Abstract 720: Downregulation of ATP6V1B1 in HER2-overexpressing human breast cancer cell line leads resistance against trastuzumab-mediated antibody-dependent cellular cytotoxicity
Authors: Nishie Mariko , Suzuki Eiji , Kawaguchi Kosuke , Sakamoto Keiko , Fukushima Yuji , Hattori Masakazu , Sugie Tomoharu , Toi Masakazu 
Published: August 2015

Abstract
Introduction: PIK3CA mutation has been becoming widely recognized as a resistant mechanism or therapeutic biomarker for trastuzumab based anti HER2 targeting therapy, however little is clear on resistant mechanism of trastuzumab mediated antibody dependent cellular cytotoxicity (ADCC) in HER2 overexpressing (HER2+) breast cancer patients. In the current study, we investigated the role of Vacuolar-ATPase subunit ATP6V1B1 on the resistant mechanism of trastuzumab mediated ADCC in HER2+ human breast cancer cell line SK-BR-3.
Methods and Results:To establish the trastuzumab mediated ADCC resistant SK-BR-3, isolated healthy PBMCs are co-cultured with 1-10 μg/ml of trastuzumab and SK-BR-3 for 4-9 hours. The cell mixture was incubated with CD45 magnetic beads and CD45 positive PBMCs were removed from the mixture using MACS cell separation system (Miltenyi Biotec, Germany). The survived SK-BR-3 thereafter co-cultured with 1-10 μg/ml of trastuzumab and PBMCs for several times. After 7 passages of the resistant cell making procedure (approximately for 6 months), cytotoxicity was measured by conventional ADCC assay (E/T ratio = 10/1) with various concentration of trastuzumab. The cytotoxicity of ADCC resistant SK-BR-3 was significantly reduced as compared with normal SK-BR-3 cytotoxicity in 10μg/ml of trastuzumab (15.9% and 42.8%, respectively). Then, we analyzed the gene expression profile of ADCC resistant SK-BR-3 by using DNA microarray to sort candidate genes for the resistance. Several genes were up- or down-regulated in ADCC resistant SK-BR-3 and we focused on ATP6V1B1 gene that was significantly reduced on the resistant cells. To clarify the result of comprehensive gene expression analysis, we investigated ATP6V1B1 mRNA level of SK-BR-3 and resistant cell by using real time PCR and found the 50% reduction of ATP6V1B1 mRNA level in ADCC resistant cells. Thus, to evaluate the role of ATP6V1B1 on the trastuzumab mediated ADCC resistant mechanism, ATP6V1B1-knocked down SK-BR-3 (sh-RNA knockdown method) were tested in trastuzumab mediated ADCC assay and found that ATP6V1B1-knocked down SK-BR-3 were significantly less ADCC activity with LDH assay as compared to control SK-BR-3 (19.1% and 40.0%, respectively).Conclusions: ATP6V1B1 might be one factor for trastuzumab mediated ADCC resistance in HER2+ breast cancer cells.
Citation Format: Mariko Nishie, Eiji Suzuki, Kosuke Kawaguchi, Keiko Sakamoto, Yuji Fukushima, Masakazu Hattori, Tomoharu Sugie, Masakazu Toi. Downregulation of ATP6V1B1 in HER2-overexpressing human breast cancer cell line leads resistance against trastuzumab-mediated antibody-dependent cellular cytotoxicity. [abstract]. In: Proceedings of the 106th Annual Meeting of the American Association for Cancer Research; 2015 Apr 18-22; Philadelphia, PA. Philadelphia (PA): AACR; Cancer Res 2015;75(15 Suppl):Abstract nr 720. doi:10.1158/1538-7445.AM2015-720