mTORC2 is a multimeric kinase composed of the mammalian target of rapamycin kinase (mTOR), mLST8, mSin1, and rictor. The complex is insensitive to acute rapamycin exposure and has shown functions in controlling cell growth and actin cytoskeletal assembly. mTORC2 has recently been shown to phosphorylate and activate Akt. Because f70% of gliomas harbor high levels of activated Akt, we investigated whether mTORC2 activity was elevated in gliomas. In this study, we found that mTORC2 activity was elevated in glioma cell lines as well as in primary tumor cells as compared with normal brain tissue (P < 0.05). Moreover, we found that rictor protein and mRNA levels were also elevated and correlated with increased mTORC2 activity. Overexpression of rictor in cell lines led to increased mTORC2 assembly and activity. These lines exhibited increased anchorage-independent growth in soft agar, increased S-phase cell cycle distribution, increased motility, and elevated integrin B 1 and B 3 expression. In contrast, small interfering RNAmediated knockdown of rictor inhibited these oncogenic activities. Protein kinase CA (PKCA) activity was shown to be elevated in rictor-overexpressing lines but reduced in rictor-knockdown clones, consistent with the known regulation of actin organization by mTORC2 via PKCA. Xenograft studies using these cell lines also supported a role for increased mTORC2 activity in tumorigenesis and enhanced tumor growth. In summary, these data suggest that mTORC2 is hyperactivated in gliomas and functions in promoting tumor cell proliferation and invasive potential due to increased complex formation as a result of the overexpression of rictor. [Cancer Res 2007;67(24):11712-20]

The translation of the cyclin D1 and c-myc mRNAs occurs via internal ribosome entry site (IRES)-mediated initiation under conditions of reduced eIF-4F complex formation and Akt activity. Here we identify hnRNP A1 as an IRES trans-acting factor that regulates cyclin D1 and c-myc IRES activity, depending on the Akt status of the cell. hnRNP A1 binds both IRESs in vitro and in intact cells and enhances in vitro IRES-dependent reporter expression. Akt regulates this IRES activity by inducing phosphorylation of hnRNP A1 on serine 199. Serine 199-phosphorylated hnRNP A1 binds to the IRESs normally but is unable to support IRES activity in vitro. Reducing expression levels of hnRNP A1 or overexpressing a dominant negative version of the protein markedly inhibits rapamycin-stimulated IRES activity in cells and correlated with redistribution of cyclin D1 and c-myc transcripts from heavy polysomes to monosomes. Importantly, knockdown of hnRNP A1 also renders quiescent Aktcontaining cells sensitive to rapamycin-induced G 1 arrest. These results support a role for hnRNP A1 in mediating rapamycin-induced alterations of cyclin D1 and c-myc IRES activity in an Akt-dependent manner and provide the first direct link between Akt and the regulation of IRES activity.A majority of eukaryotic mRNAs contain 5Ј-UTRs 2 that are relatively unstructured and typically less than 100 nucleotides in length, which allows for efficient cap-dependent translation initiation. However, the leaders of some cellular mRNAs are relatively long and highly structured and can contain multiple upstream AUG or CUG codons such that scanning ribosomes are unlikely to efficiently initiate translation. In a number of these mRNAs, translation initiation is mediated by cap-independent mechanisms via an internal ribosome entry site (1). IRES-mediated translation initiation can occur during a variety of physiological conditions and has been reported to promote initiation for several mRNAs during cell cycle progression, differentiation, and apoptosis and during stress responses (2-6). IRESs are thought to directly recruit the ribosome to within close proximity to the start codon, thus bypassing the need for cap binding and ribosome scanning (7). Our previous data have demonstrated that both the cyclin D1 and c-myc mRNAs contain IRESs whose function is markedly enhanced following the inhibition of cap-dependent initiation by rapamycin in a manner dependent on Akt activity (8). In cells containing quiescent Akt, the IRESs of the cyclin D1 and c-myc mRNAs are constitutively active and are stimulated following rapamycin treatment; however, in cells containing active Akt cyclin D1 and c-myc, IRES activity is repressed and is not induced following rapamycin exposure.Several proteins that regulate IRES activity, collectively termed IRES trans-acting factors (ITAFs), have been described (7). These ITAFs function by associating with the IRES and either facilitate direct ribosome binding with the mRNA or alter the structure of the IRES. For instance, the ITAFs PTB, Unr, and h...

mTORC2 is a multiprotein kinase composed of mTOR, mLST8, PRR5, mSIN1 and Rictor. The complex is insensitive to rapamycin and has demonstrated functions controlling cell growth, motility, invasion and cytoskeletal assembly. mTORC2 is the major hydrophobic domain kinase which renders Akt fully active via phosphorylation on serine 473. We isolated Hsp70 as a putative Rictor interacting protein in a yeast two-hybrid assay and confirmed this interaction via co-immunoprecipitation and colocalization experiments. In cells expressing an antisense RNA targeting Hsp70, mTORC2 formation and activity were impaired. Moreover, in cells lacking Hsp70 expression, mTORC2 activity was inhibited following heat shock while controls demonstrated increased mTORC2 activity. These differential effects on mTORC2 activity were specific, in that mTORC1 did not demonstrate Hsp70-dependent alterations under these conditions. These data suggest that Hsp70 is a component of mTORC2 and is required for proper assembly and activity of the kinase both constitutively and following heat shock.

BackgroundHyperactivation of the mTORC2 signaling pathway has been shown to contribute to the oncogenic properties of gliomas. Moreover, overexpression of the mTORC2 regulatory subunit Rictor has been associated with increased proliferation and invasive character of these tumor cells.Methodology/Principal FindingsTo determine whether Rictor overexpression was sufficient to induce glioma formation in mice, we inserted a Cre-lox-regulated human Rictor transgene into the murine ROSA26 locus. This floxed Rictor strain was crossed with mice expressing the Cre recombinase driven from the glial fibrillary acidic protein (GFAP) promoter whose expression is limited to the glial cell compartment. Double transgenic GFAP-Cre/RictorloxP/loxP mice developed multifocal infiltrating glioma containing elevated mTORC2 activity and typically involved the subventricular zone (SVZ) and lateral ventricle. Analysis of Rictor-dependent signaling in these tumors demonstrated that in addition to elevated mTORC2 activity, an mTORC2-independent marker of cortical actin network function, was also elevated. Upon histological examination of the neoplasms, many displayed oligodendroglioma-like phenotypes and expressed markers associated with oligodendroglial lineage tumors. To determine whether upstream oncogenic EGFRvIII signaling would alter tumor phenotypes observed in the GFAP-Cre/RictorloxP/loxP mice, transgenic GFAP-EGFRvIII; GFAP-Cre/RictorloxP/loxP mice were generated. These mice developed mixed astrocytic-oligodendroglial tumors, however glioma formation was accelerated and correlated with increased mTORC2 activity. Additionally, the subventricular zone within the GFAP-Cre/RictorloxP/loxP mouse brain was markedly expanded, and a further proliferation within this compartment of the brain was observed in transgenic GFAP-EGFRvIII; GFAP-Cre/RictorloxP/loxP mice.Conclusion/SignificanceThese data collectively establish Rictor as a novel oncoprotein and support the role of dysregulated Rictor expression in gliomagenesis via mTOR-dependent and mTOR-independent mechanisms. Furthermore, oncogenic EGFRvIII signaling appears to potentiate the in vivo proliferative capacity of GFAP-Cre/RictorloxP/loxP gliomas.

The relative activity of the AKT kinase has been demonstrated to be a major determinant of sensitivity of tumor cells to mammalian target of rapamycin (mTOR) complex 1 inhibitors. Our previous studies have shown that the multifunctional RNAbinding protein heterogeneous nuclear ribonucleoprotein (hnRNP) A1 regulates a salvage pathway facilitating internal ribosome entry site (IRES)-dependent mRNA translation of critical cellular determinants in an AKT-dependent manner following mTOR inhibitor exposure. This pathway functions by stimulating IRES-dependent translation in cells with relatively quiescent AKT, resulting in resistance to rapamycin. However, the pathway is repressed in cells with elevated AKT activity, rendering them sensitive to rapamycin-induced G 1 arrest as a result of the inhibition of global eIF-4E-mediated translation. AKT phosphorylation of hnRNP A1 at serine 199 has been demonstrated to inhibit IRES-mediated translation initiation. Here we describe a phosphomimetic mutant of hnRNP A1 (S199E) that is capable of binding both the cyclin D1 and c-MYC IRES RNAs in vitro but lacks nucleic acid annealing activity, resulting in inhibition of IRES function in dicistronic mRNA reporter assays. Utilizing cells in which AKT is conditionally active, we demonstrate that overexpression of this mutant renders quiescent AKT-containing cells sensitive to rapamycin in vitro and in xenografts. We also demonstrate that activated AKT is strongly correlated with elevated Ser(P) 199 -hnRNP A1 levels in a panel of 22 glioblastomas. These data demonstrate that the phosphorylation status of hnRNP A1 serine 199 regulates the AKT-dependent sensitivity of cells to rapamycin and functionally links IRES-transacting factor annealing activity to cellular responses to mTOR complex 1 inhibition.A broad range of tumor types have been reported to exhibit hypersensitivity to mTORC1 2 inhibition with rapalogs depending on their degree of AKT activation (1-3). The cells that have elevated AKT activity as a result of dysregulated PI3K activity, AKT gene amplification, or a loss of PTEN display markedly increased G 1 arrest following rapamycin exposure relative to cells having quiescent AKT (2, 3). Our previous studies have demonstrated that this differential sensitivity can be explained, in part, by continued IRES-initiated mRNA translation of cyclin D1 and c-MYC in the face of mTOR inhibition mediated by the ITAF hnRNP A1 (4). We have also demonstrated that direct phosphorylation of the ITAF hnRNP A1 on serine 199 by AKT regulates differential cyclin D1 and c-MYC IRES activity (5).The ability of IRES-mediated protein synthesis to contribute to aberrant gene expression in cancer and during integrated cell stress responses is well documented (6 -8); however, the processes regulating IRES function are poorly defined. Cellular IRESs require ITAFs to recruit the 40 S small ribosomal subunit leading to the formation of a competent preinitiation complex (9). Some ITAFs have been shown to directly interact with components of the ribosome to f...