Abstract 5255: The comparison between MAPK and PI3K signaling pathway inhibition in KRAS mutant NSCLC cell lines.

Abstract

Abstract Introduction: KRAS mutations are found 15-25% of patients with lung adenocarcinoma and they lead to constitutive activation of KRAS signaling pathway that results in sustained cell proliferation. Currently, there are no direct anti-KRAS therapies available. Therefore, it is rational to target the downstream molecules of KRAS signaling pathway. MAPK signaling pathway (RAF-MEK-ERK) and the PI3K pathway (PI3K-AKT-mTOR PI3K/Akt) are the activated pathways by KRAS signaling and crucial for cell survival and proliferation in non-small cell lung cancer (NSCLC). Here, we examined the inhibition of both these pathways combination and alone and analyzed the anti-proliferative and apoptotic events in KRAS mutant NCSLC cell lines, A549 and Calu-1. Materials and Methods: Cytotoxicity was determined by MTT assay after the cells were treated with LY294002 (PI3K inhibitor), U0126 (MEK inhibitor) and RAD001 (mTOR inhibitor) for 24 hour. The expression levels of p-EGFR, EGFR, p-ERK, ERK, AKT, p-AKT, p53, cyclinD1, c-myc, p27kip1, BAX, BIM and GAPDH were detected by Western blot after 6 and 24 hour treatment. Results: In proliferation assays, combination of RAD001 (100nM) with U0126 (10uM) and LY294002 (10uM) resulted in higher cytotoxic activity than single agents alone at 24 hour in both cell lines. Western blots revealed that U0126 directly inhibited the phosphorylation of ERK at 6 and 24 hour, followed by inhibiting the activations of its downstream molecules, including cyclinD1 and c-myc but increased the expression of p27kip1, BAX and BIM at 6 and 24 hour. On the other hand, LY294002 downregulated p-AKT signaling only at 6 hour, but not in 24 hour. However, LY294002 and RAD001 combination was effective on cyclinD1 and c-myc expression at 24 hour which can correlate with their cytotoxic effect. Although PI3K/mTOR inhibition is more effective in cytotoxicity, MEK/mTOR inhibition markedly decreases cell proliferation protein marker expressions. Conclusion: These findings suggest that the combination of MEK and mTOR inhibitors and PI3K and mTOR inhibitors compared with either drug alone can inhibit cell proliferation, induce apoptosis and effect downstream signaling pathways. Also, in molecular level, U0126/RAD001 combination is a better option than LY294002/RAD001 combination in A549 and Calu-1 cells. Citation Format: Irem Dogan, Ayla Cihan, Abdullah Ekmekci. The comparison between MAPK and PI3K signaling pathway inhibition in KRAS mutant NSCLC cell lines. [abstract]. In: Proceedings of the 104th Annual Meeting of the American Association for Cancer Research; 2013 Apr 6-10; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2013;73(8 Suppl):Abstract nr 5255. doi:10.1158/1538-7445.AM2013-5255 Note: This abstract was not presented at the AACR Annual Meeting 2013 because the presenter was unable to attend.