Abstract A52: Comparison of the efficacy of different mTOR inhibitors for the treatment of prostate cancer

Background: We previously showed that the mTOR pathway is activated in castration resistant prostate cancer (CRPC) cell lines while its inhibition resulted in upregulation of androgen receptor (AR) signaling, resulting in resistance to mTOR-based therapy (Wang et al., 2008, Oncogene, 27(56):7106-17). mTOR forms two complexes: activation of mTORC1 (mTOR, raptor) increases mRNA translation by phosphorylation of the downstream molecules p70-S6K (S6K) and eIF4E binding protein 1 (4E-BP1), while activation of mTORC2 (mTOR, rictor) leads to activation of Akt, which is also activated by phosphatidylinositol 3-kinase (PI3K). A number of drugs targeting these complexes are currently available, among them, RAD001, which inhibits mTORC1 alone, BEZ235, a dual mTORC1/PI3K inhibitor, and Torin1, an ATP-competitive inhibitor which prevents activation of both mTORC1 and mTORC2. The goal of the present study was to identify the drug that is most effective against PCa either alone or in combination with the AR inhibitor bicalutamide. Results: The drugs were used in a number of cell lines including C4-2 and LNCaP-AI, two CRPC sublines of androgen-dependent LNCaP cells, as well as CWR-R1 and CWR22Rv1, two lines derived from relapsed CWR22 tumors in castrated mice. Another androgen-sensitive cell line, PC-346C, was also used. RAD001 dose responsibly inhibited growth in C4-2 cells, but the other cell lines were resistant to this therapy, either alone or in combination with bicalutamide. In contrast, LNCaP-AI and CWR-R1 cells were also sensitive to BEZ235 and Torin1, but PC-346C cells were resistant to all three. Hence we investigated the molecular mechanism underlying the resistance of these cells to mTOR inhibitors. Clonogenic assays revealed that 20 nM BEZ235 and 25 nM Torin1 are optimal doses that prevent growth of PCa cells, effect comparable of that of 10 nM of RAD001 (physiological concentration in blood of patients under treatment with this drug). Comparison of the downstream targets of the various drugs at these doses revealed that 4E-BP1 and Akt are highly phosphorylated in both C4-2 and PC-346C cells despite RAD001 treatment, whereas BEZ235 and Torin1 inhibited 4E-BP1 phosphorylation. This is despite the fact that AR levels, but not AR transcriptional activity, increased in BEZ235 treated cells, but not in those treated with RAD001 and Torin1. However, phosphorylation of its binding partner eIF4E increased in PC-346C cells but not in C4-2 cells. Conclusion: Phosphorylation of 4E-BP1 and eIF4E, as well as Akt, needs to be downregulated in order for the drug to be effective. The effects of BEZ235 are likely complicated by the increase in AR levels despite the use of bicalutamide. Therefore, a stronger AR inhibitor, such as MDV3100, in combination with this drug, may be required. Acknowledgments: We would like to thank Dr. David Sabatini for the drug Torin1, Novartis Pharmaceuticals for providing BEZ235 and RAD001, and Astra Zeneca, Cheshire, UK, for the drug bicalutamide. CWR-R1 cells were generously provided by Dr Elizabeth Wilson (University of North Carolina), while PC-346C cells were from Dr. W.M. van Weerden, Josephine Nefkens Institute, Erasmus MC, Rotterdam, The Netherlands .