Abstract
Abstract Hepatocellular carcinoma (HCC) is one of the most lethal cancers. Signaling pathways including AKT-mTOR axis is often dysregulated in HCC. RAD001 principally targets at the mTOR complex 1. mTOR inhibition is reportedly associated with activation of feedback loop and parallel pathways. TKI258 is a potent tyrosine kinase inhibitor targeting the FGFRs and VEGFRs, which blocks subsequent PI3K/AKT signaling pathways in cancer cells. In the current study, we aim to study the in-vitro and in-vivo effects of the drug combination with reference to the parallel and upstream pathway of AKT-mTOR axis. We have treated the HCC cell line Hep3B with TKI258 and RAD001. RAD001 could suppress the phosphorylation of down stream signal mediators of mTOR pathway including p70S6 kinase 1 (S6K) and eukaryotic initiation factor binding protein 1 (4E-BP1) and led to G1 phase arrest. The results demonstrated a significant increase in suppression of cell proliferation in vitro with the combination of TKI258 (300nM) and RAD001 (either 200nM or 200pM) compared with either drug alone. Although the addition of the TKI258 only slightly suppressed the AKT positive feedback loop induced by RAD001, the combination of drugs could significantly suppress the phosphorylation of mTOR (Ser2448), ERK1/2 (Ser217/221) and p38 (Thr180/Tyr182) as well as their endogenous protein expression levels. The expression levels of FGFR1 and FGFR2 were also suppressed. The overall protein reduction was due to the synergistic inhibition of p70S6 (Ser240/244) and 4E-BP1 (Ser65) phosphorylation, which indicated a strong suppression in translation and protein synthesis. Pro-caspases 3 and PARP cleavage were not observed at 24 hours and only slightly detected at 48 hours post-treatment, suggesting that the drug combination did not further increase cytotoxic effects but mainly increased in cytostatic arrest ability. In Hep3B derived xenograft model, TKI258 (15mg/ml) and RAD001 (2.5mg/kg) had shown a synergistic inhibition of tumor growth in volume and weight. In addition, there was a significant reduction in microvessel density (MVD) in the xenograft, which indicated an improved effect in angiogenesis inhibition. Our data showed that the combination of RAD001 and TKI258 were active in controlling HCC proliferation via double inhibition of both AKT-mTOR axis and its parallel pathway. Acknowledge: The works are supported by Novartis Oncology. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 103rd Annual Meeting of the American Association for Cancer Research; 2012 Mar 31-Apr 4; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2012;72(8 Suppl):Abstract nr 2811. doi:1538-7445.AM2012-2811
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