Hypocretin/Orexin Interactions with Norepinephrine Contribute to the Opiate Withdrawal Syndrome.

We previously found that human heroin addicts and mice chronically exposed to morphine exhibit a significant increase in the number of detected hypocretin/orexin (Hcrt)-producing neurons. However, it remains unknown how this increase affects target areas of the hypocretin system involved in opioid withdrawal, including norepinephrine containing structures locus coeruleus (LC) and A1/A2 medullary regions. Using a combination of immunohistochemical, biochemical, imaging, and behavioral techniques, we now show that the increase in detected hypocretin cell number translates into a significant increase in hypocretin innervation and tyrosine hydroxylase (TH) levels in the LC without affecting norepinephrine-containing neuronal cell number. We show that the increase in TH is completely dependent on Hcrt innervation. The A1/A2 regions were unaffected by morphine treatment. Manipulation of the Hcrt system may affect opioid addiction and withdrawal.SIGNIFICANCE STATEMENT Previously, we have shown that the hypothalamic hypocretin system undergoes profound anatomic changes in human heroin addicts and in mice exposed to morphine, suggesting a role of this system in the development of addictive behaviors. The locus coeruleus plays a key role in opioid addiction. Here we report that the hypothalamic hypocretin innervation of the locus coeruleus increases dramatically with morphine administration to mice. This increase is correlated with a massive increase in tyrosine hydroxylase expression in locus coeruleus. Elimination of hypocretin neurons prevents the tyrosine hydroxylase increase in locus coeruleus and dampens the somatic and affective components of opioid withdrawal.

Translation of circHGF RNA encodes an HGF protein variant promoting glioblastoma growth through stimulation of c-MET.

INTRODUCTION: HGF/c-MET signaling is a significant driver of glioblastoma (GBM) growth and disease progression. Unfortunately, c-MET targeted therapies have been found to be largely ineffective suggesting additional redundant mechanisms of c-MET activation. METHODS: Utilizing RNA-sequencing (RNA-seq) and ribosome profiling analyses of circular RNAs, circ-HGF (hsa_circ_0080914) was identified as markedly upregulated in primary GBM and found to potentially encode an HGF protein variant (C-HGF) 119 amino acids in length. This candidate HGF variant was characterized and evaluated for its ability to mediate c-MET activation and regulate PDX GBM cell growth, motility and invasive potential in vitro and tumor burden in intracranial xenografts in mice. RESULTS: An internal ribosome entry site (IRES) was identified within the circ-HGF RNA which mediated translation of the cross-junctional ORF encoding C-HGF and was observed to be highly expressed in GBM relative to normal brain tissue. C-HGF was also found to be secreted from GBM cells and concentrated cell culture supernatants or recombinant C-HGF activated known signaling cascades downstream of c-MET. C-HGF was shown to interact directly with the c-MET receptor resulting in its autophosphorylation and activation in PDX GBM lines. Knockdown of C-HGF resulted in suppression of c-MET signaling and marked inhibition of cell growth, motility and invasiveness, whereas overexpression of C-HGF displayed the opposite effects. Additionally, modulation of C-HGF expression regulated tumor growth in intracranial xenografted PDX GBM models. CONCLUSIONS: These results reveal an alternative mechanism of c-MET activation via a circular RNA encoded HGF protein variant which is relevant in GBM biology. Targeting C-HGF may offer a promising approach for GBM clinical management.

Targeting the YAP-TEAD interaction interface for therapeutic intervention in glioblastoma.

INTRODUCTION: Recent studies have suggested that dysregulated Hippo pathway signaling may contribute to glioblastoma proliferation and invasive characteristics. The downstream effector of the pathway, the Yes-associated protein (YAP) oncoprotein, has emerged as a promising target in glioblastoma multiforme (GBM). METHODS: Utilizing a high-throughput yeast two-hybrid based screen, a small molecule was identified which inhibits the association of the co-transcriptional activator YAP1 and the TEA domain family member 1 (TEAD1) transcription factor protein-protein interaction interface. This candidate inhibitor, NSC682769, a novel benzazepine compound, was evaluated for its ability to affect Hippo/YAP axis signaling and potential anti-glioblastoma properties. RESULTS: NSC682769 potently blocked association of YAP and TEAD in vitro and in GBM cells treated with submicromolar concentrations. Moreover, inhibitor-coupled bead pull down and surface plasmon resonance analyses demonstrate that NSC682769 binds to YAP. NSC682769 treatment of GBM lines and patient derived cells resulted in downregulation of YAP expression levels resulting in curtailed YAP-TEAD transcriptional activity. In GBM cell models, NSC682769 inhibited proliferation, colony formation, migration, invasiveness and enhanced apoptosis. In tumor xenograft and genetically engineered mouse models, NSC682769 exhibited marked anti-tumor responses and resulted in increased overall survival and displayed significant blood-brain barrier penetration. CONCLUSIONS: These results demonstrate that blockade of YAP-TEAD association is a viable therapeutic strategy for glioblastoma. On the basis of these favorable preclinical studies further clinical studies are warranted.

Repurposing Potential of Riluzole as an ITAF Inhibitor in mTOR Therapy Resistant Glioblastoma.

Internal ribosome entry site (IRES)-mediated protein synthesis has been demonstrated to play an important role in resistance to mechanistic target of rapamycin (mTOR) targeted therapies. Previously, we have demonstrated that the IRES trans-acting factor (ITAF), hnRNP A1 is required to promote IRES activity and small molecule inhibitors which bind specifically to this ITAF and curtail IRES activity, leading to mTOR inhibitor sensitivity. Here we report the identification of riluzole (Rilutek®), an FDA-approved drug for amyotrophic lateral sclerosis (ALS), via an in silico docking analysis of FDA-approved compounds, as an inhibitor of hnRNP A1. In a riluzole-bead coupled binding assay and in surface plasmon resonance imaging analyses, riluzole was found to directly bind to hnRNP A1 and inhibited IRES activity via effects on ITAF/RNA-binding. Riluzole also demonstrated synergistic anti-glioblastoma (GBM) affects with mTOR inhibitors in vitro and in GBM xenografts in mice. These data suggest that repurposing riluzole, used in conjunction with mTOR inhibitors, may serve as an effective therapeutic option in glioblastoma.

The protein arginine methyltransferase PRMT5 confers therapeutic resistance to mTOR inhibition in glioblastoma.

INTRODUCTION: Clinical trials directed at mechanistic target of rapamycin (mTOR) inhibition have yielded disappointing results in glioblastoma (GBM). A major mechanism of resistance involves the activation of a salvage pathway stimulating internal ribosome entry site (IRES)-mediated protein synthesis. PRMT5 activity has been implicated in the enhancement of IRES activity. METHODS: We analyzed the expression and activity of PRMT5 in response to mTOR inhibition in GBM cell lines and short-term patient cultures. To determine whether PRMT5 conferred resistance we used genetic and pharmacological approaches to ablate PRMT5 activity and assessed the effects on in vitro and in vivo sensitivity. Mutational analyses of the requisite IRES-trans-acting factor (ITAF), hnRNP A1 determined whether PRMT5-mediated methylation was necessary for ITAF RNA binding and IRES activity. RESULTS: PRMT5 activity is stimulated in response to mTOR inhibitors. Knockdown or treatment with a PRMT5 inhibitor blocked IRES activity and sensitizes GBM cells. Ectopic expression of non-methylatable hnRNP A1 mutants demonstrated that methylation of either arginine residues 218 or 225 was sufficient to maintain IRES binding and hnRNP A1-dependent cyclin D1 or c-MYC IRES activity, however a double R218K/R225K mutant was unable to do so. The PRMT5 inhibitor EPZ015666 displayed synergistic anti-GBM effects in vitro and in a xenograft mouse model in combination with PP242. CONCLUSIONS: These results demonstrate that PRMT5 activity is stimulated upon mTOR inhibition in GBM. Our data further support a signaling cascade in which PRMT5-mediated methylation of hnRNP A1 promotes IRES RNA binding and activation of IRES-mediated protein synthesis and resultant mTOR inhibitor resistance.

Mechanistic Target of Rapamycin (mTOR) Inhibition Synergizes with Reduced Internal Ribosome Entry Site (IRES)-mediated Translation of Cyclin D1 and c-MYC mRNAs to Treat Glioblastoma.

Our previous work has demonstrated an intrinsic mRNA-specific protein synthesis salvage pathway operative in glioblastoma (GBM) tumor cells that is resistant to mechanistic target of rapamycin (mTOR) inhibitors. The activation of this internal ribosome entry site (IRES)-dependent mRNA translation initiation pathway results in continued translation of critical transcripts involved in cell cycle progression in the face of global eIF-4E-mediated translation inhibition. Recently we identified compound 11 (C11), a small molecule capable of inhibiting c-MYC IRES translation as a consequence of blocking the interaction of a requisite c-MYC IRES trans-acting factor, heterogeneous nuclear ribonucleoprotein A1, with its IRES. Here we demonstrate that C11 also blocks cyclin D1 IRES-dependent initiation and demonstrates synergistic anti-GBM properties when combined with the mechanistic target of rapamycin kinase inhibitor PP242. The structure-activity relationship of C11 was investigated and resulted in the identification of IRES-J007, which displayed improved IRES-dependent initiation blockade and synergistic anti-GBM effects with PP242. Mechanistic studies with C11 and IRES-J007 revealed binding of the inhibitors within the UP1 fragment of heterogeneous nuclear ribonucleoprotein A1, and docking analysis suggested a small pocket within close proximity to RRM2 as the potential binding site. We further demonstrate that co-therapy with IRES-J007 and PP242 significantly reduces tumor growth of GBM xenografts in mice and that combined inhibitor treatments markedly reduce the mRNA translational state of cyclin D1 and c-MYC transcripts in these tumors. These data support the combined use of IRES-J007 and PP242 to achieve synergistic antitumor responses in GBM.

Phosphorylation of the Hippo Pathway Component AMOTL2 by the mTORC2 Kinase Promotes YAP Signaling, Resulting in Enhanced Glioblastoma Growth and Invasiveness.

The mechanistic target of rapamycin (mTOR) and Hippo signaling pathways are two major signaling cascades that coordinately regulate cell growth and proliferation. Dysregulation of these pathways plays a critical role in gliomagenesis. Recent reports have provided evidence of cross-talk between the mTOR and Hippo pathways; however, a complete description of the signaling relationships between these pathways remains to be elucidated. Utilizing a gene-trapping strategy in a mouse glioma model, we report the identification of AMOTL2 as a candidate substrate for mTORC2. AMOTL2 is phosphorylated at serine 760 by mTORC2. Mutation of AMOTL2 mimicking constitutive Ser(760) phosphorylation blocks its ability to bind and repress YAP leading to increased relative expression of known YAP gene targets. Moreover, overexpression of AMOTL2 or a nonphosphorylatable AMOTL2-S760A mutant inhibited YAP-induced transcription, foci formation, growth, and metastatic properties, whereas overexpression of a phosphomimetic AMOTL2-S760E mutant negated these repressive effects of AMOTL2 in glioblastoma (GBM) cells in vitro. Similar effects on xenograft growth were observed in GBM cells expressing these AMOTL2 Ser(760) mutants. YAP was also shown to be required for Rictor-mediated GBM growth and survival. Finally, an analysis of mTORC2/AMOTL2/YAP activities in primary GBM samples supported the clinical relevance of this signaling cascade, and we propose that pharmacological agents cotargeting these regulatory circuits may hold therapeutic potential.

m6A-modification of cyclin D1 and c-myc IRESs in glioblastoma controls ITAF activity and resistance to mTOR inhibition.

A major mechanism conferring resistance to mTOR inhibitors is activation of a salvage pathway stimulating internal ribosome entry site (IRES)-mediated mRNA translation, driving the synthesis of proteins promoting resistance of glioblastoma (GBM). Previously, we found this pathway is stimulated by the requisite IRES-trans-acting factor (ITAF) hnRNP A1, which itself is subject to phosphorylation and methylation events regulating cyclin D1 and c-myc IRES activity. Here we describe the requirement for m6A-modification of IRES RNAs for efficient translation and resistance to mTOR inhibition. DRACH-motifs within these IRES RNAs upon m6A modification resulted in enhanced IRES activity via increased hnRNP A1-binding following mTOR inhibitor exposure. Inhibitor exposure stimulated the expression of m6A-methylosome components resulting in increased activity in GBM. Silencing of METTL3-14 complexes reduced IRES activity upon inhibitor exposure and sensitized resistant GBM lines. YTHDF3 associates with m6A-modified cyclin D1 or c-myc IRESs, regulating IRES activity, and mTOR inhibitor sensitivity in vitro and in xenograft experiments. YTHDF3 interacted directly with hnRNP A1 and together stimulated hnRNP A1-dependent nucleic acid strand annealing activity. These data demonstrate that m6A-methylation of IRES RNAs regulate GBM responses to this class of inhibitors.

Phosphomimetic substitution of heterogeneous nuclear ribonucleoprotein A1 at serine 199 abolishes AKT-dependent internal ribosome entry site-transacting factor (ITAF) function via effects on strand annealing and results in mammalian target of rapamycin complex 1 (mTORC1) inhibitor sensitivity.

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 RNA-binding 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.

Critical Role for Cap-Independent c-MYC Translation in Progression of Multiple Myeloma.

Dysregulated c-myc is a determinant of multiple myeloma progression. Translation of c-myc can be achieved by an mTOR-mediated, cap-dependent mechanism or a cap-independent mechanism where a sequence in the 5'UTR of mRNA, termed the internal ribosome entry site (IRES), recruits the 40S ribosomal subunit. This mechanism requires the RNA-binding factor hnRNP A1 (A1) and becomes critical when cap-dependent translation is inhibited during endoplasmic reticulum (ER) stress. Thus, we studied the role of A1 and the myc IRES in myeloma biology. A1 expression correlated with enhanced c-myc expression in patient samples. Expression of A1 in multiple myeloma lines was mediated by c-myc itself, suggesting a positive feedback circuit where myc induces A1 and A1 enhances myc translation. We then deleted the A1 gene in a myc-driven murine myeloma model. A1-deleted multiple myeloma cells demonstrated downregulated myc expression and were inhibited in their growth in vivo. Decreased myc expression was due to reduced translational efficiency and depressed IRES activity. We also studied the J007 inhibitor, which prevents A1's interaction with the myc IRES. J007 inhibited myc translation and IRES activity and diminished myc expression in murine and human multiple myeloma lines as well as primary samples. J007 also inhibited tumor outgrowth in mice after subcutaneous or intravenous challenge and prevented osteolytic bone disease. When c-myc was ectopically reexpressed in A1-deleted multiple myeloma cells, tumor growth was reestablished. These results support the critical role of A1-dependent myc IRES translation in myeloma.

mTORC2-mediated direct phosphorylation regulates YAP activity promoting glioblastoma growth and invasive characteristics.

The Hippo and mTOR signaling cascades are major regulators of cell growth and division. Aberrant regulation of these pathways has been demonstrated to contribute to gliomagenesis and result in enhanced glioblastoma proliferation and invasive characteristics. Several crosstalk mechanisms have been described between these two pathways, although a complete picture of these signaling interactions is lacking and is required for effective therapeutic targeting. Here we report the ability of mTORC2 to directly phosphorylate YAP at serine 436 (Ser436) positively regulating YAP activity. We show that mTORC2 activity enhances YAP transcriptional activity and the induction of YAP-dependent target gene expression while its ablation via genetic or pharmacological means has the opposite affects on YAP function. mTORC2 interacts with YAP via Sin1 and mutational analysis of serine 436 demonstrates that this phosphorylation event affects several properties of YAP leading to enhanced transactivation potential. Moreover, YAP serine 436 mutants display altered glioblastoma growth, migratory capacity and invasiveness both in vitro and in xenograft experiments. We further demonstrate that mTORC2 is able to regulate a Hippo pathway resistant allele of YAP suggesting that mTORC2 can regulate YAP independent of Hippo signaling. Correlative associations between the expression of these components in GBM patient samples also supported the presence of this signaling relationship. These results advance a direct mTORC2/YAP signaling axis driving GBM growth, motility and invasiveness.

Correction: Specific blockade of Rictor-mTOR association inhibits mTORC2 activity and is cytotoxic in glioblastoma.

[This corrects the article DOI: 10.1371/journal.pone.0176599.].

Specific blockade of Rictor-mTOR association inhibits mTORC2 activity and is cytotoxic in glioblastoma.

A small molecule which specifically blocks the interaction of Rictor and mTOR was identified utilizing a high-throughput yeast two-hybrid screen and evaluated as a potential inhibitor of mTORC2 activity in glioblastoma multiforme (GBM). In vitro, CID613034 inhibited mTORC2 kinase activity at submicromolar concentrations and in cellular assays specifically inhibited phosphorylation of mTORC2 substrates, including AKT (Ser-473), NDRG1 (Thr-346) and PKCα (Ser-657), while having no appreciable effects on the phosphorylation status of the mTORC1 substrate S6K (Thr-389) or mTORC1-dependent negative feedback loops. CID613034 demonstrated significant inhibitory effects on cell growth, motility and invasiveness in GBM cell lines and sensitivity correlated with relative Rictor or SIN1 expression. Structure-activity relationship analyses afforded an inhibitor, JR-AB2-011, with improved anti-GBM properties and blocked mTORC2 signaling and Rictor association with mTOR at lower effective concentrations. In GBM xenograft studies, JR-AB2-011 demonstrated significant anti-tumor properties. These data support mTORC2 as a viable therapeutic target in GBM and suggest that targeting protein-protein interactions critical for mTORC2 function is an effective strategy to achieve therapeutic responses.

mTORC2 modulates feedback regulation of p38 MAPK activity via DUSP10/MKP5 to confer differential responses to PP242 in glioblastoma.

Dual-specificity phosphatases (DUSPs) dephosphorylate MAP kinases (MAPKs) resulting in their inactivation. Activation of MAPK signaling leads to enhanced DUSP expression, thus establishing feedback regulation of the MAPK pathway. The DUSPs are subject to regulation at the post-translational level via phosphorylation resulting in alterations of protein stability. Here we report that mTORC2 function leads to stabilization of the p38 MAPK phosphatase, DUSP10, thereby inhibiting p38 activity. We demonstrate that mTORC2 binds DUSP10 and phosphorylates DUSP10 on serine residues 224 and 230. These phosphorylation events block DUSP10 turnover resulting in inactivation of p38 signaling. We further show that insulin-stimulated PI3K/mTORC2 signaling regulates DUSP10 stability and p38 activity. Importantly, knockdown of DUSP10 or ectopic overexpression of nonphosphorylatable or phosphomimetic DUSP10 mutants was sufficient to confer differential mTOR kinase inhibitor responses to GBM cells in vitro and in murine xenografts. Finally, DUSP10 was shown to be overexpressed in a significant number of GBM patients. These data demonstrate the ability of the mTORC2 pathway to exert regulatory effects on the DUSP10/p38 feedback loop to control the cellular effects of mTOR kinase inhibitors in GBM and support the use of DUSP10 expression as a surrogate biomarker to predict responsiveness.

Protor-2 interacts with tristetraprolin to regulate mRNA stability during stress.

The A/U-rich RNA-binding protein tristetraprolin (TTP) is an mRNA destabilizing factor which plays a role in the regulated turnover of many transcripts encoding proteins involved in immune function and cell growth control. TTP also plays a role in stress-induced destabilization of mRNAs. Here we report the interaction of TTP with a component of the mTORC2 kinase, Protor-2 (PRR5-L, protein Q6MZQ0/FLJ14213/CAE45978). Protor-2 is structurally similar to human PRR5 and has been demonstrated to bind mTORC2 via Rictor and/or Sin1 and may signal downstream events promoting apoptosis. Protor-2 dissociates from mTORC2 upon hyperactivation of the kinase and is not required for mTORC2 integrity or activity. We identified Protor-2 in a yeast two-hybrid screen as a TTP interactor using the C-terminal mRNA decay domain of TTP as bait. The interaction of Protor-2 with TTP was also confirmed in vivo in co-immunoprecipitation experiments and Protor-2 was also detected in immunoprecipitates of Rictor. Protor-2 was shown to stimulate TTP-mediated mRNA turnover of several TTP-associated mRNAs (TNF-α, GM-CSF, IL-3 and COX-2) in Jurkat cells when overexpressed while the half-lives of transcripts which do not decay via a TTP-mediated mechanism were unaffected. Knockdown of Protor-2 via RNAi inhibited TTP-mediated mRNA turnover of these TTP-associated mRNAs and inhibited association of TTP with cytoplasmic stress granules (SG) or mRNA processing bodies (P-bodies) following induction of the integrated stress response. These results suggest that Protor-2 associates with TTP to accelerate TTP-mediated mRNA turnover and functionally links the control of TTP-regulated mRNA stability to mTORC2 activity.

Conditional astroglial Rictor overexpression induces malignant glioma in mice.

BACKGROUND: Hyperactivation 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 FINDINGS: To 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/Rictor(loxP/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/Rictor(loxP/loxP) mice, transgenic GFAP-EGFRvIII; GFAP-Cre/Rictor(loxP/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/Rictor(loxP/loxP) mouse brain was markedly expanded, and a further proliferation within this compartment of the brain was observed in transgenic GFAP-EGFRvIII; GFAP-Cre/Rictor(loxP/loxP) mice. CONCLUSION/SIGNIFICANCE: These 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/Rictor(loxP/loxP) gliomas.

AP-1 regulates cyclin D1 and c-MYC transcription in an AKT-dependent manner in response to mTOR inhibition: role of AIP4/Itch-mediated JUNB degradation.

One mechanism by which AKT kinase-dependent hypersensitivity to mammalian target of rapamycin (mTOR) inhibitors is controlled is by the differential expression of cyclin D1 and c-MYC. Regulation of posttranscriptional processes has been demonstrated to be crucial in governing expression of these determinants in response to rapamycin. Our previous data suggested that cyclin D1 and c-MYC expression might additionally be coordinately regulated in an AKT-dependent manner at the level of transcription. Under conditions of relatively quiescent AKT activity, treatment of cells with rapamycin resulted in upregulation of cyclin D1 and c-MYC nascent transcription, whereas in cells containing active AKT, exposure repressed transcription. Promoter analysis identified AKT-dependent rapamycin responsive elements containing AP-1 transactivation sites. Phosphorylated c-JUN binding to these promoters correlated with activation of transcription whereas JUNB occupancy was associated with promoter repression. Forced overexpression of JunB or a conditionally active JunB-ER allele repressed cyclin D1 and c-MYC promoter activity in quiescent AKT-containing cells following rapamycin exposure. AIP4/Itch-dependent JUNB protein degradation was found to be markedly reduced in active AKT-containing cells compared with cells harboring quiescent AKT. Moreover, silencing AIP4/Itch expression or inhibiting JNK mediated AIP4 activity abrogated the rapamycin-induced effects on cyclin D1 and c-MYC promoter activities. Our findings support a role for the AKT-dependent regulation of AIP4/Itch activity in mediating the differential cyclin D1 and c-MYC transcriptional responses to rapamycin.

Inhibition of SAPK2/p38 enhances sensitivity to mTORC1 inhibition by blocking IRES-mediated translation initiation in glioblastoma.

A variety of mechanisms confer hypersensitivity of tumor cells to the macrolide rapamycin, the prototypic mTORC1 inhibitor. Several studies have shown that the status of the AKT kinase plays a critical role in determining hypersensitivity. Cancer cells in which AKT activity is elevated are exquisitely sensitive to mTORC1 inhibitors while cells in which the kinase is quiescent are relatively resistant. Our previous work has shown that a transcript-specific protein synthesis salvage pathway is operative in cells with quiescent AKT levels, maintaining the translation of crucial mRNAs involved in cell-cycle progression in the face of global eIF-4E-mediated translation inhibition. The activation of this salvage pathway is dependent on SAPK2/p38-mediated activation of IRES-dependent initiation of the cyclin D1 and c-MYC mRNAs, resulting in the maintenance of their protein expression levels. Here, we show that both genetic and pharmacologic inhibition of SAPK2/p38 in glioblastoma multiforme cells significantly reduces rapamycin-induced IRES-mediated translation initiation of cyclin D1 and c-MYC, resulting in increased G(1) arrest in vitro and inhibition of tumor growth in xenografts. Moreover, we observed that the AKT-dependent signaling alterations seen in vitro are also displayed in engrafted tumors cells and were able to show that combined inhibitor treatments markedly reduced the mRNA translational state of cyclin D1 and c-MYC transcripts in tumors isolated from mice. These data support the combined use of SAPK2/p38 and mTORC1 inhibitors to achieve a synergistic antitumor therapeutic response, particularly in rapamycin-resistant quiescent AKT-containing cells.