Regulation Of mTOR Activity Research Articles

SPAG5 upregulation predicts poor prognosis in cervical cancer patients and alters sensitivity to taxol treatment via the mTOR signaling pathway

Previously, we found that sperm-associated antigen 5 (SPAG5) was upregulated in pelvic lymph node metastasis–positive cervical cancer. The aim of this study is to examine the role of SPAG5 in the proliferation and tumorigenicity of cervical cancer and its clinical significance in tumor progression. In our study, SPAG5 expression in cervical cancer patients was detected using quantitative real-time polymerase chain reaction, western blotting, and immunohistochemistry; cervical cancer cell function with downregulated SPAG5 in vitro was explored using tetrazolium assay, flow cytometry, and colony formation and Transwell assays. SPAG5 was upregulated in tumor tissue compared with paired adjacent noncancerous tissues; SPAG5 upregulation in tumor tissues indicated poor disease-free survival, which was also an independent prognostic indicator for cervical cancer patients. In vitro study demonstrated that SPAG5 downregulation inhibited cell proliferation and growth significantly by G2/M arrest and induction of apoptosis, and hindered cell migration and invasion. Under SPAG5 downregulation, the sensitivity of cervical cancer cells differed according to taxol dose, which correlated with mammalian target of rapamycin (mTOR) signaling pathway activity. In general, SPAG5 upregulation relates to poor prognosis in cervical cancer patients, and SPAG5 is a regulator of mTOR activity during taxol treatment in cervical cancer.

Adiponectin attenuates the osteoblastic differentiation of vascular smooth muscle cells through the AMPK/mTOR pathway

Vascular calcification is common in patients with peripheral artery diseases and coronary artery diseases. The osteoblastic differentiation of vascular smooth muscle cells (VSMCs) contributes significantly to vascular calcification. Adiponectin has been demonstrated to exert a protective effect in osteoblastic differentiation of VSMCs through regulating mTOR activity. However, the upstream and downstream signaling molecules of adiponectin-regulated mTOR signaling have not been identified in VSMCs with osteoblastic differentiation. In this study, the VSMC differentiation model was established by beta-glycerophosphate (β-GP) induction. The mineralization was identified by Alizarin Red S staining. Protein expression and phosphorylation were detected by Western blot or immunofluorescence. Adiponectin attenuated osteoblastic differentiation and mineralization of β-GP-treated VSMCs. Adiponectin inhibited osteoblastic differentiation of VSMCs through increasing the level of p-AMPKα. Pretreatment of VSMCs with AMPK inhibitor blocked while AMPK activator enhanced the effect of adiponectin on osteoblastic differentiation of VSMCs. Adiponectin upregulated TSC2 expression and downregulated mTOR and S6K1 phosphorylation in β-GP-treated VSMCs. Adiponectin treatment significantly attenuates the osteoblastic differentiation and calcification of VSMCs through modulation of AMPK–TSC2–mTOR–S6K1 signal pathway.

Exploring the role of metformin in anticancer treatments: A systematic review

Many clinical and preclinical studies suggest that metformin has antitumor activity. There are two main mechanisms that justify this effect: its ability to activate AMPK, preventing the gluconeogenesis in the liver and stimulating glucose uptake in muscle (insulin-independent), and its potential to negatively regulate mTOR activity (insulin- dependent). Thus, numerous studies have evaluated its role in cancer risk, prognosis and as an antitumor therapy in different malignancies. The following is a systematic review on the clinical evidence about the effects of metformin in cancer. Uncontrolled studies suggest that metformin is associated with reduced risk of different types of cancers among patients with hyperinsulinemia conditions, such as diabetes and obesity. However, among cancer patients, the literature is conflicting about the real impact of metformin on survival and outcomes of cancer treatments. The effects of metformin in nondiabetic patients remain unknown. Ongoing randomized trials are awaited to prove the true antineoplastic activity of metformin.

The Role of Phospholipase D in Modulating the MTOR Signaling Pathway in Polycystic Kidney Disease

The mammalian target of rapamycin (mTOR) signaling pathway is aberrantly activated in polycystic kidney disease (PKD). Emerging evidence suggests that phospholipase D (PLD) and its product phosphatidic acid (PA) regulate mTOR activity. In this study, we assessed in vitro the regulatory function of PLD and PA on the mTOR signaling pathway in PKD. We found that the basal level of PLD activity was elevated in PKD cells. Targeting PLD by small molecule inhibitors reduced cell proliferation and blocked mTOR signaling, whereas exogenous PA stimulated mTOR signaling and abolished the inhibitory effect of PLD on PKD cell proliferation. We also show that blocking PLD activity enhanced the sensitivity of PKD cells to rapamycin and that combining PLD inhibitors and rapamycin synergistically inhibited PKD cell proliferation. Furthermore, we demonstrate that targeting mTOR did not induce autophagy, whereas targeting PLD induced autophagosome formation. Taken together, our findings suggest that deregulated mTOR pathway activation is mediated partly by increased PLD signaling in PKD cells. Targeting PLD isoforms with pharmacological inhibitors may represent a new therapeutic strategy in PKD.

LKB1/AMPK/mTOR Signaling Pathway in Non-small-cell Lung Cancer

Links between cancer and metabolism have been suggested for a long time but compelling evidence for this hypothesis came from the recent molecular characterization of the LKB1/AMPK signaling pathway as a tumor suppressor axis. Besides the discovery of somatic mutations in the LKB1 gene in certain type of cancers, a critical emerging point was that the LKB1/AMPK axis remains generally functional and could be stimulated by pharmacological molecules such as metformin in cancer cells. In addition, AMPK plays a central role in the control of cell growth, proliferation and autophagy through the regulation of mTOR activity, which is consistently deregulated in cancer cells. Targeting of AMPK/mTOR is thus an attractive strategy in the development of therapeutic agents against non-small-cell lung cancer (NSCLC). In this review, the LKB1/AMPK/mTOR signaling pathway is described, highlighting its protective role, and opportunities for therapeutic intervention, and clinical trials in NSCLC.

MTOR inactivation by ROS-JNK-p53 pathway plays an essential role in Psedolaric acid B induced autophagy-dependent senescence in murine fibrosarcoma L929 cells

Pseudolaric acid B (PAB), the primary biologically active compound isolated from the root bark of P. kaempferi Gordon, has been reported to exhibit anti-tumor effect primarily via cell cycle arrest and apoptosis. Our previous study demonstrated that PAB triggered mitotic catastrophe in L929 cells. In addition, a small percentage of the cells undergoing mitotic catastrophe displayed an apoptotic phenotype. Therefore, we continued to investigate the fate of the other cells. The results indicated that PAB induced senescence through p19–p53–p21 and p16–Rb pathways in L929 cells. PAB also triggered autophagy via inhibiting Akt-mammalian target of rapamycin (mTOR) activity in L929 cells. In addition, autophagy was demonstrated to reinforce senescence through regulating the senescence pathways. Thus, we focused on the detailed molecular mechanisms whereby autophagy promoted senescence. Reactive oxygen species (ROS) plays an important in autophagy and senescence. We found that PAB triggered a ROS-JNK-p53 positive feedback loop and this feedback loop played a crucial role in autophagy via repressing the activation of mTOR. Furthermore, ROS-JNK-p53 positive feedback loop was demonstrated to regulate senescence. Tuberous sclerosis proteins1 and 2, also known as TSC1 and TSC2, form a protein-complex. TSC1/TSC2 heterodimer is a downstream target of growth factor-phosphoinositide 3-kinase-Akt signaling which negatively regulates mTOR activity. Activation of mTOR by insulin or inhibition of endogenous TSC2 levels by siRNA obviously delayed PAB-induced senescence. In conclusion, mTOR inactivation by ROS-JNK-p53 pathway played an important role in autophagy-dependent senescence in PAB-treated L929 cells.

Abstract 1656: Mouse embryonic fibroblasts null for krüppel-like factor 4 show reduced autophagy and elevated mTOR activity.

Abstract Krüppel-like factor 4 (KLF4) is a zinc-finger transcription factor named for its homology to the Drosophila protein Krüppel. KLF4 negatively regulates cell-cycle progression and, cell proliferation, and is a known tumor suppressor in certain cancers. Autophagy, or self-eating, is a process through which intracellular aggregates can be degraded and their components recycled. The role of autophagy in cancer appears to be context dependent. In pre-cancerous tissue, autophagy may serve a protective purpose, removing cytotoxic aggregates and promoting a healthy intracellular environment. In cancerous tissue, however, autophagy may allow cancer cell survival. Previous studies examining gene expression in WT (KLF4+/+) and KLF4 null (KLF4-/-) mouse embryonic fibroblasts (MEFs) revealed autophagy related gene expression to be downregulated in KLF4-/- MEFs. In order to evaluate whether KLF4 is important in autophagy, we examined microtubule associated protein-1 light chain 3 protein (LC3, a marker of autophagy) and a downstream target of mTOR (p70S6K, a marker of autophagy inhibition) in KLF4+/+ and KLF4-/- MEFs. Our results show that levels of LC3 are reduced and levels of p70S6K are elevated in both KLF4-/- MEFs and MEFs in which KLF4 has been transiently silenced. Rapamycin treatment, which inhibits mTOR activity, fails to restore autophagy in KLF4 null MEFs. Collectively, these findings suggest that KLF4 may negatively regulate mTOR activity and that KLF4 may also be necessary for induction of autophagy either downstream or entirely independent of mTOR. Citation Format: Hagos Engda, Elise DeRoo. Mouse embryonic fibroblasts null for krüppel-like factor 4 show reduced autophagy and elevated mTOR activity. [abstract]. In: Proceedings of the 104th Annual Meeting of the American Association for Cancer Research; 2013 Apr 6-10; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2013;73(8 Suppl):Abstract nr 1656. doi:10.1158/1538-7445.AM2013-1656 Note: This abstract was not presented at the AACR Annual Meeting 2013 because the presenter was unable to attend.

XLID CUL4B mutants are defective in promoting TSC2 degradation and positively regulating mTOR signaling in neocortical neurons

Truncating or missense mutation of cullin 4B (CUL4B) is one of the most prevalent causes underlying X-linked intellectual disability (XLID). CUL4B-RING E3 ubiquitin ligase promotes ubiquitination and degradation of various proteins. Consistent with previous studies, overexpression of wild-type CUL4B in 293 cells enhanced ubiquitylation and degradation of TSC2 or cyclin E. The present study shows that XLID mutant (R388X), (R572C) or (V745A) CULB failed to promote ubiquitination and degradation of TSC2 or cyclin E. Adenoviruses-mediated expression of wild-type CUL4B decreased protein level of TSC2 or cyclin E in cultured neocortical neurons of frontal lobe. Furthermore, shRNA-mediated CUL4B knockdown caused an upregulation of TSC2 or cyclin E. XLID mutant (R388X), (R572C) or (V745A) CUL4B did not downregulate protein expression of TSC2 or cyclin E in neocortical neurons. By promoting TSC2 degradation, CUL4B could positively regulate mTOR activity in neocortical neurons of frontal cortex. Consistent with this hypothesis, CUL4B knockdown-induced upregulation of TSC2 in neocortical neurons resulted in a decreased protein level of active phospho-mTORSer2448 and a reduced expression of active phospho-p70S6KThr389 and phospho-4E-BP1Thr37/46, two main substrates of mTOR-mediated phosphorylation. Wild-type CUL4B also increased protein level of active phospho-mTORSer2448, phospho-p70S6KThr389 or phospho-4E-BP1Thr37/46. XLID CUL4B mutants did not affect protein level of active phospho-mTORSer2448, phospho-p70S6KThr389 or phospho-4E-BP1Thr37/46. Our results suggest that XLID CUL4B mutants are defective in promoting TSC2 degradation and positively regulating mTOR signaling in neocortical neurons.

Negative regulation of mTOR activity by LKB1-AMPK signaling in non-small cell lung cancer cells

To investigate the role of LKB1 in regulation of mTOR signaling in non-small cell lung cancer (NSCLC) cells. LKB1 protein expression and phosphorylation of AMPK, 4E-BP1 and S6K in the cells were assessed using Western blotting in various NSCLC cell lines (A549, H460, H1792, Calu-1 and H1299). Energy stress was mimicked by treating the cells with 2-deoxyglucose (2-DG). Compound C was used to inhibit AMPK activity. Cell growth was measured using the MTS assay. LKB1 protein was expressed in LKB1 wild-type Calu-1, H1299 and H1792 cells, but it was undetected in LKB1 mutant A549 and H460 cells. Treatment of the LKB1 wild-type cells with 2-DG (5, 10 and 25 mmol/L) augmented the phosphorylation of AMPK in dose- and time-dependent manners. In the LKB1 wild-type cells, 2-DG dramatically suppressed the phosphorylation of two mTOR targets, 4E-BP1 and S6K, whereas the LKB1 mutant A549 and H460 cells were highly resistant to 2-DG-induced inhibition on mTOR activity. In addition, stable knockdown of LKB1 in H1299 cells impaired 2-DG-induced inhibition on mTOR activity. Pretreatment of H1299 and H1792 cells with the AMPK inhibitor compound C (10 μmol/L) blocked 2-DG-induced inhibition on mTOR activity. 2-DG inhibited the growth of H1299 cells more effectively than that of H460 cells; stable knockdown of LKB1 in H1299 cells attenuated the growth inhibition caused by 2-DG. In non-small cell lung cancer cells, LKB1/AMPK signaling negatively regulates mTOR activity and contributes to cell growth inhibition in response to energy stress.

PI3K/mTOR Inhibition Upregulates NOTCH-MYC Signalling Leading to an Impaired Cytotoxic Response

Abstract 1358The PI3K/mTOR and NOTCH pathways are promising therapeutic targets for the treatment of T-ALL. Hyperactivation of the PI3K/mTOR pathway occurs frequently, predominantly due to loss of PTEN function through deletion, mutation, microRNA induced downregulation or post-translational modification. NOTCH signalling is aberrantly activated in the majority of patients, most commonly due to mutation of Notch-1. Activation of NOTCH signalling can also positively regulate mTOR activity and increase PI3K/Akt signalling via downregulation of PTEN expression.We examined the effects of PI3K/mTOR blockade, using the dual inhibitor PI-103, on the proliferation and survival of T-ALL cell lines with various combinations of NOTCH and PTEN abnormalities. There was marked reduction in the proliferation of all T-ALL cell lines tested, regardless of their PTEN status or level of activated Akt. However, using Annexin-V/PI staining, we observed significant induction of cell death (<50% survival) in only 3/15 cell lines.Blockade of NOTCH signalling, using a gamma secretase inhibitor (GSI), had no effect on cell survival and only a modest effect on cell proliferation: only 5/13 NOTCH deregulated cell lines, all of which expressed wild-type PTEN, showed a clear reduction in cell number. By contrast only 1 of the 8 GSI-resistant cell lines expressed wild-type PTEN.We tested the effect of combined blockade of PI3K/mTOR and NOTCH pathways using PI-103 + GSI to determine the extent of any non-overlapping effects. In NOTCH-mutant/PTEN-WT cells, combined blockade led to a reduction in cell size and number with a more rapid and marked increase in cell cycle arrest and reduced levels of Cdk4 and Cyclin D3, than achieved with either agent alone. Further, 8/13 cell lines with deregulated Notch showed a significant reduction in cell survival with PI-103+GSI compared with PI-103 alone. In 5/6 NOTCH-mutant/PTEN-WT cell lines survival fell to ≤50% below control, with rapid commitment to cell death (48–72 hours). This mutational context represents the majority of primary T-ALL samples at presentation - confirmation of the enhanced effect of dual PI3K/mTOR and NOTCH blockade was obtained in primary T-ALLs cultured in suspension or on stromal support. Utilizing selective inhibitors of the PI3K (PIK90) or mTOR (rapamycin) enzymes in combination with GSI we found that blockade of both enzymes was required to achieve maximal levels of cell death.c-MYC is an important oncogene in T-ALL - it is a direct transcriptional target of Notch signaling and protein stability is modulated via the PI3K/Akt/GSK3 module. Therefore, we examined the effects of PI3K/mTOR and NOTCH blockade on levels of nuclear c-MYC in cells sensitive to inhibition of both pathways. c-MYC was downregulated by GSI but, surprisingly, increased after 48h of PI-103 treatment. This followed increased levels of nuclear Notch intracellular domain and could be abolished by the addition of GSI, indicating a NOTCH-dependent mechanism. Gene expression microarray analysis confirmed global upregulation of NOTCH signalling in PI-103 treated cells and the suppression of this effect by addition of GSI. These findings were confirmed for Notch target genes (Deltex-1, c-MYC, Hes-1, CD21 and GIMAP5) by qPCR and flow cytometry (CD21).Upregulation of Notch-MYC proliferation and survival signals could explain why T-ALL cells survive after PI3K/mTOR blockade. We found that maintenance of c-MYC levels by retroviral expression counteracted the effects of PI3K/mTOR/NOTCH blockade and comparable levels of cell death, to those seen with PI-103 plus GSI, were seen in cells treated with PI-103 + the c-MYC inhibitor, 10058-F4.Our data show that targeting PI3K/mTOR can upregulate NOTCH-MYC activity in T-ALL cells with aberrant NOTCH signalling. These finding have implications for the use of PI3K inhibitors for the treatment of T-ALL, and other malignancies with activated NOTCH signaling, and provide a rational basis for the use of drug combinations that target both pathways. Disclosures:No relevant conflicts of interest to declare.

Leucine Stimulates Insulin Secretion via Down-regulation of Surface Expression of Adrenergic α2A Receptor through the mTOR (Mammalian Target of Rapamycin) Pathway: IMPLICATION IN NEW-ONSET DIABETES IN RENAL TRANSPLANTATION

The amino acid leucine is a potent secretagogue, capable of inducing insulin secretion. It also plays an important role in the regulation of mTOR activity, therefore, providing impetus to investigate if a leucine-sensing mechanism in the mTOR pathway is involved in insulin secretion. We found that leucine-induced insulin secretion was inhibited by both the mTOR inhibitor rapamycin as well as the adrenergic α2 receptor agonist clonidine. We also demonstrated that leucine down-regulated the surface expression of adrenergic α2A receptor via activation of the mTOR pathway. The leucine stimulatory effect on insulin secretion was attenuated in diabetic Goto-Kakizaki rats that overexpress adrenergic α2A receptors, confirming the role of leucine in insulin secretion. Thus, our data demonstrate that leucine regulates insulin secretion by modulating adrenergic α2 receptors through the mTOR pathway. The role of the mTOR pathway in metabolic homeostasis led us to a second important finding in this study; retrospective analysis of clinical data showed that co-administration of rapamycin and clonidine was associated with an increased incidence of new-onset diabetes in renal transplantation patients over those receiving rapamycin alone. We believe that inhibition of mTOR by rapamycin along with activation of adrenergic α2 receptors by clonidine represents a double-hit to pancreatic islets that synergistically disturbs glucose homeostasis. This new insight may have important implications for the clinical management of renal transplant patients.

Ubiquitin-specific Peptidase 9, X-linked (USP9X) Modulates Activity of Mammalian Target of Rapamycin (mTOR)

The mammalian target of rapamycin (mTOR) is an atypical serine/threonine kinase that responds to extracellular environment to regulate a number of cellular processes. These include cell growth, proliferation, and differentiation. Although both kinase-dependent and -independent functions of mTOR are known to be critical modulators of muscle cell differentiation and regeneration, the signaling mechanisms regulating mTOR activity during differentiation are still unclear. In this study we identify a novel mTOR interacting protein, the ubiquitin-specific protease USP9X, which acts as a negative regulator of mTOR activity and muscle differentiation. USP9X can co-immunoprecipitate mTOR with both Raptor and Rictor, components of mTOR complexes 1 and 2 (mTORC1 and -2), respectively, suggesting that it is present in both mTOR complexes. Knockdown of USP9X leads to increased mTORC1 activity in response to growth factor stimulation. Interestingly, upon initiation of differentiation of C2C12 mouse skeletal myoblasts, knockdown of USP9X increases mTORC2 activity. This increase in mTORC2 activity is accompanied by accelerated differentiation of myoblasts into myotubes. Taken together, our data describe the identification of the deubiquitinase USP9X as a novel mTORC1 and -2 binding partner that negatively regulates mTOR activity and skeletal muscle differentiation.

Golgi apparatus location and regulation of mTOR is regulated by novel Golgi recruiting protein GRP

Mammalian target of rapamycin (mTOR) is a central protein involved in the control of cell growth and metabolism. Dysregulation of mTOR signaling has been closely linked to many human diseases such as cancer, diabetes, obesity cardiovascular diseases, neurological disorders and age related diseases. Although many of the components in the mTOR pathway have been identified, little is known regarding the mechanism of the spatial arrangement of mTOR signaling. Previously, our lab reported that in several common cell lines, a significant amount of mTOR is localized to the membrane of particular sites called the endoplasmic reticulum (ER) and Golgi apparatus. mTOR localization to the ER and Golgi is mediated by novel motifs called the Golgi localization sequence (GLS) and an overlapping endoplasmic reticulum localization sequence (ELS), respectively. Mutation of the GLS or ELS, or over‐expression of these sequences alone, results in inhibition of both mTOR complexes. These results suggest that subcellular localization of mTOR to the Golgi and ER is essential for mTOR signaling. Because GLS lacks apparent membrane association property, we hypothesized that a Golgi membrane protein(s) is responsible for anchoring mTOR to Golgi through the interaction with GLS. We identified a protein that possesses a strong interaction with the GLS. This novel protein carries a Golgi membrane localization domain and directly interacts with and regulates mTOR activity. We therefore named it GLS Receptor Protein (GRP). My data exhibits strong evidence that GRP mediates mTOR localization to the Golgi and is involved in mTOR regulation. Support for this research was provided by the New Jersey Commission on Cancer Research and the National Institute of Health.

Antileukemic effects of AMPK activators on BCR-ABL–expressing cells

AbstractThe mammalian target of rapamycin (mTOR) signaling pathway plays a critical role in growth and survival of BCR-ABL transformed cells. AMPK kinase is a metabolic sensor that exhibits suppressive effects on the mTOR pathway and negatively regulates mTOR activity. We report that AMPK activators, such as metformin and 5-aminoimidazole-4-carboxamide ribonucleotide, suppress activation of the mTOR pathway in BCR-ABL–expressing cells. Treatment with these inhibitors results in potent suppression of chronic myeloid leukemia leukemic precursors and Ph+ acute lymphoblastic leukemia cells, including cells expressing the T315I-BCR-ABL mutation. Altogether, our data suggest that AMPK is an attractive target for the treatment of BCR-ABL–expressing malignancies and raise the potential for use of AMPK activators in the treatment of refractory chronic myeloid leukemia and Ph+ acute lymphoblastic leukemia.

Intake of branched-chain amino acids influences the levels of MAFbx mRNA and MuRF-1 total protein in resting and exercising human muscle

Resistance exercise and amino acids are two major factors that influence muscle protein turnover. Here, we examined the effects of resistance exercise and branched-chain amino acids (BCAA), individually and in combination, on the expression of anabolic and catabolic genes in human skeletal muscle. Seven subjects performed two sessions of unilateral leg press exercise with randomized supplementation with BCAA or flavored water. Biopsies were collected from the vastus lateralis muscle of both the resting and exercising legs before and repeatedly after exercise to determine levels of mRNA, protein phosphorylation, and amino acid concentrations. Intake of BCAA reduced (P < 0.05) MAFbx mRNA by 30 and 50% in the resting and exercising legs, respectively. The level of MuRF-1 mRNA was elevated (P < 0.05) in the exercising leg two- and threefold under the placebo and BCAA conditions, respectively, whereas MuRF-1 total protein increased by 20% (P < 0.05) only in the placebo condition. Phosphorylation of p70(S6k) increased to a larger extent (∼2-fold; P < 0.05) in the early recovery period with BCAA supplementation, whereas the expression of genes regulating mTOR activity was not influenced by BCAA. Muscle levels of phenylalanine and tyrosine were reduced (13-17%) throughout recovery (P < 0.05) in the placebo condition and to a greater extent (32-43%; P < 0.05) following BCAA supplementation in both resting and exercising muscle. In conclusion, BCAA ingestion reduced MAFbx mRNA and prevented the exercise-induced increase in MuRF-1 total protein in both resting and exercising leg. Further-more, resistance exercise differently influenced MAFbx and MuRF-1 mRNA expression, suggesting both common and divergent regulation of these two ubiquitin ligases.

Resveratrol Inhibits mTOR Signaling by Promoting the Interaction between mTOR and DEPTOR

Resveratrol (RSV) is a naturally occurring polyphenol that has been found to exert antioxidant, anti-inflammatory, and neuroprotective properties. However, how RSV exerts its beneficial health effects remains largely unknown. Here, we show that RSV inhibits insulin- and leucine-stimulated mTOR signaling in C2C12 fibroblasts via a Sirt1-independent mechanism. Treating C2C12 cells with RSV dramatically inhibited insulin-stimulated Akt, S6 kinase, and 4E-BP1 phosphorylation but had little effect on tyrosine phosphorylation of the insulin receptor and activation of the p44/42 MAPK signaling pathway. RSV treatment also partially blocked mTOR and S6 kinase phosphorylation in TSC1/2-deficient mouse embryonic fibroblasts, suggesting the presence of an inhibitory site downstream of TSC1/2. Knocking out PDK1 or suppressing AMP-activated protein kinase had little effect on leucine-stimulated mTOR signaling. On the other hand, RSV significantly increased the association between mTOR and its inhibitor, DEPTOR. Furthermore, the inhibitory effect of RSV on leucine-stimulated mTOR signaling was greatly reduced in cells in which the expression levels of DEPTOR were suppressed by RNAi. Taken together, our studies reveal that RSV inhibits leucine-stimulated mTORC1 activation by promoting mTOR/DEPTOR interaction and thus uncover a novel mechanism by which RSV negatively regulates mTOR activity.

Abstract 5091: Lymphovascular tumor emboli exhibit a quiescent state refractory to chemotherapy and targeted therapies because of endogenously low metabolism

Abstract Previous studies with a human xenograft model of inflammatory breast cancer, MARY-X, demonstrated that overexpressed E-cadherin moderated the formation of the lymphovascular embolus in vivo and tumoral spheroids of super-high density in vitro. In vitro, MARY-X cells grow very slowly as spheroids and the consumption of nutrients is markedly decreased, suggesting that the metabolism in the MARY-X spheroids is also very low. This low metabolism may also be exhibited in vivo within the lymphovascular embolus and may be responsible for its resistance to chemotherapy. This study investigated the mechanisms which lead to a low level of metabolism by initially analyzing AMPK, a metabolic checkpoint coordinating cell growth with energy status and the mTOR pathway, a pathway that is a highly conserved nutrient-responsive regulator of cell growth found in all eukaryotes. The levels of phosphorylated AMPK proteins (AMPKα and β subunits) decrease gradually with the formation of MARY-X spheroids in vitro. Since it had been reported that E-cadherin inhibits the activity of LKB1, a protein upstream of AMPK, which activates both AMPK and several AMPK-related kinases, the decrease in AMPK activity in the MARY-X spheroids could be the result of the homophilic E-cadherin interactions mediating the tight spheroid aggregates. mTOR activity, on the other hand, increases within the MARY-X spheroids. Because AMPK inversely regulates mTOR activity, it is easy to understand why mTOR activity increases. Using rapamycin on the spheroids led to the down-regulation in the activity of p70 S6 kinase but less effects on the activity of 4E-PB1. Most importantly, the treated spheroids did not dissociate nor undergo apoptosis. Use of Ly294002, a PI3K/Akt inhibitor, completely abolished the activities of both p70 S6 kinase and 4E-BP1, the substrates of mTOR. The treated spheroids dissociated and the cells underwent apoptosis. Compound C (AMPK inhibitor) blocked the activity of AMPK, which resulted in a marked upregulation of the mTOR pathway with great increases in both p70 S6 kinase and 4E-BP1 and an increase in resulting metabolism. However the treated spheroids, just like with Ly294002, dissociated and underwent apoptosis. Increasing the cellular metabolism leads to cell death even in enriched medium. Growing MARY-X in serum free media results in the activation of AMPK, when intracellular ATP levels decline and intracellular AMP increases, as occurs during nutrient deprivation or hypoxia. AMPK activation normally inhibits the mTOR pathway but in MARY-X spheroids grown in serum-free medium, the mTOR pathway is not inhibited, cellular metabolism remains low and the spheroids do not dissociate or undergo apoptosis. If these observations can be confirmed in in vivo studies, we may need to rethink our targeting strategies in the face of a quiescent tumor embolus exhibiting low metabolism. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 101st Annual Meeting of the American Association for Cancer Research; 2010 Apr 17-21; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2010;70(8 Suppl):Abstract nr 5091.

The mTOR Kinase Determines Effector versus Memory CD8+ T Cell Fate by Regulating the Expression of Transcription Factors T-bet and Eomesodermin

The mechanisms underpinning integration of instructions that program naive CD8+ T cells for effector and/or memory differentiation are not well understood. Herein, we demonstrate that interleukin-12 (IL-12) enhanced and sustained antigen and costimulatory molecule (B7.1)-induced mTOR kinase activity in naive CD8+ (OT-I) T cells via phosphoinositide 3-kinase and STAT4 transcription factor pathways. Blocking mTOR activity by rapamycin reversed IL-12-induced effector functions because of loss of persistent expression of the transcription factor T-bet. Rapamycin treatment of IL-12-conditioned OT-I cells promoted persistent Eomesodermin expression and produced memory cell precursors that demonstrated enhanced sustenance and antigen-recall responses upon adoptive transfer. The memory cell precursors showed greater tumor efficacy than IL-12-conditioned effector OT-I cells. These results identify mTOR as the central regulator of transcriptional programs that determine effector and/or memory cell fates in CD8+ T cells. Targeting mTOR activity offers new opportunities to regulate CD8+ T cell-mediated immunity.

RIP1 Activates PI3K-Akt via a Dual Mechanism Involving NF-κB–Mediated Inhibition of the mTOR-S6K-IRS1 Negative Feedback Loop and Down-regulation of PTEN

Therapeutic inhibition of mammalian target of rapamycin (mTOR) in cancer is complicated by the existence of a negative feedback loop linking mTOR to the phosphatidylinositol 3-kinase (PI3K)-Akt pathway. Thus, mTOR inhibition by rapamycin or TSC1/2 results in increased PI3K-Akt activation. The death domain kinase receptor interacting protein 1 (RIP1) plays a key role in nuclear factor-kappaB (NF-kappaB) activation and also activates the PI3K-Akt pathway through unknown mechanisms. RIP1 has recently been found to be overexpressed in glioblastoma multiforme, the most common adult primary malignant brain tumor, but not in grade II to III glioma. Our data suggest that RIP1 activates PI3K-Akt using dual mechanisms by removing the two major brakes on PI3K-Akt activity. First, increased expression of RIP1 activates PI3K-Akt by interrupting the mTOR negative feedback loop. However, unlike other signals that regulate mTOR activity without affecting its level, RIP1 negatively regulates mTOR transcription via a NF-kappaB-dependent mechanism. The second mechanism used by RIP1 to activate PI3K-Akt is down-regulation of cellular PTEN levels, which appears to be independent of NF-kappaB activation. The clinical relevance of these findings is highlighted by the demonstration that RIP1 levels correlate with activation of Akt in glioblastoma multiforme. Thus, our study shows that RIP1 regulates key components of the PTEN-PI3K-Akt-mTOR pathway and elucidates a novel negative regulation of mTOR signaling at the transcriptional level by the NF-kappaB pathway. Our data suggest that the RIP1-NF-kappaB status of tumors may influence response to treatments targeting the PTEN-PI3K-mTOR signaling axis.

PIM1 Protein Kinase regulates PRAS40 phosphorylation and mTOR activity in FDCP1 cells

PIM1 is a serine /threonine kinase that has diverse biological roles in cell survival, proliferation and differentiation. PIM1 has been implicated in early transformation and tumor progression in haematopoietic malignancies and prostate carcinomas. The ability of PIM1 to regulate these processes is thought to be in part secondary to its activity in stimulating 4EBP1 phosphorylation and enhancement of protein synthesis. Because 4EBP1 is an mTOR substrate, we have investigated how PIM1 might regulate this latter enzyme. We have examined the ability of PIM1 to modulate PRAS40, a protein known to negatively regulate mTOR activity in FDP1 cells. Upon phosphorylation, PRAS40 dissociates from the mTOR complex and increases mTOR kinase activity. We find that enforced overexpression of PIM1 increases PRAS40 phosphorylation at Thr246, an AKT phosphorylation site, whether grown in complete media or deprived of IL-3 and serum. The increase in PRAS40 phosphorylation was independent of AKT activation and not inhibited by wortmannin. In vitro kinase assays indicate that the PIM1 protein kinase is capable of directly phosphorylating Thr246 in PRAS40. PIM1 protein kinase overexpression reduced the association of PRAS40 with mTOR, and increased the mTOR directed phosphorylation of 4EBP1 and p70S6Kinase. Treatment of FDCP1 cells transfected with PIM1 (FD/mpim44) with small molecule inhibitors of PIM1 kinase activity reduced both PRAS40 and 4EBP1 phosphorylation. These results suggest that PIM1 regulates mTOR activity through phosphorylation of PRAS40. Thus, increases in mTOR activity mediated by the PIM protein kinase may have the potential to control cell growth.