Abstract 1137: Mutation and function of PIK3CA and HRAS in human oral squamous cell carcinoma cells
Authors: Akiyama Hitoshi , Nakashiro Koichi , Tokuzen Norihiko , Tanaka Hiroshi , Hino Satoshi , Hamakawa Hiroyuki 
Published: July 2016

Abstract
Whole-exome sequencing studies showed the mutational landscape of head and neck squamous cell carcinoma including oral cavity. Recent several reports also demonstrated the somatic mutations of oncogenes and tumor suppressor genes in oral squamous cell carcinoma (OSCC), and identified the mutations of HRAS, BRAF, FGFR3, SMAD4, KIT, PTEN, NOTCH1, AKT1, CTNNB1, and PTPN11 to predict disease-free survival of OSCC patients. In this study, we have attempted to elucidate somatic mutations of these genes and their functions in human OSCC cells. We isolated and cultured tumor cells from the resected primary tumor (KT-T), metastatic lymph node (KT-N), and cancerous pleural effusion (KT-M) in the same patient with OSCC. KT-T cells showed epithelial-like shape, whereas both KT-N and KT-M cells indicated the morphological changes to fibroblast-like spindle shape. Genomic DNA was extracted from these cells, and then we performed the mutational analysis by ultra-deep targeted sequencing using Haloplex Cancer Research Panel. All types of KT cells had the mutation of TP53 (R141P, C135S, and E182*) and PIK3CA (H1047R), and an additional active mutation of HRAS (Q61R) was detected in only metastatic KT-N and KT-M cells. Subsequently, we transfected all KT cells with synthetic small interfering RNA (siRNA) specific for PIK3CA (siPIK3CA) and/or HRAS (siHRAS) at the concentration of 10 nM complexed with Lipofectamine RNAiMAX. We confirmed the knockdown effects of these siRNAs in KT cells by quantitative RT-PCR. After transfection of these siRNAs for 72 hours, the growth of KT cells was evaluated by WST-8 assay. Knockdown of PIK3CA or HRAS expression significantly suppressed the growth of KT-T cells only, whereas double knockdown of PIK3CA and HRAS inhibited the growth of KT-N and KT-M cells as well. Next, we investigated the function of mutant HRAS (Q61R) in KT-T cells. Overexpression of mutant HRAS did not change the growth rate and morphology of KT-T cells. Finally, we examined the effects of BEZ-235 (dual PI3K/mTOR inhibitor) and trametinib (MEK1/2 inhibitor) on the growth of KT-T, KT-N, and KT-M cells. BEZ-235 (100 nM) almost completely suppressed the growth of these cells. Trametinib (2 μM) also reduced the growth rate by 16.9% in KT-T, 13.5% in KT-N, and 45.7% in KT-M cells. Furthermore, BEZ-235 combined with trametinib inhibited the cell growth more effectively even at low concentrations. These results suggest that constitutive active mutations of HRAS and PIK3CA support the growth of human OSCC cells and these signaling pathways may be useful therapeutic targets for the patients with OSCC.
Citation Format: Hitoshi Akiyama, Koichi Nakashiro, Norihiko Tokuzen, Hiroshi Tanaka, Satoshi Hino, Hiroyuki Hamakawa. Mutation and function of PIK3CA and HRAS in human oral squamous cell carcinoma cells. [abstract]. In: Proceedings of the 107th Annual Meeting of the American Association for Cancer Research; 2016 Apr 16-20; New Orleans, LA. Philadelphia (PA): AACR; Cancer Res 2016;76(14 Suppl):Abstract nr 1137.