Abstract 5335: Elucidation of initial resistance mechanisms in EGFR mutation-positive lung cancer focusing on YAP1 and cancer stem cells
Authors: Ogimoto Tatsuya , Ozasa Hiroaki , Hashimoto Kentaro , Yoshida Hiroshi , Hosoya Kazutaka , Yamazoe Masatoshi , Ajimizu Hitomi , Funazo Tomoko , Tsuji Takahiro , Yoshida Hironori , Itotani Ryo , Sakamori Yuichi , Hirai Toyohiro 
Published: June 2022

Abstract
Background: EGFR mutation-positive lung cancer has high response rates to EGFR-tyrosine kinase inhibitors (TKIs), but eventually acquires resistance to EGFR-TKIs. Acquired resistance mechanisms are diverse because of tumor heterogeneity, which makes it difficult to overcome acquired resistance. Initial resistance is the resistance mechanism of tumor cells that survive the initial treatment. If we could overcome the initial resistance, we could prevent cancer cells from developing various types of acquired resistance. We are now focusing on YAP1 and cancer stem cells, which have been also reported as acquired resistance mechanisms in various types of cancers.
Methods: PC-9 cells and HCC827 cells which were EGFR mutation-positive lung cancer cell lines were mainly utilized in our experiments; they were purchased from ECACC and ATCC, respectively. We mainly utilized osimertinib as an EGFR-TKI and verteporfin as a YAP1 inhibitor. The nuclear translocation of YAP1 was confirmed by fluorescence immunostaining. siRNA was utilized to knock down YAP1. Cell viability assays were performed using CellTiter-Glo reagent. qRT-PCR was performed to confirm the gene expression of cancer stem cells.
Results: YAP1 is activated by nuclear translocation. In PC-9 cells, YAP1 was localized in the nucleus after osimertinib exposure. In contrast, YAP1 was localized in the nucleus of HCC827 cells before osimertinib exposure and remained in the nucleus even after osimertinib exposure. These data suggest that YAP1 activation is correlated with initial resistance. Cell viability assay showed that both PC-9 cells and HCC827 cells became more sensitive to osimertinib with the knocking down of YAP1. In addition, cell viability assays using PC-9 cells with verteporfin and osimertinib showed increased sensitivity to osimertinib. The gene expression of ALDH1A1 and SOX2 which are involved in cancer stem cells were increased in PC-9 cells after osimertinib exposure. On the other hand, the gene expression of ALDH1A1 was up-regulated but SOX2 was down-regulated in HCC827 cells after osimertinib exposure. We hypothesized that increased gene expression of cancer stem cells occurred downstream of YAP1 followed by initial resistance, thus we are currently confirming the gene alteration of cancer stem cells associated with osimertinib exposure with the knocking down YAP1. We are also considering the investigation of combination therapy of EGFR-TKIs and YAP1 inhibitors in vivo studies.
Conclusions: YAP1 and cancer stem cells may be involved in initial resistance mechanisms to EGFR-TKIs in EGFR mutation-positive lung cancer. Our results suggest that especially YAP1 can be a potential therapeutic target, thus we will continue additional studies of combination therapy of EGFR-TKIs and YAP1 inhibitors. In addition, we will continue to investigate whether the gene for cancer stem cells acts downstream of YAP1.
Citation Format: Tatsuya Ogimoto, Hiroaki Ozasa, Kentaro Hashimoto, Hiroshi Yoshida, Kazutaka Hosoya, Masatoshi Yamazoe, Hitomi Ajimizu, Tomoko Funazo, Takahiro Tsuji, Hironori Yoshida, Ryo Itotani, Yuichi Sakamori, Toyohiro Hirai. Elucidation of initial resistance mechanisms in EGFR mutation-positive lung cancer focusing on YAP1 and cancer stem cells [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2022; 2022 Apr 8-13. Philadelphia (PA): AACR; Cancer Res 2022;82(12_Suppl):Abstract nr 5335.