ABSTRACT
Selective autophagy mediates the removal of harmful material from the cytoplasm. This cargo material is selected by cargo receptors, which orchestrate its sequestration within double-membrane autophagosomes and subsequent lysosomal degradation. The cargo receptor p62/SQSTM1 is present in cytoplasmic condensates, and a fraction of them are constantly delivered into lysosomes. However, the molecular composition of the p62 condensates is incompletely understood. To obtain insights into their composition, we develop a method to isolate these condensates and find that p62 condensates are enriched in components of the translation machinery. Furthermore, p62 interacts with translation initiation factors, and eukaryotic initiation factor 2α (eIF2α) and eIF4E are degraded by autophagy in a p62-dependent manner. Thus, p62-mediated autophagy may in part be linked to down-regulation of translation initiation. The p62 condensate isolation protocol developed here may facilitate the study of their contribution to cellular quality control and their roles in health and disease.
Subject(s)
Biomolecular Condensates , Eukaryotic Initiation Factor-2 , Eukaryotic Initiation Factor-4E , RNA-Binding Proteins , Humans , HEK293 Cells , RNA-Binding Proteins/metabolism , Biomolecular Condensates/drug effects , Biomolecular Condensates/metabolism , Eukaryotic Initiation Factor-2/antagonists & inhibitors , Eukaryotic Initiation Factor-2/metabolism , Eukaryotic Initiation Factor-4E/antagonists & inhibitors , Eukaryotic Initiation Factor-4E/metabolism , Autophagy/drug effects , Autophagy/genetics , Wortmannin/pharmacologyABSTRACT
eIF4E plays an important role in regulating tumor growth and angiogenesis, and eIF4E is highly expressed in a variety of lung cancer cell lines. siRNA eIF4E can significantly inhibit the proliferation of lung cancer cells, indicating that inhibition of eIF4E may become a novel anti-tumor target. In the previous study, we synthesized a series of small molecule compounds with the potential to inhibit eIF4E. Among them, the compound EGPI-1 significantly inhibited the proliferation of a variety of lung cancer cells such as A549, NCI-H460, NCI-H1650 and 95D without inhibiting the proliferation of HUVEC cells. Further studies found that EGPI-1 interfered with the eIF4E/eIF4G interaction and inhibited the phosphorylation of eIF4E in NCI-H460 cells. The results of flow cytometry showed that EGPI-1 induced apoptosis and G0/G1 cycle arrest in NCI-H460 cell. Interestingly, we also found that EGPI-1 induced autophagy and DNA damage in NCI-H460 cells. The mechanism results showed that EGPI-1 inhibited the Ras/MNK/ERK/eIF4E signaling pathway. Moreover, EGPI-1 inhibited tube formation of HUVECs, as well as inhibited the neovascularization of CAM, proving the anti-angiogenesis activity of EGPI-1. The NCI-H460 xenograft studies showed that EGPI-1 inhibited tumor growth and angiogenesis in vivo by regulating Ras/MNK/ERK/eIF4E pathway. Our studies proved that eIF4E was a novel target for regulating tumor growth, and the eIF4E/eIF4G interaction inhibitor EGPI-1 was promising to develop into a novel anti-lung cancer drug.
Subject(s)
Angiogenesis Inhibitors/pharmacology , Antineoplastic Agents/pharmacology , Benzylidene Compounds/pharmacology , Eukaryotic Initiation Factor-4E/antagonists & inhibitors , Eukaryotic Initiation Factor-4G/antagonists & inhibitors , Hydrazines/pharmacology , Lung Neoplasms/drug therapy , Thiazoles/pharmacology , A549 Cells , Angiogenesis Inhibitors/chemistry , Angiogenesis Inhibitors/therapeutic use , Animals , Antineoplastic Agents/chemistry , Antineoplastic Agents/therapeutic use , Apoptosis/drug effects , Benzylidene Compounds/chemistry , Benzylidene Compounds/therapeutic use , Cell Cycle Checkpoints/drug effects , Cell Line, Tumor , Cell Proliferation/drug effects , Chick Embryo , Eukaryotic Initiation Factor-4E/genetics , Eukaryotic Initiation Factor-4E/metabolism , Gene Knockdown Techniques , Humans , Hydrazines/chemistry , Hydrazines/therapeutic use , Lung Neoplasms/metabolism , Lung Neoplasms/pathology , Mice , Mice, Inbred BALB C , Mice, Nude , Neovascularization, Pathologic/prevention & control , Signal Transduction/drug effects , Thiazoles/chemistry , Thiazoles/therapeutic use , Up-Regulation , Xenograft Model Antitumor AssaysABSTRACT
Chemoresistance has become a primary hurdle in the therapeutic outcome of hepatocellular carcinoma. Substantial evidences have demonstrated that microRNAs (miRNAs) are closely associated with the chemoresistance of hepatocellular carcinoma (HCC). Our investigation is aimed at testifying the influence of microRNA-15a-5p (miR-15a-5p)/eukaryotic translation initiation factor 4E (eIF4E) on hepatocellular carcinoma resistance to pirarubicin (THP). In our study, miR-15a-5p expression was increased in THP-treated HepG2 cells. Downregulation of miR-15a-5p blocked cell growth and elevated cell apoptosis of HepG2 cells treated with THP. Moreover, eIF4E was verified as a direct target of miR-15a-5p by binding its 3'-UTR, which was confirmed by luciferase report experiment. Additionally, eIF4E was negatively associated with the miR-15a-5p expression in HepG2 cells. Mechanically, eIF4E was proven as a specific downstream of miR-15a-5p and mediated the effects of miR-15a-5p on cell viability and apoptosis of HepG2 cells treated with THP. These findings supported that miR-15a-5p facilitated THP resistance of hepatocellular carcinoma cells by modulating eIF4E, thus providing an experimental basis that miR-15a-5p might act as a novel diagnostic target in hepatocellular carcinoma resistance to THP.
Subject(s)
Carcinoma, Hepatocellular/drug therapy , Carcinoma, Hepatocellular/genetics , Doxorubicin/analogs & derivatives , Eukaryotic Initiation Factor-4E/antagonists & inhibitors , Liver Neoplasms/drug therapy , Liver Neoplasms/genetics , MicroRNAs/antagonists & inhibitors , MicroRNAs/genetics , 3' Untranslated Regions , Antineoplastic Agents/pharmacology , Apoptosis/drug effects , Apoptosis/genetics , Binding Sites/genetics , Carcinoma, Hepatocellular/metabolism , Cell Proliferation/drug effects , Cell Proliferation/genetics , Computational Biology , Doxorubicin/pharmacology , Drug Resistance, Neoplasm/genetics , Eukaryotic Initiation Factor-4E/metabolism , Gene Expression Regulation, Neoplastic/drug effects , Hep G2 Cells , Humans , Liver Neoplasms/metabolism , MicroRNAs/metabolismABSTRACT
OBJECTIVES: Eukaryotic translation initiation factor 4E (eIF4E) is activated in cancers in response to stress. This is regulated by MAP kinase interacting serine/threonine kinase (MNK) in cancerous but not normal cells. Chemoresistance causes treatment failure in advanced cervical cancer. In this study, we addressed chemotherapy effects on eIF4E for cervical cancer and reversal effects by MNK inhibitor cercosporamide for chemo-resistance mitigation. METHODS: Cell assays and mouse tumour models were used to determine the efficacy of cercosporamide. Western blotting was applied to understand the affected cell signaling after cercosporamide treatment. KEY FINDINGS: Cercosporamide spared normal cervical epithelial cells. On cervical cancer cell lines, it showed inhibition of cell growth and migration, and induced apoptosis. Cercosporamide was effective on chemoresistant cancer cells and augmented the efficiency of doxorubicin and cisplatin both in vitro and in vivo. Cercosporamide suppressed eIF4E signaling. Of note, chemotherapy increased p-eIF4E. Cercosporamide abolished chemotherapy-induced eIF4E activation. The higher level of p-eIF4E in cancer cells compared with normal cervical epithelial cells explains the preferential toxicity of cercosporamide. CONCLUSIONS: This work demonstrates the ability of cercosporamide to overcome chemoresistance and highlight preferential inhibition of eIF4E via MNK inhibition in cervical cancer.
Subject(s)
Antineoplastic Agents/pharmacology , Benzofurans/pharmacology , Drug Resistance, Neoplasm/drug effects , Eukaryotic Initiation Factor-4E/antagonists & inhibitors , Intracellular Signaling Peptides and Proteins/antagonists & inhibitors , Molecular Targeted Therapy , Protein Serine-Threonine Kinases/antagonists & inhibitors , Uterine Cervical Neoplasms/metabolism , Animals , Antineoplastic Agents/therapeutic use , Benzofurans/therapeutic use , Cell Line, Tumor , Female , Humans , Mice, Nude , Uterine Cervical Neoplasms/drug therapy , Xenograft Model Antitumor AssaysABSTRACT
The eukaryotic translation initiation factor 4E (eIF4E) is the master regulator of cap-dependent protein synthesis. Overexpression of eIF4E is implicated in diseases such as cancer, where dysregulation of oncogenic protein translation is frequently observed. eIF4E has been an attractive target for cancer treatment. Here we report a high-resolution X-ray crystal structure of eIF4E in complex with a novel inhibitor (i4EG-BiP) that targets an internal binding site, in contrast to the previously described inhibitor, 4EGI-1, which binds to the surface. We demonstrate that i4EG-BiP is able to displace the scaffold protein eIF4G and inhibit the proliferation of cancer cells. We provide insights into how i4EG-BiP is able to inhibit cap-dependent translation by increasing the eIF4E-4E-BP1 interaction while diminishing the interaction of eIF4E with eIF4G. Leveraging structural details, we designed proteolysis targeted chimeras (PROTACs) derived from 4EGI-1 and i4EG-BiP and characterized these on biochemical and cellular levels. We were able to design PROTACs capable of binding eIF4E and successfully engaging Cereblon, which targets proteins for proteolysis. However, these initial PROTACs did not successfully stimulate degradation of eIF4E, possibly due to competitive effects from 4E-BP1 binding. Our results highlight challenges of targeted proteasomal degradation of eIF4E that must be addressed by future efforts.
Subject(s)
Biphenyl Compounds/metabolism , Eukaryotic Initiation Factor-4E/metabolism , Binding Sites , Biphenyl Compounds/chemistry , Biphenyl Compounds/pharmacology , Cell Line, Tumor , Cell Survival/drug effects , Drug Design , Eukaryotic Initiation Factor-4E/antagonists & inhibitors , Eukaryotic Initiation Factor-4E/genetics , Humans , Kinetics , Molecular Docking Simulation , Prodrugs/chemical synthesis , Prodrugs/chemistry , Prodrugs/metabolism , Prodrugs/pharmacology , Protein Interaction Maps/drug effects , Proteolysis/drug effects , Proteomics , Recombinant Proteins/biosynthesis , Recombinant Proteins/chemistry , Recombinant Proteins/isolation & purificationABSTRACT
The clinical efficacy of sorafenib in hepatocellular carcinoma (HCC) is disappointing due to its low response rate and high rates of adverse effects. The eukaryotic translation initiation factor 4F (eIF4F) complex, mainly consisting of eIF4E-eukaryotic translation initiation factor 4G (eIF4G) interaction, is involved in the induction of drug resistance. Herein, we aimed to demonstrate that eIF4E-eIF4G complex inhibition enhanced the effect of sorafenib. The antiproliferation effect of combined treatment was evaluated by MTT assay and colony formation assay. Flow cytometry was used to detect the early cell apoptosis and cell cycle. The specific mechanism was demonstrated using western blot and lentivirus transfection. The combination of sorafenib with eIF4E-eIF4G inhibitors 4E1RCat (structural) or 4EGI-1 (competitive) synergistically inhibited the cell viability and colony formation ability of HCC cells. Moreover, the combined treatment induced more early apoptosis than sorafenib alone through downregulating the Bcl-2 expression. Besides, the coadministration of sorafenib and 4E1RCat or 4EGI-1 synergistically inhibited the expressions of eIF4E, eIF4G and phospho-4E-BP1 in HCC cells while blocking the phosphorylation of 4E-BP1 with lentiviral transfection failed to increase the sensitivity of HCC cells to sorafenib treatment. PI3K-AKT-mTOR signaling was also inhibited by the combined treatment. In a word, eIF4E-eIF4G complex inhibition synergistically enhances the effect of sorafenib in HCC treatment.
Subject(s)
Antineoplastic Agents/pharmacology , Carcinoma, Hepatocellular/pathology , Eukaryotic Initiation Factor-4F/antagonists & inhibitors , Liver Neoplasms/pathology , Sorafenib/pharmacology , Apoptosis/drug effects , Cell Line, Tumor , Cell Movement/drug effects , Down-Regulation , Drug Combinations , Eukaryotic Initiation Factor-4E/antagonists & inhibitors , Eukaryotic Initiation Factor-4G/antagonists & inhibitors , Humans , Phosphatidylinositol 3-Kinases/drug effects , Proto-Oncogene Proteins c-bcl-2/drug effects , TOR Serine-Threonine Kinases/drug effectsABSTRACT
eIF4E plays key roles in protein synthesis and tumorigenesis. It is phosphorylated by the kinases MNK1 and MNK2. Binding of MNKs to eIF4G enhances their ability to phosphorylate eIF4E. Here, we show that mTORC1, a key regulator of mRNA translation and oncogenesis, directly phosphorylates MNK2 on Ser74. This suppresses MNK2 activity and impairs binding of MNK2 to eIF4G. These effects provide a novel mechanism by which mTORC1 signaling impairs the function of MNK2 and thereby decreases eIF4E phosphorylation. MNK2[S74A] knock-in cells show enhanced phosphorylation of eIF4E and S6K1 (i.e., increased mTORC1 signaling), enlarged cell size, and increased invasive and transformative capacities. MNK2[Ser74] phosphorylation was inversely correlated with disease progression in human prostate tumors. MNK inhibition exerted anti-proliferative effects in prostate cancer cells in vitro. These findings define a novel feedback loop whereby mTORC1 represses MNK2 activity and oncogenic signaling through eIF4E phosphorylation, allowing reciprocal regulation of these two oncogenic pathways.
Subject(s)
Eukaryotic Initiation Factor-4E/metabolism , Intracellular Signaling Peptides and Proteins/metabolism , Mechanistic Target of Rapamycin Complex 1/metabolism , Protein Serine-Threonine Kinases/metabolism , Animals , Cell Cycle Checkpoints/drug effects , Cell Line , Cell Proliferation/drug effects , Eukaryotic Initiation Factor-4E/antagonists & inhibitors , Humans , Intracellular Signaling Peptides and Proteins/antagonists & inhibitors , Intracellular Signaling Peptides and Proteins/genetics , Male , Mechanistic Target of Rapamycin Complex 1/antagonists & inhibitors , Mice , Mice, Inbred BALB C , Mice, Transgenic , Morpholines/pharmacology , Mutagenesis, Site-Directed , Phosphorylation/drug effects , Prostatic Neoplasms/metabolism , Prostatic Neoplasms/pathology , Protein Binding , Protein Kinase Inhibitors/pharmacology , Protein Serine-Threonine Kinases/antagonists & inhibitors , Protein Serine-Threonine Kinases/genetics , Signal Transduction/drug effects , Tuberous Sclerosis Complex 2 Protein/genetics , Tuberous Sclerosis Complex 2 Protein/metabolismABSTRACT
Skin squamous cell carcinomas (SCCs) are a major cause of death in patients who have undergone or will undergo organ transplantation. Moreover, these neoplasms cause significant disease and economic burden and diminish patients' life quality. However, no effective treatment or intervention strategies are available. In this study, we investigated the pathologic role of 5'-cap translation, which is regulated by the formation of a ternary initiation factor complex involving eIF4E, eIF4G, and eIF4A1. We detected increased expression of phosphorylated eIF4E, eIF4G, and eIF4A1 in human and murine skin SCCs. The increase in these ternary initiation factor complex proteins was associated with enhanced eIF4E translation targets cyclin D1 and c-Myc. Conversely, small interfering RNA-mediated depletion of eIF4E in human SCC cells (A431 and SCC-13) reduced eIF4G and proteins that regulate the cell cycle and proliferation. Notably, inhibition of Raf/MAPK/extracellular signal-regulated kinase signaling decreased eIF4E and phosphorylated eIF4E accumulation and significantly diminished cell-cycle gene expression and tumor volume of A431-derived xenograft tumors. Furthermore, disrupting the eIF4E with an allosteric inhibitor of eIF4E and eIF4G binding, 4EGI-1, decreased the eIF4E/eIF4G expression and reduced the proliferation. Finally, combined inhibition of the Raf/MAPK/extracellular signal-regulated kinase axis and eIF4E impaired 5'-capâdependent translation and abrogated tumor cell proliferation. These data demonstrate that 5'-capâdependent translation is a potential therapeutic target for abrogating lethal skin SCCs in patients who have undergone or will undergo organ transplantation.
Subject(s)
Carcinoma, Squamous Cell/genetics , Eukaryotic Initiation Factor-4E/antagonists & inhibitors , Gene Expression Regulation, Neoplastic/drug effects , RNA, Small Interfering/pharmacology , Skin Neoplasms/genetics , Allosteric Regulation/drug effects , Animals , Carcinoma, Squamous Cell/drug therapy , Carcinoma, Squamous Cell/pathology , Cell Cycle/drug effects , Cell Cycle/genetics , Cell Line, Tumor , Cell Proliferation/drug effects , Cell Proliferation/genetics , Cyclin D1/genetics , Eukaryotic Initiation Factor-4A/metabolism , Eukaryotic Initiation Factor-4E/genetics , Eukaryotic Initiation Factor-4E/metabolism , Eukaryotic Initiation Factor-4G/metabolism , Humans , MAP Kinase Signaling System/drug effects , MAP Kinase Signaling System/genetics , Mice , Peptide Chain Initiation, Translational/drug effects , Phosphorylation , Proto-Oncogene Proteins c-myc/genetics , RNA Caps/metabolism , RNA, Small Interfering/therapeutic use , Skin/pathology , Skin Neoplasms/drug therapy , Skin Neoplasms/pathology , Xenograft Model Antitumor AssaysABSTRACT
BACKGROUND: The BCL2 inhibitor venetoclax has shown efficacy in several hematologic malignancies, with the greatest response rates in indolent blood cancers such as chronic lymphocytic leukaemia. There is a lower response rate to venetoclax monotherapy in diffuse large B-cell lymphoma (DLBCL). METHODS: We tested inhibitors of cap-dependent mRNA translation for the ability to sensitise DLBCL and mantle cell lymphoma (MCL) cells to apoptosis by venetoclax. We compared the mTOR kinase inhibitor (TOR-KI) MLN0128 with SBI-756, a compound targeting eukaryotic translation initiation factor 4G1 (eIF4G1), a scaffolding protein in the eIF4F complex. RESULTS: Treatment of DLBCL and MCL cells with SBI-756 synergised with venetoclax to induce apoptosis in vitro, and enhanced venetoclax efficacy in vivo. SBI-756 prevented eIF4E-eIF4G1 association and cap-dependent translation without affecting mTOR substrate phosphorylation. In TOR-KI-resistant DLBCL cells lacking eIF4E binding protein-1, SBI-756 still sensitised to venetoclax. SBI-756 selectively reduced translation of mRNAs encoding ribosomal proteins and translation factors, leading to a reduction in protein synthesis rates in sensitive cells. When normal lymphocytes were treated with SBI-756, only B cells had reduced viability, and this correlated with reduced protein synthesis. CONCLUSIONS: Our data highlight a novel combination for treatment of aggressive lymphomas, and establishes its efficacy and selectivity using preclinical models.
Subject(s)
Antineoplastic Combined Chemotherapy Protocols/therapeutic use , Drug Resistance, Neoplasm/drug effects , Eukaryotic Initiation Factor-4E/antagonists & inhibitors , Lymphoma, B-Cell/drug therapy , Molecular Targeted Therapy , Animals , Apoptosis , Bridged Bicyclo Compounds, Heterocyclic/administration & dosage , Cell Proliferation , Female , Humans , Lactams/administration & dosage , Lymphoma, B-Cell/metabolism , Lymphoma, B-Cell/pathology , Mice , Mice, Inbred NOD , Mice, SCID , Quinolones/administration & dosage , Sulfonamides/administration & dosage , Tumor Cells, Cultured , Xenograft Model Antitumor AssaysABSTRACT
In order to suppress 5' cap-mediated translation a highly available inhibitor of the interaction between the 5' mRNA cap and the eIF4E complex has been developed. 4Ei-10 is a member of the class of ProTide compounds and has elevated membrane permeability and is a strong active chemical antagonist for eIF4E. Once taken up by cells it is converted by anchimeric activation of the lipophilic 2-(methylthio) ethyl protecting group and after that Hint1 P-N bond cleavage to N7-(p-chlorophenoxyethyl) guanosine 5'-monophosphate (7-Cl-Ph-Ethyl-GMP). Using this powerful interaction, it has been demonstrated that 4Ei-10 inhibits non-small cell lung cancer (NSCLC) cell growth. In addition, treatment of NSCLC cells with 4Ei-10 results in suppression of translation and diminished expression of a cohort of cellular proteins important to maintaining the malignant phenotype and resisting apoptosis such as Bcl-2, survivin, and ornithine decarboxylase (ODC). Finally, as a result of targeting the translation of anti-apoptotic proteins, NSCLC cells are synergized to be more sensitive to the existing anti-neoplastic treatment gemcitabine currently used in NSCLC therapy.
Subject(s)
Antineoplastic Agents , Carcinoma, Non-Small-Cell Lung , Eukaryotic Initiation Factor-4E , Lung Neoplasms , Nucleotides , Prodrugs , Humans , Antineoplastic Agents/pharmacology , Antineoplastic Agents/therapeutic use , Apoptosis/drug effects , Carcinoma, Non-Small-Cell Lung/drug therapy , Cell Line, Tumor , Cell Survival/drug effects , Deoxycytidine/analogs & derivatives , Deoxycytidine/pharmacology , Drug Interactions , Eukaryotic Initiation Factor-4E/antagonists & inhibitors , Lung Neoplasms/drug therapy , Prodrugs/pharmacology , Nucleotides/pharmacology , Nucleotides/therapeutic use , GemcitabineABSTRACT
Eukaryotic translation initiation factor 4E (eIF4E) is deregulated in patients with renal cell carcinoma (RCC) and associated with poor prognosis, and is activated and regulated by Mnk kinases. In this study, we investigated the anti-RCC potential of a unique Mnk inhibitor cercosporamide. We showed that cercosporamide is active against RCC cells via suppressing growth, survival and migration. Combination indices value indicated that the combination of cercosporamide with sunitinib or temsirolimus are synergistic in RCC. In two independent RCC xenograft mouse models, complete tumor growth arrest or reverse was observed throughout the duration of drug treatment in the combination of cercosporamide with sunitinib or temsirolimus groups. Of note, cercosporamide inhibited RCC angiogenesis via negatively regulating a number of RCC endothelial cellular events including morphogenesis, migration, growth and survival. Mechanistically, we found that cercosporamide suppressed pro-angiogenic factors VEGF and HIFα, inhibited EMT and reduced pro-survival and cell cycle proteins; and furthermore this was attributed to cercosporamide's ability in inhibiting eIF4E. This work demonstrates the anti-RCC activity of cercosporamide through targeting both RCC tumor cells and angiogenesis, and provides the first preclinical proof-of-concept of evidence of Mnk inhibition for RCC treatment.
Subject(s)
Angiogenesis Inhibitors/therapeutic use , Antineoplastic Agents/therapeutic use , Benzofurans/therapeutic use , Carcinoma, Renal Cell/drug therapy , Kidney Neoplasms/drug therapy , Protein Kinase Inhibitors/therapeutic use , Angiogenesis Inhibitors/pharmacology , Animals , Antineoplastic Agents/pharmacology , Benzofurans/pharmacology , Carcinoma, Renal Cell/metabolism , Carcinoma, Renal Cell/pathology , Cell Line, Tumor , Drug Synergism , Eukaryotic Initiation Factor-4E/antagonists & inhibitors , Eukaryotic Initiation Factor-4E/metabolism , Humans , Intracellular Signaling Peptides and Proteins/antagonists & inhibitors , Intracellular Signaling Peptides and Proteins/metabolism , Kidney Neoplasms/metabolism , Kidney Neoplasms/pathology , Mice, SCID , Protein Kinase Inhibitors/pharmacology , Protein Serine-Threonine Kinases/antagonists & inhibitors , Protein Serine-Threonine Kinases/metabolism , Sirolimus/analogs & derivatives , Sirolimus/pharmacology , Sirolimus/therapeutic use , Sunitinib/pharmacology , Sunitinib/therapeutic use , TOR Serine-Threonine Kinases/antagonists & inhibitors , TOR Serine-Threonine Kinases/metabolismABSTRACT
Eukaryotic translation initiation factor 4E (eIF4E) binds the m7GTP cap structure at the 5'-end of mRNAs, stimulating the translation of proteins implicated in cancer cell growth and metastasis. eIF4E is a notoriously challenging target, and most of the reported inhibitors are negatively charged guanine analogues with negligible cell permeability. To overcome these challenges, we envisioned a covalent targeting strategy. As there are no cysteines near the eIF4E cap binding site, we developed a covalent docking approach focused on lysine. Taking advantage of a "make-on-demand" virtual library, we used covalent docking to identify arylsulfonyl fluorides that target a noncatalytic lysine (Lys162) in eIF4E. Guided by cocrystal structures, we elaborated arylsulfonyl fluoride 2 to 12, which to our knowledge is the first covalent eIF4E inhibitor with cellular activity. In addition to providing a new tool for acutely inactivating eIF4E in cells, our computational approach may offer a general strategy for developing selective lysine-targeted covalent ligands.
Subject(s)
Eukaryotic Initiation Factor-4E/antagonists & inhibitors , Lysine/chemistry , Sulfonamides/pharmacology , Binding Sites , Drug Discovery , Eukaryotic Initiation Factor-4E/chemistry , Eukaryotic Initiation Factor-4E/metabolism , HEK293 Cells , Humans , Molecular Docking Simulation , Protein Binding , Sulfonamides/metabolismABSTRACT
Dysregulation of translation initiation factor 4E (eIF4E) activity occurs in various cancers. Mitogen-activated protein kinase (MAPK) interacting kinases 1 and 2 (MNK1 and MNK2) play a fundamental role in activation of eIF4E. Structure-activity relationship-driven expansion of a fragment hit led to discovery of dual MNK1 and MNK2 inhibitors based on a novel pyridine-benzamide scaffold. The compounds possess promising in vitro and in vivo pharmacokinetic profiles and show potent on target inhibition of eIF4E phosphorylation in cells.
Subject(s)
Intracellular Signaling Peptides and Proteins/antagonists & inhibitors , Protein Kinase Inhibitors/pharmacology , Protein Serine-Threonine Kinases/antagonists & inhibitors , Cell Line, Tumor , Crystallography, X-Ray , Drug Discovery , Eukaryotic Initiation Factor-4E/antagonists & inhibitors , Humans , Models, Molecular , Phosphorylation , Protein Kinase Inhibitors/chemical synthesis , Protein Kinase Inhibitors/pharmacokinetics , Structure-Activity RelationshipABSTRACT
BACKGROUND: Targeting ß-catenin has been shown to have great potential therapeutic value in cervical cancer. Because ß-catenin is also essential for normal cells, strategies to specifically target cancer will require identification of druggable factors capable of distinguishing ß-catenin signaling pathways between cancer and normal cells. METHODS: Expression of p-eIF4E and p-ß-catenin was analyzed in malignant and normal cervical tissues and cells. The effects and its underlying mechanisms of targeting MNK and eukaryotic translation initiation factor 4E (eIF4E) were determined in cervical cancer and normal cells. RESULTS: Inhibiting MNK/eIF4E axis selectively targets cervical cancer without affecting normal cervical cells, via suppressing eIF4E-mediated ß-catenin activation. We found that eIF4E phosphorylation was upregulated in cervical cancer cells and tissues but not normal cervical counterparts, and its phosphorylation at Ser 209 activates Wnt/ß-catenin signaling, promotes growth and migration in cervical cancer, in an MNK-dependent manner. MNK inhibition via genetic small interfering RNA (siRNA) knockdown or pharmacologic inhibitor effectively decreased phosphorylation of eIF4E and ß-catenin, leading to reduced ß-catenin activity and transcript levels of Wnt target genes in cervical cancer cells. Consistently, we found that MNK kinase inhibitor is effective in inhibiting proliferation and migration, and inducing apoptosis in cervical cancer but not normal cervical cells. The combination of MNK kinase inhibitor with paclitaxel achieved greater efficacy in cervical cancer cells than paclitaxel alone. CONCLUSIONS: Our work identifies MNK-eIF4E axis as a specific and critical regulator of ß-catenin activity in cervical cancer but not normal cervical cells, and suggests that targeting MNK is a useful therapeutic strategy in cervical cancer.
Subject(s)
Antineoplastic Agents/therapeutic use , Copper-Transporting ATPases/antagonists & inhibitors , Eukaryotic Initiation Factor-4E/antagonists & inhibitors , Protein Kinase Inhibitors/therapeutic use , Uterine Cervical Neoplasms/drug therapy , beta Catenin/metabolism , Antineoplastic Agents/pharmacology , Cell Line, Tumor , Cell Proliferation , Eukaryotic Initiation Factor-4E/metabolism , Female , Humans , Phosphorylation , Protein Kinase Inhibitors/pharmacology , Uterine Cervical Neoplasms/enzymology , Uterine Cervical Neoplasms/pathologyABSTRACT
The eukaryotic initiation factor 4E (eIF4E) is an emerging anticancer drug target for specific anticancer therapy as a promising approach to overcome drug resistance and promote chemotherapy antitumor efficacy. A series of bromophenol-thiazolylhydrazone hybrids were designed, synthesized and evaluated for their antitumor activities. Among of them, the most potent compound 3e (EGPI-1) could inhibit the eIF4E/eIF4G interaction. Further mechanism study demonstrated EGPI-1 played an antitumor role in multiple modes of action including regulating the activity of eIF4E by inhibiting the phosphorylation of eIF4E and 4EBP1, disrupting mitochondrial function through the mTOR/4EBP1 signaling pathway, and inducing autophagy, apoptosis and ROS generation. Moreover, EGPI-1 showed good safety and favorable pharmacokinetic properties in vivo. These observations demonstrate that EGPI-1 may serve as an excellent lead compound for the development of new anticancer drugs that target the eIF4E/eIF4G interface and as a chemical genetic probe to investigate the role of the eIF4E in biological processes and human diseases.
Subject(s)
Antineoplastic Agents/pharmacology , Eukaryotic Initiation Factor-4E/antagonists & inhibitors , Eukaryotic Initiation Factor-4G/antagonists & inhibitors , Hydrazones/pharmacology , Thiazoles/pharmacology , Animals , Antineoplastic Agents/chemical synthesis , Antineoplastic Agents/pharmacokinetics , Antineoplastic Agents/toxicity , Apoptosis/drug effects , Autophagy/drug effects , Binding Sites , Cell Line, Tumor , Cell Proliferation/drug effects , Drug Design , Eukaryotic Initiation Factor-4E/chemistry , Eukaryotic Initiation Factor-4E/metabolism , Eukaryotic Initiation Factor-4G/metabolism , Female , Human Umbilical Vein Endothelial Cells , Humans , Hydrazones/chemical synthesis , Hydrazones/pharmacokinetics , Hydrazones/toxicity , Male , Mice , Molecular Docking Simulation , Phosphorylation , Protein Binding , Rats, Sprague-Dawley , Reactive Oxygen Species/metabolism , Signal Transduction/drug effects , Thiazoles/chemical synthesis , Thiazoles/pharmacokinetics , Thiazoles/toxicity , Xenograft Model Antitumor AssaysABSTRACT
Resistance to adjuvant chemotherapy remains therapeutic challenge in nasopharyngeal carcinoma (NPC). In this work, we demonstrate that targeting eukaryotic translation initiation factor 4E (eIF4E) is a potential sensitizing strategy to overcome chemoresistance in NPC. We observe the aberrant activation of eIF4E and translational upregulation of eIF4E-regulated oncogenes in NPC cell after pro-longed exposure of cisplatin. Functional analysis demonstrates that eIF4E depletion effectively inhibits proliferation and induces apoptosis in cisplatin-resistant NPC cells. Consistently, eIF4E knockdown significantly enhances cisplatin efficacy in cisplatin-sensitive cells. We identify eIF4E as a therapeutically actionable targets by showing that ribavirin, an anti-viral drug, phenocopies the effects of eIF4E knockdown in NPC. We further demonstrate that ribavirin acts on chemoresistant NPC cells through suppressing eIF4E activity and oncogenic protein translation. Using two independent NPC xenograft mouse models, we show that ribavirin not only is effective in inhibiting chemoresistant NPC growth but also significantly augments the inhibitory effects of cisplatin efficacy in vivo without causing significant toxicity in mice. Taken together, our work shows an activation of eIF4E-mediated growth and survival mechanisms in response to chemotherapy and suggests that inhibition of eIF4E activity represents an attractive sensitizing strategy for NPC treatment. Our findings also suggest that ribavirin is a useful addition to the treatment armamentarium for NPC.
Subject(s)
Drug Resistance, Neoplasm/genetics , Eukaryotic Initiation Factor-4E/metabolism , Nasopharyngeal Carcinoma/drug therapy , Animals , Antineoplastic Combined Chemotherapy Protocols/pharmacology , Cell Line, Tumor , Cisplatin/administration & dosage , Cisplatin/pharmacology , Eukaryotic Initiation Factor-4E/antagonists & inhibitors , Eukaryotic Initiation Factor-4E/genetics , Humans , Mice, SCID , Molecular Targeted Therapy/methods , Nasopharyngeal Carcinoma/genetics , Oncogenes , Phosphorylation/drug effects , Protein Biosynthesis , Ribavirin/administration & dosage , Ribavirin/pharmacology , Serine/metabolismABSTRACT
Protein disorder plays a crucial role in signal transduction and is key for many cellular processes including transcription, translation, and cell cycle. Within the intrinsically disordered protein interactome, the α-helix is commonly used for binding, which is induced via a disorder-to-order transition. Because the targeting of protein-protein interactions (PPIs) remains an important challenge in medicinal chemistry, efforts have been made to mimic this secondary structure for rational inhibitor design through the use of stapled peptides. Cap-dependent mRNA translation is regulated by two disordered proteins, 4E-BP1 and eIF4G, that inhibit or stimulate the activity of the m7G cap-binding translation initiation factor, eIF4E, respectively. Both use an α-helical motif for eIF4E binding, warranting the investigation of stapled peptide mimics for manipulating eIF4E PPIs. Herein, we describe our efforts toward this goal, resulting in the synthesis of a cell-active stapled peptide for further development in manipulating aberrant cap-dependent translation in human diseases.
Subject(s)
Adaptor Proteins, Signal Transducing/chemistry , Cell Cycle Proteins/chemistry , Drug Design , Eukaryotic Initiation Factor-4E/chemistry , Eukaryotic Initiation Factor-4G/chemistry , Peptide Fragments/chemical synthesis , Adaptor Proteins, Signal Transducing/antagonists & inhibitors , Adaptor Proteins, Signal Transducing/genetics , Amino Acid Sequence , Cell Cycle Proteins/antagonists & inhibitors , Cell Cycle Proteins/genetics , Cell Line, Tumor , Cell Proliferation/drug effects , Cell Survival/drug effects , Eukaryotic Initiation Factor-4E/antagonists & inhibitors , Eukaryotic Initiation Factor-4E/genetics , Eukaryotic Initiation Factor-4G/antagonists & inhibitors , Eukaryotic Initiation Factor-4G/genetics , Humans , Inhibitory Concentration 50 , Kinetics , Peptide Fragments/chemistry , Peptide Fragments/pharmacology , Plasmids , Protein BindingABSTRACT
Rbm38 is a p53 target and an RNA-binding protein known to suppress p53 translation by preventing eukaryotic translation initiation factor 4E (eIF4E) from binding to p53 mRNA. In this study, we show that synthetic peptides corresponding to the binding interface between Rbm38 and eIF4E, including an 8 amino acid peptide (Pep8) derived from Rbm38, are effective in relieving Rbm38-mediated repression of p53. Molecular simulations showed that Ser-6 in Pep8 forms a hydrogen bond with Asp-202 in eIF4E. Substitution of Ser-6 with Lys, but not with Asp, enhanced the ability of Pep8 to inhibit the Rbm38-eIF4E complex. Importantly, Pep8 alone or together with a low dose of doxorubicin potently induced p53 expression and suppressed colony and tumor sphere formation and xenograft tumors in Rbm38- and p53-dependent manners. Together, we conclude that modulating the Rbm38-eIF4E complex may be explored as a therapeutic strategy for cancers that carry wild-type p53. SIGNIFICANCE: Disruption of the Rbm38-eIF4E complex via synthetic peptides induces wild-type p53 expression, suppresses tumor growth and progression, and may serve as a novel cancer therapeutic strategy.
Subject(s)
Breast Neoplasms/drug therapy , Eukaryotic Initiation Factor-4E/antagonists & inhibitors , Gene Expression Regulation, Neoplastic/drug effects , Peptide Fragments/pharmacology , Protein Interaction Domains and Motifs/drug effects , RNA-Binding Proteins/antagonists & inhibitors , Tumor Suppressor Protein p53/metabolism , Animals , Antibiotics, Antineoplastic/pharmacology , Apoptosis , Breast Neoplasms/enzymology , Breast Neoplasms/pathology , Cell Proliferation , Doxorubicin/pharmacology , Eukaryotic Initiation Factor-4E/genetics , Eukaryotic Initiation Factor-4E/metabolism , Female , Humans , Mice , Mice, Nude , RNA-Binding Proteins/genetics , RNA-Binding Proteins/metabolism , Tumor Cells, Cultured , Tumor Suppressor Protein p53/genetics , Xenograft Model Antitumor AssaysABSTRACT
BET inhibitors (BETi), which target transcription of key oncogenic genes, are currently being evaluated in early-phase clinical trials. However, because BETis show limited single-agent activity, there is increasing interest in identifying signaling pathways to enhance the efficacy of BETis. Here, we demonstrate increased MNK kinase-dependent eIF4E phosphorylation following treatment with BETis, indicating activation of a prosurvival feedback mechanism in response to BETis. BET PROTACs, which promote degradation of BET proteins, also induced eIF4E phosphorylation in cancer cells. Mechanistically, we show that the effect of BETis on MNK-eIF4E phosphorylation was mediated by p38 MAPKs. We also show that BETis suppressed RacGAP1 to induce Rac signaling-mediated eIF4E phosphorylation. Significantly, MNK inhibitors and MNK1/2 knockdown enhanced the efficacy of BETis in suppressing proliferation of cancer cells in vitro and in a syngeneic mouse model. Together, these results demonstrate a novel prosurvival feedback signaling induced by BETis, providing a mechanistic rationale for combination therapy with BET and MNK inhibitors for synergistic inhibition of cancer cells.
Subject(s)
Acetanilides/administration & dosage , Aniline Compounds/administration & dosage , Azepines/administration & dosage , Eukaryotic Initiation Factor-4E/metabolism , Heterocyclic Compounds, 3-Ring/administration & dosage , Protein Serine-Threonine Kinases/metabolism , Purines/administration & dosage , Thyroid Neoplasms/drug therapy , Triazoles/administration & dosage , Acetanilides/pharmacology , Aniline Compounds/pharmacology , Animals , Azepines/pharmacology , Cell Culture Techniques , Cell Line, Tumor , Cell Proliferation/drug effects , Drug Synergism , Eukaryotic Initiation Factor-4E/antagonists & inhibitors , Heterocyclic Compounds, 3-Ring/pharmacology , Humans , Mice , Phosphorylation/drug effects , Purines/pharmacology , Signal Transduction , Thyroid Neoplasms/metabolism , Triazoles/pharmacology , Xenograft Model Antitumor AssaysABSTRACT
Although cancer patients initially respond well to chemotherapy, they eventually develop resistance and relapse. In this work, we demonstrate that eIF4E-targeting therapy is a potential sensitizing strategy for overcoming chemoresistance and progression in cancer. We show that ribavirin, an anti-viral drug and pharmacological eIF4E inhibitor, effectively inhibits proliferation and decreases viability of paclitaxel-resistant cervical cancer and 5-FU-resistant colon cancer cells while is less toxic to human fibroblast cells. Importantly, oral administration of ribavirin significantly inhibits paclitaxel-resistant colon and 5-FU-resistant cervical cancer growth in xenograft mouse cancer model without causing significant toxicity in mice. Consistently, combination of ribavirin with paclitaxel or 5-FU sensitizes colon and cervical cancer cells to chemotherapeutic agents treatment in vitro and in vivo. We further confirm that the mechanism of the action of ribavirin in chemoresistant cancer cells is through suppressing eIF4E function. In addition, specific eIF4E knockdown via two independent siRNA mimics the effects of ribavirin in chemoresistant colon and cervical cancer cells. Cell cycle analysis indicate that ribavirin enhances the anti-proliferative effect of 5-FU by additionally arresting cells at G2/M phase via increasing cyclin B1, p-histone H3(Ser10) and Mad2 levels. Our work demonstrates that eIF4E inhibition using inhibitor or siRNA, either as single agent or in combination, could sensitize chemoresistant cancer cells to paclitaxel or 5-FU treatment and thereby improving the efficacy of chemodrug. Our findings demonstrate the therapeutic value of inhibiting eIF4E, particularly in chemoresistant cancers. Our findings also suggest ribavirin as a promising sensitizing drug for cancer treatment.