Abstract B59: Dual mTOR kinase inhibitor reverses rapamycin resistance in prostate cancer cells

Abstract

Abstract The mTOR pathway is frequently over-activated in human cancers. However, classic allosteric mTOR inhibitors rapamycin and its analogs only exert limited clinical benefits in patients. It has been shown that many tumors either fail to respond to rapamycin initially or are able to acquire resistance after the initial treatment of rapamycin. Such primary and secondary resistances remain as major concerns in rapamycin-based anti-cancer therapies that may inevitably lead to therapeutic failure. We have previously shown that the ATP-competitive dual mTOR kinase inhibitor AZD8055 can significantly inhibit malignant behaviors in parental cells however its functional efficacies in the rapamycin-resistance setting remains unclear. Cancer cells with wildtype PTEN were selected as models of primary rapamycin resistance since the mTOR pathway is usually less abnormal in these cells. To establish cells with acquired rapamycin resistance, PTEN-null prostate cancer PC-3 cells were gradually treated with increasing concentrations of rapamycin (up to 10 μM) until the cells were completely unresponsive to rapamycin (PC-3RR). Cell proliferation was determined by MTT assay and clonogenic survival was tested by colony formation assay. m7G pull-down assay was employed to detect the cap-dependent translation. mTOR downstream pathways were detected by Western blotting. PTEN wildtype and PC-3RR cells essentially failed to respond to rapamycin; IC50 values were greater than 100 nM in PTEN wildtype cells and 1 μM in PC-3RR cells, respectively. However, for both cell types AZD8055 suppressed proliferation at the nanomolar level in a dose dependent manner, with IC50 values less than 30 nM. Similarly, cellular clonogenic capacity was impaired by AZD8055 to a greater extent than was the case for rapamycin. At the molecular level, AZD8055, but not rapamycin, strongly prevents the dissociation of 4E-BP1 from eIF4E, therefore blocking cap-dependent translation. Further, while the p-S6K (T389) and p-S6 (S235/236) pathway was potently inhibited by both agents, AZD8055 displayed more striking inhibition on p-4E-BP1 (T37/46) and p-Akt (S473) than did rapamycin in all cells tested. Interestingly, similar to rapamycin, AZD8055 slightly increased p-Akt (T308), suggesting the existence of the PI3K-mediated feedback loop. However, the PI3K inhibitor wortmannin failed to sensitize cell response to AZD8055, implying such feedback-dependent activation has little impact on the drug efficacy. These findings indicate that dual mTOR kinase inhibitor AZD8055 significantly inhibits malignant functions in cancer cells that are highly resistant to rapamycin. Such effects are likely to be associated with deactivation of rapamycin-insensitive mTOR downstream components such as Akt and 4E-BP1, but not the upstream feedback pathway. These data provide in vitro evidence in support of future evaluation of ATP-competitive mTOR inhibitors in cancer models that are resistant to rapamycin analogs. Citation Format: Yao Dai, Li Zhao, Dietmar W. Siemann. Dual mTOR kinase inhibitor reverses rapamycin resistance in prostate cancer cells. [abstract]. In: Proceedings of the AACR-NCI-EORTC International Conference: Molecular Targets and Cancer Therapeutics; 2015 Nov 5-9; Boston, MA. Philadelphia (PA): AACR; Mol Cancer Ther 2015;14(12 Suppl 2):Abstract nr B59.