Increased Phosphorylation Of 4E-BP1 Research Articles

Analysis of the stimulative effect of arginine on translation initiation of protein synthesis in skeletal muscle

Introduction: Amino acids are not only important precursors for the synthesis of proteins, but also participate in the regulation of major metabolic pathways. Specific amino acids stimulate protein synthesis. Previous studies have shown that arginine (Arg) stimulates protein synthesis at the level of translation initiation through the activation of the mechanistic target of rapamycin complex 1 (mTORC1) in cell culture system; however, little is known about the effect of Arg on protein synthesis in vivo. Here, we evaluated whether Arg stimulates protein synthesis at the level of translation initiation and compared the effects of Arg on translation initiation with those of leucine (Leu), which is well-studied the detail of its stimulative mechanism, in mouse skeletal muscle. Methods: Eighteen hour-fasted mice were intraperitoneally injected with saline or 4.6 mmol/ kg/ body weight of Arg or Leu and subsequently sacrificed and excised gastrocnemius muscles at 1 h after injection. In addition, we used mouse-derived C2C12 myotubes treated with Arg to examine the mechanism by which Arg stimulates translation initiation in mouse skeletal muscle. The cells were preincubated with several kinds of inhibitors for appropriate time and then treated with Arg or Leu. Using homogenate of skeletal muscle and cell lysate, the phosphorylation states of eukaryotic translation initiation factor 4E-binding protein 1 (4E-BP1) and ribosomal protein S6 kinase 1 (S6K1), key regulatory factors involved in the translation initiation were measured by immunoblotting as indicators of translation initiation activity. Results: Intraperitoneal injection of either Arg or Leu significantly increased S6K1 phosphorylation compared to the saline-injection, but the increase in 4E-BP1 phosphorylation was observed in Leu-injected mice not in Arg-injected mice. These results suggest that Arg can stimulate translation initiation in skeletal muscle of fasted mice. In C2C12 myotubes, as with Leu, Arg increased both 4E-BP1 and S6K phosphorylation, and these increases were abolished by the treatment of rapamycin, a mTORC1 inhibitor. These data indicate that Arg directly activates mTORC1 pathway as well as Leu. In addition, the treatment of a phosphoinositide 3-kinase (PI3K) inhibitor and a AKT inhibitor completely cancelled Arg-dependent 4E-BP1 and S6K1 phosphorylation; however, the inhibition of Leu-induced 4E-BP1 and S6K1 phosphorylation by these inhibitors were in partial. Furthermore, the inhibitor of the G protein‐coupled receptor family C, group 6, subtype A (GPRC6a), which has been shown to associate with regulation of PI3K/AKT pathway and mTORC1 activation, completely suppressed the increases of 4E-BP1 and S6K1 phosphorylation by Arg. Conclusion:Our data indicate that Arg has the ability to activate translation initiation in mouse skeletal muscle. In addition, the results of our experiments using skeletal muscle cells support the idea that Arg stimulates translation initiation via GPRC6a/PI3K/AKT/mTORC1 pathway in mouse skeletal muscle. Unlike the effects of Arg, it is likely that besides the PI3K/AKT pathway, Leu activates another signaling pathways upstream of mTORC1 to stimulate translation initiation in skeletal muscle. This research was funded by JSPS KAKENHI, grant number 18K05501. This is the full abstract presented at the American Physiology Summit 2024 meeting and is only available in HTML format. There are no additional versions or additional content available for this abstract. Physiology was not involved in the peer review process.

Parkin is a critical player in the effects of caffeine over mitochondrial quality control pathways during skeletal muscle regeneration in mice.

This study aimed to investigate the effects of caffeine on pathways associated with mitochondrial quality control and mitochondrial capacity during skeletal muscle regeneration, focusing on the role of Parkin, a key protein involved in mitophagy. We used invitro C2C12 myoblast during differentiation with and without caffeine in the medium, and we evaluated several markers of mitochondrial quality control pathways and myotube growth. Invivo experiments, we used C57BL/6J (WT) and Parkintm 1Shn lineage (Parkin-/- ) mice and injured tibial anterior muscle. The mice regenerated TA muscle for 3, 10, and 21 days with or without caffeine ingestion. TA muscle was used to analyze the protein content of several markers of mitochondrial quality pathways, muscle satellite cell differentiation, and protein synthesis. Furthermore, it analyzed mtDNA, mitochondrial respiration, and myofiber growth. C2C12 differentiation experiments showed that caffeine decreased Parkin content, potentially leading to increased DRP1 and PGC-1α content and altered mitochondrial population, thereby enhancing growth capacity. Using Parkin-/- mice, we found that caffeine intake during the regenerative process induces an increase in AMPKα phosphorylation and PGC-1α and TFAM content, changes that were partly Parkin-dependent. In addition, the absence of Parkin potentiates the ergogenic effect of caffeine by increasing mitochondrial capacity and myotube growth. Those effects are related to increased ATF4 content and activation of protein synthesis pathways, such as increased 4E-BP1 phosphorylation. These findings demonstrate that caffeine ingestion changes mitochondrial quality control during skeletal muscle regeneration, and Parkin is a central player in those mechanisms.

Insulin potentiates essential amino acids effects on mechanistic target of rapamycin complex 1 signaling in MAC-T cells

Different models of lactation offer conflicting evidence as to whether insulin signaling is required for AA to stimulate mechanistic target of rapamycin complex 1 (mTORC1) activity. We hypothesized that insulin potentiates essential AA stimulation of mTORC1 activity in the MAC-T mammary epithelial cell line. Here, our objective was to assess mTORC1 signaling activity in response to insulin and individual or grouped essential AA. Insulin and essential AA concentrations in the treatment medium ranged from normo- to supraphysiological, with insulin at 0, 1, 10, or 100 nmol/L and essential AA at approximately 0, 0.01, 0.05, 0.1, 1, or 3× reference plasma levels. Effects and interaction of insulin and total essential AA were tested in a 3 × 5 factorial design (n = 3 replicates/treatment); insulin and the individual AA Leu, Met, Ile, and Arg were likewise tested in 3 × 4 factorials (n = 4). As the remaining individual AA His, Lys, Phe, Thr, Trp, and Val were expected to not affect mTORC1, these were tested only at the highest insulin level, 100 nmol/L (n = 4). For all of these, linear and quadratic effects of total and individual AA were evaluated. Essential AA were subsequently grouped by their positive (Leu, Met, Ile, Arg, and Thr; TOR-AA) or absent-to-negative effects (His, Lys, Phe, Trp, and Val; NTOR-AA), and tested for interaction in a 2 × 2 factorial design (n = 4), with each AA at its respective 1× plasma level, and insulin held at 100 nmol/L. All experiments consisted of 1 h treatment incubation, followed by Western blotting of cell lysates to measure phosphorylation and abundance of the mTORC1 pathway proteins Akt (Ser473); ribosomal protein S6 kinase p70 (S6K1, Thr389); eukaryotic translation initiation factor 4E-binding protein 1 (4E-BP1, Ser65); and ribosomal protein S6 (S6, Ser240/244). The Akt phosphorylation was overall increased by insulin, with a possible negative interaction with both total essential AA and the individual AA Leu. Total essential AA also increased S6K1 and 4E-BP1 phosphorylation in an insulin-dependent manner. The individual AA Leu, Met, Ile, and Arg increased S6K1 phosphorylation in an insulin-dependent manner. Similarly, Met and Arg increased 4E-BP1 phosphorylation in an insulin-dependent manner. Histidine, Lys, Trp, and Val did not affect S6K1 phosphorylation. However, S6K1 phosphorylation was linearly increased by Thr and quadratically decreased by Phe. Relative to the phosphorylation of S6K1 when cells were incubated with no essential AA, the NTOR-AA group had no effect, whereas the TOR-AA increased phosphorylation to the same degree observed with all 10 essential AA. Overall, we have found that insulin is required for essential AA to stimulate mTORC1 activity in MAC-T cells. In addition, the AA responsible for the bulk of mTORC1 activation in MAC-T are limited to Leu, Met, Ile, Arg, and Thr.

Ethanol acutely antagonizes the refeeding-induced increase in mTOR-dependent protein synthesis and decrease in autophagy in skeletal muscle.

The purpose of this study was to determine the impact of acute ethanol administration on the major signal transduction pathways in skeletal muscle responsible for regulating the protein synthetic and degradative response to refeeding. Adult male C57Bl/6 mice were fasted overnight; mice were then either refed normal rodent chow for 30min or a separate group of mice remained food deprived (i.e., fasted). Thereafter, mice were administered either 3g/kg ethanol or saline. Gastrocnemius/plantaris was collected 1h later and analyzed. Acute ethanol decreased basal and prevented the refeeding-induced increase in muscle protein synthesis. While ethanol prevented a nutrient-stimulated increase in S6K1 phosphorylation, it did not alter the increase in 4E-BP1 phosphorylation. Downstream of S6K1, ethanol also attenuated the refeeding-induced increase in S6 and eIF4B phosphorylation, as well as the decrease in eEF2 phosphorylation. Although ethanol decreased ERK and p90 RSK phosphorylation, activation of this signaling pathway was not altered by refeeding in either control or ethanol-treated mice. Related to protein degradation, in vitro-determined proteasome activity and the content of total ubiquitinated proteins were not altered by ethanol and/or refeeding. Control mice appeared to exhibit a refeeding-induced decrease in autophagy as suggested by the increased FoxO3 and ULK1 phosphorylation and total p62 protein as well as decreased LC3B-II; however, ethanol blunted these refeeding-induced changes. These data suggest that ethanol can acutely prevent the normally observed mTOR-dependent increase in protein synthesis and reduction in autophagy in response to nutrient stimulation, but does not appear to acutelyalter proteasome activity.

The oncoprotein Myc controls the phosphorylation of S6 kinase and AKT through protein phosphatase 2A.

This study focuses on the effects of Myc oncoprotein on the translational apparatus of the cell. Translation is an energy consuming process that involves a large number of accessory factors. The production of components of the protein synthesis machinery can be regulated at the transcriptional level by specific factors. It has been shown that the product of the oncogene Myc, a transcription factor frequently activated in cancer, can control translational activity through an increase in the transcription of the eIF4F complex components (eIF4E, eIF4AI, and eIF4GI). However, additional effects at the posttranslational level have also been described. For instance, it has been shown that Myc upregulation can induce mammalian target of rapamycin (mTOR)-dependent 4E-binding protein 1 (4E-BP1) hyperphosphorylation. We induced overexpression or inhibition of Myc through transfection of complementary DNA constructs or specific small interfering RNA in PC3 (prostate carcinoma) and HeLa (cervical carcinoma) cells. We have observed that overexpression of Myc causes an increase in 4E-BP1 phosphorylation and activation of protein synthesis. Unexpectedly, we detected a parallel decrease in the phosphorylation level of S6 kinase (in PC3 and HeLa) and AKT (in HeLa). We report evidence that these changes are mediated by an increase in protein phosphatase 2A activity.

Abstract 3515: Down the rabbit hole with structural effects of FK866 in primary CLL B cells

Abstract FK866 (Daporinad or APO866) is a specific inhibitor of NAMPT (nicotinamide phosphoribosyltransferase), a rate limiting enzyme involved in NAD generation. It is a prospective cancer treatment utilized in several clinical trials and has generated much interest. It has been previously demonstrated that FK866 (10nM) induces cell death in CLL patient derived B-cells (CLL-B cells) by early depletion of NAD (1 day after treatment) followed by a subsequent decrease in cellular ATP levels (2 days after treatment). Upon further study we observed that CLL-B cells have structural changes after treatment with FK866 for either 6 or 18-20 hours via electron microscopy. There is a presence of bound structures as well as nuclear invaginations. These structures are only observed in cells that were treated with FK866 and not with any other drugs used (fludarabine, bendamustine, chlorambucil) nor with the DMSO or untreated controls. We stained f-actin (a cytoskeletal protein) with FITC labelled phalloidin in CLL-B cells with and without FK866 treatment and then viewed with confocal microscopy to exclude the chance that these structures were artifacts. Changes in the f-actin were only observed in the FK866 treated cells. Phalloidin staining was performed at 6 and 18 hours. We observed holes in the f-actin framework that passed through the entire cell and in some instances, went through the nuclei too. A comparison of the phalloidin staining was performed in non-CLL based cell lines that were known to be sensitive to treatment with FK866 though structural differences had not been observed at the same time points (6 and 18-20 hours). Two Ewing's sarcoma cell lines and two small cell lung cancer cell lines were used. All four cell lines showed no changes in the f-actin. Labeled dextran beads were observed via confocal microscopy to be taken up more readily by the FK866 treated CLL -B cells as compared to the vehicle control. This would imply that the treated cells are actively removing nutrients from the environment. A lysosome stain, LysoTracker, was employed to determine if there was any involvement of lysosomes in this phenomenon. No change was observed between the untreated, vehicle control and the FK866 treated CLL patient derived B-cells. We were curious about the mitochondrial dynamics in the FK866 treated cells so they were stained with MitoTracker. We were able to detect mitochondria in the cytoplasm of the cells in all treatments but the FK866 treated cells displayed a more distinct punctate pattern. Interestingly, we also observed the mitochondria were appearing at or near the holes in the cytoskeleton. As the FK866 treated cells are deprived of their NAD, the CLL cells may be undergoing macropinocytosis, an mTOR mediated phenomenon used by cells to extract nutrients from the environment. We have shown that FK866 causes an increase in 4EBP1 phosphorylation compared to controls. In conclusion, FK866 may mediate macropinocytosis in CLL cells in an mTOR dependent manner. Citation Format: Cheryl Peltier, Eileen McMillan-Ward, Elizabeth Henson, Nabanita Chatterje, Donna Hewitt, Danielle Desautels, Matthieu Bourrier, Cornelia Mutz, Iris Gehrke, Eric Bouchard, Heinrich Kovar, Shantanu Banerji, Spencer Gibson, Sara Israels, Versha Banerji. Down the rabbit hole with structural effects of FK866 in primary CLL B cells [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2018; 2018 Apr 14-18; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2018;78(13 Suppl):Abstract nr 3515.

BRD7 regulates the insulin-signaling pathway by increasing phosphorylation of GSK3β.

Reduced hepatic expression levels of bromodomain-containing protein 7 (BRD7) have been suggested to play a role in the development of glucose intolerance in obesity. However, the molecular mechanism by which BRD7 regulates glucose metabolism has remained unclear. Here, we show that BRD7 increases phosphorylation of glycogen synthase kinase 3β (GSK3β) in response to activation of the insulin receptor-signaling pathway shortly after insulin stimulation and the nutrient-sensing pathway after feeding. BRD7 mediates phosphorylation of GSK3β at the Serine 9 residue and this effect on GSK3β occurs even in the absence of AKT activity. Using both in vitro and in vivo models, we further demonstrate that BRD7 mediates phosphorylation of ribosomal protein S6 kinase (S6K) and leads to increased phosphorylation of the eukaryotic translation initiation factor 4E-binding protein 1 (4E-BP1) and, therefore, relieves its inhibition of the eukaryotic translation initiation factor 4E (eIF4E). However, the increase in phosphorylation of 4E-BP1 with BRD7 overexpression is blunted in the absence of AKT activity. In addition, using liver-specific BRD7 knockout (LBKO)mice, we show that BRD7 is required for mTORC1 activity on its downstream molecules. These findings show a novel basis for understanding the molecular dynamics of glucose metabolism and suggest the unique function of BRD7 in the regulation of glucose homeostasis.

Effect of phytoecdysteroids on protein synthesis and Akt/mTOR signaling after downhill running in skeletal muscle of mice

Phytoecdysteroids are natural plant steroids synthesized by a variety of hardy plants. Phytoecdysteroids, such as 20‐hydroxyecdysone (20E), possess anabolic properties and previous research indicates that 20E stimulates protein synthesis in cultured muscle cells and increases grip strength in young rats after 28 days of supplementation. The purpose of this study was to investigate the extent to which 20E stimulates protein synthesis and Akt/mTOR signaling in skeletal muscle after an acute bout of downhill running (DHR). Male C57BL6 mice (3–6 mo old) were assigned to four groups. Mice in the DHR groups performed an acute bout of DHR to exhaustion on a rodent treadmill at 17 m•min−1 and −20° decline. After completion of the DHR bout and recovery, mice received an oral gavage treatment of either 50 mg•kg−1 body mass (BM) of 20E (DHR + 20E; n=5) or vehicle (DHR + vehicle; n=6), and then treated daily for the next four consecutive days. Two groups that did not perform DHR, but were treated for five consecutive days with either 50 mg•kg−1 BM of 20E (No DHR + 20E; n=8) or vehicle (No DHR + vehicle; n=6), were used as controls. On the fifth day post‐DHR, mice were not treated with 20E or vehicle; however, an IP injection of puromycin (0.040 μmol•g−1 BM) was administered 30 min prior to sacrifice to assess protein synthesis. Skeletal muscles were harvested and activation of protein synthesis was assessed using the SUnSET method and Western blot and Akt/mTOR signaling activation was measured via Western blot. Mice in the DHR + vehicle group ran for 58.5 min, while mice in the DHR + 20E group ran for 57.0 min. Compared to No DHR + vehicle, DHR + vehicle increased skeletal muscle puromycin incorporation 14%, but DHR + 20E increased puromycin incorporation 32%, five days post‐DHR. Puromycin incorporation was not different between No DHR + vehicle and No DHR + 20E groups. DHR resulted in ~25% increase in phosphorylation of both Akt and p70S6K, compared to No DHR + vehicle; but there was no difference in Akt or p70S6K phosphorylation between DHR + 20E and DHR + vehicle groups, five days post‐DHR. Compared to No DHR + vehicle, DHR + vehicle increased 4EBP1 phosphorylation 35%, but DHR + 20E increased 4EBP1 phosphorylation 70%, and DHR + vehicle increased ribosomal protein S6 (rpS6) phosphorylation 40%, but DHR + 20E increased rpS6 phosphorylation 119%, five days post‐DHR. In conclusion, these findings suggest that DHR stimulates protein synthesis and activates Akt/mTOR signaling five days after DHR, but 20E treatment enhances the pattern of response to an acute bout of DHR in skeletal muscle of young mice.

Citrulline directly modulates muscle protein synthesis via the PI3K/MAPK/4E-BP1 pathway in a malnourished state: evidence from in vivo, ex vivo, and in vitro studies.

Citrulline (CIT) is an endogenous amino acid produced by the intestine. Recent literature has consistently shown CIT to be an activator of muscle protein synthesis (MPS). However, the underlying mechanism is still unknown. Our working hypothesis was that CIT might regulate muscle homeostasis directly through the mTORC1/PI3K/MAPK pathways. Because CIT undergoes both interorgan and intraorgan trafficking and metabolism, we combined three approaches: in vivo, ex vivo, and in vitro. Using a model of malnourished aged rats, CIT supplementation activated the phosphorylation of S6K1 and 4E-BP1 in muscle. Interestingly, the increase in S6K1 phosphorylation was positively correlated (P < 0.05) with plasma CIT concentration. In a model of isolated incubated skeletal muscle from malnourished rats, CIT enhanced MPS (from 30 to 80% CIT vs. Ctrl, P < 0.05), and the CIT effect was abolished in the presence of wortmannin, rapamycin, and PD-98059. In vitro, on myotubes in culture, CIT led to a 2.5-fold increase in S6K1 phosphorylation and a 1.5-fold increase in 4E-BP1 phosphorylation. Both rapamycin and PD-98059 inhibited the CIT effect on S6K1, whereas only LY-294002 inhibited the CIT effect on both S6K1 and 4E-BP1. These findings show that CIT is a signaling agent for muscle homeostasis, suggesting a new role of the intestine in muscle mass control.

Alternative splicing of S6K1 promotes non-small cell lung cancer survival

Ribosomal S6 kinase 1 (S6K1) that acts downstream of the mammalian target of rapamycin (mTOR) plays an important role in cell proliferation, protein translation, and cell survival. The gene RPS6KB1 encoding for S6K1 had been found to be alternatively spliced to form different isoforms. In this study, we identified that short isoforms of S6K1 splice variant were overproduced in non-small cell lung cancer (NSCLC). Moreover, suppression of S6K1 short isoforms inhibited NSCLC cell growth and induced apoptosis via upregulation of the BH3-only protein Bim in vitro and in vivo. Additionally, short isoforms of S6K1 activated mTORC1, leading to increased 4E-BP1 phosphorylation. Taken together, our findings suggested that S6K1 short isoforms were deregulated in NSCLC and promoted cell survival. Altogether, our study opens possibilities of new therapeutic approaches for NSCLC that selectively downregulate S6K1 shorter isoforms.

Abstract 892: Functional characterization of EIF1AX mutations in thyroid cancer predicts for gain of function by increasing translational rate with concomitant derepression of upstream inputs from mTOR

Abstract EIF1AX (eukaryotic translation initiation factor 1A) is a component of the translation pre-initiation complex (PIC). Recurrent EIF1AX mutations, first reported in uveal melanomas, are found in ∼1% of papillary thyroid cancers in a mutually exclusive manner with other oncogenic driver events (BRAF, RAS, and oncogenic fusions). By contrast, they are enriched in advanced thyroid cancers (9% of anaplastic and 11% poorly-differentiated thyroid cancers), and are strongly associated with RAS mutations (p&amp;lt;0.0001). EIF1AX mutations cluster in the N-terminal (NTT) or C-terminal tails (CTT). EIF1AX NTT missense mutations in thyroid cancer occur within the first 15 amino acids, whereas the CTT mutation disrupts a splice acceptor site at exon 6 (A113splice). A113splice is the most prevalent defect and is private to this disease, and results in two differentially spliced mRNAs: (1) Cryptic splice variant: by use of a cryptic acceptor site in exon 6 that leads to a 132 AA protein that excludes 12 AA; (2) Truncated splice variant: by retaining intron 5, leading to a 115 AA truncated protein. EIF1AX mutants retain the ability to recruit the ternary complex, as shown by co-IP with EIF2 after ectopic expression of NTT or A113splice EIF1AX mutants in HEK293T cells, or after CRISPR-mediated knock-in of A113splice into Cal62 RAS-mutant thyroid cancer cells. The EIF1AX mutants had greater affinity to EIF5 compared to WT, consistent with a more stable PIC. As translation initiation is a rate-limiting step, the altered affinity of EIF1AX mutants to PIC components could impact the rate of protein synthesis. We tested this by L-azidohomoalanine labeling, which showed contrasting roles of the two A113-generated splice variants expressed at endogenous levels: i.e. the cryptic splice variant increased protein synthetic rate, whereas the truncated splice variant strongly inhibited protein translation. Despite inhibiting translation, the truncated splice variant showed a paradoxical increase in 4EBP1 phosphorylation. Upon A113splice knock-in, where both variants are expressed, translation is increased, which we hypothesize results from the combined effects of 4EBP1 phosphorylation, caused by relief of negative feedback events upstream in the pathway, with increased PIC assembly caused by the cryptic splice variant. We are currently determining whether the altered rate of protein synthesis is global or selective. Of note, cells expressing the cryptic, but not the truncated splice form, showed a transforming phenotype as assessed by soft agar colony formation. As EIF1AX mutations co-occur with RAS mutations in advanced thyroid cancers, it is likely that RAS-induced PI3K-AKT/mTOR signaling may provide a further cooperative benefit and play a key role in disease progression, and in generating specific tumor cell dependencies. Citation Format: Gnana P. Krishnamoorthy, Inigo Landa, Jeffrey A. Knauf, James Nagarajah, Gunnar Rätsch, Hans-Guido Wendel, James A. Fagin. Functional characterization of EIF1AX mutations in thyroid cancer predicts for gain of function by increasing translational rate with concomitant derepression of upstream inputs from mTOR. [abstract]. In: Proceedings of the 107th Annual Meeting of the American Association for Cancer Research; 2016 Apr 16-20; New Orleans, LA. Philadelphia (PA): AACR; Cancer Res 2016;76(14 Suppl):Abstract nr 892.

Targeting the Serine-Threonine Kinase PIM2: Implications in IL-6 Mediated Survival in Myeloma

Despite major advances in chemotherapy, multiple myeloma remains incurable and in need of new therapies that target novel pathways. Insufficient understanding of the molecular pathways that regulate survival in myeloma is a major impediment towards designing better therapies to prolong survival in patients or even cure the disease. This necessitates the identification of new protein targets that are crucial for the growth and survival of multiple myeloma. Just like normal plasma cells, MM cells also depend on their interactions with bone marrow stromal cells (BMSC) for survival and production of essential growth factors. We have previously shown that MM cells interact with dendritic cells (DC) in the microenvironment and in vitro can stimulate DC to produce IL-6 (ASH2010#132, ASH2011 #147, ASH2012#722). Our recent publications show that when MM cells are not in direct contact with DC, the IL-6 produced by DC can protect MM cells against dexamethasone induced cell death, while neutralizing the IL-6 with antibodies can reverse that effect (Nair et al., 2011). Unfortunately, exactly how this survival response is mediated in MM is not very clear.PIM2, a serine threonine kinase, part of the proto-oncogene group of PIM kinases has been implicated in survival in several types of cancers including prostate cancer and multiple myeloma. In our lab, microarray gene expression analysis of publicly available datasets (Figure 1) show a trend towards increased expression of PIM2 in plasma cells from myeloma patients (left panel), and significantly in the poor prognosis subgroup MAF (Zhan et al., 2006) (right panel). For the first time we show that IL-6 produced by DC may be protecting myeloma cells by up regulating PIM2 and inactivating a major protein translation inhibitor 4EBP1, which also happens to be a PIM2 target. We show that silencing PIM2 with siRNA down regulates PIM2 activity and reverses the inactivation of 4EBP1, while the latter is known to cause cell death in myeloma. We also demonstrate that neutralizing IL-6 in MM cells that either don’t produce IL-6 on their own (MM.1S) or those that do (U266), abrogates extraneous DC-IL6 ability to induce PIM2 and its downstream target 4EBP1. Recombinant IL-6 also provided similar induction of PIM2 in myeloma and increased 4EBP1 phosphorylation, which was again reversed by neutralizing the antibody against IL-6.In myeloma patients, the use of dexamethasone in frontline therapies is often complicated by the ability of the bone marrow environment to produce IL-6 that not only induce increased proliferation of MM but also help resist dexamethasone mediated cell death in myeloma. Interestingly, when we used a novel PIM2 inhibitor, JP_11646 (kindly provided by Jasco Pharmaceuticals, LLC), it not only arrested IL-6 induced proliferation even at sub-lethal doses, but also prevented IL-6 mediated rescue of myeloma cells (Figure 2). This suggests that PIM2 might be a major player in IL-6 mediated drug resistance in myeloma and targeting it may help to subvert IL-6 mediated survival in myeloma. Through RT-PCR and westerns, we also show that IL-6 modulates PIM2 expression and activity resulting in increased 4EBP1 phosphorylation (Figure 3). This was abrogated when PIM2 activity was inhibited by JP_11646 (Figure 3). We also present data that suggests IL-6 via PIM2 may be regulating other anti-apoptotic molecules downstream of IL-6 receptors including MCL-1, that is vital to MM survival. Developing PIM2 targeted therapies provides an exciting opportunity to affect the myeloma tumor microenvironment where MM induced IL-6 production from BM could be inducing drug resistance. [Display omitted] [Display omitted] [Display omitted] DisclosuresCaserta:Jasco Pharmaceuticals LLC: Equity Ownership. Baldino:Jasco Pharmaceuticals LLC: Equity Ownership.

MNK1 pathway activity maintains protein synthesis in rapalog-treated gliomas

High levels of mammalian target of rapamycin complex 1 (mTORC1) activity in malignant gliomas promote tumor progression, suggesting that targeting mTORC1 has potential as a therapeutic strategy. Remarkably, clinical trials in patients with glioma revealed that rapamycin analogs (rapalogs) have limited efficacy, indicating activation of resistance mechanisms. Targeted depletion of MAPK-interacting Ser/Thr kinase 1 (MNK1) sensitizes glioma cells to the mTORC1 inhibitor rapamycin through an indistinct mechanism. Here, we analyzed how MNK1 and mTORC1 signaling pathways regulate the assembly of translation initiation complexes, using the cap analog m7GTP to enrich for initiation complexes in glioma cells followed by mass spectrometry-based quantitative proteomics. Association of eukaryotic translation initiation factor 4E (eIF4E) with eIF4E-binding protein 1 (4EBP1) was regulated by the mTORC1 pathway, whereas pharmacological blocking of MNK activity by CGP57380 or MNK1 knockdown, along with mTORC1 inhibition by RAD001, increased 4EBP1 binding to eIF4E. Furthermore, combined MNK1 and mTORC1 inhibition profoundly inhibited 4EBP1 phosphorylation at Ser65, protein synthesis and proliferation in glioma cells, and reduced tumor growth in an orthotopic glioblastoma (GBM) mouse model. Immunohistochemical analysis of GBM samples revealed increased 4EBP1 phosphorylation. Taken together, our data indicate that rapalog-activated MNK1 signaling promotes glioma growth through regulation of 4EBP1 and indicate a molecular cross-talk between the mTORC1 and MNK1 pathways that has potential to be exploited therapeutically.

Role for the Phosphatidylinositol 3-Kinase-Akt-TOR Pathway during Sindbis Virus Replication in Arthropods

The efficient transmission of alphaviruses requires the establishment of a persistent infection in the arthropod vector; however, the nature of the virus-arthropod host interaction is not well understood. The phosphatidylinositol 3-kinase (PI3K)-Akt-TOR pathway is a signaling pathway with which viruses interact to manipulate cellular functions. The viral activation of this pathway can enhance translation and inhibit apoptosis, potentially promoting viral replication; conversely, repression can enhance cell death. Using a system to study Sindbis virus RNA replication in Drosophila melanogaster, we found that the overexpression of Akt enhanced Sindbis virus replication. In contrast, a decrease in viral replication was observed for flies hypomorphic for the Akt gene. Infection of cultured Drosophila cells led to the phosphorylation and activation of Akt. The chemical inhibition of PI3K, Akt, and TOR in mosquito cells reduced virus replication, suggesting that this pathway is proviral. Early after infection, there was an increase in the TOR-dependent phosphorylation of 4E-BP1 in mosquito cells and a consequent increase in the translation of a capped reporter mRNA. In contrast, no change in 4E-BP1 phosphorylation was seen in mammalian cells, and the level of translation of the reporter decreased following infection. Finally, we found that the increase in the phosphorylation of 4E-BP1 was stimulated by replicon RNA but not by UV-inactivated virus. Our data indicate that Sindbis virus replication complex formation in mosquito cells activates the PI3K-Akt-TOR pathway, causing the phosphorylation of 4E-BP1 and increasing the formation of eukaryotic initiation factor 4F (eIF4F), which promote cap-dependent translation. This virus-induced increase in cap-dependent translation allows the efficient translation of viral mRNA while minimizing the burden on the cell.

Abstract 2510: eIF4E is a potential tumor specific target for radiosensitization

Abstract The translational control of gene expression has been implicated as a component of the cellular response to ionizing radiation (IR). However, the mechanisms mediating this process and its specific role in radiosensitivity have not been defined. eIF4E, an mRNA 5’ cap-binding protein, is a critical protein involved in regulating gene translation in response to a variety of environmental signals. In the study described here, we have determined the effects of IR on eIF4E activity. Moreover, using 3 tumor cell lines (MDA-MB-231, DU145 and A549) and 2 normal cell lines (MRC9 and HMEC), the potential role of eIF4E as a molecular determinant of radiosensitivity was investigated. eIF4E activity is regulated by eIF4E binding protein 1 (4E-BP1); 4E-BP1 phosphorylation releases eIF4E to enhance eIF4F complex formation and cap-dependent translation. Exposure of MDA-MB-231 or MRC9 cells to IR (2 Gy, 1h) resulted in an increase in 4E-BP1 phosphorylation. Consistent with this effect, m7-GTP batch chromatography showed that IR induced an increase in cap-bound eIF4G and a decrease in cap-bound 4E-BP1 proteins. Thus, these results indicate that IR, via 4E-BP1 phosphorylation, enhances eIF4E activity and cap-dependent translation. Whereas phosphorylation of eIF4E has also been shown to influence its translational activity, IR had no effect on eIF4E phosphorylation in any of the cell lines evaluated. To determine the significance of eIF4E as a regulator of radiosensitivity, tumor and normal cell lines were treated with siRNA to eIF4E and radiation survival curves were generated based on the colony formation assay. Knockdown of eIF4E in each of the 3 tumor cell lines resulted in an increase in radiosensitivity with DEFs (dose enhancement factors at 0.1 surviving fraction) ranging from 1.24 to 1.44. In contrast, eIF4E knockdown had no effect on the radiosensitivity of normal fibroblasts (MRC9) or normal mammary epithelial cells (HMEC). To investigate the mechanism through which eIF4E influences tumor cell radiosensitivity, we initially focused on MDA-MB-231 cells. Knockdown of eIF4E had no significant effect on MDA-MB-231 cell cycle distribution indicating that a loss of S-phase cells does not account for the observed radiosensitization. In addition, eIF4E knockdown had no effect on apoptotic cell death after IR (6 Gy, 24 and 48h). However, knockdown of eIF4E significantly enhanced the level of mitotic catastrophe induced by IR (2 Gy). These initial results suggest that eIF4E may influence the ability of tumor cells to respond to genotoxic injury. Whereas the specific mechanisms remain to be determined, these data suggest that eIF4E selectively regulates the radiosensitivity of tumor cells over normal cells. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 102nd Annual Meeting of the American Association for Cancer Research; 2011 Apr 2-6; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2011;71(8 Suppl):Abstract nr 2510. doi:10.1158/1538-7445.AM2011-2510

Olfactomedin 4 is a novel target gene of retinoic acids and 5-aza-2′-deoxycytidine involved in human myeloid leukemia cell growth, differentiation, and apoptosis

AbstractClinical application of retinoic acids (RAs) and demethylation agents has proven to be effective in treating certain myeloid leukemia patients. However, the target genes that mediate these antileukemia activities are still poorly understood. In this study, we identified olfactomedin 4 (OLFM4), a myeloid-lineage–specific gene from the olfactomedin family, as a novel target gene for RAs and the demethylation agent, 5-aza-2′-deoxycytidine. We demonstrated that the retinoic acid receptor alpha/retinoic X receptor alpha heterodimer binds to a retinoic acid response-element (DR5) site in the OLFM4 promoter and mediates all-trans-retinoic acid (ATRA)–induced transactivation of the OLFM4 gene. OLFM4 overexpression in HL-60 cells led to growth inhibition, differentiation, and apoptosis, and potentiated ATRA induction of these effects. Conversely, down-regulation of endogenous OLFM4 in acute myeloid leukemia-193 cells compromised ATRA-induced growth inhibition, differentiation, and apoptosis. Overexpression of OLFM4 in HL-60 cells inhibited constitutive and ATRA-induced phosphorylation of the eukaryote initiation factor 4E-binding protein 1 (4E-BP1), whereas down-regulation of OLFM4 protein in acute myeloid leukemia-193 cells increased 4E-BP1 phosphorylation, suggesting that OLFM4 is a potent upstream inhibitor of 4E-BP1 phosphorylation/deactivation. Thus, our study demonstrates that OLFM4 plays an important role in myeloid leukemia cellular functions and induction of OLFM4-mediated effects may contribute to the therapeutic value of ATRA.

Abstract 1702: Androgen receptor antagonist bicalutamide sensitizes androgen-independent prostate cancer cells to mTOR inhibitor rapamycin by regulating phosphorylation of translation initiation suppressor eIF4E-binding protein 1 (4E-BP1)

Abstract We previously showed that resistance of prostate cancer cells to the mTOR inhibitor rapamycin results from its ability to induce androgen receptor (AR) transcriptional activity, whereas co-treatment with the anti-androgen bicalutamide prevented this effect. Based on our data, collaborators at UC Davis Cancer Center are conducting a clinical trial to test the efficacy of this combination in patients with advanced prostate cancer. However, the mechanism by which the combination of rapamycin and bicalutamide work remains unknown. Investigation of the cause for resistance of prostate cancer cells to rapamycin showed that rapamycin-induced growth inhibition as well as AR transcriptional activity is mediated primarily by p70S6 kinase (S6K1), a downstream target of mTOR, but not by the 4E-BP1/eIF4E/eIF4G pathway, another downstream target of mTOR. Additionally, 4E-BP1 phosphorylation at Ser 65 was dose dependently inhibited by rapamycin in androgen-dependent LNCaP, but not in androgen-independent C4-2 cells, although rapamycin equally affected S6K1phosphorylation in both cells. Comparison of AR expression and 4E-BP1 phosphorylation in various prostate cancer cell lines demonstrated that those expressing high levels of the full-length AR also had increased 4E-BP1 phosphorylation, whereas no such relationship existed with S6K1. Hence, we investigated whether increased AR activity contributed to resistance of C4-2 cells to rapamycin-induced 4E-BP1 dephosphorylation. Bicalutamide treatment as well as culture in medium containing decreased levels of androgens repressed 4E-BP1 phosphorylation, showing that the AR is a positive regulator of 4E-BP1 phosphorylation. The combination of bicalutamide and rapamycin, but not rapamycin alone, inhibited 4E-BP1 phosphorylation in C4-2 cells. Additionally, bicalutamide sensitized PC-3 cells stably transfected with the AR, but not wild-type PC-3 cells that are AR null, to rapamyin-induced 4E-BP1 dephosphorylation. We also compared the effect of bicalutamide in two cell lines derived from CWR22 relapsed tumors: in CWR22R1 cell, which expressed significant levels of the full-length AR, 4E-BP1 phosphorylation was inhibited by bicalutamide, whereas in CWR22Rv1 cells, where the AR lack the ligand-binding domain (LBD), bicalutamide was unable to affect 4E-BP1 phosphorylation. Thus, in cells where bicalutamide is able to bind the AR, this drug sensitizes the cells to the effects of rapamycin. These results were verified in a CWR22 mouse xenograft model of prostate cancer progression. Taken together, our data show that rapamycin treatment increased AR activity by inhibiting S6K1, and that this increase in AR activity in turn induced resistance to rapamycin's ability to repress 4E-BP1 phosphorylation. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 101st Annual Meeting of the American Association for Cancer Research; 2010 Apr 17-21; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2010;70(8 Suppl):Abstract nr 1702.

Oral Leucine Enhances Myocardial Protein Synthesis in Rats Acutely Administered Ethanol ,

Acute alcohol ingestion induces an inhibition of myocardial protein synthesis by impairing mRNA translation initiation. Elevating plasma leucine (Leu) concentrations via oral gavage stimulates mRNA translation initiation in several tissues, although the effect in heart has not been well defined. The experiments described herein were designed to test the effects of a gavage solution containing Leu on protein synthesis and potential mechanisms important in accelerating mRNA translation initiation in cardiac muscle of rats given ethanol acutely to mimic “binge” dinking. Gavage with Leu stimulated protein synthesis and enhanced the assembly of the active eukaryotic initiation factor (eIF)4G·eIF4E complex. Increased assembly of the active eIF4G·eIF4E complex was associated with a 130% rise in phosphorylation of eIF4G(Ser1108) and a decreased assembly (∼30%) of inactive eIF4E-binding protein1 (4EBP1)·eIF4E complex in rats-administered ethanol. The reduced assembly of the 4EBP1·eIF4E complex was associated with an increase in phosphorylation of 4EBP1 in the hyperphosphorylated γ-form following Leu gavage. Phosphorylation of mammalian target of rapamycin on Ser2448, an upstream regulator of phosphorylation of 4EBP1, was elevated following Leu gavage. Neither the phosphorylation of 70-kDa ribosomal protein S6 kinase on Thr389 nor eIF4E phosphorylation was increased following Leu gavage under any condition. Leu gavage accelerates myocardial protein synthesis following acute ethanol intoxication by enhancing eIF4G·eIF4E complex assembly through increased phosphorylation of eIF4G and decreased association of 4EBP1 with eIF4E.

Phosphorylation of translation factors in response to anoxia in turtles, Trachemys scripta elegans: role of the AMP-activated protein kinase and target of rapamycin signalling pathways

Long-term survival of oxygen deprivation by animals with well-developed anoxia tolerance depends on multiple biochemical adaptations including strong metabolic rate depression. We investigated whether the AMP-activated protein kinase (AMPK) could play a regulatory role in the suppression of protein synthesis that occurs when turtles experience anoxic conditions. AMPK activity and the phosphorylation state of ribosomal translation factors were measured in liver, heart, red muscle and white muscle of red-eared slider turtles (Trachemys scripta elegans) subjected to 20 h of anoxic submergence. AMPK activity increased twofold in white muscle of anoxic turtles compared with aerobic controls but remained unchanged in liver and red muscle, whereas in heart AMPK activity decreased by 40%. Immunoblotting with phospho-specific antibodies revealed that eukaryotic elongation factor-2 phosphorylation at the inactivating Thr56 site increased six- and eightfold in red and white muscles from anoxic animals, respectively, but was unchanged in liver and heart. The phosphorylation state of the activating Thr389 site of p70 ribosomal protein S6 kinase was reduced under anoxia in red muscle and heart but was unaffected in liver and white muscle. Exposure to anoxia decreased 40S ribosomal protein S6 phosphorylation in heart and promoted eukaryotic initiation factor 4E-binding protein-1 (4E-BP1) dephosphorylation in red muscle, but surprisingly increased 4E-BP1 phosphorylation in white muscle. The changes in phosphorylation state of translation factors suggest that organ-specific patterns of signalling and response are involved in achieving the anoxia-induced suppression of protein synthesis in turtles.

Glycogen Synthase Kinase 3β Is a Novel Regulator of High Glucose- and High Insulin-induced Extracellular Matrix Protein Synthesis in Renal Proximal Tubular Epithelial Cells

High glucose (30 mM) and high insulin (1 nM), pathogenic factors of type 2 diabetes, increased mRNA expression and synthesis of lamininbeta1 and fibronectin after 24 h of incubation in kidney proximal tubular epithelial (MCT) cells. We tested the hypothesis that inactivation of glycogen synthase kinase 3beta (GSK3beta) by high glucose and high insulin induces increase in synthesis of laminin beta1 via activation of eIF2Bepsilon. Both high glucose and high insulin induced Ser-9 phosphorylation and inactivation of GSK3beta at 2 h that lasted for up to 48 h. This was associated with dephosphorylation of eIF2Bepsilon and eEF2, and increase in phosphorylation of 4E-BP1 and eIF4E. Expression of the kinase-dead mutant of GSK3beta or constitutively active kinase led to increased and diminished laminin beta1 synthesis, respectively. Incubation with selective kinase inhibitors showed that high glucose- and high insulin-induced laminin beta1 synthesis and phosphorylation of GSK3beta were dependent on PI 3-kinase, Erk, and mTOR. High glucose and high insulin augmented activation of Akt, Erk, and p70S6 kinase. Dominant negative Akt, but not dominant negative p70S6 kinase, inhibited GSK3beta phosphorylation induced by high glucose and high insulin, suggesting Akt but not p70S6 kinase was upstream of GSK3beta. Status of GSK3beta was examined in vivo in renal cortex of db/db mice with type 2 diabetes at 2 weeks and 2 months of diabetes. Diabetic mice showed increased phosphorylation of renal cortical GSK3beta and decreased phosphorylation of eIF2Bepsilon, which correlated with renal hypertrophy at 2 weeks, and increased laminin beta1 and fibronectin protein content at 2 months. GSK3beta and eIF2Bepsilon play a role in augmented protein synthesis associated with high glucose- and high insulin-stimulated hypertrophy and matrix accumulation in renal disease in type 2 diabetes.