PEBP4 Directs the Malignant Behavior of Hepatocellular Carcinoma Cells via Regulating mTORC1 and mTORC2

Phosphatidylethanolamine binding protein 4 (PEBP4) is an understudied multifunctional small protein. Previous studies have shown that the expression of PEBP4 is increased in many cancer specimens, which correlates to cancer progression. The present study explored the mechanism by which PEBP4 regulates the growth and progression of hepatocellular carcinoma cells. Thus, we showed that knockdown of PEBP4 in MHCC97H cells, where its expression was relatively high, diminished activities of serine/threonine protein kinase B (PKB, also known as Akt), mammalian target of rapamycin complex 1(mTORC1), and mTORC2, events that were not restored by insulin-like growth factor 1 (IGF-1). Conversely, overexpression of PEBP4 in MHCC97L cells with the low endogenous level yielded opposite effects. Furthermore, physical association of PEBP4 with Akt, mTORC1, and mTORC2 was observed. Interestingly, introduction of AktS473D mutant, bypassing phosphorylation by mTORC2, rescued mTORC1 activity, but without effects on mTORC2 signaling. In contrast, the effect of PEBP4 overexpression on the activity of mTORC1 but not that of mTORC2 was suppressed by MK2206, a specific inhibitor of Akt. In conjunction, PEBP4 knockdown-engendered reduction of cell proliferation, migration and invasion was partially rescued by Akt S473D while increases in these parameters induced by overexpression of PEBP4 were completely abolished by MK2206, although the expression of epithelial mesenchymal transition (EMT) markers appeared to be fully regulated by the active mutant of Akt. Finally, knockdown of PEBP4 diminished the growth of tumor and metastasis, whereas they were enhanced by overexpression of PEBP4. Altogether, our study suggests that increased expression of PEBP4 exacerbates malignant behaviors of hepatocellular cancer cells through cooperative participation of mTORC1 and mTORC2.     

Effects of Chronic LY341495 on Hippocampal mTORC1 Signaling in Mice with Chronic Unpredictable Stress-Induced Depression

In several rodent models, acute administration of the metabotropic glutamate 2/3 (mGlu2/3) receptor antagonist LY341495 induced antidepressant-like effects via a mechanism of action similar to that of ketamine. However, the effects of chronic mGlu2/3 antagonism have not yet been explored. Therefore, we investigated the effects of chronic LY341495 treatment on the mechanistic target of rapamycin complex 1 (mTORC1) signaling and the levels of synaptic proteins in mice subjected to chronic unpredictable stress (CUS). LY341495 (1 mg/kg) was administered daily for 4 weeks to mice with and without CUS exposure. After the final treatment, the forced swimming test (FST) was used to assess antidepressant-like effects. The hippocampal levels of mTORC1-related proteins were derived by Western blotting. Chronic LY341495 treatment reversed the CUS-induced behavioral effects of FST. CUS significantly reduced the phosphorylation of mTORC1 and downstream effectors [eukaryotic translation initiation factor 4E-binding protein 1 (4E-BP-1) and small ribosomal protein 6 (S6)], as well as the expression of synaptic proteins postsynaptic density-95 (PSD-95) and AMPA receptor subunit GluR1 (GluA1) in the hippocampus. However, chronic LY341495 treatment rescued these deficits. Our results suggest that the activation of hippocampal mTORC1 signaling is related to the antidepressant effect of chronic LY341495 treatment in an animal model of CUS-induced depression.     

The Characterization of a Subependymal Giant Astrocytoma-Like Cell Line from Murine Astrocyte with mTORC1 Hyperactivation

Tuberous sclerosis complex (TSC) is a genetic disorder caused by inactivating mutations in TSC1 (hamartin) or TSC2 (tuberin), crucial negative regulators of the mechanistic target of rapamycin complex 1 (mTORC1) signaling pathway. TSC affects multiple organs including the brain. The neurologic manifestation is characterized by cortical tubers, subependymal nodules (SEN), and subependymal giant cell astrocytoma (SEGA) in brain. SEGAs may result in hydrocephalus in TSC patients and mTORC1 inhibitors are the current recommended therapy for SEGA. Nevertheless, a major limitation in the research for SEGA is the lack of cell lines or animal models for mechanistic investigations and development of novel therapy. In this study, we generated TSC1-deficient neural cells from spontaneously immortalized mouse astrocytes in an attempt to mimic human SEGA. The TSC1-deficient cells exhibit mTORC1 hyperactivation and characteristics of transition from astrocytes to neural stem/progenitor cell phenotypes. Rapamycin efficiently decreased mTORC1 activity of these TSC1-deficient cells in vitro. In vivo, TSC1-deficient cells could form SEGA-like tumors and Rapamycin treatment decreased tumor growth. Collectively, our study generates a novel SEGA-like cell line that is invaluable for studying mTORC1-driven molecular and pathological alterations in neurologic tissue. These SEGA-like cells also provide opportunities for the development of novel therapeutic strategy for TSC patients with SEGA.     

Tribbles Pseudokinase 2 (TRIB2) Regulates Expression of Binding Partners in Bovine Granulosa Cells

Members of the Tribbles (TRIB) family of pseudokinases are critical components of intracellular signal transduction pathways in physiological and pathological processes. TRIBs, including TRIB2, have been previously shown as signaling mediators and scaffolding proteins regulating numerous cellular events such as proliferation, differentiation and cell death through protein stability and activity. However, the signaling network associated with TRIB2 and its binding partners in granulosa cells during ovarian follicular development is not fully defined. We previously reported that TRIB2 is differentially expressed in growing dominant follicles while downregulated in ovulatory follicles following the luteinizing hormone (LH) surge or human chorionic gonadotropin (hCG) injection. In the present study, we used the yeast two-hybrid screening system and in vitro coimmunoprecipitation assays to identify and confirm TRIB2 interactions in granulosa cells (GCs) of dominant ovarian follicles (DFs), which yielded individual candidate binding partners including calmodulin 1 (CALM1), inhibin subunit beta A (INHBA), inositol polyphosphate phosphatase-like 1 (INPPL1), 5′-nucleotidase ecto (NT5E), stearoyl-CoA desaturase (SCD), succinate dehydrogenase complex iron sulfur subunit B (SDHB) and Ras-associated protein 14 (RAB14). Further analyses showed that all TRIB2 binding partners are expressed in GCs of dominant follicles but are differentially regulated throughout the different stages of follicular development. CRISPR/Cas9-driven inhibition along with pQE-driven overexpression of TRIB2 showed that TRIB2 differently regulates expression of binding partners, which reveals the importance of TRIB2 in the control of gene expression linked to various biological processes such as proliferation, differentiation, cell migration, apoptosis, calcium signaling and metabolism. These data provide a larger view of potential TRIB2-regulated signal transduction pathways in GCs and provide strong evidence that TRIB2 may act as a regulator of target genes during ovarian follicular development.     

Pharmacological Inhibition of mTORC2 Reduces Migration and Metastasis in Melanoma

Despite recent advances in therapy, liver metastasis from melanoma is still associated with poor prognosis. Although targeting the mTOR signaling pathway exerts potent anti-tumor activity, little is known about specific mTORC2 inhibition regarding liver metastasis. Using the novel mTORC2 specific inhibitor JR-AB2-011, we show significantly reduced migration and invasion capacity by impaired activation of MMP2 in melanoma cells. In addition, blockade of mTORC2 induces cell death by non-apoptotic pathways and reduces tumor cell proliferation rate dose-dependently. Furthermore, a significant reduction of liver metastasis was detected in a syngeneic murine metastasis model upon therapy with JR-AB2-011 as determined by in vivo imaging and necropsy. Hence, our study for the first time highlights the impact of the pharmacological blockade of mTORC2 as a potent novel anti-cancer approach for liver metastasis from melanoma.     

Activation of ERK and p38 Reduces AZD8055-Mediated Inhibition of Protein Synthesis in Hepatocellular Carcinoma HepG2 Cell Line

Protein synthesis is important for maintaining cellular homeostasis under various stress responses. In this study, we screened an anticancer drug library to select compounds with translational repression functions. AZD8055, an ATP-competitive mechanistic target of rapamycin complex 1/2 (mTORC1/2) inhibitor, was selected as a translational suppressor. AZD8055 inhibited protein synthesis in mouse embryonic fibroblasts and hepatocellular carcinoma HepG2 cells. Extracellular signal-regulated kinase (ERK) and p38 mitogen-activated protein kinase (MAPK) were activated during the early phase of mTORC1/2 inhibition by AZD8055 treatment. Combined treatment of AZD8055 with the MAPK kinase1/2 (MEK1/2) inhibitor refametinib or the p38 inhibitor SB203580 markedly decreased translation in HepG2 cells. Thus, the inhibition of ERK1/2 or p38 may enhance the efficacy of AZD8055-mediated inhibition of protein synthesis. In addition, AZD8055 down-regulated the phosphorylation of eukaryotic initiation factor 4E-binding protein 1 (4E-BP1), and AZD8055-induced phosphorylation of ERK1/2 and p38 had no effect on phosphorylation status of 4E-BP1. Interestingly, AZD8055 modulated the 4E-BP1 mRNA pool by up-regulating ERK1/2 and p38 pathways. Together, these results suggest that AZD8055-induced activation of MAPKs interferes with inhibition of protein synthesis at an early stage of mTORC1/2 inhibition, and that it may contribute to the development of resistance to mTORC1/2 inhibitors.     

Identification of an AMPK Phosphorylation Site in Drosophila TSC2 (gigas) that Regulate Cell Growth

AMP-activated protein kinase (AMPK) is an important metabolic regulator that mediates cellular adaptation to diverse stresses. One of the AMPK substrates, tuberous sclerosis complex 2 (TSC2), was suggested to mediate AMPK-induced silencing of mTOR complex 1 (mTORC1) signaling that is critical for cell growth. However, it is not known whether the AMPK-dependent TSC2 phosphorylation, originally observed in mammalian cells, is conserved in invertebrates. Here we show that energy depletion inhibits mTORC1 signaling through the AMPK-TSC2 axis in Drosophila S2 cells. We have discovered an AMPK phosphorylation site in TSC2-like genes from many different invertebrate species including Drosophila. The site (Ser1338 in Drosophila TSC2) is specifically and efficiently phosphorylated by AMPK in vitro. To evaluate the functional role of this phosphorylation site in vivo, we generated transgenic flies that can express identical amount of either wild-type or phosphorylation-resistant mutant Drosophila TSC2 in a tissue-specific manner. In response to transgenic Sestrin induction, which causes ectopic AMPK activation and subsequent mTORC1 inhibition, wild-type Drosophila TSC2 synergistically reduced tissue growth in the dorsal epithelium of Drosophila wings. However, phosphorylation-resistant mutant Drosophila TSC2 was unable to show such a growth-inhibiting effect, suggesting that this phosphorylation is important for AMPK-dependent regulation of cell growth.     

Caloric Restriction Normalizes Obesity-Induced Alterations on Regulators of Skeletal Muscle Growth Signaling

The objective of this study was to establish the impact of caloric restriction on high fat diet‐induced alterations on regulators of skeletal muscle growth. We hypothesized that caloric restriction would reverse the negative effects of high fat diet‐induced obesity on REDD1 and mTOR‐related signaling. Following an initial 8 week period of HF diet‐induced obesity, caloric restriction (CR ~30 %) was employed while mice continued to consume either a low (LF) or high fat (HF) diet for 8 weeks. Western analysis of skeletal muscle showed that CR reduced (p < 0.05) the obesity‐related effects on the lipogenic protein, SREBP1. Likewise, CR reduced (p < 0.05) the obesity‐related effects on the hyperactivation of mTORC1 and ERK1/2 signaling to levels comparable to the LF mice. CR also reduced (p < 0.05) obesity‐induced expression of negative regulators of growth, REDD1 and cleaved caspase 3. These findings have implications for on the reversibility of dysregulated growth signaling in obese skeletal muscle, using short‐term caloric restriction.     

Cardiac Ablation of Rheb1 Induces Impaired Heart Growth,Endoplasmic Reticulum-Associated Apoptosis and Heart Failure in Infant Mice

Ras homologue enriched in brain 1 (Rheb1) plays an important role in a variety of cellular processes. In this study, we investigate the role of Rheb1 in the post-natal heart. We found that deletion of the gene responsible for production of Rheb1 from cardiomyocytes of post-natal mice resulted in malignant arrhythmias, heart failure, and premature death of these mice. In addition, heart growth impairment, aberrant metabolism relative gene expression, and increased cardiomyocyte apoptosis were observed in Rheb1-knockout mice prior to the development of heart failure and arrhythmias. Also, protein kinase B (PKB/Akt) signaling was enhanced in Rheb1-knockout mice, and removal of phosphatase and tensin homolog (Pten) significantly prolonged the survival of Rheb1-knockouts. Furthermore, signaling via the mammalian target of rapamycin complex 1 (mTORC1) was abolished and C/EBP homologous protein (CHOP) and phosphorylation levels of c-Jun N-terminal kinase (JNK) were increased in Rheb1 mutant mice. In conclusion, this study demonstrates that Rheb1 is important for maintaining cardiac function in post-natal mice via regulation of mTORC1 activity and stress on the endoplasmic reticulum. Moreover, activation of Akt signaling helps to improve the survival of mice with advanced heart failure. Thus, this study provides direct evidence that Rheb1 performs multiple important functions in the heart of the post-natal mouse. Enhancing Akt activity improves the survival of infant mice with advanced heart failure.     

Tetraspanin 8-Rictor-Integrin α3 Complex Is Required for Glioma Cell Migration

The malignant glioma remains one of the most aggressive human malignancies with extremely poor prognosis. Glioma cell invasion and migration are the main causes of death. In the current study, we studied the expression and the potential functions of tetraspanin 8 (Tspan8) in malignant gliomas. We found that Tspan8 expression level is high in both malignant glioma tissues and in several human glioma cell lines, where it formed a complex integrin α3 and rictor, the latter is a key component of mammalian target of rapamycin (mTOR) complex 2 (mTORC2). Disruption of this complex, through siRNA-mediated knockdown of anyone of these three proteins, inhibited U251MG glioma cell migration in vitro. We further showed that Tspan8-rictor association appeared required for mTORC2 activation. Knockdown of Tspan8 by the targeted siRNAs prevented mTOR-rictor (mTORC2) assembly as well as phosphorylation of AKT (Ser-473) and protein kinase C α (PKCα) in U251MG cells. Together, these results demonstrate that over-expressed Tspan8 in malignant glioma forms a complex with rictor and integrin α3 to mediate mTORC2 activation and glioma cell migration. Therefore, targeting Tspan8-rictor-integrin α3 complex may provide a potential therapeutic intervention for malignant glioma.     

Inhibition of Glutamine Uptake Resensitizes Paclitaxel Resistance in SKOV3-TR Ovarian Cancer Cell via mTORC1/S6K Signaling Pathway

Ovarian cancer is a carcinoma that affects women and that has a high mortality rate. Overcoming paclitaxel resistance is important for clinical application. However, the effect of amino acid metabolism regulation on paclitaxel-resistant ovarian cancer is still unknown. In this study, the effect of an amino acid-deprived condition on paclitaxel resistance in paclitaxel-resistant SKOV3-TR cells was analyzed. We analyzed the cell viability of SKOV3-TR in culture conditions in which each of the 20 amino acids were deprived. As a result, the cell viability of the SKOV3-TR was significantly reduced in cultures deprived of arginine, glutamine, and lysine. Furthermore, we showed that the glutamine-deprived condition inhibited mTORC1/S6K signaling. The decreased cell viability and mTORC1/S6K signaling under glutamine-deprived conditions could be restored by glutamine and α-KG supplementation. Treatment with PF-4708671, a selective S6K inhibitor, and the selective glutamine transporter ASCT2 inhibitor V-9302 downregulated mTOR/S6K signaling and resensitized SKOV3-TR to paclitaxel. Immunoblotting showed the upregulation of Bcl-2 phosphorylation and a decrease in Mcl-1 expression in SKOV3-TR via the cotreatment of paclitaxel with PF-4708671 and V-9302. Collectively, this study demonstrates that the inhibition of glutamine uptake can resensitize SKOV3-TR to paclitaxel and represents a promising therapeutic target for overcoming paclitaxel resistance in ovarian cancer.     

Dysregulation of mTOR Signaling after Brain Ischemia

In this review, we provide recent data on the role of mTOR kinase in the brain under physiological conditions and after damage, with a particular focus on cerebral ischemia. We cover the upstream and downstream pathways that regulate the activation state of mTOR complexes. Furthermore, we summarize recent advances in our understanding of mTORC1 and mTORC2 status in ischemia–hypoxia at tissue and cellular levels and analyze the existing evidence related to two types of neural cells, namely glia and neurons. Finally, we discuss the potential use of mTORC1 and mTORC2 as therapeutic targets after stroke.     

Identification of New Potential Interaction Partners for Human Cytoplasmic Copper Chaperone Atox1: Roles in Gene Regulation?

The human copper (Cu) chaperone Atox1 delivers Cu to P_1B type ATPases in the Golgi network, for incorporation into essential Cu-dependent enzymes. Atox1 homologs are found in most organisms; it is a 68-residue ferredoxin-fold protein that binds Cu in a conserved surface-exposed Cys-X-X-Cys (CXXC) motif. In addition to its well-documented cytoplasmic chaperone function, in 2008 Atox1 was suggested to have functionality in the nucleus. To identify new interactions partners of Atox1, we performed a yeast two-hybrid screen with a large human placenta library of cDNA fragments using Atox1 as bait. Among 98 million fragments investigated, 25 proteins were found to be confident interaction partners. Nine of these were uncharacterized proteins, and the remaining 16 proteins were analyzed by bioinformatics with respect to cell localization, tissue distribution, function, sequence motifs, three-dimensional structures and interaction networks. Several of the hits were eukaryotic-specific proteins interacting with DNA or RNA implying that Atox1 may act as a modulator of gene regulation. Notably, because many of the identified proteins contain CXXC motifs, similarly to the Cu transport reactions, interactions between these and Atox1 may be mediated by Cu.     

Proteomic Analysis Identifies an NADPH Oxidase 1 (Nox1)-Mediated Role for Actin-Related Protein 2/3 Complex Subunit 2 (ARPC2) in Promoting Smooth Muscle Cell Migration

A variety of vascular pathologies, including hypertension, restenosis and atherosclerosis, are characterized by vascular smooth muscle cell (VSMC) hypertrophy and migration. NADPH oxidase 1 (Nox1) plays a pivotal role in these phenotypes via distinct downstream signaling. However, the mediators differentiating these distinct phenotypes and their precise role in vascular disease are still not clear. The present study was designed to identify novel targets of VSMC Nox1 signaling using 2D Differential In-Gel Electrophoresis and Mass Spectrometry (2D-DIGE/MS). VSMC treatment with scrambled (Scrmb) or Nox1 siRNA and incubation with the oxidant hydrogen peroxide (H₂O₂; 50 μM, 3 h) followed by 2D-DIGE/MS on cell lysates identified 10 target proteins. Among these proteins, actin-related protein 2/3 complex subunit 2 (ARPC2) with no previous link to Nox isozymes, H₂O₂, or other reactive oxygen species (ROS), was identified and postulated to play an intermediary role in VSMC migration. Western blot confirmed that Nox1 mediates H₂O₂-induced ARPC2 expression in VSMC. Treatment with a p38 MAPK inhibitor (SB203580) resulted in reduced ARPC2 expression in H₂O₂-treated VSMC. Additionally, wound-healing “scratch” assay confirmed that H₂O₂ stimulates VSMC migration via Nox1. Importantly, gene silencing of ARPC2 suppressed H₂O₂-stimulated VSMC migration. These results demonstrate for the first time that Nox1-mediated VSMC migration involves ARPC2 as a downstream signaling target.