Abstract B141: Effects of AZD8055, a novel mTOR kinase inhibitor, in human cancer cells developing resistance to rapamycin

Abstract

Abstract Background: Cancer cells are thought to adapt to rapamycin, an inhibitor of mTORC1, by activating AKT via mTORC2 and others mTORC1-independent survival pathways. In this study, we explored the effects of AZD8055, an ATP competitive inhibitor of mTOR, inhibiting both mTORC1 and mTORC2, in cancer cells with intrinsic and developing resistance to rapamycin. Materials and Methods: Antiproliferative effects and cellular growth inhibition of AZD8055 and rapamycin were evaluated in a panel of 20 human cancer cell lines by MTT assay. Baseline and phosphorylated protein levels were assessed usingWestern blot. mRNA expression was studied by qRT-PCR. Results: AZD8055 and rapamycin (48h exposures) displayed antiproliferative effects in our cell panel at IC50s ranging 0.005–>3µM and 0.04–20µM, respectively. CAKI1 and SK-HEP1 cells expressed high mRNA levels of mesenchymal markers (vimentin, N-cadherin, ZEB1, SIP1, SLUG, ACTA2, and HMGA2) and low levels of E-cadherin associated with intrinsic resistance to several receptor tyrosine kinase inhibitors. AZD8055 induced antiproliferative effects in CAKI1 and SK-HEP1 cells at concentrations <0.1µM. In CAKI1 and SK-HEP1 cells exposed to 0.1µM rapamycin for 5–24h, we observed the inhibition of p-S6 Ser235/236 but an activation of p-AKTser473. Consistent with its mechanism of action, exposure to 0.1µM AZD8055 led to the inhibition to p-S6, and p-4EBP1Ser65, without p-AKT ser473 activation. Downstream to mTORC2, PKC Ser657 was transiently activated by both rapamycin and AZD8055 in CAKI1 cells but not in SK-HEP1 cells. To study the acquired resistance to rapamycin, SK-HEP1 cells were grown with increased concentrations of rapamycin for >6 months, making these cells tolerant to ≥4-fold IC50 concentrations. In SK-RAP cells, we observed the transcriptional activation of IGF1R, TGFB2, the anti-apoptotic factors BCL2 and CDKN1Ap21, and the multidrug resistance ABCB1mdr1. In SK-RAP cells, sustained exposure to rapamycin increased pAKTser473 and decreased p-S6 and p-PKC. Consistent with the inhibition of mTOR kinase activity, exposure to AZD8055 for 5h almost completely abrogated rapamycin-induced AKTser473 activation in SK-RAP cells. Furthermore, SK-RAP cells were more sensitive to AZD8055 than to the parental SK-HEP1 cells (IC50: 0.1 versus >3µM, p=0.044), possibly due to the extended functions of AZD8055 against mTORC1 and inhibition of mTORC2. Conclusions: AZD8055, by inhibiting mTOR kinase activity, decreases mTORC1 substrates pS6 and p-4EBP1Ser65 and mTORC2 substrate AKTser473. AZD8055 decreases cancer cell proliferation in cell lines expressing mesenchymal markers displaying intrinsic and developing resistance to rapamycin, abrogating the hyperactivation of AKT observed in these cells after rapamycin exposure. Citation Information: Mol Cancer Ther 2009;8(12 Suppl):B141.