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Implication of 4E-BP1 protein dephosphorylation and accumulation in pancreatic cancer cell death induced by combined gemcitabine and TRAIL.

ABSTRACT: Pancreatic cancer cells show varying sensitivity to the anticancer effects of gemcitabine. However, as a chemotherapeutic agent, gemcitabine can cause intolerably high levels of toxicity and patients often develop resistance to the beneficial effects of this drug. Combination studies show that use of gemcitabine with the pro-apoptotic cytokine TRAIL can enhance the inhibition of survival and induction of apoptosis of pancreatic cancer cells. Additionally, following combination treatment there is a dramatic increase in the level of the hypophosphorylated form of the tumour suppressor protein 4E-BP1. This is associated with inhibition of mTOR activity, resulting from caspase-mediated cleavage of the Raptor and Rictor components of mTOR. Use of the pan-caspase inhibitor Z-VAD-FMK indicates that the increase in level of 4E-BP1 is also caspase-mediated. ShRNA-silencing of 4E-BP1 expression renders cells more resistant to cell death induced by the combination treatment. Since the levels of 4E-BP1 are relatively low in untreated pancreatic cancer cells these results suggest that combined therapy with gemcitabine and TRAIL could improve the responsiveness of tumours to treatment by elevating the expression of 4E-BP1.

SUBMITTER: Elia A

PROVIDER: S-EPMC5870593 | biostudies-literature | 2017 Dec

REPOSITORIES: biostudies-literature

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Implication of 4E-BP1 protein dephosphorylation and accumulation in pancreatic cancer cell death induced by combined gemcitabine and TRAIL.

Elia Androulla A Henry-Grant Ricky R Adiseshiah Charlotte C Marboeuf Catherine C Buckley Rebecca J RJ Clemens Michael J MJ Mudan Satvinder S Pyronnet Stéphane S

Cell death & disease 20171212 12

Pancreatic cancer cells show varying sensitivity to the anticancer effects of gemcitabine. However, as a chemotherapeutic agent, gemcitabine can cause intolerably high levels of toxicity and patients often develop resistance to the beneficial effects of this drug. Combination studies show that use of gemcitabine with the pro-apoptotic cytokine TRAIL can enhance the inhibition of survival and induction of apoptosis of pancreatic cancer cells. Additionally, following combination treatment there is ...[more]

PMID: 29233971

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Project description:ObjectiveCyclin-dependent kinase 1 (CDK1)/cyclin B1 phosphorylates many of the same substrates as mTORC1 (a key regulator of glucose metabolism), including the eukaryotic initiation factor 4E-binding protein 1 (4E-BP1). Only mitotic CDK1 phosphorylates 4E-BP1 at residue S82 in mice (S83 in humans), in addition to the common 4E-BP1 phospho-acceptor sites phosphorylated by both CDK1 and mTORC1. We examined glucose metabolism in mice having a single aspartate phosphomimetic amino acid knock in substitution at the 4E-BP1 serine 82 (4E-BP1S82D) mimicking constitutive CDK1 phosphorylation.MethodsKnock-in homozygous 4E-BP1S82D and 4E-BP1S82A C57Bl/6N mice were assessed for glucose tolerance testing (GTT) and metabolic cage analysis on regular and on high-fat chow diets. Gastrocnemius tissues from 4E-BP1S82D and WT mice were subject to Reverse Phase Protein Array analysis. Since the bone marrow is one of the few tissues typically having cycling cells that transit mitosis, reciprocal bone-marrow transplants were performed between male 4E-BP1S82D and WT mice, followed by metabolic assessment, to determine the role of actively cycling cells on glucose homeostasis.ResultsHomozygous knock-in 4E-BP1S82D mice showed glucose intolerance that was markedly accentuated with a diabetogenic high-fat diet (p = 0.004). In contrast, homozygous mice with the unphosphorylatable alanine substitution (4E-BP1S82A) had normal glucose tolerance. Protein profiling of lean muscle tissues, largely arrested in G0, did not show protein expression or signaling changes that could account for these results. Reciprocal bone-marrow transplantation between 4E-BP1S82D and wild-type littermates revealed a trend for wild-type mice with 4E-BP1S82D marrow engraftment on high-fat diets to become hyperglycemic after glucose challenge.Conclusions4E-BP1S82D is a single amino acid substitution that induces glucose intolerance in mice. These findings indicate that glucose metabolism may be regulated by CDK1 4E-BP1 phosphorylation independent from mTOR and point towards an unexpected role for cycling cells that transit mitosis in diabetic glucose control.

Project description:BackgroundMutations 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.MethodsMDA-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.ResultsWe 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.ConclusionThe current findings suggest that REDD1 downregulation might improve the anticancer activity of aspirin in a subset of breast tumors.

Project description:BackgroundSarcopenia is the loss of muscle mass/function that occurs during the aging process. The links between mechanistic target of rapamycin (mTOR) activity and muscle development are largely documented, but the role of its downstream targets in the development of sarcopenia is poorly understood. Eukaryotic initiation factor 4E-binding proteins (4E-BPs) are targets of mTOR that repress mRNA translation initiation and are involved in the control of several physiological processes. However, their role in skeletal muscle is still poorly understood. The goal of this study was to assess how loss of 4E-BP1 and 4E-BP2 expression impacts skeletal muscle function and homeostasis in aged mice and to characterize the associated metabolic changes by metabolomic and lipidomic profiling.MethodsTwenty-four-month-old wild-type and whole body 4E-BP1/4E-BP2 double knockout (DKO) mice were used to measure muscle mass and function. Protein homeostasis was measured ex vivo in extensor digitorum longus by incorporation of l-[U-14 C]phenylalanine, and metabolomic and lipidomic profiling of skeletal muscle was performed by Metabolon, Inc.ResultsThe 4E-BP1/2 DKO mice exhibited an increase in muscle mass that was associated with increased grip strength (P < 0.05). Protein synthesis was higher under both basal (+102%, P < 0.05) and stimulated conditions (+65%, P < 0.05) in DKO skeletal muscle. Metabolomic and complex lipid analysis of skeletal muscle revealed robust differences pertaining to amino acid homeostasis, carbohydrate abundance, and certain aspects of lipid metabolism. In particular, levels of most free amino acids were lower within the 4E-BP1/2 DKO muscle. Interestingly, although glucose levels were unchanged, differences were observed in the isobaric compound maltitol/lactitol (33-fold increase, P < 0.01) and in several additional carbohydrate compounds. 4E-BP1/2 depletion also resulted in accumulation of medium-chain acylcarnitines and a 20% lower C2/C0 acylcarnitine ratio (P < 0.01) indicative of reduced β-oxidation.ConclusionsTaken together, these findings demonstrate that deletion of 4E-BPs is associated with perturbed energy metabolism in skeletal muscle and could have beneficial effects on skeletal muscle mass and function in aging mice. They also identify 4E-BPs as potential targets for the treatment of sarcopenia.

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Implication of 4E-BP1 protein dephosphorylation and accumulation in pancreatic cancer cell death induced by combined gemcitabine and TRAIL.

Publications

Implication of 4E-BP1 protein dephosphorylation and accumulation in pancreatic cancer cell death induced by combined gemcitabine and TRAIL.

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