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Amplification of the gene encoding the epidermal growth factor receptor (EGFR) occurs commonly in glioblastoma (GBM), leading to activation of downstream kinases, including phosphatidylinositol 3′-kinase (PI3K), Akt, and mammalian target of rapamycin (mTOR). A serine-threonine kinase, mTOR controls cell growth by regulating mRNA translation, metabolism, and autophagy; acting as both a downstream effector and upstream regulator of PI3K. These signaling functions are distributed between at least two distinct complexes, mTORC1 and mTORC2 with respect to pathway specificity. We have investigated mTOR signaling in glioma cells with the allosteric mTORC1 inhibitor rapamycin, the mTORC1/2 inhibitor Ku-0063794, a dual PI3K/mTORC1/2 kinase inhibitor PI-103, and siRNA against raptor, rictor, or mTOR, and evaluated the value of mTOR inhibitors for the treatment of glioblastoma.
Gliomas represent the most common primary brain tumor and are among the most lethal of all cancers. EGFR is commonly mutated in GBM, leading to overexpression and activation of downstream signaling pathways. EGFR signals through a complex network of intermediates, including PI3K/AKT/mTOR, MAPK, and PLCγ. Inactivation of PTEN and activating mutations in PI3K itself collectively occur in a majority of GBM tumors, effectively uncoupling PI3K from upstream control by EGFR (1).
PI3Ks are lipid kinases activated by a wide range of RTKs to generate the second messenger phosphatidylinositol-3,4,5-trispho- couples PI3K to downstream effectors, such as sphate (PIP3). PIP3 Akt, a serine-threonine kinase that suppresses apoptosis, promotes growth, and drives proliferation. PIP3 also indirectly activates the mammalian target of rapamycin (mTOR), a protein kinase critical for cell growth (1). The mTOR kinase contains a PI3K homology domain (making mTOR a PIK-related kinase – PIKK), although mTOR itself has no lipid kinase activity (2).
Signaling functions of mTOR are distributed between at least two distinct mTOR protein complexes: mTORC1 and mTORC2. In mTORC1, mTOR is associated with a number of proteins, including PRAS40 and the rapamycin-sensitive adapter protein of mTOR (Raptor), whereas in mTORC2, mTOR is associated with a separate protein complex, including the rapamycin-insensitive companion of mTOR (Rictor). Stimulation of PI3K in response to growth factors leads to phosphorylation and activation of Akt. In addition, phospho-Akt phosphorylates and separately inhibits PRAS40 and the Tsc1/2 (hamartin-tuberin) complex. PRAS40 is inhibitory to mTORC1, while tuberin is inhibitory to GTPase RHEB, which in turn is inhibitory to mTORC1. The detailed signaling leading to activation of mTORC2 is less clearly understood (3).
The activated mTORC1 complex phosphorylates substrates, including Thr-389 S6K, Ser-209 eIF4E, and 4EBP1. The mTORC2 complex phosphorylates Akt on Ser-473, and also phosphorylates additional substrates, including serum glucocorticoid-induced protein kinase (SGK) and PKCα Inhibition of mTORC1/S6K1 by allosteric inhibitors, including rapamycin (Sirolimus) (Fig. 1), CCI-779 (Tensirolimus), RAD001 (4), or other similar agents triggers a negative feedback loop through an IRS-I-dependent mechanism, resulting in increase phosphorylation of Akt. This negative feedback loop is prominent in glioma, however the robustness with which inhibition of mTORC1 activates Akt varies across multiple cancer types (5–7). Unlike rapamycin, ATP-competitive inhibitors of mTORC1/mTORC2 by mTOR inhibitors, including Torin, Ku-0063794 (8, 9), and pp242 (10) blocks the phosphorylation of Akt at Ser473. As an integrator of cell growth and proliferation, mTOR also regulates autophagy, a program of cellular self-digestion activated during periods of nutrient and growth factor deprivation (11). The signaling linking activation of mTOR signaling to blockade of autophagy in metazoan cells is poorly understood.
Preclinical evaluation of dual PI3K/mTOR inhibitors, such as PI-103 and NVP-BEZ235 have demonstrated efficacy for these agents in blocking the growth of glioblastoma (GBM) cells in vitro and in vivo (5, 12). NVP-BEZ235 and other dual inhibitors are therefore being evaluated in early clinical trials. Thus, inhibitors of mTOR and of PI3K/mTOR provide a new class of agents and therapeutic for glioma. Pure ATP-competitive inhibitors of mTORC1/2 inhibit both mTOR complexes, and have been evaluated in less detail in glioma. We have directly compared rapamycin, Ku-0063794, PI-103, or siRNA against mTOR in glioma cells. In contrast to rapamycin, both Ku-0063794 and PI-103 blocked the phosphorylation of Akt and prevented its activation. Ku-0063794 and PI-103 also decreased the phosphorylation of the mTORC1 target 4EBP1 and induced autophagy more effectively in comparison with the allosteric mTORC1 inhibitor rapamycin (Figs. 2 and and33).
We thank Zachary Knight, Benjamin Houseman, Morri Feldman, and Kevan Shokat for providing PI-103, PIK-90, and Ku-0063794. We acknowledge support from NIH grants PCA133091, NS055750, CA102321, CA097257, CA128583, CA148699 P01 CA081403, Burroughs Wellcome Fund, American Brain Tumor Association, The Brain Tumor Society, Accelerate Brain Cancer Cure; Alex’s Lemonade Stand, Children’s National Brain Tumor, Katie Dougherty, Pediatric Brain Tumor, Samuel G. Waxman and V Foundations.
1Acridine orange is light sensitive.
2MOPS SDS running buffer for NOVEX-NuPAGE 4–12% BT SDS-PAGE gel, Tris-acetate running buffer for NOVEX-NuPAGE 3–8% Tris-acetate gel (for high molecular weight protein detection, such as mTOR, p-mTOR (MW 289 kDa)), and Tris-glycine running buffer for NOVEX-NuPAGE 16% Tris-cyclin gel (for low molecular weight protein detection, such as LC3 (MW 18 kDa for LC3I and MW 16 kDa for LC3II)).
3For NOVEX-NuPAGE 16% Tris-cyclin gel, two gels run at 125 V constant for 1.5 h. 30–40 mA (start); 8–12 mA (end).
41× NOVEX-NuPAGE transfer buffer with 10% methanol for NOVEX-NuPAGE 4–12% gel and 3–8% gel. 1× NOVEX-NuPAGE Tris-glycine transfer buffer with 20% methanol for NOVEX-NuPAGE 16% Tris-cyclin gel.
5For NOVEX-NuPAGE 16% Tris-cyclin gel, run at 20 V constant for 1.5 h.
6For the primary antibodies purchased from cell signaling was used at 1:1,000 dilution, for LC3 at 1:500 dilution, and for β-tubulin at 1:2,000 dilution.
7Transfecting cells at lower density can minimize the loss of cell viability due to cell overgrowth after 3 days.
8Removing the complexes after 6 h without loss of transfection activity, but can reduce the toxicity to cells.