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In Human Breast Cancer Cell Lines Chain-Related Molecule a and B 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 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 Supplementary http://www.jimmunol.org/content/suppl/2012/02/01/jimmunol.110223 http://www.jimmunol.org/ Material 7.DC1 Why The JI? Submit online. • Rapid Reviews! 30 days* from submission to initial decision • No Triage! Every submission reviewed by practicing scientists by guest on September 25, 2021 • Fast Publication! 4 weeks from acceptance to publication *average Subscription Information about subscribing to The Journal of Immunology is online at: http://jimmunol.org/subscription Permissions Submit copyright permission requests at: http://www.aai.org/About/Publications/JI/copyright.html Email Alerts Receive free email-alerts when new articles cite this article. Sign up at: http://jimmunol.org/alerts 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 protein 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 cytokine pro- induction of MICA/B proteins 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 Gene 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 oncogene 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 leukemia 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 oncogenes 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 tyrosine kinase 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 protein kinase; 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-
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