Cancer Research Institute, Seoul National University College of Medicine, Seoul, Republic of Korea.
Abstract
Aberrations of Phosphoinositide 3-kinase (PI3K)/AKT signaling are frequently observed in many types of cancer, promoting its emergence as a promising target for cancer treatment. PI3K can become activated by various pathways, one of which includes RAS. RAS can not only directly activate the PI3K/AKT pathway via binding to p110 of PI3K, but also regulates mTOR via ERK or RSK independently of the PI3K/AKT pathway. Thus, actively mutated RAS can constitutively activate PI3K signaling. Additionally, in RAS tumorigenic transformation, signal transducer and activator of transcription 3 (STAT3) has been known also to be required. In this study, we examined the efficacy of NVP-BKM120, a pan-class I PI3K inhibitor in human gastric cancer cells and hypothesized that the combined inhibition of PI3K and STAT3 would be synergistic in KRAS mutant gastric cancer cells. NVP-BKM120 demonstrated anti-proliferative activity in 11 human gastric cancer cell lines by decreasing mTOR downstream signaling. But NVP-BKM120 treatment increased p-AKT by subsequent abrogation of feedback inhibition by stabilizing insulin receptor substrate-1. In KRAS mutant gastric cancer cells, either p-ERK or p-STAT3 was also increased upon treatment of NVP-BKM120. The synergistic efficacy study demonstrated that dual PI3K and STAT3 blockade showed a synergism in cells harboring mutated KRAS by inducing apoptosis. The synergistic effect was not seen in KRAS wild-type cells. Together, these findings suggest for the first time that the dual inhibition of PI3K and STAT3 signaling may be an effective therapeutic strategy for KRAS mutant gastric cancer patients.
Figures
Figure 1NVP-BKM120 effectively suppresses cell viability in human gastric cancer cells. (A) Panels of 11 human gastric cancer cell lines were incubated in medium containing 10% serum, then harvested 24 h after plating and probed with the indicated antibodies. (B) Chemical structures of NVP-BKM120, a pan-class I PI3K inhibitor. (C) Proliferation and biological activity were measured by tetrazolium dye assay. Data shown are means ± SD of four replicate wells. Representative experimental data from three independent experiments.Figure 2NVP-BKM120 inhibits mTOR signaling but induces STAT and/or ERK signaling. SNU-1, SNU-601 and SNU-638 were incubated in medium containing 10% serum for 24 h in the presence of various concentrations of NVP-BKM120 (A) or to 1 μmol/l NVP-BKM120 (B) from 12 to 48 h. Cells were then lysed and whole-cell lysates were immunoprecipitated with indicated antibodies. α-tubulin was used as a loading control. Representative experimental data from three independent experiments.Figure 3Combination of NVP-BKM120 and AG490 shows synergism in cells harboring mutated KRAS. The combination of NVP-BKM120 and AG490 was mixed in the molar ratio of 10:1 in SNU-1, SNU-601, SNU-668 and SNU-638 cells. Four cell lines were exposed to treatments for 72 h, and cell viability was determined. CI<1, synergistic effect; CI=1, additive effect; CI>1, antagonistic effect. Representative experimental data from three independent experiments.Figure 4Combination of NVP-BKM120 and AG490 inhibits progression of SNU-1 and SNU-601 cells through apoptosis. (A) Cells were incubated in medium containing 10% serum for 72 h in the presence of NVP-BKM120, AG490, or combination of NVP-BKM120 and AG490 in indicated concentrations, after which they were fixed, stained with propidium iodide, and analyzed for cell cycle distribution by flow cytometry. (B) Cells were incubated in medium containing 10% serum for 72 h in the presence of NVP-BKM120, AG490 alone or combination of NVP-BKM120 and AG490. Cells were then lysed and whole-cell lysates were immunoprecipitated with indicated antibodies. α-tubulin was used as a loading control. Representative experimental data from three independent experiments; bars, ± SE. *P<0.05; **P<0.005.Figure 5Combination of NVP-BKM120 and AG490 on the signaling of SNU-1, SNU-601 and SNU-638. Cells were incubated in medium containing 10% serum for 48 h in the presence of 1 μmol/l NVP-BKM120, 20 μmol/l AG490 alone or combination of NVP-BKM120 and AG490. Cells were then lysed and whole-cell lysates were immunoprecipitated with antibodies indicated. Actin was used as a loading control. Representative experimental data from three independent experiments.Figure 6Schematic representation of RAS/PI3K/mTOR/STAT3 signaling. Constitutively active RAS can regulate the STAT3 pathway in dependent of the canonical PI3K/AKT pathway. In addition, RAS can directly activate mTOR via RSK-mediated phosphorylation of Raptor. mTOR as a kinase fully activates STAT3, which is essential for RAS oncogenic transformation. In RAS mutant cancer cells, therefore, despite suppression of PI3K by NVP-BKM120, the RAS signaling still enables to maintain oncogenic transformation through activation of the mTOR/STAT3 signaling. Combination of NVP-BKM120 and AG490 may result in a synergistic effect on gastric cancer harboring KRAS mutation.
그림 출처: PubMed Central 공개 논문 (PMC3584651)
그 밖의 정보
출판 형태
Journal Article, Research Support, Non-U.S. Gov't
저널
International journal of oncology · eISSN 1791-2423
