI, relative mean fluorescence vasion, and survival (9–11). The HER2/HER3 dimer is considered intensity; siRNA, small interfering RNA. to be the most active HER signaling dimer and is crucial for sig- Copyright Ó 2012 by The American Association of Immunologists, Inc. 0022-1767/12/$16.00 naling in tumors containing amplification of HER2 (12–14). HER3

www.jimmunol.org/cgi/doi/10.4049/jimmunol.1102237 2 HER2/HER3 SIGNALING REGULATES THE EXPRESSION OF MICA/B has several ligands, including the neuregulins (NRG) 1–4, but it mean fluorescence intensity (DMFI) was calculated as: (MFI with specific lacks intrinsic tyrosine kinase activity. In contrast, HER2 has no mAb 2 MFI with isotype control)/MFI with isotype control. Relative MFI defined ligand but possesses an active tyrosine kinase domain (15, (rMFI) values were calculated to compare the differences between DMFI of a specific treatment and control as: 100 3 (DMFI of specific treatment/ 16). Ligand-induced heterodimerization of HER3 with HER2 DMFI of control treatment). activates cell growth and cell proliferation via PI3K/AKT and RAS/MAPK signaling pathways (17, 18). HER2 overexpression is Western blot analysis one of the poor prognostic factors in breast cancer (19, 20), and Cells were washed in PBS (2), lysed during 15 min shaking in CelLytic HER3 is often expressed together with HER2 in this disease (21– lysis buffer (Sigma-Aldrich) with a protease inhibitor cocktail and a phos- 23). Both molecules are considered promising targets for cancer phatase inhibitor cocktail (both from Sigma-Aldrich). Cell debris was therapy, with HER2 targeting therapies already in clinical practice cleared at 20,000 3 g for 15 min. The supernatants were collected while (24, 25), and HER3 targeting therapies currently in clinical trials cell lysates and the protein concentrations were analyzed using a BCA protein assay (Thermo Scientific, Rockford, IL) according to the manu- (26, 27). facturer’s protocol. Equal amounts of protein were separated on 4–12% Although HER2 and HER3 are considered potential targets for NuPage Bis-Tris acrylamide gels (Invitrogen) and transferred to polyvi- cancer therapy, they have also been associated with an “immune nylidene difluoride membranes (Immobilon-P; Millipore, Bedford, MA). 2 escape” phenotype. Thus, several groups reported that overex- Blots were blocked for 30 min in PBS ( ) with 0.05% Tween 20 (Sigma- Aldrich) buffer (PBST) with 2% nonfat dry milk, followed by incubation pression of the HER2 oncogene can lead to tumor escape from the overnight at 4˚C in PBST with 2% nonfat dry milk and primary Abs against host immune system via downregulation of MHC class I (28–30), HER2 (Calbiochem), p-HER2 (Tyr1221/1222) (Cell Signaling Technology, and overexpression of HER2 severely impaired MHC class I- Beverly, MA), HER3 (Upstate Biotechnology, Lake Placid, NY), p-HER3 1222 473 202 (Tyr ), AKT, p-AKT (Ser ), p44/42 MAPK, p-p44/42 MAPK (Thr / Downloaded from restricted recognition by CTLs (31, 32). These findings suggest 204 that activation of the HER2 signaling pathways leads to tumor es- Try ) (Cell Signaling Technology), and b-actin (Sigma-Aldrich). After two washes in PBST, membranes were incubated with HRP-linked goat cape from the adaptive immune system. However, because MHC anti-rabbit or anti-mouse IgG Abs (Cell Signaling Technology) for 1 h at class I molecules activate the inhibitory signaling in NK cells (33), room temperature. After blots were washed three times in PBST, enhanced HER2-induced downregulation of MHC class I may instead make chemiluminescence (GE Healthcare, Fairfield, CT) was performed, and them suitable targets for NK cell-mediated tumor rejection by images were captured using an LAS-1000 camera system (Fujifilm, Tokyo, Japan). Membranes were stripped in strip buffer (PBS (2) with 2% SDS a direct NK cell–tumor interaction or by NK cell-mediated Ab- [Invitrogen] and 0.7% 2-ME [Sigma-Aldrich]) for 30 min at 50˚C and http://www.jimmunol.org/ dependent cellular cytotoxicity. Because NK cell-mediated cyto- reprobed up to three times. Densitometric analyses for protein quantifica- toxicity is regulated by the balance between activating and inhibit- tion of each blot were calculated using ImageJ 1.42q (http://rsb.info.nih. ing signals (34), it is of particular interest to understand whether gov/ij). The experiments were repeated three times. HER2/HER3 signaling also would affect the expression of activat- Cell proliferation assays ing NK cell receptors. In this study, we demonstrate that HER2/ HER3 signaling regulates the expression of MICA/B molecules in Cells were collected and stimulated with 2–250 ng/ml NRG1b for 15 min, 3 human breast carcinoma cell lines, resulting in enhanced sensitivity then seeded in triplicate into 96-well flat-bottom plates at 5 3 10 cells per 100 ml per well in RPMI 1640 with 10% FBS and 2 mM L-glutamine to NK cell-mediated recognition. and incubated for 3 d. The relative percentage of metabolically active cells compared with that of untreated cells was determined on the basis by guest on September 25, 2021 of mitochondrial conversion of MTT (Molecular Probes, Eugene, OR) Materials and Methods according the manufacturer’s protocol. In brief, after incubation, 10 ml Cell culture and reagents MTT solution (5 mg/ml) was added to each well and incubated for 4 h. Thereafter, 50 ml DMSO was added to 25 ml cell supernatant and incu- Human breast cancer cell lines MDA-MB-231, MDA-MB-453, and T47D bated for 10 min and plates were read at 540 nm using Versa Max (Mo- were obtained from the American Type Culture Collection. The genotypes lecular Devices, Sunnyvale, CA). of all cell lines were identified by short tandem repeat method using STR Identifier (Applied Biosystems, Foster City, CA) and authenticated. All cell lines were maintained in culture in RPMI 1640 medium with 2 mM Apoptotic assays L-glutamine (Invitrogen, Carlsbad, CA) supplemented with 10% FBS Cells were seeded into 24-well plates at 104 cells per well and treated with (Invitrogen) and 50 U/ml penicillin and streptomycin (Sigma-Aldrich, St. 2–250 ng/ml NRG1b for 2 d. Both nonadherent and adherent cells were Louis, MO) at 37˚C in a humidified atmosphere with 5% CO2. For cell washed in PBS (+), trypsinized, and collected. Cells were pelleted at culture work, LY294002 (Calbiochem, San Diego, CA), PD98059 (Cal- 450 3 g, washed by PBS (–), repelleted, and labeled according to the man- biochem), and lapatinib (GlaxoSmithKline, Brentford, U.K.) were dis- ufacturer’s protocol. Briefly, each sample was stained with 50 ml mixture solved in DMSO (Sigma-Aldrich). NRG1b (R&D Systems, Minneapolis, regent containing annexin V binding buffer with 2.5 ml FITC-labeled MN), caffeine (Sigma-Aldrich), and doxorubicin hydrochloride (Sigma- annexin V (eBioscience) in a V-bottom 96-well plate at room tempera- Aldrich) were dissolved in PBS (2). ture for 15 min. The cells were resuspended in 300 ml annexin V binding buffer and transferred to FACS tubes for analysis. Cells were acquired on Analysis of cell surface molecules by flow cytometry a FACSCalibur cytometer and analyzed using FlowJo software 6.4.7. The experiments were repeated three times. Tumor cells were washed in PBS (2), collected with EDTA (Sigma- Aldrich), and analyzed for cell surface expression of HER family pro- DNA transfection teins, MICA/B, MICA, MICB, and HLA class I by direct (for EGFR, HER2, HER3, HLA-A,B,C, MICA/B, MICA, and MICB) or indirect (for The vector pRK5/HER3 was a gift from Prof. Axel Ullrich (Max Planck HER4) immunofluorescence using mAbs, including PE-labeled EGFR Institute, Martinsried, Germany). Tumor cells were grown in 12-well plates (EGFR.1; BD Biosciences, San Diego, CA), allophycocyanin-labeled and transfected upon reaching ∼70–90% confluence with pcDNA3/empty HER2 (Neu 24.7; BD Biosciences), PE-labeled HER3 (1B4C3; Bio- (Invitrogen) or pRK5/HER3. Cells were transfected in Opti-MEM I cell Legend, San Diego, CA), FITC-labeled HLA-A,B,C (G46-2.6; BD Bio- culture medium (Invitrogen) with 4.0 mg plasmid using 10 ml Lipofect- sciences), PE/allophycocyanin-labeled MICA/B (6D4; BioLegend), PE- amine 2000 (Invitrogen) according to the manufacturer’s protocol. After labeled MICA (159227; R&D Systems), allophycocyanin-labeled MICB 24 or 48 h, transfected cells were collected for further experiments. (236511; R&D Systems), or purified HER4 (H4.77.16; Calbiochem) as first Ab, with PE-labeled anti-mouse Igs (RPE; affinity-isolated F(ab9)2; Small interfering RNA assay Dako, Humburg, Germany) as secondary Ab, and FITC-, PE- or allophycocyanin-labeled anti-mouse IgG1k (MOPC-21; BD Biosciences) Cell lines were grown in 12-well plates and transfected upon reaching 50% or IgG2bk (27-35; BD Biosciences) as isotype controls. Stained cells were confluency with small interfering RNA (siRNA) targeting HER2 or HER3 acquired on a FACSCalibur cytometer (BD Biosciences) and analyzed (SMARTpools; Dharmacon, Lafayette, CO) along with the control siRNA using FlowJo software 6.4.7 (Tree Star, Ashland, OR). The increase in (Dharmacon). Cells were transfected in Opti-MEM I cell culture medium The Journal of Immunology 3 using Lipofectamine 2000 according the manufacturer’s protocol. After Statistical analyses 48 h, transfected cells were used for further experiments. Differences in means were evaluated with a Student t test or Fisher exact NK cell-mediated cytotoxicity assay test. All analyses were performed at a significance level of 5% (p , 0.05) using GraphPad Prism 5 (GraphPad Software, La Jolla, CA). This work was approved by the Institutional Review Board of Karolinska University Hospital. PBMCs were collected from healthy donor buffy coats through density centrifugation. PBMCs cultured with 100 IU/ml IL-2 Results (Proleukin; Chiron, Emeryville, CA) for 3 d or negatively purified NK cells (NK cell isolation II; Miltenyi Biotec, Auburn, CA) rested overnight HER3 influences expression of MICA/B in breast carcinoma served as the source of effector NK cells. The phenotype of the cells (CD32 cell lines CD56+) was confirmed by flow cytometric analysis using PE-labeled anti- CD3 (HIT3a) and allophycocyanin-labeled anti-CD56 (HCD56) Abs (both The ability of HER2/HER3 signaling to influence MICA/B ex- from BioLegend). Treated or untreated target cells were tested for sensitivity pression levels was analyzed in breast cancer cell lines by flow to NK cell-mediated lysis in a standard chromium release assay (35). [51Cr] cytometry. MDA-MB-231 (low HER2/HER3 levels; Supplemental release in the supernatants was measured by a gamma counter (Wallac Fig. 1) and T47D and MDA-MB-453 (high HER2/HER3 levels; Sverige, Upplands Va¨sby, Sweden). The percentage of specific lysis was Supplemental Fig. 1) cell lines were chosen for these experiments. calculated as follows: 100 3 [(experimental release 2 spontaneous release)/ (maximum release 2 spontaneous release)]. To analyze the involvement of First, tumor cells were treated for 48 h with lapatinib, an EGFR NKG2D in the cytolytic activity of NK cells, effector cells were coincubated and HER2 tyrosine kinase inhibitor (36) that also can inhibit HER2/ with 20 mg/ml anti-NKG2D blocking Ab (1D11; BioLegend) or an isotype- HER3 signaling (37). Expression of MICA/B was significantly matched control Ab (11711; R&D Systems) at 4˚C for 30 min and washed downregulated by lapatinib in the HER2/HER3 high-expressing before the cytolytic assay. To analyze the involvement of MICA/B, anti- MICA/B (6D4; BioLegend) or isotype-matched control Ab (MOPC-173; T47D and MDA-MB-453 cell lines but was not affected in the Downloaded from BioLegend) was added to the target cells at 4˚C for 30 min before the co- HER2/HER3 low-expressing MDA-MB-231 cell line (Fig. 1A). culture with NK cells to a final concentration of 10 mg/ ml. These findings suggest that HER2/HER3 signaling is involved in http://www.jimmunol.org/ by guest on September 25, 2021

FIGURE 1. The expressions of MICA/B, MICA, and MICB are regulated by HER2/HER3 signaling in HER2/HER3 high-expressing breast carcinoma lines. (A) Breast carcinoma cell lines were treated with 1 mM lapatinib (LAP) or as a control with 0.01% DMSO for 48 h. (i) The effects on the MICA/B, MICA, and MICB expression were assessed by flow cytometry as shown in the representative histograms. (ii) The rMFI of MICA/B, MICA, and MICB were calculated based on at least three independent experiments and evaluated with a Student t test. (B) T47D cells were transfected with siRNA targeting HER2 (siHER2), HER3 (siHER3), or control siRNA (siCtr). (i) Forty-eight hours after the transfection, the HER2 and HER3 levels were assessed by Western blot analyses. (ii) The blots were semiquantified, and the columns represent the mean values of three independent experiments. The silencing effect was evaluated with a Fisher exact test. (iii) Representative histograms demonstrating the MICA/B, MICA, and MICB expression in cells upon genetic silencing (as in- dicated), and mean values of (iv) MICA/B and (v) MICB rMFI of three independent experiments were evaluated with a Fisher exact test. Bars indicate SEM. *p , 0.05, ***p , 0.001. A.U., Arbitrary units; ISO, isotype control. 4 HER2/HER3 SIGNALING REGULATES THE EXPRESSION OF MICA/B the regulation of MICA/B expression in breast cancer cells, when suggest that HER2/HER3 regulates the expression of MICA/B HER2/HER3 are expressed at high levels. molecules predominantly by the PI3K/AKT pathway. Next, we silenced either HER2 or HER3 in the high-expressing T47D breast carcinoma line using siRNA. Forty-eight hours after Activation of HER2/HER3 signaling with the HER3 ligand b siRNA transfection, the expression of HER2 or HER3 in T47D NRG1 enhances the expression of MICA/B cells was found to be attenuated equally, as measured in Western Next, we asked whether a ligand-induced HER2/HER3 signaling blot (Fig. 1Bi, 1Bii). Interestingly, the expression of MICA/B was will impact MIC expression as tested with Abs both against total significantly downregulated by siRNA of HER3 but not by siRNA MICA/B and as tested separately against MICA and MICB. To of HER2 in T47D cells (Fig. 1Biii, 1Biv). MDA-MB-453 cells study this, NRG1b, the most important HER3 ligand known also showed similar results (Supplemental Fig. 2Ai). to activate signaling via heterodimerization of the HER2/HER3 Next, MICA and MICB were assessed separately after lapatinib complex (12, 40), was used. A significant upregulation of total or siRNA treatment. Whereas lapatinib had a strong effect on MICA/B could be achieved in the HER3 high-expressing MDA- MICB and no effect on MICA in T47D cells, the opposite was true MB-453 and T47D cells following a 24-h exposure to NRG1b. in MDA-MB-453 cells (Fig. 1Ai). The upregulation was mainly accounted for by an increase in Both HER2 and HER3 siRNA downregulated MICB expression MICB for T47D cells and in MICA in MDA-MB-453 cells (Fig. in T47D cells (Fig 1Biii, 1Bv). The effect, however, was signifi- 4A). In contrast, no effect of NRG1b was observed in the HER3 cantly stronger for siRNA of HER3 as compared with siRNA of low-expressing MDA-MB-231 cells. Ligation with NRG1b was HER2. Only siRNA of HER3, but not siRNA of HER2, was able confirmed to induce phosphorylation of HER3 and to activate both to downregulate MICA in MDA-MB-453 cells (Supplemental Fig. AKT and MAPK downstream signaling pathways in HER3 high- Downloaded from 2A). These findings suggested that the expression level of HER3, expressing T47D cells, but not or only marginally in HER3 low- but not of HER2, is important for the expression of MICA/B expressing MDA-MB-231 cells, although the MAPK pathway was molecules, which is why for the subsequent experiments we fo- already constitutively activated in the latter cell line (Fig. 4B). No cused on the role of HER3 in regulating MICA/B expression. effect of NRG1b was found on tumor cell proliferation or tumor To investigate this further, the expressions of MICA and MICB cell apoptosis in T47D cells (Supplemental Fig. 3A, 3B).

were analyzed after transient transfection with HER3 in the HER2/ http://www.jimmunol.org/ HER3 low-expressing MDA-MB-231 cells. In MDA-MB-231 cells NRG1b-induced MICA/B are regulated by HER2/HER3 transfected with HER3, but not in empty vector transfected cells, signaling and not by genotoxic stress HER3 was strongly expressed and phosphorylated (Fig. 2A). In It has been demonstrated that NKG2D ligand expression can be line with the aforementioned observations, marked increases in induced by activation of the DNA stress-sensing ataxia-telan- MICA/B and MICA were observed in the HER3 transfected cells giectasia–mutated (ATM) and ATM and Rad3-related protein compared with empty vector transfected ones (Fig. 2B), whereas kinases (ATR) (5). We therefore tested whether the NRG1b in- no increase in MICB expression was noticed (Fig. 2Bi). duced upregulation of MICA/B was mediated directly by HER2/ HER3 signaling or via activation of ATM and ATR. We use MICA/B are regulated mainly by the PI3K/AKT pathway in NRG1b as an activator of HER2/HER3 signaling, while topo- by guest on September 25, 2021 breast carcinomas II inhibitor doxorubicin, known to activate ATM, was The downstream signaling of HER2/HER3 is mainly mediated used as an inducer of MICA/B expression via DNA stress (41–43). via the PI3K/AKT and RAS/MAPK pathways (17, 18). To fur- Lapatinib was used as an inhibitor of HER2/HER3 signaling (37), ther evaluate whether these pathways regulate the MIC expres- while caffeine was used as inhibitor of ATM and ATR (43, 44). sion, three cell lines (T47D, MDA-MB-453, and MDA-MB-231) The baseline expression of MICA/B was enhanced by doxorubi- were treated with the PI3K/AKT signal inhibitor LY294002 (38) cin, while being downregulated by caffeine in T47D cells (Fig. and the RAS/MAPK signal inhibitor PD98059 (39). LY294002 5Ai, 5Aii). As expected, doxorubicin-induced MICA/B expression strongly downregulated the expression of MICA/B in T47D and was blocked by caffeine in T47D cells (Fig. 5Aiii, Supplemental MDA-MB-231 cells at concentrations between 10 and 30 mM and Fig. 4Ai). Next, T47D cells were pretreated with 1 mM caffeine or moderately in the MDA-MB-453 cells. In contrast, treatment with 1 mM lapatinib for 2 h prior stimulation with 50 ng/ml NRG1b for PD98059 only resulted in minor changes (Fig. 3). These findings 24 h. Interestingly, NRG1b-induced MICA/B expression was not

FIGURE 2. Overexpression of HER3 results in enhanced MICA/B and MICA expression in MDA-MB-231 cells. (A) Expression and phosphorylation of HER3 in HER3 low-expressing MDA-MB-231 breast carcinoma cells transfected with an empty vector (Ctr) or HER3 vector were determined by Western blot analysis. (Bi) Histograms demonstrating MICA/B, MICA, and MICB expression in cells transfected with empty vector or HER3 vector as assessed by flow cytometry, and (ii) the according rMFI mean values of MICA/B and MICA were evaluated with a Student t test as a result from three independent experiments. Bars indicate SEM. *p , 0.05, **p , 0.01. ISO, Isotype control. The Journal of Immunology 5

NRG-induced NK cytotoxicity is NKG2D-dependent Because NRG1b enhanced the expression of MICA/B in HER3 high-expressing cells, but not in HER3 low-expressing ones, we next evaluated whether NRG1b treatment would affect their sensitivity to NK cell-mediated cytotoxicity. Tumor cells were treated with 50 ng/ml NRG1b for 24 h, and thereafter their sen- sitivity to NK cell cytotoxicity was evaluated using IL-2–activated PBMCs or purified NK cells as effector cells. As a consequence of NRG1b treatment, a strong increase in sensitivity for both IL-2– activated PBMCs or purified NK cells was noted in HER3 high- expressing T47D cells (Fig. 6Ai, 6Aii), but not in HER3 low- expressing MDA-MB-231 cells (Fig. 6Aiii, 6Aiv). We next ana- lyzed whether lapatinib-induced suppression of MICA/B expres- sion would result in decreased sensitivity to NK cell-mediated killing in HER2/HER3 overexpressing T47D and MDA-MB-453 cells. This was shown to be the case, as a significant decrease in sensitivity to purified NK cells was found as a consequence of lapatinib treatment of both the T47D and the MDA-MB-453 tu- mor cell lines (Fig. 6B). Because neither treatment with NRG1b Downloaded from nor with lapatinib affected MHC class I expression on any of the tested target cells (data not shown), this argues for a direct causal relationship between the upregulation of MICA/B expression by NRG1b and the increase in tumor NK sensitivity. MICA/B are recognized by the NKG2D receptor expressed on NK cells and CD8+ T cells (1, 45). To verify that this receptor is http://www.jimmunol.org/ involved in the NRG1b-induced sensitivity to NK cell killing, purified NK cells were pretreated with anti-NKG2D blocking Ab or target cells were pretreated with anti-MICA/B blocking Ab before the NK assay. The anti-NKG2D blocking Ab inhibited NRG1b-induced NK cytotoxicity against the T47D tumor tar- get, whereas treatment with an isotype control Ab had no effect (Fig. 6C). Additionally, anti-MICA/B blocking Ab also inhibited FIGURE 3. The PI3K/AKT pathway regulates the expression of MICA/ NRG1b-induced NK cytotoxicity in T47D cells (Fig. 6D), indi- A B C by guest on September 25, 2021 B. Breast carcinoma cells—( ) T47D, ( ) MDA-MB-453, and ( )MDA- cating that the NRG1b-induced NK cell-mediated cytotoxicity MB-231—were cultured with various concentration of the PI3K inhibitor was dependent on NKG2D–MICA/B interaction. LY294002 (LY), MEK1 inhibitor PD98059 (PD), or DMSO (0.03%) as control. The effects on the expression of MICA/B treated with each in- hibitor are shown as representative histograms, and the rMFI mean values Discussion of three independent experiments were evaluated with a Fisher exact test. In the present study, we report several findings supporting an Open histograms show isotype control, gray histograms show cells treated with DMSO, and black histograms show cells treated with 30 mM each important role for HER2/HER3 in regulating MICA/B expression reagent. Bars indicate SEM. *p , 0.05, **p , 0.01. ISO, Isotype control. in breast carcinoma cell lines. Our observations support the notion that oncogenes directly can induce the expression of NKG2DL. This is in line with the report by Boissel et al. (6), who reported that the BCR/ABL oncogene can regulate the expression of MICA in blocked by caffeine (Fig. 5Bi, Supplemental Fig. 4Aii), but was chronic myelogenous leukemia, and extends these findings to solid clearly blocked by lapantinib (Fig. 5Ci, Supplemental Fig. 4Aiii). tumors expressing the HER2/HER3 oncogenes. MICA/B proteins Immunobloting showed that lapatinib, but not caffeine, attenuated induced by oncogenes such as HER2/HER3 and BCR/ABL could NRG1b-induced phosphorylation of both HER2 and HER3, re- be one mechanism whereby the innate immune system is alerted sulting in inhibition of the downstream pathways of HER2/HER3 to the presence of a developing tumor. The regulation of MICA/B signaling (Fig. 5Bii, 5Cii). expression by oncogenes has not previously been investigated in We also assessed whether siRNA of HER2 and HER3 could solid tumors, which is why we evaluated in breast carcinoma cell block the NRG1b-induced upregulation of MICA/B expression. lines the HER2/HER3 signaling pathways involved in MICA/B The results showed that silencing of HER3 almost completely regulation as well as their impact on NK cell-mediated killing. blocked NRG1b-induced upregulation of MICA/B, whereas si- We demonstrated that lapatinib downregulates the expression of lencing of HER2 had no effect (Fig. 5Di, Supplemental Fig. 4B). MICA/B in HER2/HER3 high-expressing breast cancer cell lines. Furthermore, silencing HER3 significantly blocked NRG1b-in- Although lapatinib is mainly recognized as a dual inhibitor of duced phosphorylation of both AKT and MAPK, whereas HER2 EGFR and HER2 (36), lapatinib was also shown to block the silencing had no effect on these proteins (Fig. 5Dii). MDA-MB- ligand-induced phosphorylation of HER3, induced by the heter- 453 cells also showed similar results (Supplemental Fig. 2B). odimerization of HER3 and HER2, and as a consequence to in- These findings suggested that NRG1b-induced upregulation of hibit p-AKT and p-MAPK (37). These initial findings suggested MICA/B was not enhanced by genotoxic stress but by a direct that HER2/HER3 signaling may be involved in the regulation of activation of HER2/HER3 signaling. Additionally, they under- the expression of MICA/B. score that HER3 is a more important receptor partner than HER2 Several of our observations suggested a dominant role for HER3 for the ligand-induced MICA/B upregulation. as compared with HER2 in enhancing the expression of MICA/B. 6 HER2/HER3 SIGNALING REGULATES THE EXPRESSION OF MICA/B Downloaded from http://www.jimmunol.org/ by guest on September 25, 2021

FIGURE 4. Effects of NRG1b on the expression of MICA/B, MICA, and MICB. (A) Cells were treated with 50 ng/ml NRG1b for 24 h. (i) MICA/B, MICA, and MICB expression with or without NRG1b treatment were assessed in HER3 high-expressing (T47D and MDA-MB-453) and HER3 low- expressing (MDA-MB-231) cell lines, and (ii) the rMFI of MICA/B, MICA, and MICB in cells with or without NRG1b treatment were evaluated with a Student t test. Open histograms show isotype control (ISO), gray histograms show no treatment (NT), and black histograms show cells treated with NRG1b (NRG). Bars indicate SEM. *p , 0.05, **p , 0.01, ***p , 0.001. (B) Phosphorylation of HER2, HER3, and their downstream signaling molecules in T47D and MDA-MB-231 cells untreated or treated with 50 ng NRG1b for various intervals is shown. A comparable amount of cell lysate- derived protein was loaded in each lane, and Western blot analysis was performed with Abs specific for p-HER3, p-HER2, p-AKT, p-p44/42 MAPK with anti–b-actin Ab as an internal control. The experiments were repeated three times.

Thus, we found a marked increase of MICA in HER3 transfected factor for the regulation of both MICA and MICB expression in HER2/HER3 low-expressing MDA-MB-231 cells. We also dem- breast carcinoma cell lines. In support of this interpretation of our onstrated that silencing HER3 in cells expressing high levels of data, the expression level of HER2 is much higher than that of HER2/HER3 significantly attenuated the expression of MICA in HER3 in both T47D and MDA-MB-453 cells. As an alternative MDA-MB-453 cells and that of MICB in T47D cells, whereas explanation to the seemingly dominant role of HER3 as compared silencing of HER2 had less impact. Taken together, this suggested with HER2, other receptors may take over as heterodimerization that HER3 but not HER2 levels represent an important rate-limiting partners to HER3 when HER2 is silenced. Thus, also EGFR can The Journal of Immunology 7 Downloaded from http://www.jimmunol.org/ by guest on September 25, 2021

FIGURE 5. Effects of caffeine, lapatinib, and HER2 or HER3 silencing on doxorubicin- or NRG1b-induced upregulation of MICA/B in T47D cells. (A) Histograms demonstrating MICA/B expression in cells treated with (i) 0.05 mM doxorubicin or (ii) 1 mM caffeine for 24 h were assessed by flow cytometry. The cells were pretreated with (Aiii) 1 mM caffeine for 2 h followed by doxorubicin (0.05 mM), (Bi) 1 mM caffeine for 2 h followed by NRG1b (50 ng/ml), (Ci)1mM lapatinib for 2 h followed by NRG1b (50 ng/ml), (Di) underwent gene silencing (as indicated) for 48 h followed by NRG1b (50 ng/ ml) for 24 h. Then the cells were collected and MICA/B surface levels (expressed as rMFI) were assessed by flow cytometric analysis and evaluated with a Fisher exact test. (Bii, Cii, and Dii) Western blot analyses were performed with the cell lysates from the indicated treatment groups with/without NRG1b treatment for an hour using Abs specific for p-HER2, HER2, p-HER3, HER3, p-AKT, p-p44/42 MAPK, and b-actin. Representative data and the according mean values from the semiquantative analysis (expressed in arbitrary units are shown and evaluated with a Student t test. The experiments were repeated three times. Bars indicate SEM. *p , 0.05, **p , 0.01, ***p , 0.001. A.U., Arbitrary unit; CAF, caffeine; CTR, control siRNA; DOX, doxorubicin; ISO, isotype control; LAP, lapatinib; NT, no treatment control. undergo heterodimerization with HER3 (46), although arguing may depend on a more efficient silencing of HER3, although this against this the expression of EGFR is relatively low in T47D was not apparent from our Western blot analysis. There is evi- cells. Finally, we cannot entirely exclude that the greater effect on dence for a differential regulation between MICA and MICB and signaling as a result of silencing HER3 as compared with HER2 that they could respond differently depending on the type of 8 HER2/HER3 SIGNALING REGULATES THE EXPRESSION OF MICA/B

FIGURE 6. Effects of HER2/HER3 signaling on NK cell-mediated cytotoxicity. (A) T47D (HER3 high-expressing) and MDA-MB-231 (HER3 low- expressing) cells treated with/without NRG1b (50 ng/ml) were subjected to 51Cr-release assay for 4 h using IL-2–activated PBMCs (upper graphs) or pu- rified NK cells (lower graphs) as effector cells. (B) NK cell-mediated cytotoxicity against target cell pre- treated with or without 1 mM lapatinib were evalu- ated in both T47D cells for 4 h (left graph) and MDA-MB-453 cells for 18 h (right graph). (C) Pu- rified NK cells were pretreated with blocking Ab of NKG2D or isotype control before cytotoxicity assay with NRG1b-stimulated T47D cells for 4 h and cy- toxicity was evaluated. Data are presented as mean of triplicate samples and are representative of three in- Downloaded from dependent experiments. (D) T47D cells were treated with/without NRG1b for 24 h and pretreated with blocking Ab of MICA/B or isotype control before the cytotoxicity assay with NK cells for 4 h at an E:T ratio of 5:1. Data are presented as mean of triplicate samples and are representative of two independent experiments. Bars indicate SEM. *p , 0.05. ISO, http://www.jimmunol.org/ Isotype control; LAP, lapatinib; NRG, NRG1b;NT, no treatment.

stimuli (47). Our results are in line with this, where we find breast cancer cell lines. In contrast, when analyzing the NRG1b by guest on September 25, 2021 a differential regulation of MICA and MICB as a result of lapa- ligand-induced MICA/B in breast carcinoma cell lines, this was tinib or silencing HER3 in the MDA-MB-453 versus the T47D found not to be blocked by caffeine but only by lapatinib. These cell lines. findings indicated that NRG1b-induced HER2/HER3 activation The two downstream pathways of the HER2/HER3 signaling are does not regulate MICA/B through genotoxic stress but does so the PI3K/AKT and RAS/MAPK pathways (26), and thus we next directly mainly via the PI3K/AKT pathway. Because also BCL/ assessed which of these is more important for baseline expression ABL regulates MICA expression mainly via PI3K/AKT signaling of MICA/B in breast cancer lines. Based on the results from (6), the role of this signaling pathway for the induction of MICA/ experiments with signal inhibitors, we demonstrate that PI3K/ B in other types of cancer merits further investigations. We also AKT is the dominant pathway in the regulation of MICA/B ex- assessed the effect of silencing HER2 or HER3 in the NRG1b- pression. These findings are in line with the studies on the BCR/ induced MICA/B. In line with our findings related to the regula- ABL-dependent MICA regulation (6), where exposure of BCR/ tion of basal MICA and MICB expression, HER3 and not HER2 ABL+ K562 cells to the same PI3K inhibitor LY294002 resulted was found to be important for NRG1b-induced upregulation of in a dose-dependent (in ranges similar to ours) MICA down- MICA/B. Also, we found that NRG1b-induced phosphorylation of modulation. Also in line with our findings, inhibition of MAPK by AKT was blocked more by siRNA of HER3 than by that of HER2. the MEK1 inhibitor PD98059 did not affect MICA expression in These findings suggested that HER3 is an important rate-limiting the same study. Therefore, the PI3K/AKT pathway seems to be factor for both basal and NRG1b-induced expression of MICA/B. the dominant one in inducing MICA/B molecules for the HER2/ A seemingly contradictory finding was that treatment with HER3 oncogenes and MICA molecule for the BCR/ABL onco- NRG1b promoted less changes in MICA/B expression in MDA- gene. MB-453 cells than in T47D cells, whereas MDA-MB-453 cells Signaling via BCR/ABL and HER2/HER3 is different in that expressed more HER2/HER3 as well as MICA/B than did T47D HER2/HER3 signaling can be activated by ligands whereas BCR/ cells. A possible reason for this could be that the high HER2/ ABL cannot. This gave us the possibility of studying both the HER3 expression in MDA-MB-453 cells has already activated baseline and ligand-induced MICA/B expression in HER2/HER3- close to a maximum of downstream signaling, whereas the lower expressing breast carcinomas. ATM–ATR, which plays a key role HER2/HER3 expression in T47D cells leaves room for additional in the DNA damage response pathway, is frequently activated in activation. Alternatively, the intensity of HER2/HER3 signaling tumor cells and was found to regulate NKG2DLs via transcrip- depends not only on HER2/HER3 expression levels but also on tional regulation (5). Our results in T47D cells treated with caf- activating of downstream signaling molecules (48), or feine, an inhibitor of the ATM–ATR pathway, are in line with on hitherto unknown mechanisms. a role also for this pathway in maintaining baseline levels as Because MICA/B are important ligands for the NK-activating well as DNA stress-induced MICA/B in HER2/HER3-expressing receptor NKG2D, it was of primary importance for us to deter- The Journal of Immunology 9 mine the functional consequences of HER2/HER3-mediated MICA expressed in gastrointestinal epithelium. Proc. Natl. Acad. Sci. USA 93: 12445– 12450. and MICB regulations on tumor target cells. As anticipated, ac- 5. Gasser, S., S. Orsulic, E. J. Brown, and D. H. Raulet. 2005. The DNA damage tivation of HER2/HER3 signaling by NRG1b enhanced sensitivity pathway regulates innate immune system ligands of the NKG2D receptor. Nature to NK cell-mediated cytotoxicity in HER3 overexpressing breast 436: 1186–1190. 6. Boissel, N., D. Rea, V. Tieng, N. Dulphy, M. Brun, J. M. Cayuela, P. Rousselot, carcinoma cells. Conversely, inactivation of HER2/HER3 signal- R. Tamouza, P. Le Bouteiller, F. X. Mahon, et al. 2006. BCR/ABL oncogene ing by lapatinib attenuated the sensitivity to NK cell-mediated directly controls MHC class I chain-related molecule A expression in chronic cytotoxicity. These findings implicated a role for the NKG2D– myelogenous leukemia. J. Immunol. 176: 5108–5116. 7. Bryceson, Y. T., M. E. March, H. G. Ljunggren, and E. O. Long. 2006. Acti- MICA/B interaction in the modulation of the NK cell sensitivity in vation, coactivation, and costimulation of resting human natural killer cells. these breast cancer lines. To more directly prove this, we dem- Immunol. Rev. 214: 73–91. 8. Cerwenka, A., J. L. Baron, and L. L. Lanier. 2001. Ectopic expression of retinoic onstrated that NRG1b-enhanced NK cell-mediated cytotoxicity acid early inducible-1 gene (RAE-1) permits natural killer cell-mediated rejec- could be blocked with an anti-NKG2D Ab. This result could not tion of a MHC class I-bearing tumor in vivo. Proc. Natl. Acad. Sci. USA 98: rule out that the interaction between NKG2D ligands other than 11521–11526. 9. Gschwind, A., O. M. Fischer, and A. Ullrich. 2004. The discovery of receptor MICA/B could be of importance for the observed NRG1b-induced tyrosine kinases: targets for cancer therapy. Nat. Rev. Cancer 4: 361–370. enhancement of NK cell activity. Arguing for a predominant role 10. Guy, P. M., J. V. Platko, L. C. Cantley, R. A. Cerione, and K. L. Carraway, III. for MICA and MICB as compared with other NKG2D ligands in 1994. Insect cell-expressed p180erbB3 possesses an impaired tyrosine kinase activity. Proc. Natl. Acad. Sci. 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