Abstract 4671: Inhibition of phosphatidylinositol 3-kinase suppresses propagation of drug-tolerant cancer cell subpopulations enriched by 5-fluorouracil
Authors: Nishizuka Satoshi S. , Ishida Kaoru , Ohmori Yukimi , Kume Kohei , Ito Chie , Konta Akari , Koizumi Yuka , Nukatsuka Mamoru , Kobunai Takashi , Takechi Teiji , Iwaya Takeshi 
Published: July 2016

Abstract
The majority of advanced gastric cancer has been treated with 5-fluorouracil (5-FU)-based chemotherapy after complete resection, but a considerable number of patients experience relapse within 5 years after the resection. It has been considered that extremely small drug-tolerant cancer cell subpopulations are responsible for the relapse after chemotherapy. Previously, we established a 5-FU-tolerant gastric cancer cell line, MKN45T, which requires a higher drug concentration to suppress its growth relative to its parental, MKN45. Using the pair of cell lines, we developed an orthotopic xenograft (OX) model that mimics advanced human gastric cancer by transplantation of the cells into the gastric submucosal layer of nude mice. The OX of the 5-FU-tolerant cell line, MKN45T, showed an aggressive tumorigenicity, metastasis, and peritoneal dissemination, in contrast to the parental cell line, MKN45. Proteomic screening using a colony lysate array, a special format of reverse-phase protein array that allows us to quantify the protein levels in individual drug-tolerant subpopulations (i.e., colonies), revealed the level of phosphatidylinositol 3-kinase (PI3K) phosphorylation was increased according to the 5-FU dose-escalation. The prevalence of cells with active PI3K also increased with 5-FU treatment in OX. In contrast, phosphorylation of Akt and mTOR proteins were slightly decreased by the 5-FU treatment. Interestingly, PTEN protein expression was almost completely suppressed in OX after the 5-FU treatment. To see whether PI3K inhibitors have potency in the reduction of tumors with persistent growth after 5-FU treatment, PI3K inhibitors, including LY294002, Queracetin, Wortmannin, GNE493, and GDC0941, were tested by means of colony formation assay. Among them, simultaneous administration of 5-FU and GDC0941 significantly suppressed colony formation, which led us to examine the inhibitory effect of GDC0941 for tumor growth in the OX model. Sequential administration of 5-FU and GDC0941 prohibited the propagation of 83% (5/6) of OX tumors in the stomach as well as other organs. Western blot was then performed to clarify the growth suppression mechanisms by GDC0941 for PI3K-activated tumors triggered by 5-FU. The Akt/PI3K/mTOR and PTEN pathway analysis in response to single/combinational drug administration revealed that the majority (75%, 6/8) of gastric cancer cell lines tested exhibited an increased level of PI3K phosphorylation with 5-FU administration. The increased level of PI3K phosphorylation with 5-FU, which subsequently induces Akt and mTOR activation, was effectively suppressed by GDC0941 administration, particularly with a considerably decreased level of p70 S6 kinase phosphorylation. These results suggest that administration of 5-FU followed by GDC0941 may suppress relapse after complete resection with 5-FU-based chemotherapy for gastric cancer.
Citation Format: Satoshi S. Nishizuka, Kaoru Ishida, Yukimi Ohmori, Kohei Kume, Chie Ito, Akari Konta, Yuka Koizumi, Mamoru Nukatsuka, Takashi Kobunai, Teiji Takechi, Takeshi Iwaya. Inhibition of phosphatidylinositol 3-kinase suppresses propagation of drug-tolerant cancer cell subpopulations enriched by 5-fluorouracil. [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 4671.