Abstract C60: In vitro efficacy of the dual PI3-kinase/mTOR inhibitor NVP-BEZ235 in hepatocellular carcinoma

Abstract

Abstract Background: The PI3-Kinase/Akt/mTOR pathway has been shown to be involved in the development of hepatocellular carcinoma (HCC). Allosteric inhibitors of mTORC1, such as everolimus (RAD001), have shown preliminary activity in patients with advanced HCC. The PI3-K pathway affects cell growth and survival through the activation of numerous other kinases besides mTOR. Therefore, disruption of this pathway upstream of mTOR may be more effective therapeutically than inhibition of mTOR alone. To investigate this hypothesis, the effects of NVP-BEZ235, a dual inhibitor of PI3-K and mTOR, were compared to rapamycin in HCC cell lines in vitro. Methods: Four HCC cell lines (PLC, HepG2, SKHep1, and Hep3B) were exposed to NVP-BEZ235 at increasing concentrations (50nM-250nM) for 4 hours. Based upon results of western blots and MTS proliferation assays, a dose of 100nM was chosen for further in vitro studies. The same HCC cell lines were treated with DMSO, Rapamycin (100nM) or NVP-BEZ235 (100nM) and either lysed after 24hours for western blot analysis or assayed for proliferation by MTS after 48 hours. Expression of substrates of interest such as phospho-Akt, phospho-S6, phospho-4EBP, and survivin were determined by western blot. Results: NVP-BEZ235 effectively suppressed tumor cell proliferation and inhibited its biologic targets, as determined by phosphorylation of S6 and Akt [Ser473], at low nanomolar concentrations. No significant apoptosis was detected at any concentration as determined by propidium iodide staining. In addition, NVP-BEZ235 was more effective than either rapamycin or DMSO in reducing proliferation in HCC cell lines. Both rapamycin and NVP-BEZ235 inhibited S6 phosphorylation; however only NVP-BEZ235 diminished Akt or PRAS40 (proline-rich Akt substrate) phosphorylation. NVP-BEZ235 was also significantly more effective than rapamycin in suppressing the phosphorylation of 4EBP, a well recognized substrate of TORC1. In keeping with this, NVP-BEZ235 was more effective than rapamycin in suppressing the expression of proteins such as survivin, suggesting a more profound inhibition of cap-dependent translation. Conclusions: Dual inhibition of PI3-K/mTOR induced greater arrest of tumor cell proliferation compared to inhibition of mTORC1 alone in HCC. The ability to more effectively suppress cap-dependent translation may be central to the efficacy of drugs such as NVP-BEZ235 in HCC. Xenograft studies are underway to confirm these effects in vivo. Inhibition of PI3-K/mTOR may be a promising therapeutic strategy for patients with advanced HCC. Citation Information: Mol Cancer Ther 2009;8(12 Suppl):C60.