Mammalian target of rapamycin (mTOR), which functions in two distinct multiprotein complexes, termed mTORC1 and mTORC2, is a downstream signaling kinase in the PI3K/AKT pathway. mTOR is often deregulated in glioblastoma (GBM), the most aggressive primary brain tumor in adults, due to the frequent loss of tumor suppressor, PTEN. mTORC1 and mTORC2 control cell growth, proliferation, migration, survival, and stem cell regulation in response to nutrients and growth factors. The presence of stem cells within the tumor mass and the surrounding areas precludes the possibility for elimination of GBM. Furthermore, the substrate of activated AKT, the proline-rich AKT substrate of 40 kDa (PRAS40), is a surrogate marker of activated AKT in response to mTOR inhibition therapy in GBM patients. PRAS40 regulates mTORC1 and has recently been considered as a target for cancer therapy. Rapamycin and its analogs failed in clinical trials for GBM patients due to their incomplete inhibition of mTORC1 as well as the activation of mitogenic pathways via negative feedback loops. The aim of this study is to provide evidence that mTORC1 and mTORC2 inhibitors [Torin 1 (tricyclic benzonaphthyridinone inhibitor), Torin 2, and XL 388 ([7-(6-Amino-3-pyridinyl)-2,3-dihydro-1,4-benzoxazepin-4(5H)-yl][3-fluoro-2-methyl-4-(methylsulfonyl)phenyl]-methanone)] effectively target mTORC1/2, suppressing cell growth, dissemination, and altering self-renewal properties of GBM stem cells. Results demonstrated that: 1) A significant number of tumors showed an increased expression of pAKTSer473 and pmTORSer2448 using immunohistochemistry, suggesting an overactivation of mTOR pathway; 2) Co-expression of mTOR and stem cell marker, nestin was evident in GBM tumors as shown by immunofluorescence analysis, implying its role in stem cell regulation; 3) Torin 2, but not Torin 1 or XL 388, was able to suppress mTORC1 activity completely as shown by the reduced expression of its downstream substrate pS6KSer235/236 in a dose-dependent manner; 4) Torin 1, Torin 2, and XL 388 inhibited the phosphorylation of 4E-BP1 and PRAS40; 5) Torin 1, Torin 2, and XL 388 suppressed the mTORC2 activity as reported by complete dephosphorylation of pAKTSer473; 6) Functional analysis revealed that Torin 1, Torin 2, and XL 388 effectively suppressed proliferation as shown by MTT analysis and S-phase entry as evidenced by EdU incorporation; 7) Both Torin 1 and Torin 2 inhibited GBM cell migration, however XL 388 was ineffective; 8) Torin 1 and Torin 2 suppressed the self-renewal of GBM stem cells and inhibited their proliferation, XL 388 remained ineffective. These results suggested that Torin 1 and Torin 2, but not XL 388 are useful in suppressing mTORC1 and mTORC2, thereby inhibiting GBM cell proliferation and dissemination. Moreover, Torin 1 and Torin 2 were effective in inhibiting stem cell self-renewal, underscoring their potential use for the treatment of GBM.
Citation Format: John L. Gillick, Zachary E. Thwing, Sudeepta Sridhara, Raj Murali, Meena Jhanwar-Uniyal. Targeting glioblastoma and its stem cells using novel small molecule inhibitors of the mTOR pathway. [abstract]. In: Proceedings of the 106th Annual Meeting of the American Association for Cancer Research; 2015 Apr 18-22; Philadelphia, PA. Philadelphia (PA): AACR; Cancer Res 2015;75(15 Suppl):Abstract nr 686. doi:10.1158/1538-7445.AM2015-686