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Novel therapeutic approaches are needed for the treatment of Ewing sarcoma tumors. We previously identified that Ewing sarcoma cell lines are sensitive to drugs that inhibit protein translation. However, translational and therapeutic approaches to inhibit protein synthesis in tumors are limited. In this work, we identified that reactive oxygen species, which are generated by a wide range of chemotherapy and other drugs, inhibit protein synthesis and reduce the level of critical proteins that support tumorigenesis in Ewing sarcoma cells. In particular, we identified that both hydrogen peroxide and auranofin, an inhibitor of thioredoxin reductase and regulator of oxidative stress and reactive oxygen species, activate the repressor of protein translation 4E-BP1 and reduce the levels of the oncogenic proteins RRM2 and PLK1 in Ewing and other sarcoma cell lines. These results provide novel insight into the mechanism of how ROSinducing drugs target cancer cells via inhibition of protein translation and identify a mechanistic link between ROS and the DNA replication (RRM2) and cell cycle regulatory (PLK1) pathways. [ABSTRACT FROM AUTHOR]

The 78-kDa glucose regulated protein (GRP78) commonly upregulated in a wide variety of tumors is an important prognostic marker and a promising target for suppressing tumorigenesis and treatment resistance. While GRP78 is well established as a major endoplasmic reticulum (ER) chaperone with anti-apoptotic properties and a master regulator of the unfolded protein response, its new role as a regulator of oncoprotein expression is just emerging. MYC is dysregulated in about 70 % of human cancers and is the most commonly activated oncoprotein. However, despite recent advances, therapeutic targeting of MYC remains challenging. Here we identify GRP78 as a new target for suppression of MYC expression. Using multiple MYC-dependent cancer models including head and neck squamous cell carcinoma and their cisplatin-resistant clones, breast and pancreatic adenocarcinoma, our studies revealed that GRP78 knockdown by siRNA or inhibition of its activity by small molecule inhibitors (YUM70 or HA15) reduced c-MYC expression, leading to onset of apoptosis and loss of cell viability. This was observed in 2D cell culture, 3D spheroid and in xenograft models. Mechanistically, we determined that the suppression of c-MYC is at the post-transcriptional level and that YUM70 and HA15 treatment potently upregulated the eukaryotic translation inhibitor 4E-BP1, which targets eIF4E critical for c-MYC translation initiation. Furthermore, knock-down of 4E-BP1 via siRNA rescued YUM70-mediated c-MYC suppression. As YUM70 is also capable of suppressing N-MYC expression, this study offers a new approach to suppress MYC protein expression through knockdown or inhibition of GRP78.

Abstract Background Local recurrence after surgery and radiochemotherapy seriously affects the prognosis of locally advanced rectal cancer (LARC) patients. Studies on molecular markers related to the radiochemotherapy sensitivity of cancers have been widely carried out, which might provide valued information for clinicians to carry out individual treatment. Aim To find potential biomarkers of tumors for predicting postoperative recurrence. Methods In this study, LARC patients undergoing surgery and concurrent radiochemotherapy were enrolled. We focused on clinicopathological factors and PTEN, SIRT1, p-4E-BP1, and pS6 protein expression assessed by immunohistochemistry in 73 rectal cancer patients with local recurrence and 76 patients without local recurrence. Results The expression of PTEN was higher, while the expression of p-4E-BP1 was lower in patients without local recurrence than in patients with local recurrence. Moreover, TNM stage, lymphatic vessel invasion (LVI), PTEN and p-4E-BP1 might be independent risk factors for local recurrence after LARC surgery combined with concurrent radiochemotherapy. Conclusions This study suggests that PTEN and p-4E-BP1 might be potential biomarkers for prognostic prediction and therapeutic targets for LARC.

Background: Local recurrence after surgery and radiochemotherapy seriously affects the prognosis of locally advanced rectal cancer (LARC) patients. Studies on molecular markers related to the radiochemotherapy sensitivity of cancers have been widely carried out, which might provide valued information for clinicians to carry out individual treatment. Aim: To find potential biomarkers of tumors for predicting postoperative recurrence. Methods: In this study, LARC patients undergoing surgery and concurrent radiochemotherapy were enrolled. We focused on clinicopathological factors and PTEN, SIRT1, p-4E-BP1, and pS6 protein expression assessed by immunohistochemistry in 73 rectal cancer patients with local recurrence and 76 patients without local recurrence. Results: The expression of PTEN was higher, while the expression of p-4E-BP1 was lower in patients without local recurrence than in patients with local recurrence. Moreover, TNM stage, lymphatic vessel invasion (LVI), PTEN and p-4E-BP1 might be independent risk factors for local recurrence after LARC surgery combined with concurrent radiochemotherapy. Conclusions: This study suggests that PTEN and p-4E-BP1 might be potential biomarkers for prognostic prediction and therapeutic targets for LARC. [ABSTRACT FROM AUTHOR]

The mechanistic target of rapamycin (mTOR) is a serine/threonine kinase that functions via its discrete binding partners to form two multiprotein complexes, mTOR complex 1 and 2 (mTORC1 and mTORC2). Rapamycin-sensitive mTORC1, which regulates protein synthesis and cell growth, is tightly controlled by PI3K/Akt and is nutrient-/growth factor-sensitive. In the brain, mTORC1 is also sensitive to neurotransmitter signaling. mTORC2, which is modulated by growth factor signaling, is associated with ribosomes and is insensitive to rapamycin. mTOR regulates stem cell and cancer stem cell characteristics. Aberrant Akt/mTOR activation is involved in multistep tumorigenesis in a variety of cancers, thereby suggesting that the inhibition of mTOR may have therapeutic potential. Rapamycin and its analogues, known as rapalogues, suppress mTOR activity through an allosteric mechanism that only suppresses mTORC1, albeit incompletely. ATP-catalytic binding site inhibitors are designed to inhibit both complexes. This review describes the regulation of mTOR and the targeting of its complexes in the treatment of cancers, such as glioblastoma, and their stem cells. [ABSTRACT FROM AUTHOR]

Novel therapeutic approaches are needed for the treatment of Ewing sarcoma tumors. We previously identified that Ewing sarcoma cell lines are sensitive to drugs that inhibit protein translation. However, translational and therapeutic approaches to inhibit protein synthesis in tumors are limited. In this work, we identified that reactive oxygen species, which are generated by a wide range of chemotherapy and other drugs, inhibit protein synthesis and reduce the level of critical proteins that support tumorigenesis in Ewing sarcoma cells. In particular, we identified that both hydrogen peroxide and auranofin, an inhibitor of thioredoxin reductase and regulator of oxidative stress and reactive oxygen species, activate the repressor of protein translation 4E-BP1 and reduce the levels of the oncogenic proteins RRM2 and PLK1 in Ewing and other sarcoma cell lines. These results provide novel insight into the mechanism of how ROS-inducing drugs target cancer cells via inhibition of protein translation and identify a mechanistic link between ROS and the DNA replication (RRM2) and cell cycle regulatory (PLK1) pathways.

Insight into the molecular mechanisms governing the development and maintenance of pluripotency is important for understanding early development and the use of stem cells in regenerative medicine. We demonstrate the selective inhibition of mTORC1 signaling is important for developing the inner cell mass (ICM) and the self-renewal of human embryonic stem cells. S6K suppressed the expression and function of pluripotency-related transcription factors (PTFs) OCT4, SOX2, and KLF4 through phosphorylation and ubiquitin proteasome-mediated protein degradation, indicating that S6K inhibition is required for pluripotency. PTFs inhibited mTOR signaling. The phosphorylation of S6 was decreased in PTF-positive cells of the ICM in embryos. Activation of mTORC1 signaling blocked ICM formation and the selective inhibition of S6K by rapamycin increased the ICM size in mouse blastocysts. Thus, selective inhibition of mTORC1 signaling supports the development and maintenance of pluripotency. [ABSTRACT FROM AUTHOR]

It is known that activation of protein synthesis and hypertrophy of muscle fibers in response to mechanical stress is realized through an anabolic mTORC1-dependent signaling pathway. However, mechanosensors through which a mechanical signal can be perceived and further transmitted to the mTORC1-dependent signaling pathway (mechanotransduction) are poorly identified. Mechanically activated (MA) ion channels are candidates for the role of such sarcolemmal mechanosensors. In this regard, the aim of this study was to investigate the potential role of MA channels (Piezo1) in the activation of the mTORC1-dependent pathway in the isolated rat soleus muscle in response to mechanical stress. Wistar rats were divided into 3 groups: (1) "Control" (animal muscles were not exposed to MA channel inhibitor or Piezo1 channel activator); (2) "Gadolinium" (animal muscles were incubated with gadolinium chloride, MA channel inhibitor), and (3) "Yoda" (animal muscles were incubated with Yoda1, Piezo1 MA channel activator). In rats from each group, m. soleus was isolated from the left limb and incubated in the appropriate solution without mechanical stress in the form of a series of stretching ("Rest"); m. soleus from the right limb was subjected to a series of stretching ("Stretch") and then incubated in the appropriate solution. Phosphorylation of mTORC1 targets (p70S6K, rpS6, and 4E-BP1) in rat m. soleus was determined by PAAG electrophoresis and immunoblotting. A series of passive stretches of isolated m. soleus led to an increase in the phosphorylation of p70S6K, its substrate rpS6, and 4E-BP1 by 38.5, 168 and 112%, respectively, compared to the muscle that was not subjected to mechanical stress. Incubation of the muscles with gadolinium completely prevented the activation of mTORC1 markers caused by a series of stretches. Incubation of m. soleus in a solution with Yoda1 resulted in a decrease in the mechano-dependent phosphorylation of p70S6K, rpS6 and 4E-BP1 compared to the muscle that was not exposed to Yoda1. Thus, the methodological approach used in this work did not reveal the participation of Piezo1 in mechano-anabolic coupling in rat m. soleus. [ABSTRACT FROM AUTHOR]

The pathogenesis of idiopathic pulmonary fibrosis (IPF) is quite similar to that of cancer pathogenesis, and several pathways appear to be involved in both disorders. The mammalian target of the rapamycin (mTOR) pathway harbors several established oncogenes and tumor suppressors. The same signaling molecules and growth factors, such as vascular endothelial growth factor (VEGF), contributing to cancer development and progression play a part in fibroblast proliferation, myofibroblast differentiation, and the production of extracellular matrix in IPF development as well. The expression of candidate genes acting upstream and downstream of mTORC1, as well as Vegf and low-density lipoprotein receptor related protein 1(Lrp1), was assessed using specific primers and quantitative polymerase chain reaction (qPCR) within the lung tissues of bleomycin (BLM)-induced IPF mouse models. Lung fibrosis was evaluated by histological examinations and hydroxyproline colorimetric assay. BLM-exposed mice developed lung injuries characterized by inflammatory manifestations and fibrotic features, along with higher levels of collagen and hydroxyproline. Gene expression analyses indicated a significant elevation of regulatory associated protein of mTOR (Raptor), Ras homolog enriched in brain (Rheb), S6 kinase 1, and Eukaryotic translation initiation factor 4E-binding protein 1 (4Ebp1), as well as a significant reduction of Vegfa, Tuberous sclerosis complex (Tsc2), and Lrp1; no changes were observed in the Tsc1 mRNA level. Our findings support the elevation of S6K1 and 4EBP1 in response to the TSC/RHEB/mTORC1 axis, which profoundly encourages the development and establishment of IPF and cancer. In addition, this study suggests a possible preventive role for VEGF-A and LRP1 in the development of IPF. [ABSTRACT FROM AUTHOR]

Although the mTOR-4E-BP1 signaling pathway is implicated in aging and aging-related disorders, the role of 4E-BP1 in regulating human stem cell homeostasis remains largely unknown. Here, we report that the expression of 4E-BP1 decreases along with the senescence of human mesenchymal stem cells (hMSCs). Genetic inactivation of 4E-BP1 in hMSCs compromises mitochondrial respiration, increases mitochondrial reactive oxygen species (ROS) production, and accelerates cellular senescence. Mechanistically, the absence of 4E-BP1 destabilizes proteins in mitochondrial respiration complexes, especially several key subunits of complex III including UQCRC2. Ectopic expression of 4E-BP1 attenuates mitochondrial abnormalities and alleviates cellular senescence in 4E-BP1-deficient hMSCs as well as in physiologically aged hMSCs. These f indings together demonstrate that 4E-BP1 functions as a geroprotector to mitigate human stem cell senescence and maintain mitochondrial homeostasis, particularly for the mitochondrial respiration complex III, thus providing a new potential target to counteract human stem cell senescence. [ABSTRACT FROM AUTHOR]

The mechanistic target of rapamycin (mTOR) is a serine/threonine kinase that functions via its discrete binding partners to form two multiprotein complexes, mTOR complex 1 and 2 (mTORC1 and mTORC2). Rapamycin-sensitive mTORC1, which regulates protein synthesis and cell growth, is tightly controlled by PI3K/Akt and is nutrient-/growth factor-sensitive. In the brain, mTORC1 is also sensitive to neurotransmitter signaling. mTORC2, which is modulated by growth factor signaling, is associated with ribosomes and is insensitive to rapamycin. mTOR regulates stem cell and cancer stem cell characteristics. Aberrant Akt/mTOR activation is involved in multistep tumorigenesis in a variety of cancers, thereby suggesting that the inhibition of mTOR may have therapeutic potential. Rapamycin and its analogues, known as rapalogues, suppress mTOR activity through an allosteric mechanism that only suppresses mTORC1, albeit incompletely. ATP-catalytic binding site inhibitors are designed to inhibit both complexes. This review describes the regulation of mTOR and the targeting of its complexes in the treatment of cancers, such as glioblastoma, and their stem cells.

The pathogenesis of idiopathic pulmonary fibrosis (IPF) is quite similar to that of cancer pathogenesis, and several pathways appear to be involved in both disorders. The mammalian target of the rapamycin (mTOR) pathway harbors several established oncogenes and tumor suppressors. The same signaling molecules and growth factors, such as vascular endothelial growth factor (VEGF), contributing to cancer development and progression play a part in fibroblast proliferation, myofibroblast differentiation, and the production of extracellular matrix in IPF development as well. The expression of candidate genes acting upstream and downstream of mTORC1, as well as Vegf and low-density lipoprotein receptor related protein 1(Lrp1), was assessed using specific primers and quantitative polymerase chain reaction (qPCR) within the lung tissues of bleomycin (BLM)-induced IPF mouse models. Lung fibrosis was evaluated by histological examinations and hydroxyproline colorimetric assay. BLM-exposed mice developed lung injuries characterized by inflammatory manifestations and fibrotic features, along with higher levels of collagen and hydroxyproline. Gene expression analyses indicated a significant elevation of regulatory associated protein of mTOR (Raptor), Ras homolog enriched in brain (Rheb), S6 kinase 1, and Eukaryotic translation initiation factor 4E-binding protein 1 (4Ebp1), as well as a significant reduction of Vegfa, Tuberous sclerosis complex (Tsc2), and Lrp1; no changes were observed in the Tsc1 mRNA level. Our findings support the elevation of S6K1 and 4EBP1 in response to the TSC/RHEB/mTORC1 axis, which profoundly encourages the development and establishment of IPF and cancer. In addition, this study suggests a possible preventive role for VEGF-A and LRP1 in the development of IPF.

The present study was designed to test the hypothesis that upregulating protein synthesis attenuates the loss of muscle mass in a model of disuse atrophy. The studies compared the effect of unilateral hindlimb immobilization in wild-type (WT) mice and double-knockout (DKO) mice lacking the translational regulators 4E-BP1 and 4E-BP2. Immobilization-induced downregulation of protein synthesis occurred in both groups of mice, but protein synthesis was higher in gastrocnemius muscle from the immobilized hindlimb of fasted DKO compared with WT mice. Surprisingly, although protein synthesis was partially elevated in DKO compared with WT mice, atrophy occurred to the same extent in both groups of animals. This may be partially due to impaired leucine-induced stimulation of protein synthesis in DKO compared with WT mice due to downregulated eukaryotic initiation factor eIF4E expression in muscle of DKO compared with WT mice. Expression of the E3 ubiquitin ligases MAFbx and MuRF-1 mRNAs and total protein ubiquitylation was upregulated in the immobilized compared with the nonimmobilized hindlimb of both WT and DKO mice, with little difference in the magnitude of the upregulation between genotypes. Analysis of newly synthesized proteins revealed downregulation of several glycolytic enzymes in the gastrocnemius of DKO mice compared with WT mice, as well as in the immobilized compared with the nonimmobilized hindlimb. Overall, the results suggest that the elevated rate of protein synthesis during hindlimb immobilization in fasted DKO mice is insufficient to prevent disuse-induced muscle atrophy, probably due to induction of compensatory mechanisms including downregulation of eIF4E expression. [ABSTRACT FROM AUTHOR]

• The interaction between mmu_circ_0005373 and 4E-BP1 protein may be inhibited to promote lung fibrosis. • The low expression of mmu_circ_0005373 activates the mTOR/4E-BP1 pathway to promote lung fibrosis. • Hsa_circ_0136255 is reduced in SSc peripheral blood mononuclear cells, and predicted to interact with 4E-BP1 protein. • Hsa_circ_0136255/hsa-miR-330-3p/TNFAIP3 ceRNA network had biological significance in patients with SSc. • Hsa_circ_0136255, hsa-miR-330-3p, and TNFAIP3 may be used as biomarkers and therapeutic targets for SSc. The pathogenesis of SSc pulmonary fibrosis is complex and prognosis is poor. In order to find biomarkers to provide assistance in the diagnosis and treatment of systemic sclerosis (SSc), this study explored the role of SSc-related differentially expressed circRNAs in the fibrosis process. This study explored whether circular RNA (circRNA) mediated the mTOR signaling pathway by interacting with the eukaryotic translation initiation factor eIF4E-binding protein 1 (4E-BP1), participated in a competing endogenous RNA (ceRNA) network, and regulated the mechanism of pulmonary fibrosis in systemic sclerosis (SSc). The results showed that the expression of mmu_circ_0005373 was reduced, and mmu_circ_0005373 may regulate the mTOR signaling pathway by inhibiting the interacting with 4E-BP1 protein in the lung of SSc mice, and promote fibrosis in SSc. Hsa_circ_0136255, which is homologous to mmu_circ_0005373, is also reduced in SSc peripheral blood mononuclear cells, and predicted to interact with 4E-BP1 protein. Hsa_circ_0136255/hsa-miR-330-3p/TNFAIP3 ceRNA network had biological significance in SSc, and correlated with clinical data, including high-resolution CT, average expiratory flow at 25% vital capacity, neutrophil count, lymphocyte percentage, standard deviation of red blood cell distribution width, coefficient of variation of red blood cell distribution width, platelet distribution width, glutamic transaminase, γ-glutamyl transpeptidase, lymphocyte percentage, basophils percentage, red blood cell, plateletcrit, cholinesterase, and mean corpuscular hemoglobin concentration. Hsa_circ_0136255, hsa-miR-330-3p, and TNFAIP3 may be used as biomarkers for clinical diagnosis and treatment of SSc. [ABSTRACT FROM AUTHOR]

The 78-kDa glucose regulated protein (GRP78) commonly upregulated in a wide variety of tumors is an important prognostic marker and a promising target for suppressing tumorigenesis and treatment resistance. While GRP78 is well established as a major endoplasmic reticulum (ER) chaperone with anti-apoptotic properties and a master regulator of the unfolded protein response, its new role as a regulator of oncoprotein expression is just emerging. MYC is dysregulated in about 70 % of human cancers and is the most commonly activated oncoprotein. However, despite recent advances, therapeutic targeting of MYC remains challenging. Here we identify GRP78 as a new target for suppression of MYC expression. Using multiple MYC-dependent cancer models including head and neck squamous cell carcinoma and their cisplatin-resistant clones, breast and pancreatic adenocarcinoma, our studies revealed that GRP78 knockdown by siRNA or inhibition of its activity by small molecule inhibitors (YUM70 or HA15) reduced c-MYC expression, leading to onset of apoptosis and loss of cell viability. This was observed in 2D cell culture, 3D spheroid and in xenograft models. Mechanistically, we determined that the suppression of c-MYC is at the post-transcriptional level and that YUM70 and HA15 treatment potently upregulated the eukaryotic translation inhibitor 4E-BP1, which targets eIF4E critical for c-MYC translation initiation. Furthermore, knock-down of 4E-BP1 via siRNA rescued YUM70-mediated c-MYC suppression. As YUM70 is also capable of suppressing N-MYC expression, this study offers a new approach to suppress MYC protein expression through knockdown or inhibition of GRP78. [ABSTRACT FROM AUTHOR]

Manganese (Mn) exerts serious neurotoxic effects, among which, the disruption of the glutamate/glutamine (Glu/Gln) cycle, leads to an excitotoxic insult. The molecular mechanisms mediating Mn-induced neurotoxicity, have not yet been fully understood. Glu, the major excitatory neurotransmitter in the nervous system, activates a variety of signal transduction cascades involved in protein synthesis regulation. Although protein translation is an exquisitely regulated process, translational dysregulation has been observed in many neurodegenerative disorders. Hence, we investigated the effect of a short-term Mn exposure in signaling pathways critically involved in protein synthesis, such as the phosphatidylinositol 3 kinase (PI3K)/protein kinase B (Akt) cascade. To this end, we used the well-characterized chick cerebellar Bergmann glial cells (BGC) primary culture. Confluent BGC monolayers were exposed to different MnCl2 concentrations (50–500 μM) for different time periods. The phosphorylation patterns of Akt, the eukaryotic translation initiation factor 4E-binding protein 1 (4E-BP1) as well as the adenosine monophosphate-dependent protein kinase (AMPK) were measured. A time and dose-dependent increase in the phosphorylation status of these proteins was found, thus the involvement of a Ca2+/ PI3K/mTOR pathway could be demonstrated. Accordingly, a modulation of [35S]-methionine incorporation into newly synthesized polypeptides was found upon Mn acute exposure. These results demonstrate that Mn exerts triggers a change in the protein repertoire of glia cells that support their involvement in Mn neurotoxicity. Summary Statement: We demonstrate herein that short-term exposure of radial glia cells to Manganese, a neurotoxic metal, induces an effect on protein synthesis, altering the protein repertoire of these cells. [ABSTRACT FROM AUTHOR]

The polyamines putrescine, spermidine and spermine are nutrient-like polycationic molecules involved in metabolic processes and signaling pathways linked to cell growth and cancer. One important pathway is the PI3K/Akt pathway where studies have shown that polyamines mediate downstream growth effects. Downstream of PI3K/Akt is the mTOR signaling pathway, a nutrient-sensing pathway that regulate translation initiation through 4EBP1 and p70S6K phosphorylation and, along with the PI3K/Akt, is frequently dysregulated in breast cancer. In this study, we investigated the effect of intracellular polyamine modulation on mTORC1 downstream protein and general translation state in two breast cancer cell lines, MCF-7 and MDA-MB-231. The effect of mTORC1 pathway inhibition on the growth and intracellular polyamines was also measured. Results showed that polyamine modulation alters 4EBP1 and p70S6K phosphorylation and translation initiation in the breast cancer cells. mTOR siRNA gene knockdown also inhibited cell growth and decreased putrescine and spermidine content. Co-treatment of inhibitors of polyamine biosynthesis and mTORC1 pathway induced greater cytotoxicity and translation inhibition in the breast cancer cells. Taken together, these data suggest that polyamines promote cell growth in part through interaction with mTOR pathway. Similarly intracellular polyamine content appears to be linked to mTOR pathway regulation. Finally, dual inhibition of polyamine and mTOR pathways may provide therapeutic benefits in some breast cancers. [ABSTRACT FROM AUTHOR]

Tyrosine kinase inhibitors (TKIs) such as imatinib, nilotinib, dasatinib, and ponatinib have significantly improved the life expectancy of Philadelphia chromosome-positive (Ph+) acute lymphocytic leukemia (ALL) patients; however, resistance to TKIs remains a major clinical challenge. Point mutations in the tyrosine kinase domain (TKD) of BCR-ABL1 have emerged as the predominant cause of acquired resistance. In approximately 30% of patients, the mechanism of resistance to TKIs remains elusive. This study aimed to investigate mechanisms of nonmutational resistance in Ph+ ALL. Here we report the development of a nonmutational resistance cell line SupB15-RT; conferring resistance to approved ABL kinase inhibitors (AKIs) and allosteric inhibitors GNF-2, ABL001, and crizotinib, except for dasatinib (IC90 50nM), a multitarget kinase inhibitor. We found that the AKT/mTOR pathway is activated in these cells and their proliferation inhibited by Torin-1 with an IC50 of 24.7 nM. These observations were confirmed using 3 different ALL patient-derived long term cultures (PDLTCs): (1) HP (BCR-ABL1 negative), (2) PH (BCR-ABL1 positive and responsive to TKIs) and (3) BV (BCR-ABL1 positive and nonmutational resistant to TKIs). Furthermore, Torin-1 and NVP-BEZ235 induced apoptosis in PH and BV cells but not in HP cells.Our experiments provide evidence of the involvement of AKT/mTOR pathway in the evolution of nonmutational resistance in Ph+ ALL which will assist in developing novel targeted therapy for Ph+ ALL patients with BCR-ABL1 independent nonmutational resistance.

Aistė Savukaitytė,1 Greta Gudoitytė,1 Agnė Bartnykaitė,1 Rasa Ugenskienė,1,2 Elona Juozaitytė3 1Oncology Research Laboratory, Institute of Oncology, Lithuanian University of Health Sciences, Kaunas, Lithuania; 2Institute of Biology Systems and Genetic Research, Lithuanian University of Health Sciences, Kaunas, Lithuania; 3Department of Oncology and Hematology, Hospital of Lithuanian University of Health Sciences Kaunas Clinics, Kaunas, LithuaniaCorrespondence: Aistė Savukaitytė Email aiste.savukaityte@lsmuni.ltBackground: Mutations within genes encoding components of the PI3K/AKT/mTOR (phosphoinositide 3-kinase/protein kinase B/mechanistic target of rapamycin) signaling axis frequently activate the pathway in breast cancer, making it an attractive therapeutic target. Inhibition of mTORC1 (mechanistic target of rapamycin complex 1) activity upon aspirin treatment has been reported in breast cancer cells harboring PI3KCA (phosphatidylinositol-4,5-bisphosphate 3-kinase catalytic subunit alpha) mutation and is considered to account for anticancer action.Methods: MDA-MB-468 (harbors mutated PTEN (phosphatase and TENsin homolog)), MCF-7 (PI3KCA-mutated), MDA-MB-231 (no PI3K pathway mutations) cancer cell lines and MCF10A non-cancerous breast epithelial cells were employed for the assessment of modulation of mTORC1 signaling by aspirin. Targeted amplicon-based next-generation sequencing using the Ion Torrent technology was carried out to determine gene expression changes following drug treatment. Western blot was performed to analyze the expression and phosphorylation of proteins. Knockdown by siRNA approach was applied to assess the role of REDD1/DDIT4 (DNA damage-inducible transcript 4) in mTORC1 inhibition by aspirin.Results: We show a decline in phosphorylation of mTORC1 downstream substrate 4E-BP1 (eukaryotic translation initiation factor 4E-binding protein 1) in response to treatment with aspirin and its metabolite salicylic acid in MDA-MB-468, MCF-7, MDA-MB-231, and MCF10A cell lines. We further demonstrate a novel molecular response to aspirin in breast cancer cells. Specifically, we found that aspirin and salicylic acid increase the expression of REDD1 protein, that is known for its suppressive function towards mTORC1. Unexpectedly, we observed that siRNA knockdown of REDD1 expression facilitated aspirin-mediated suppression of mTORC1 downstream substrate 4E-BP1 phosphorylation in the MDA-MB-468 cell line. REDD1 downregulation slightly encouraged reduction in 4E-BP1 phosphorylation by aspirin in MCF-7 cells but did not elicit a reproducible effect in the MDA-MB-231 cell line. siRNA knockdown of REDD1 did not affect the expression of phosphorylated form of 4E-BP1 following aspirin treatment in MCF10A non-cancerous breast epithelial cells.Conclusion: The current findings suggest that REDD1 downregulation might improve the anticancer activity of aspirin in a subset of breast tumors.Keywords: aspirin, breast cancer, REDD1, mTORC1 signaling, 4E-BP1

Prothioconazole (PTC) is a new broad-spectrum triazole antibacterial agent that is being widely used in agriculture. PTC has been linked to a number of reproductive outcomes including embryo implantation disorder; however, the exact mechanism underlying this relationship has yet to be determined. Proper trophoblast proliferation and migration is a prerequisite for successful embryo implantation. To elucidate the underlying molecular perturbations, we detect the effect of PTC on extravillous trophoblast cells proliferation and migration, and investigate its potential mechanisms. Exposure to different concentrations of PTC (0–500 μM) significantly inhibited the cell viability and migration ability (5 μM PTC exposure), and also caused the cell cycle arrest at the lowest dose (1 μM PTC exposure). Transcriptome analysis revealed that PTC exposure disturbed multiple biological processes including cell cycle and apoptosis, consistent with cell phenotype. Specifically, eukaryotic translation initiation factor 4E binding protein 1 (EIF4EBP1, 4E-BP1) was identified as up-regulated in PTC exposure group and knockdown of EIF4EBP1, and attenuated the G1 phase arrest induced by PTC exposure. In summary, our data demonstrated that 4E-BP1 participated in PTC-induced cell cycle arrest in extravillous trophoblast cells by regulating cyclin D1. These findings shed light on the potential adverse effect of PTC exposure on the embryo implantation. [ABSTRACT FROM AUTHOR]

Weimin Dong,1 Yong Luo,2 Guian Zhang,1 Hui Zhang,1 Yuxiang Liang,1 Yangjia Zhuo,1 Yingke Liang,1 Fen Zou,1 Weide Zhong1,3,4 1Department of Urology, Guangdong Key Laboratory of Clinical Molecular Medicine and Diagnostics, Guangzhou First People’s Hospital, School of Medicine, South China University of Technology, Guangzhou, Guangdong 510180, People’s Republic of China; 2Department of Urology, The Second People’s Hospital of Foshan, Affiliated Foshan Hospital of Southern Medical University, Foshan 528000, People’s Republic of China; 3Department of Urology, Huadu District People’s Hospital, Southern Medical University, Guangzhou 510800, People’s Republic of China; 4Urology Key Laboratory of Guangdong Province, The First Affiliated Hospital of Guangzhou Medical University, Guangzhou Medical University, Guangzhou 510230, People’s Republic of ChinaCorrespondence: Weide Zhong; Fen ZouDepartment of Urology, Guangdong Key Laboratory of Clinical Molecular Medicine and Diagnostics, Guangzhou First People’s Hospital, School of Medicine, South China University of Technology, Guangzhou 510180, People’s Republic of ChinaTel +86 20-81048312Fax +86 20-8337332Email zhongwd2009@live.cn; zoufen2008@126.comIntroduction: Although carbon nanospheres (CNPs) are promising nanomaterials in cancer treatment, how they affect prostate cancer (PCa) remains unclear.Methods: In this study, scanning electron microscopy (SEM), X-ray diffraction (XRD), and Raman spectroscopy were used to confirm the successful synthesis of CNPs. CCK-8, flow cytometry, Transwell, wound healing, Western blot and immunohistochemistry (IHC) assays were performed to evaluate the antitumor effect of CNPs toward the two kinds of prostate cancer cell lines PC3 and DU145.Results: Our results showed that CNPs inhibited cell growth, invasion, and migration and induced apoptosis and autophagy in PCa cells. Multifactor detection of a single Akt phosphorylation pathway and Western blot results suggested the suppression of 4E-BP1 in PCa cells after incubation with CNPs. The results from animal experiments also suggested the antitumor effect of CNPs and reduced 4E-BP1 expression in PCa tissue samples from BALB/c nude mice administered a local subcutaneous injection of CNPs.Keywords: carbon nanosphere, prostate cancer, antitumor, 4E-BP1

Signal transducer and activator of transcription 3 (STAT3) regulates cell growth, cell survival, angiogenesis, metastasis of cancer cells, and cancer immune evasion by regulating gene expression as a transcription factor. However, the effect of STAT3 on translation is almost unknown. We demonstrated that STAT3 acts as a trans‐acting factor for MLST8 gene expression and the protein level of mLST8, a core component of mechanistic target of rapamycin complex 1 and 2 (mTORC1/2), positively regulates the mTORC1/2 downstream pathways. Suppression of STAT3 by siRNA attenuated 4E‐BP1 phosphorylation, cap‐dependent translation, and cell proliferation in a variety of cancer cells. In HCT116 cells, STAT3 knockdown‐induced decreases in 4E‐BP1 and AKT phosphorylation levels were further attenuated by MLST8 knockdown or recovered by mLST8 overexpression. STAT3 knockdown‐induced G2/M phase arrest was partially restored by co‐knockdown of 4EBP1, and the attenuation of cell proliferation was enhanced by the expression of an mTORC1‐mediated phosphorylation‐defective mutant of 4E‐BP1. ChIP and promoter mapping using a luciferase reporter assay showed that the −951 to −894 bp of MLST8 promoter seems to include STAT3‐binding site. Overall, these results suggest that STAT3‐driven MLST8 gene expression regulates cap‐dependent translation through 4E‐BP1 phosphorylation in cancer cells.

Eukaryotic translation initiation factor 4E-binding protein 1 (4E-BP1) is a translation repressor downstream of mTORC1 pathway, and plays a pivotal role in the adaptive immune response. However, whether 4E-BP1 participates in the regulation of adaptive immunity of early vertebrates is still unclear. In present study, using Nile tilapia Oreochromis niloticus as a model, we investigated the regulatory roles of 4E-BP1 (On-4E-BP1) on lymphocyte-mediated adaptive immune response of fish species. On-4E-BP1 is highly conserved compared with the homologues from other vertebrates, and it is widely distributed in the immune-related tissues of Nile tilapia with the highest expression level in the gill. Once the animals were infected by Aeromonas hydrophila, transcription level of On-4E-BP1 in spleen lymphocytes was significantly induced on day 5 after infection. Meanwhile, phosphorylation of On-4E-BP1 in lymphocytes was also enhanced during the primary adaptive immune response, suggesting that 4E-BP1 is involved in the adaptive immunity of Nile tilapia. Furthermore, when lymphocytes were activated by agonist PMA or T cell specific mitogen PHA in vitro, phosphorylation of On-4E-BP1 was obviously up-regulated. More importantly, once mTORC1/4E-BP1 activity was blocked by specific inhibitor, the inducible expression of T cell activation markers IFN-γ and CD122 was severely impaired during PHA-induced T cell activation, suggesting this signaling regulates T lymphocyte activation of Nile tilapia. In addition, blockade of mTORC1/4E-BP1 axis also resulted in a crippled proliferation of lymphocyte during the primary response of anti-bacterial adaptive immunity. Collectively, we found that mTORC1/4E-BP1 signaling participates in the adaptive immune response by regulating lymphocyte activation and proliferation in Nile tilapia. This study enriches our current knowledge on teleost adaptive immunity, and provides novel perspective to understand the evolution of adaptive immune system.

Tyrosine kinase inhibitors (TKIs) such as imatinib, nilotinib, dasatinib, and ponatinib have significantly improved the life expectancy of Philadelphia chromosome-positive (Ph+) acute lymphocytic leukemia (ALL) patients; however, resistance to TKIs remains a major clinical challenge. Point mutations in the tyrosine kinase domain (TKD) of BCR-ABL1 have emerged as the predominant cause of acquired resistance. In approximately 30% of patients, the mechanism of resistance to TKIs remains elusive. This study aimed to investigate mechanisms of nonmutational resistance in Ph+ ALL. Here we report the development of a nonmutational resistance cell line SupB15-RT; conferring resistance to approved ABL kinase inhibitors (AKIs) and allosteric inhibitors GNF-2, ABL001, and crizotinib, except for dasatinib (IC90 50nM), a multitarget kinase inhibitor. We found that the AKT/mTOR pathway is activated in these cells and their proliferation inhibited by Torin-1 with an IC50 of 24.7 nM. These observations were confirmed using 3 different ALL patient-derived long term cultures (PDLTCs): (1) HP (BCR-ABL1 negative), (2) PH (BCR-ABL1 positive and responsive to TKIs) and (3) BV (BCR-ABL1 positive and nonmutational resistant to TKIs). Furthermore, Torin-1 and NVP-BEZ235 induced apoptosis in PH and BV cells but not in HP cells. Our experiments provide evidence of the involvement of AKT/mTOR pathway in the evolution of nonmutational resistance in Ph+ ALL which will assist in developing novel targeted therapy for Ph+ ALL patients with BCR-ABL1 independent nonmutational resistance. [ABSTRACT FROM AUTHOR]

The polyamines putrescine, spermidine and spermine are nutrient-like polycationic molecules involved in metabolic processes and signaling pathways linked to cell growth and cancer. One important pathway is the PI3K/Akt pathway where studies have shown that polyamines mediate downstream growth effects. Downstream of PI3K/Akt is the mTOR signaling pathway, a nutrient-sensing pathway that regulate translation initiation through 4EBP1 and p70S6K phosphorylation and, along with the PI3K/Akt, is frequently dysregulated in breast cancer. In this study, we investigated the effect of intracellular polyamine modulation on mTORC1 downstream protein and general translation state in two breast cancer cell lines, MCF-7 and MDA-MB-231. The effect of mTORC1 pathway inhibition on the growth and intracellular polyamines was also measured. Results showed that polyamine modulation alters 4EBP1 and p70S6K phosphorylation and translation initiation in the breast cancer cells. mTOR siRNA gene knockdown also inhibited cell growth and decreased putrescine and spermidine content. Co-treatment of inhibitors of polyamine biosynthesis and mTORC1 pathway induced greater cytotoxicity and translation inhibition in the breast cancer cells. Taken together, these data suggest that polyamines promote cell growth in part through interaction with mTOR pathway. Similarly intracellular polyamine content appears to be linked to mTOR pathway regulation. Finally, dual inhibition of polyamine and mTOR pathways may provide therapeutic benefits in some breast cancers.

Sigma-1 receptors (Sig-1Rs) are endoplasmic reticulum (ER) chaperones implicated in neuropathic pain. Here we examine if the Sig-1R may relate to neuropathic pain at the level of dorsal root ganglia (DRG). We focus on the neuronal excitability of DRG in a "spare nerve injury" (SNI) model of neuropathic pain in rats and find that Sig-1Rs likely contribute to the genesis of DRG neuronal excitability by decreasing the protein level of voltage-gated Cav2.2 as a translational inhibitor of mRNA. Specifically, during SNI, Sig-1Rs translocate from ER to the nuclear envelope via a trafficking protein Sec61β. At the nucleus, the Sig-1R interacts with cFos and binds to the promoter of 4E-BP1, leading to an upregulation of 4E-BP1 that binds and prevents eIF4E from initiating the mRNA translation for Cav2.2. Interestingly, in Sig-1R knockout HEK cells, Cav2.2 is upregulated. In accordance with those findings, we find that intra-DRG injection of Sig-1R agonist (+)pentazocine increases frequency of action potentials via regulation of voltage-gated Ca2+ channels. Conversely, intra-DRG injection of Sig-1R antagonist BD1047 attenuates neuropathic pain. Hence, we discover that the Sig-1R chaperone causes neuropathic pain indirectly as a translational inhibitor. [ABSTRACT FROM AUTHOR]

Abstract Background Eukaryotic Initiation Factor 4E-Binding Protein (EIF4EBP1, 4EBP1) is overexpressed in many human cancers including breast cancer, yet the role of 4EBP1 in breast cancer remains understudied. Despite the known role of 4EBP1 as a negative regulator of cap-dependent protein translation, 4EBP1 is predicted to be an essential driving oncogene in many cancer cell lines in vitro, and can act as a driver of cancer cell proliferation. EIF4EBP1 is located within the 8p11-p12 genomic locus, which is frequently amplified in breast cancer and is known to predict poor prognosis and resistance to endocrine therapy. Methods Here we evaluated the effect of 4EBP1 targeting using shRNA knock-down of expression of 4EBP1, as well as response to the mTORC targeted drug everolimus in cell lines representing different breast cancer subtypes, including breast cancer cells with the 8p11-p12 amplicon, to better define a context and mechanism for oncogenic 4EBP1. Results Using a genome-scale shRNA screen on the SUM panel of breast cancer cell lines, we found 4EBP1 to be a strong hit in the 8p11 amplified SUM-44 cells, which have amplification and overexpression of 4EBP1. We then found that knock-down of 4EBP1 resulted in dramatic reductions in cell proliferation in 8p11 amplified breast cancer cells as well as in other luminal breast cancer cell lines, but had little or no effect on the proliferation of immortalized but non-tumorigenic human mammary epithelial cells. Kaplan-Meier analysis of EIF4EBP1 expression in breast cancer patients demonstrated that overexpression of this gene was associated with reduced relapse free patient survival across all breast tumor subtypes. Conclusions These results are consistent with an oncogenic role of 4EBP1 in luminal breast cancer and suggests a role for this protein in cell proliferation distinct from its more well-known role as a regulator of cap-dependent translation.

Feng Li,1,2,* Qingshui Wang,3,* Xiaoxue Xiong,3 Chenyi Wang,3 Xiaohua Liu,4 Ziqiang Liao,3 Ke Li,3 Bifeng Xie,5 Yao Lin3 1Department of Pathology, Provincial Clinical Medical College, Fujian Medical University, Fuzhou, Fujian Province, People’s Republic of China; 2Department of Pathology, Fujian Provincial Hospital, Fuzhou, Fujian Province, People’s Republic of China; 3Provincial University Key Laboratory of Cellular Stress Response and Metabolic Regulation, College of Life Sciences, Fujian Normal University, Fuzhou, Fujian Province, People’s Republic of China; 4Department of Obstetrics, Anxi County Hospital, Anxi, Fujian Province, People’s Republic of China; 5College of Life Sciences, Fujian Normal University, Fuzhou, Fujian Province, People’s Republic of China *These authors contributed equally to this work Background: Eukaryotic translation initiation factor 4E (eIF4E) is a key regulator of protein synthesis. Changes in eIF4E activity disproportionally affect the translation of a subset of oncogenic mRNAs in some cancers. Materials and methods: We have assessed the expression levels of vascular endothelial growth factor C (VEGFC), eIF4E, eIF4E-binding proteins (4E-BPs) and phospho-4E-BP1 in clear cell renal carcinoma (ccRCC; n=101) using immunohistochemistry and analyzed the relevant mRNA levels and survival using online databases. Results: The protein levels of VEGFC, an eIF4E-regulated gene, were upregulated in ccRCC tissues compared with adjacent normal renal tissues, indicating an enhanced eIF4E activity in ccRCC. The expression of eIF4E had no significant changes in ccRCC tissues. However, 4E-BP1 and phospho-4E-BP1 were found to be overexpressed in ccRCC tissues (P

Decline of protein quality control in neurons contributes to age-related neurodegenerative disorders caused by misfolded proteins. 4EBP1 is a key node in the regulation of protein synthesis, as activated 4E-BP1 represses global protein translation. Overexpression of 4E-BP1 mediates the benefits of dietary restriction and can counter metabolic stress, and 4E-BP1 disinhibition on mTORC1 repression may be neuroprotective; however, whether 4E-BP1 overexpression is neuroprotective in mammalian neurons is yet to be fully explored. To address this question, we generated 4E-BP1-overexpressing transgenic mice and confirmed marked reductions in protein translation in 4E-BP1-overexpressing primary neurons. After documenting that 4E-BP1-overexpressing neurons are resistant to proteotoxic stress elicited by brefeldin A treatment, we exposed primary neurons to three different Parkinson's disease (PD)-linked toxins (rotenone, maneb, or paraquat) and documented significant protection in neurons from newborn male and female 4E-BP1-OE transgenic mice. We observed 4E-BP1-dependent upregulation of genes encoding proteins that comprise the mitochondrial unfolded protein response, and noted 4E-BP1 overexpression required activation of the mitochondrial unfolded protein response for neuroprotection against rotenone toxicity. We also tested whether 4E-BP1 could prevent a-synuclein neurotoxicity by treating 4E-BP1- overexpressing primary neurons with a-synuclein preformed fibrils, and we observed marked reductions in a-synuclein aggregation and neurotoxicity, thus validating that 4E-BP1 is a powerful suppressor of PD-linked pathogenic insults. Our results indicate that increasing 4E-BP1 expression or enhancing 4E-BP1 activation can robustly induce the mitochondrial unfolded protein response and thus could be an appealing strategy for treating a variety of neurodegenerative diseases, including especially PD. [ABSTRACT FROM AUTHOR]

Eukaryotic ribosome and cap-dependent translation are attractive targets in the antitumor, antiviral, anti-inflammatory, and antiparasitic therapies. Currently, a broad array of small-molecule drugs is known that specifically inhibit protein synthesis in eukaryotic cells. Many of them are well-studied ribosome-targeting antibiotics that block translocation, the peptidyl transferase center or the polypeptide exit tunnel, modulate the binding of translation machinery components to the ribosome, and induce miscoding, premature termination or stop codon readthrough. Such inhibitors are widely used as anticancer, anthelmintic and antifungal agents in medicine, as well as fungicides in agriculture. Chemicals that affect the accuracy of stop codon recognition are promising drugs for the nonsense suppression therapy of hereditary diseases and restoration of tumor suppressor function in cancer cells. Other compounds inhibit aminoacyl-tRNA synthetases, translation factors, and components of translation-associated signaling pathways, including mTOR kinase. Some of them have antidepressant, immunosuppressive and geroprotective properties. Translation inhibitors are also used in research for gene expression analysis by ribosome profiling, as well as in cell culture techniques. In this article, we review well-studied and less known inhibitors of eukaryotic protein synthesis (with the exception of mitochondrial and plastid translation) classified by their targets and briefly describe the action mechanisms of these compounds. We also present a continuously updated database (http://eupsic.belozersky.msu.ru/) that currently contains information on 370 inhibitors of eukaryotic protein synthesis. [ABSTRACT FROM AUTHOR]

Signal transducer and activator of transcription 3 (STAT3) regulates cell growth, cell survival, angiogenesis, metastasis of cancer cells, and cancer immune evasion by regulating gene expression as a transcription factor. However, the effect of STAT3 on translation is almost unknown. We demonstrated that STAT3 acts as a trans‐acting factor for MLST8 gene expression and the protein level of mLST8, a core component of mechanistic target of rapamycin complex 1 and 2 (mTORC1/2), positively regulates the mTORC1/2 downstream pathways. Suppression of STAT3 by siRNA attenuated 4E‐BP1 phosphorylation, cap‐dependent translation, and cell proliferation in a variety of cancer cells. In HCT116 cells, STAT3 knockdown‐induced decreases in 4E‐BP1 and AKT phosphorylation levels were further attenuated by MLST8 knockdown or recovered by mLST8 overexpression. STAT3 knockdown‐induced G2/M phase arrest was partially restored by co‐knockdown of 4EBP1, and the attenuation of cell proliferation was enhanced by the expression of an mTORC1‐mediated phosphorylation‐defective mutant of 4E‐BP1. ChIP and promoter mapping using a luciferase reporter assay showed that the −951 to −894 bp of MLST8 promoter seems to include STAT3‐binding site. Overall, these results suggest that STAT3‐driven MLST8 gene expression regulates cap‐dependent translation through 4E‐BP1 phosphorylation in cancer cells. [ABSTRACT FROM AUTHOR]

Eukaryotic initiation factor 4E (eIF4E)‐binding protein 1 (4E‐BP1) binds eIF4E and represses protein translation by displacing it from the mRNA. In this study, we investigated the influence of 4E‐BP1 translational apparatus on the regulation of transforming growth factor‐beta 1 (TGF‐β1)‐induced anabolic signaling in chondrocytes. The level of 4E‐BP1 expression was significantly higher in human OA cartilage than normal cartilage. TGF‐β1 increased total protein synthesis, including aggrecan (ACAN) and collagen type II (Col II), together with activation of Akt/mTOR signaling pathway. mTOR silencing significantly suppressed ACAN and Col II expressions through decreasing TGF‐β1‐induced phosphorylation of 4E‐BP1. On the contrary, 4E‐BP1 knockdown promoted total protein synthesis but suppressed Col II and ACAN expressions with decreased expression of Smad2/3 and Smad4 and increased expression of inhibitory Smad6 and Smad7. TGF‐β1 suppressed the interaction of 4E‐BP1 and eIF4E and subsequently enhanced protein synthesis. Furthermore, 4E‐BP1 regulated translation levels of inhibitory Smads, which decreased the accumulation of nuclear Smad2/3 complexes on the promoter of ACAN and Col II genes, subsequently affecting transcription of ACAN and Col II. These results demonstrated that TGF‐β1‐modulated phosphorylation of 4EBP1 plays a role in the expression of Col II and ACAN through differential alteration of Smad signaling pathway. [ABSTRACT FROM AUTHOR]

Translational regulation has invited considerable interest consequent of its circumstantial dysregulation during cancer genesis. eIF4E (Eukaryotic Initiation Factor 4E) has been identified as an important factor involved in tumor progression by way of instrumenting the convergence of oncogenic signals for up-regulation of Cap-dependent translation. In the backdrop of dramatic eIF4E over-expression in a large population of human cancers, we suggest that the tumorigenic property of eIF4E is strictly attributed to its phosphorylation state. We provide evidence that while phosphorylated eIF4E fails to be sequestered by 4E-BP1, its dephosphorylated form shows overwhelming binding with 4E-BP1 without any consideration to the state of 4E-BP1 phosphorylation to suggest that eIF4E-4EBP1 binding is governed by eIF4E phosphorylation instead of 4E-BP1. We also show that eIF4E engages in Cap-assembly formation preferably in a phosphorylation-dependent manner to suggest that eIF4E phosphorylation rather than 4E-BP1 regulates its availability for Cap-assembly. • Altering the eIF4E/4EBP1 ratio in cells could determine the cellular response of 4E-BP1 to rapamycin. • 4E-BP1 abundance confines the phosphorylation of eIF4E. • The binding between eIF4E and 4EBP1 is regulated at the level of eIF4E whereby the dynamics of eIF4E phosphorylation state dictate the binding. • Loss of 4E-BP1 phosphorylation in response to rapamycin may result only as a consequence of its binding with eIF4E. • Phosphorylation of eIF4E at Ser209 strongly inhibits its binding to 4E-BP1 but increases its ability to bind 5′ cap of eukaryotic mRNA indicating that the two events are discrete. [ABSTRACT FROM AUTHOR]

Protein deprivation has been shown to induce fatty liver in humans and animals, but the molecular mechanisms underlying such induction are largely unknown. Our previous studies have shown that a low-protein diet increases eukaryotic translation initiation factor 4E-binding protein 1 (4E-BP1) protein and triglyceride (TG) levels in rat liver. 4E-BP1 is known to repress translation by binding to eIF4E. There is also evidence indicating that 4E-BP1 regulates lipid metabolism. Here, we examined the role of 4E-BP1 on TG accumulation in the livers of rats under protein deprivation. The low-protein diet rapidly increased the hepatic 4E-BP1 mRNA level within 1 day, followed by the induction of hepatic TG accumulation. The knockdown of hepatic 4E-BP1 attenuated the TG accumulation in rat liver induced by the low-protein diet. 4E-BP1 knockdown also increased the protein level of carnitine palmitoyltransferase 1A (CPT1A), a regulator of fatty acid oxidation, in the liver of rats fed a low-protein diet. These results indicate that a low-protein diet increases the amount of 4E-BP1, leading to TG accumulation in rat liver. We thus conclude that 4E-BP1 plays an important role in inducing hepatic steatosis under protein deprivation. [ABSTRACT FROM AUTHOR]

Phosphorylation of translational repressor eukaryotic translation initiation factor 4E (eIF4E)‐binding protein 1 (4E‐BP1) controls the initiation of cap‐dependent translation, a type of protein synthesis that is frequently upregulated in human diseases such as cancer. Because of its critical cellular function, it is not surprising that multiple kinases can post‐translationally modify 4E‐BP1 to drive aberrant cap‐dependent translation. We recently reported a site‐selective chemoproteomic method for uncovering kinase–substrate interactions, and using this approach, we discovered the cyclin‐dependent kinase (CDK)4 as a new 4E‐BP1 kinase. Herein, we describe our extension of this work and reveal the role of CDK4 in modulating 4E‐BP1 activity in the transition from mitosis to G1, thereby demonstrating a novel role for this kinase in cell cycle regulation. [ABSTRACT FROM AUTHOR]

Caffeine is a popular psychostimulant, which is frequently consumed with ethanol. However, the effects of caffeine on neuronal cells constantly exposed to ethanol have not been investigated. Apoptosis and oxidative stress occurring in ethanol-induced neurotoxicity were previously associated with decreased phosphorylation of the mTOR/p70S6K/4E-BP1 signaling proteins. Evidence also suggested that caffeine inhibits the mTOR pathway. In this study, human SH-SY5Y neuroblastoma cells were exposed to caffeine after pretreatment for 24 hours with ethanol. Results indicated that both ethanol and caffeine caused neuronal cell death in a dose- and time-dependent manner. Exposure to 20-mM caffeine for 24 hours magnified reduced cell viability and enhanced apoptotic cell death induced by 200 mM of ethanol pretreatment. The phosphorylation of mTOR, p70S6K, and 4E-BP1 markedly decreased in cells exposed to caffeine after ethanol pretreatment, associated with a decrease of the mitochondrial membrane potential (ΔΨm). These findings suggested that caffeine treatment after neuronal cells were exposed to ethanol resulted in marked cell damages, mediated through enhanced inhibition of mTOR/p70S6K/4E-BP1 signaling leading to impaired ΔΨm and, eventually, apoptotic cell death. [ABSTRACT FROM AUTHOR]

Cellular signals that influence Cap-dependent translation have assumed significant relevance in the backdrop of their enforced dysregulation during oncogenesis. Eukaryotic initiation factor 4E(eIF4E), the mRNA cap-binding protein, has emerged as a key player to facilitate tumor progression through upregulated cap-dependent translation synchronized with enhanced cell division. We provide evidence that eIF4E phosphorylation is regulated by mTORC1 by virtue of its interaction with Raptor through a novel TPTPNPP motif and consequent phosphorylation invitro and in vivo in a Rapamycin-sensitive manner. While we show that phosphorylation pattern of eIF4E responds faithfully to Rapamycin inhibition, the prolonged exposure to Rapamycin rescues the loss of eIF4E phosphorylation through Mnk1 activation. We also present evidence that eIF4E interacts with the amino terminal domain of S6K1 in a phospho-dependent manner, and this interaction is instrumental in overriding Rapamycin inhibition of S6K1. The data endorses eIF4E as a regulatory subunit that modulates the functional attributes of mTOR effectors to synchronize cap-dependent translation with growth assertion. [ABSTRACT FROM AUTHOR]

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.

Leaky gut is a condition of increased paracellular permeability of the intestine due to compromised tight junction barriers. In recent years, this affliction has drawn the attention of scientists from different fields, as a myriad of studies prosecuted it to be the silent culprit of various immune diseases. Due to various controversies surrounding its culpability in the clinic, approaches to leaky gut are restricted in maintaining a healthy lifestyle, avoiding irritating factors, and practicing alternative medicine, including the consumption of supplements. In the current study, we investigate the tight junction-modulating effects of processed Aloe vera gel (PAG), comprising 5–400-kD polysaccharides as the main components. Our results show that oral treatment of 143 mg/kg PAG daily for 10 days improves the age-related leaky gut condition in old mice, by reducing their individual urinal lactulose/mannitol (L/M) ratio. In concordance with in vivo experiments, PAG treatment at dose 400 μg/mL accelerated the polarization process of Caco-2 monolayers. The underlying mechanism was attributed to enhancement in the expression of intestinal tight junction-associated scaffold protein zonula occludens (ZO)-1 at the translation level. This was induced by activation of the MAPK/ERK signaling pathway, which inhibits the translation repressor 4E-BP1. In conclusion, we propose that consuming PAG as a complementary food has the potential to benefit high-risk patients.

Background and Purpose: It is urgently required to develop promising candidates to permeate across blood-brain barrier (BBB) efficiently with simultaneous disrupting vasculogenic mimicry capability of gliomas. Previously, a series of iron (II) complexes were synthesized through a modified method. Hence, the aim of this study was to evaluate anticancer activity of Fe(PIP)3SO4 against glioma cancer cells.Methods: Cytotoxic effects were determined via MTT assay, and IC50 values were utilized to evaluate the cytotoxicity. Cellular uptake of Fe(PIP)3SO4 between U87 and HEB cells was conducted by subtracting content of the complex remaining in the cell culture supernatants. Propidium Iodide (PI)-flow cytometric analysis was used to analyze cell cycle proportion of U87 cells treated with Fe(PIP)3SO4. The reactive oxygen species levels induced by Fe(PIP)3SO4 were measured by 2'-deoxycoformycin (DCF) probe; ABTS assay was utilized to examine the radical scavenge capacity of Fe(PIP)3SO4. To study the bind efficiency to thioredoxin reductase (TrxR), Fe(PIP)3SO4 was introduced into solution containing TrxR. To verify if Fe(PIP)3SO4 could penetrate BBB, HBMEC/U87 coculture as BBB model was established, and penetrating capability of Fe(PIP)3SO4 was tested. In vitro U87 tumor spheroids were formed to test the permeating ability of Fe(PIP)3SO4. Acute toxicity and biodistribution of Fe(PIP)3SO4 were tested on mice for 72 h. Protein profiles associated with U87 cells treated with Fe(PIP)3SO4 were determined by Western blotting analysis.Results: Results showed that Fe(PIP)3SO4 could suppress cell proliferation by inducing G2/M phase cycle retardation and apoptotic pathways, which was related with expression of p53 and initiation factor 4E binding protein 1. In addition, Fe complex could suppress cell proliferation by downregulating reactive oxygen species levels via scavenging free radicals and interaction with TrxR. Furthermore, Fe(PIP)3SO4 could permeate across BBB and simultaneously inhibited the vasculogenic mimicry-channel of U87 cells, suggesting favorable antiglioblastoma efficacy. Acute toxicity manifested lower degree of the complex compared with cisplatin and temozolomide.Conclusion: Fe(PIP)3SO4 exhibited favorable anticancer activity against glioma cells associated with p53 and 4E binding protein 1, accompanied with negligible toxic effects on normal tissues. Herein, Fe(PIP)3SO4 could be developed as a promising metal-based chemotherapeutic agent to overcome BBB and antagonize glioblastomas.

The extent and dynamics of animal cell biomass accumulation during mitosis are unknown, primarily because growth has not been quantified with sufficient precision and temporal resolution. Using the suspended microchannel resonator and protein synthesis assays, we quantify mass accumulation and translation rates between mitotic stages on a single-cell level. For various animal cell types, growth rates in prophase are commensurate with or higher than interphase growth rates. Growth is only stopped as cells approach metaphase-to-anaphase transition and growth resumes in late cytokinesis. Mitotic arrests stop growth independently of arresting mechanism. For mouse lymphoblast cells, growth in prophase is promoted by CDK1 through increased phosphorylation of 4E-BP1 and cap-dependent protein synthesis. Inhibition of CDK1-driven mitotic translation reduces daughter cell growth. Overall, our measurements counter the traditional dogma that growth during mitosis is negligible and provide insight into antimitotic cancer chemotherapies.

Huntington's disease is a neurodegenerative disorder caused by a CAG repeat expansion in exon 1 of the huntingtin gene. Striatal projection neurons are mainly affected, leading to motor symptoms, but molecular mechanisms involved in their vulnerability are not fully characterized. Here, we show that eIF4E binding protein (4E-BP), a protein that inhibits translation, is inactivated in Huntington's disease striatum by increased phosphorylation. Accordingly, we detected aberrant de novo protein synthesis. Proteomic characterization indicates that translation specifically affects sets of proteins as we observed upregulation of ribosomal and oxidative phosphorylation proteins and downregulation of proteins related to neuronal structure and function. Interestingly, treatment with the translation inhibitor 4EGI-1 prevented R6/1 mice motor deficits, although corticostriatal long-term depression was not markedly changed in behaving animals. At the molecular level, injection of 4EGI-1 normalized protein synthesis and ribosomal content in R6/1 mouse striatum. In conclusion, our results indicate that dysregulation of protein synthesis is involved in mutant huntingtin-induced striatal neuron dysfunction. [ABSTRACT FROM AUTHOR]

Background and Purpose: It is urgently required to develop promising candidates to permeate across blood-brain barrier (BBB) efficiently with simultaneous disrupting vasculogenic mimicry capability of gliomas. Previously, a series of iron (II) complexes were synthesized through a modified method. Hence, the aim of this study was to evaluate anticancer activity of Fe(PIP)3SO4 against glioma cancer cells. Methods: Cytotoxic effects were determined via MTT assay, and IC50 values were utilized to evaluate the cytotoxicity. Cellular uptake of Fe(PIP)3SO4 between U87 and HEB cells was conducted by subtracting content of the complex remaining in the cell culture supernatants. Propidium Iodide (PI)-flow cytometric analysis was used to analyze cell cycle proportion of U87 cells treated with Fe(PIP)3SO4. The reactive oxygen species levels induced by Fe(PIP)3SO4 were measured by 2'-deoxycoformycin (DCF) probe; ABTS assay was utilized to examine the radical scavenge capacity of Fe(PIP)3SO4. To study the bind efficiency to thioredoxin reductase (TrxR), Fe(PIP)3SO4 was introduced into solution containing TrxR. To verify if Fe(PIP)3SO4 could penetrate BBB, HBMEC/U87 coculture as BBB model was established, and penetrating capability of Fe(PIP)3SO4 was tested. In vitro U87 tumor spheroids were formed to test the permeating ability of Fe(PIP)3SO4. Acute toxicity and biodistribution of Fe(PIP)3SO4 were tested on mice for 72 h. Protein profiles associated with U87 cells treated with Fe(PIP)3SO4 were determined by Western blotting analysis. Results: Results showed that Fe(PIP)3SO4 could suppress cell proliferation by inducing G2/M phase cycle retardation and apoptotic pathways, which was related with expression of p53 and initiation factor 4E binding protein 1. In addition, Fe complex could suppress cell proliferation by downregulating reactive oxygen species levels via scavenging free radicals and interaction with TrxR. Furthermore, Fe(PIP)3SO4 could permeate across BBB and simultaneously inhibited the vasculogenic mimicry-channel of U87 cells, suggesting favorable antiglioblastoma efficacy. Acute toxicity manifested lower degree of the complex compared with cisplatin and temozolomide. Conclusion: Fe(PIP)3SO4 exhibited favorable anticancer activity against glioma cells associated with p53 and 4E binding protein 1, accompanied with negligible toxic effects on normal tissues. Herein, Fe(PIP)3SO4 could be developed as a promising metal-based chemotherapeutic agent to overcome BBB and antagonize glioblastomas. [ABSTRACT FROM AUTHOR]

L-Glutamine (L-Gln) supplementation has been pointed out as an anticatabolic intervention, but its effects on protein synthesis and degradation signaling in skeketal muscle are still poorly known. The effects of L-Gln pretreatment (1 g kg-1 day-1 body weight for 10 days) on muscle fiber cross-sectional area (CSA), amino acid composition (measured by LC-MS/MS) and protein synthesis (Akt-mTOR) and degradation (ubiquitin ligases) signaling in soleus and extensor digitorum longus (EDL) muscles in 24-h-fasted mice were investigated. The fiber CSA of EDL muscle was not different between the L-Gln-fasted and L-Gln-fed groups. This finding was associated with reduced contents of L-Leu and L-Iso and activation of protein synthesis signaling (p-RPS6Ser240/244 and Akt-mTOR). The spectrum of soleus muscle fiber CSA distribution was larger in L-Gln-fasted as compared with placebo-fasted mice. This effect of L-Gln pretreatment was associated with changes in red fibers L-Gln metabolism as indicated by increased intracellular L-glutamine/L-glutamate ratio, L-aspartate and GABA levels. L-Gln supplementation reduced fasting-induced mass loss in tibialis anterior and gastrocnemius muscles. Evidence is presented that pretreatment with L-glutamine attenuates skeletal muscle atrophy induced by 24-h fasting through mechanisms that vary with the muscle fiber type. [ABSTRACT FROM AUTHOR]

Purpose of Review: To highlight recent results in targeting mRNA translation and discuss the results and prospects of translation inhibitors in cancer therapy. Recent Findings: Until recently, inhibitors of mRNA translation have been thought to likely lack a therapeutic window. In 2012, the Food and Drug Administration (FDA) approved omacetaxine mepesuccinate (homoharringtonine) for the treatment of adults with chronic myelogenous leukemia (CML) who are resistant to at least two tyrosine kinase inhibitors. Since then, a few drugs, notably tomivosertib (eFT-508), selinexor (KPT-330), and ribavirin, have entered clinical trials. These drugs are known to inhibit mRNA translation. More recently, a number of interesting studies report that discrete subsets of proteins in cancer cells may be selectively targeted at the translation step, through inhibiting signals such as phospho-4E-BP1, eIF4A, and eIF4E. Promising therapies using these strategies have demonstrated potent anti-tumor activity in preclinical cancer models. Summary: The growing number of translation inhibitors with diverse mechanisms, coupled with emerging insights into translational regulation of different cancer-promoting genes, suggests a bright new horizon for the field of therapeutic targeting of mRNA translation in cancer. [ABSTRACT FROM AUTHOR]

The aim of the present work was to identify the role of stretch-activated ion channels (SAC) in the transmission of mechanical signals to the key mTORC1/p70S6k anabolic pathways in the soleus muscle in rats after seven days of functional unloading of the hindlimbs. Functional unloading of the hindlimb muscles was by antiorthostatic suspension (hindlimb suspension, HS). The rats were divided into four groups: 1) group C (animal-house control; 2) C + Gd (controls whose muscles were incubated with the SAC inhibitor GdCl3; 3) HS (functional unloading for seven days); and 4) HS + Gd (a group with seven days of antiorthostatic suspension followed by incubation of muscles with GdCl3). After completion of antiorthostatic suspension, isolated soleus muscles were subjected to a series of eccentric contractions (EC). Muscles from intact animals responded to EC with increases in the phosphorylation of key markers of mTORC1 signaling: p70S6k and rpS6. The extent of mechanically induced increases in the levels of phosphorylation of these protein markers were significantly reduced to essentially identical extents in the "pure" suspension (HS) group and the HS group given the SAC blocker (HS + Gd) as compared with the increase in muscles from intact control group. These results lead to the conclusion that decreases in the mechanically dependent responses of the mTORC1 signal pathway in the soleus muscle in rats after seven days of functional unloading may be linked with inactivation of stretch-activated ion channels. [ABSTRACT FROM AUTHOR]

Acute lower extremity ischemia (ALEXI) is known worldwide as an urgent condition, occurring when there is an abrupt interruption in blood flow into an extremity. This study aims to investigate whether microRNA‐224 (miR‐224) affects the ALEXI mice and the underlying mechanism. The miR‐224 expression and C/EBP homologous protein (CHOP), mammalian target of rapamycin (mTOR), translation initiation factor 4E‐binding protein 1 (4E‐BP1), and phosphoprotein 70 ribosomal protein S6 kinase (p70S6K) messenger RNA (mRNA), as well as protein expressions, were determined. The target gene of miR‐224 was also verified by using a luciferase reporter gene assay. The vascular endothelial cells from the ALEXI mice were transfected with miR‐224 mimics, miR‐224 inhibitors, or small‐interfering RNA against CHOP. Cell proliferation was assessed using a 3‐(4,5‐dimethylthiazol‐2‐yl)‐2,5‐diphenyltetrazolium bromide (MTT) assay. The cell cycle distribution along with the cell apoptosis were both evaluated by using a flow cytometry. The muscle fibers of the lower extremities found in the ALEXI mice were evidently swollen and rounded, presenting with a remarkably narrowed gap. The positive CHOP expression increased in ALEXI mice than normal mice, while the miR‐224 expression and mTOR, 4E‐BP1, and p70S6K mRNA, as well as the protein expression, decreased. Luciferase reporter gene assay validated that the miR‐224 gene directly targeted CHOP. MiR‐224 facilitated cell proliferation but inhibited cell apoptosis; by contrast, CHOP increased cell apoptosis. Moreover, the cells transfected along with miR‐224 mimic exhibited a lower CHOP expression as well as increased mTOR, 4E‐BP1, and p70S6K expression. Our study provided evidence that miR‐224 could alleviate the occurrence and development of ALEXI in mice through activation of the mTOR signaling pathway by downregulating CHOP. MicroRNA‐224 could alleviate the occurrence and development of acute lower extremity ischemia (ALEXI) in mice through activation of the mammalian target of rapamycin (mTOR) signaling pathway by downregulating C/EBP homologous protein (CHOP). [ABSTRACT FROM AUTHOR]

Background: Eukaryotic Initiation Factor 4E-Binding Protein (EIF4EBP1, 4EBP1) is overexpressed in many human cancers including breast cancer, yet the role of 4EBP1 in breast cancer remains understudied. Despite the known role of 4EBP1 as a negative regulator of cap-dependent protein translation, 4EBP1 is predicted to be an essential driving oncogene in many cancer cell lines in vitro, and can act as a driver of cancer cell proliferation. EIF4EBP1 is located within the 8p11-p12 genomic locus, which is frequently amplified in breast cancer and is known to predict poor prognosis and resistance to endocrine therapy.Methods: Here we evaluated the effect of 4EBP1 targeting using shRNA knock-down of expression of 4EBP1, as well as response to the mTORC targeted drug everolimus in cell lines representing different breast cancer subtypes, including breast cancer cells with the 8p11-p12 amplicon, to better define a context and mechanism for oncogenic 4EBP1.Results: Using a genome-scale shRNA screen on the SUM panel of breast cancer cell lines, we found 4EBP1 to be a strong hit in the 8p11 amplified SUM-44 cells, which have amplification and overexpression of 4EBP1. We then found that knock-down of 4EBP1 resulted in dramatic reductions in cell proliferation in 8p11 amplified breast cancer cells as well as in other luminal breast cancer cell lines, but had little or no effect on the proliferation of immortalized but non-tumorigenic human mammary epithelial cells. Kaplan-Meier analysis of EIF4EBP1 expression in breast cancer patients demonstrated that overexpression of this gene was associated with reduced relapse free patient survival across all breast tumor subtypes.Conclusions: These results are consistent with an oncogenic role of 4EBP1 in luminal breast cancer and suggests a role for this protein in cell proliferation distinct from its more well-known role as a regulator of cap-dependent translation. [ABSTRACT FROM AUTHOR]

Theobromine is a caffeine derivative and the primary methylxanthine in Theobroma cacao. We have shown previously that theobromine inhibits the Akt‐mammalian target of rapamycin (mTOR) signal in vitro. In this study, we investigated whether orally administered theobromine could inhibit mTOR activity in rats. mTOR is phosphorylated by Akt. Thus, the level of phosphorylated mTOR was used as an index of mTOR activity. Male Wistar rats were divided into two groups. The control group (CN) was fed a normal diet, while the theobromine group (TB) was fed a diet supplemented with 0.05% theobromine for 40 days. We measured body‐weights and tissue weights, food and water intake, blood count, concentrations of theobromine in the plasma, liver and brain, and the levels of phosphorylated mTOR in the liver and brain. Orally administered theobromine did not affect the body‐weights and tissue weights, food and water intake, and blood count as determined by comparison with levels in rats that were fed standard chow. Theobromine was detected in the plasma, liver and brain obtained from TB rats, but was not detected in tissues obtained from CN rats. The phosphorylated mTOR levels in the liver and brain were significantly lower in TB rats than in CN rats. The results suggest that oral theobromine inhibits mTOR signalling in vivo. [ABSTRACT FROM AUTHOR]