Abstract 3468: Antitumor effect by inhibition of PI3K/AKT/mTOR and RAS/MAPK pathways in human oral squamous cell carcinoma cells
Authors: Tokuzen Norihiko , Nakashiro Koh-ichi 
Published: August 2018

Abstract
Molecular target drugs are used in the treatment of a variety of malignant tumors, but this approach to developing novel therapies for oral squamous cell carcinoma (OSCC) has lagged behind the progress seen for other cancers. The PI3K/AKT/mTOR and RAS/MAPK pathways are important pathways for the growth and survival of cancer cells. These pathways also have been activated such as AKT and ERK overexpression in OSCC. In this study, we investigated the antitumor effect of PI3K/mTOR inhibitor, NVP-BEZ235 and MEK1/2 inhibitor, Trametinib, in human OSCC cells. First, we confirmed the inhibitory effect of NPV-BEZ235 and Trametinib in PI3K/AKT/mTOR and RAS/MAPK pathways by Western blotting. NPV-BEZ235 and Trametinib were reduced phosphorylation of AKT, p70S6K, and MEK1/2 in OSCC cells. Next, we examined the anti-proliferative effect of NVP-BEZ235 and Trametinib using 4 human OSCC cell lines by WST-8 assay. NVP-BEZ235 and Trametinib significantly inhibited the growth of all OSCC cells in a dose-dependent manner in vitro. Further, we investigated potential synergistic effects of inhibiting both the PI3K/AKT/mTOR and RAS/MAPK pathways, by treating with NVP-BEZ235 and Trametinib. The anti-proliferative effect of this combination was measured in OSCC cells by calculating the Combination Index. Combination treatment with NVP-BEZ235 and Trametinib showed a synergistic effect on the cell growth of all OSCC cells in vitro. Subsequently, we analyzed the effect of NBP-BEZ235 and Trametinib on the cell cycle of green fluorescent protein (GFP)-SAS cells by flow cytometry. Treated with NVP-BEZ235 showed no enhanced effect compared to control. Treatment of Trametinib resulted in the decrease in percentage of cells in G2 phase and increase in G1 phase. This indicated that the anti-proliferative effect of Trametinib was due to G1 arrest. Furthermore, treatment of Trametinib increased apoptosis cells in GFP-SAS cells. Next, we assessed the effect of NVP-BEZ235 and Trametinib on the in vivo growth of GFP-SAS cells. GFP-SAS tumor-bearing mice were treated with single, combination, or vehicle by oral daily. Treatment with NVP-BEZ235 and Trametinib significantly reduced the size of tumors compared with vehicle control. However, combination treatment showed no synergistic effect compared to single treatment. Finally, we investigated the gene expression changes with combination treatment using microarray analysis. The suppression of PI3K/AKT/mTOR and RAS/MAPK pathways caused overexpression of genes related to cell cycle and DNA-repair. These results suggest that activation of PI3K/AKT/mTOR and RAS/MAPK pathways support the growth of OSCC cells and NVP-BEZ235 and Trametinib may be a useful therapeutic strategy for OSCC.
Citation Format: Norihiko Tokuzen, Koh-ichi Nakashiro. Antitumor effect by inhibition of PI3K/AKT/mTOR and RAS/MAPK pathways in human oral squamous cell carcinoma cells [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2018; 2018 Apr 14-18; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2018;78(13 Suppl):Abstract nr 3468.