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...

Increased expression of several osteoblastic proteases and MEPE (a bone matrix protein) occurs in X-linked hypophosphatemic rickets (hyp). This is associated with an increased release of a protease-resistant MEPE peptide (ASARM peptide), a potent inhibitor of mineralization. Cathepsin B cleaves MEPE releasing ASARM peptide and hyp osteoblast/osteocyte cells hypersecrete cathepsin D, an activator of cathepsin B. Our aims were to determine whether cathepsin inhibitors correct the mineralization defect in vivo and whether hyp-bone ASARM peptide levels are reduced after protease treatment. Normal littermates and hyp mice (n = 6) were injected intraperitoneally once a day for 4 weeks with pepstatin, CAO74 or vehicle. Animals were then sacrificed and bones plus serum removed for comprehensive analysis. All hyp mice groups (treated and untreated) remained hypophosphatemic with serum 1,25 vitamin D3 inappropriately normal. Serum PTH was significantly elevated in all hyp mice groups relative to normal mice (P = 0.0017). Untreated hyp mice had six-fold elevated levels of serum alkaline-phosphatase and twofold elevated levels of ASARM peptides relative to normal mice (P < 0.001). In contrast, serum alkaline phosphatase and serum ASARM peptides were significantly reduced (normalized) in hyp mice treated with CA074 or pepstatin. Serum FGF23 levels remained high in all hyp animal groups (P < 0.0001). Hyp mice treated with protease inhibitors showed dramatic reductions in unmineralized osteoid (femurs) compared to control hyp mice (Goldner staining). Also, hyp animals treated with protease inhibitors showed marked and significant improvements in growth plate width (42%), osteoid thickness (40%) and cortical area (40%) (P < 0.002). The mineralization apposition rate, bone formation rate and mineralization surface were normalized by protease-treatment. High-resolution pQCT mineral histomorphometry measurements and uCT also confirmed a marked mineralization improvement. Finally, the growth plate and cortical bone of hyp femurs contained a massive accumulation of osteoblast-derived ASARM peptide(s) that was reduced in hyp animals treated with CA074 or pepstatin. This study confirms in vivo administration of cathepsin inhibitors improves bone mineralization in hyp mice. This may be due to a protease inhibitor mediated decrease in proteolytic degradation of the extracellular matrix and a reduced release of ASARM peptides (potent mineralization inhibitors).

The differential expression of the critical cell cycle control proteins cyclin D1 and c-myc has been shown to result in Akt-dependent hypersensitivity of tumor cells to mTOR inhibitors. We have previously demonstrated that the differential utilization of internal ribosome entry sites within the mRNAs of these transcripts allows maintenance of protein synthesis in the face of rapamycin (rapa) exposure in an Akt-dependent manner. Here, we demonstrate that in addition to this mechanism, cyclin D1 and c-myc mRNA stability is also coordinately regulated following rapa treatment depending on Akt activity status. We identify A/U-rich response elements within the 3 0 untranslated regions (UTRs) of these transcripts, which confer the observed differential stabilities and show that the RNA-binding protein, tristetraprolin (TTP), interacts with these elements. We also present evidence that TTP accumulates in response to rapa exposure, binds to the cis-acting elements within the cyclin D1 and c-myc 3 0 UTRs and is differentially serine phosphorylated in an Akt-dependent manner. Furthermore, the differential phosphorylation status of TTP results in its sequestration by 14-3-3 proteins in quiescent Akt-containing cells. Finally, siRNA-mediated knockdown of TTP expression or inhibiting a known regulator of TTP phosphorylation, p38 MAP kinase, abolishes the effects on cyclin D1 and c-myc mRNA stability. We assume that the differential control of cyclin D1 and c-myc mRNA stability and translational efficiency constitutes a coordinate response to rapa contributing to the maintenance of expression of these determinants in rapa-resistant quiescent Aktcontaining cells following exposure.