Abstract 4151: Down regulation of ATP6V1B1 in HER2+ breast cancer leads resistance against trastuzumab mediated antibody dependent cellular cytotoxicity and it might be associated with less efficacy of trastuzumab clinically

Abstract

Abstract Among several resistant mechanisms of trastuzumab for HER2+ breast cancer patients, little is clear on resistant mechanism of trastuzumab mediated ADCC. 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. To establish the trastuzumab mediated ADCC resistant SK-BR-3, PBMCs are co-cultured with 1µg/ml of trastuzumab and SK-BR-3 for 4 hours. We removed CD45 positive PBMC by using MACS separation system and obtained ADCC resistant SK-BR-3. After we expanded the resistant cells and repeated these processes several times, we established the ADCC resistant SK-BR-3. The cytotoxicity of ADCC resistant SK-BR-3, which was evaluated by LDH assay, was significantly reduced as compared with that of normal SK-BR-3 (38.7% and 67.4%, respectively). To sort candidate genes for the resistance, we analyzed the gene expression profile of ADCC resistant SK-BR-3 by using DNA microarray and we focused on ATP6V1B1 gene that was significantly reduced on the resistant cells. 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. To evaluate the role of ATP6V1B1 on the trastuzumab mediated ADCC resistant mechanism, we established ATP6V1B1 knockout SKBR3 by using CRISPR/Cas9 system. We cultured from small numbers of the knock out cells and expand to obtain completely knock out cells. We confirmed that the ATP6V1B1 knockout cell line was knocked out by western blotting and immunohistochemistry. We found that ATP6V1B1-knockout SK-BR-3 were significantly less ADCC activity as compared to control SK-BR-3(67.4% and 21.2%, respectively). Furthermore, as the same as the result of ADCC activity, we found that ATP6V1B1-knockout SK-BR-3 was less AICC (antibody-independent cellular cytotoxicity) activity as compared to control SK-BR-3(41.2% and 12.5%, respectively). Based on the in vitro findings, we evaluated the role of ATP6V1B1 expression in HER2 positive breast cancer patients who are treated with neoadjuvant chemotherapy (NAC) containing trastuzumab. Biopsy samples before NAC were stained with ATP6V1B1 immunohistochemically. Four pathological complete response (pCR) cases after NAC and 4 non pCR cases were included. We found that non pCR cases showed significantly week expression of the ATP6V1B1 protein as compared to that of pCR cases. ATP6V1B1 gene is one of the isoforms of the Vacuolar (V)-ATPase which is located in intracellular membrane that regulate the cytosolic pH and membrane trafficking. It is hypothesized that ATP6V1B1 is involved in somewhere through the process of cytotoxicity mediated by perforin and granzymes and now we are trying to explore the mechanisms further. Citation Format: Mariko Nishie, Eiji Suzuki, Kosuke Kawaguchi, Ayane Yamaguchi, Moe Tsuda, Takeshi Kotake, Masahiro Hirata, Fengling Pu, Keiko Sakamoto, Masakazu Hattori, Tomoharu Sugie, Masakazu Toi. Down regulation of ATP6V1B1 in HER2+ breast cancer leads resistance against trastuzumab mediated antibody dependent cellular cytotoxicity and it might be associated with less efficacy of trastuzumab clinically [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2017; 2017 Apr 1-5; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2017;77(13 Suppl):Abstract nr 4151. doi:10.1158/1538-7445.AM2017-4151