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NOTCH3 Signaling Pathway Plays Crucial Roles in the Proliferation of Erbb2-Negative Human Breast Cancer Cells

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NOTCH3 Signaling Pathway Plays Crucial Roles in the Proliferation of Erbb2-Negative Human Breast Cancer Cells Research Article NOTCH3 Signaling Pathway Plays Crucial Roles in the Proliferation of ErbB2-Negative Human Breast Cancer Cells Noritaka Yamaguchi,1,4 Tetsunari Oyama,10 Emi Ito,6 Hitoshi Satoh,5 Sakura Azuma,4 Mitsuhiro Hayashi,11 Ken Shimizu,13 Reiko Honma,6 Yuka Yanagisawa,6 Akira Nishikawa,6,8,9 Mika Kawamura,6,9 Jun-ichi Imai,6,7 Susumu Ohwada,12 Kuniaki Tatsuta,2 Jun-ichiro Inoue,4 Kentaro Semba,1,4,3 and Shinya Watanabe6,7 1Consolidated Research Institute for Advanced Science and Medical Care, Departments of 2Applied Chemistry and 3Life Science and Medical Bioscience, School of Science and Engineering, Waseda University; 4Department of Cellular and Molecular Biology, Institute of Medical Science and 5Department of Medical Genome Sciences, Graduate School of Frontier Sciences, University of Tokyo; 6Department of Clinical Informatics, Graduate School of Medicine and Dentistry, Tokyo Medical and Dental University; 7Expression Profiles Team, Biological Information Research Center, National Institute of Advanced Industrial Science and Technology; 8Nippon Gene, Co., Ltd.; and 9Medicrome, Inc., Tokyo, Japan; Departments of 10Pathology and 11Surgical Oncology, Dokkyo Medical University School of Medicine, Tochigi, Japan; 12Department of Surgery, Gunma University, Graduate School of Medicine, Gunma, Japan; and 13Department of Pathology, Saitama Social Insurance Hospital, Saitama, Japan Abstract Introduction ErbB2-negative breast tumors represent a significant thera- Breast cancer is the most common type of cancer in women. peutic hurdle because of a lack of effective molecular targets. This disease often progresses to a malignant phenotype with highly Although NOTCH proteins are known to be involved in invasive and metastatic growth properties (1). Continued research mammary tumorigenesis, the functional significance of these on the molecular mechanisms that underlie this disease has proteins in ErbB2-negative breast tumors is not clear. In revealed that a receptor tyrosine kinase, ErbB2, is overexpressed the present study, we examined the expression of activated in 20% to 30% of progressed breast tumors. This research resulted NOTCH receptors in human breast cancer cell lines, including in the development of Herceptin, a humanized antibody against ErbB2-negative and ErbB2-positive cell lines. Activated NOTCH1 and NOTCH3 proteins generated by ;-secretase were ErbB2. Although treatment of ErbB2-overexpressing breast detected in most of the cell lines tested, and both proteins tumors with Herceptin is one of the few examples of a successful activated CSL-mediated transcription. Down-regulation of molecular-based therapy for breast cancer (2, 3), its efficacy is NOTCH1 by RNA interference had little or no suppressive limited because ErbB2-negative breast cancers do not respond to effect on the proliferation of either ErbB2-positive or ErbB2- the medication. Therefore, an improved understanding of the negative cell lines. In contrast, down-regulation of NOTCH3 molecular pathways involved in breast cancer development may significantly suppressed proliferation and promoted apoptosis facilitate the development of novel targeted molecular therapies. of the ErbB2-negative tumor cell lines. Down-regulation of NOTCH proteins belong to a family of conserved transmem- NOTCH3 did not have a significant effect on the ErbB2-positive brane receptors that play fundamental roles in cell fate decisions tumor cell lines. Down-regulation of CSL also suppressed the such as cell proliferation, differentiation, and apoptosis. In proliferation of ErbB2-negative breast tumor cell lines, indi- mammals, there are four NOTCH receptors (termed NOTCH1, cating that the NOTCH-CSL signaling axis is involved in cell NOTCH2, NOTCH3, and NOTCH4) and five ligands (termed proliferation. Finally, NOTCH3 gene amplification was detected Jagged1 and Jagged2 and Delta-like 1, Delta-like 3, and Delta-like in a breast tumor cell line and one breast cancer tissue speci- 4). NOTCH signaling is activated through receptor-ligand inter- men even though the frequency of NOTCH3 gene amplification actions between neighboring cells resulting in successive proteo- was low (<1%). Taken together, these findings indicate that lytic cleavages by tumor necrosis factor-a–converting enzyme and NOTCH3-mediated signaling rather than NOTCH1-mediated the g-secretase complex. This processing releases the NOTCH signaling plays an important role in the proliferation of ErbB2- intracellular domain (NICD), allowing it to enter the nucleus. Once negative breast tumor cells and that targeted suppression within the nucleus, NICD binds to the CSL (also termed CBF-1 and of this signaling pathway may be a promising strategy for RBP-Jn) DNA-binding protein to generate a large transcriptional the treatment of ErbB2-negative breast cancers. [Cancer Res activator complex containing transcriptional coactivators of the 2008;68(6):1881–8] mastermind-like (MAML) family. Formation of this complex results in the expression of various target genes, such as Hes/Hey family genes, which are involved in cell growth, differentiation, and survival (4). There is increasing evidence supporting the association of one or Note: Supplementary data for this article are available at Cancer Research Online more NOTCH pathways with breast cancer development (5, 6). (http://cancerres.aacrjournals.org/). Ectopic expression of active forms of NOTCH1 and NOTCH4 T. Oyama and E. Ito contributed equally to this work. transforms both normal human and mouse mammary epithelial Requests for reprints: Kentaro Semba, Division of Cellular and Molecular Biology, Department of Cancer Biology, The Institute of Medical Science, University of Tokyo, cells in vitro (7–9). Transgenic mice that overexpress the NICD of 4-6-1 Shirokanedai, Minato-ku, Tokyo 108-8639, Japan. Phone: 81-35449-5278; Fax: 81- NOTCH1 or NOTCH3 in mammary glands develop mammary 35449-5278; E-mail: [email protected]. I2008 American Association for Cancer Research. tumors (10), suggesting the transforming potentials of NOTCH1 doi:10.1158/0008-5472.CAN-07-1597 and NOTCH3 in vivo. In human breast cancer, aberrant activation www.aacrjournals.org 1881 Cancer Res 2008; 68: (6). March 15, 2008 Downloaded from cancerres.aacrjournals.org on September 23, 2021. © 2008 American Association for Cancer Research. Cancer Research Figure 1. Expression of NOTCH receptors in human breast cancer cell lines. A, Western blot analysis of NICD of NOTCH1 and NOTCH3 and ErbB2 in eight human breast cancer cell lines. These cell lines are classified by ErbB2 protein expression status, ErbB2-negative cell lines (HCC1143, BT-549, HCC1937, MDA-MB-468, and MCF7) and ErbB2-positive cell lines (BT-474, HCC1419, and SK-BR-3). NICD of NOTCH1 or NOTCH3 is elevated in five of five or four of five ErbB2-negative cell lines, respectively. Expression of a-tubulin is shown as a loading control. B, Western blot analysis of NICD of NOTCH1 and NOTCH3 in ErbB2-negative HCC1143 and HCC1937 cells cultured in the presence or absence of g-secretase inhibitor for 5 h. Treatment with g-secretase inhibitor causes a slight shift of both bands to a higher molecular weight, indicating that these two bands represent the NICDs of NOTCH1 and NOTCH3. C, luciferase activity in ErbB2-negative HCC1143, HCC1937, and MDA-MB-468 cells cotransfected with a Hes1 promoter luciferase construct normal or mutant CSL-binding sites and NOTCH1-, NOTCH3-, or mock siRNA. Columns, mean from experiments done in triplicate; bars, SD. *, P < 0.05 relative to mock-siRNA transfected samples. of NOTCH1 has been observed (11), and elevated expression of the temperature. Immunocomplexes were visualized with an enhanced NOTCH1 and Jagged1 mRNAs correlates with poor prognosis (12). chemiluminescence kit (GE Healthcare). However, it is currently not known which, if any, of the NOTCH Primary antibodies were anti-NOTCH1, -NOTCH3, -NOTCH4, -ErbB2, receptors are involved in the development of ErbB2-negative breast -Erk1/2, and -Akt antibodies (Santa Cruz Biotechnology), anti-NOTCH2 and -Jagged2 antibodies (Orbigen), anti–a-tubulin antibody (Calbiochem), cancers. anti–phosphorylated Erk1/2, phosphorylated-Akt, Jagged1, and cyclin D1 In the present study, we investigated the significance of NOTCH antibodies (Cell Signaling Technologies), and anti-poly(ADP-ribose) poly- signaling in ErbB2-negative human breast cancer cell lines. We merase (PARP) antibody (a kind gift from Dr. T Suzuki, Institute of Medical found that RNA interference (RNAi)–mediated NOTCH3 knock- Science, University of Tokyo). All secondary antibodies were purchased from down inhibits cell proliferation and that the NOTCH3 gene is GE Healthcare. amplified in a breast cancer specimen. These results suggest that Proliferation assay. Cells were plated at 20% to 30% confluency in NOTCH3 is crucial for ErbB2-negative breast tumor development 12-well plates and grown 8 h before siRNA transfection (day 0). On days 2, 4, and may therefore be a promising therapeutic target for these and 6 after siRNA transfection, cells were trypsinized, and the viable cell cancers. number was counted with the Trypan blue exclusion method. Reporter constructs and luciferase assay. The Hes1 promoter-driven luciferase reporter construct was generated by insertion of the Hes1 promoter (nucleotides À93 to À41) containing normal or mutated CSL- Materials and Methods binding sites (CBS; ref. 13) into SphI- and XbaI-digested 3xnB-luc luciferase Cell culture and transfections. All cell lines were purchased from vector (14). In this construct,
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