Computational inference of eIF4F complex function and structure in human cancers.

The canonical eukaryotic initiation factor 4F (eIF4F) complex, composed of eIF4G1, eIF4A1, and the cap-binding protein eIF4E, plays a crucial role in cap-dependent translation initiation in eukaryotic cells. An alternative cap-independent initiation can occur, involving only eIF4G1 and eIF4A1 through internal ribosome entry sites (IRESs). This mechanism is considered complementary to cap-dependent initiation, particularly in tumors under stress conditions. However, the selection and molecular mechanism of specific translation initiation remains poorly understood in human cancers. Thus, we analyzed gene copy number variations (CNVs) in TCGA tumor samples and found frequent amplification of genes involved in translation initiation. Copy number gains in EIF4G1 and EIF3E frequently co-occur across human cancers. Additionally, EIF4G1 expression strongly correlates with genes from cancer cell survival pathways including cell cycle and lipogenesis, in tumors with EIF4G1 amplification or duplication. Furthermore, we revealed that eIF4G1 and eIF4A1 protein levels strongly co-regulate with ribosomal subunits, eIF2, and eIF3 complexes, while eIF4E co-regulates with 4E-BP1, ubiquitination, and ESCRT proteins. Utilizing Alphafold predictions, we modeled the eIF4F structure with and without eIF4E binding. For cap-dependent initiation, our modeling reveals extensive interactions between the N-terminal eIF4E-binding domain of eIF4G1 and eIF4E. Furthermore, the eIF4G1 HEAT-2 domain positions eIF4E near the eIF4A1 N-terminal domain (NTD), resulting in the collaborative enclosure of the RNA binding cavity within eIF4A1. In contrast, during cap-independent initiation, the HEAT-2 domain directly binds the eIF4A1-NTD, leading to a stronger interaction between eIF4G1 and eIF4A1, thus closing the mRNA binding cavity without the involvement of eIF4E.

The role of eIF4F-driven mRNA translation in regulating the tumour microenvironment.

Cells can rapidly adjust their proteomes in dynamic environments by regulating mRNA translation. There is mounting evidence that dysregulation of mRNA translation supports the survival and adaptation of cancer cells, which has stimulated clinical interest in targeting elements of the translation machinery and, in particular, components of the eukaryotic initiation factor 4F (eIF4F) complex such as eIF4E. However, the effect of targeting mRNA translation on infiltrating immune cells and stromal cells in the tumour microenvironment (TME) has, until recently, remained unexplored. In this Perspective article, we discuss how eIF4F-sensitive mRNA translation controls the phenotypes of key non-transformed cells in the TME, with an emphasis on the underlying therapeutic implications of targeting eIF4F in cancer. As eIF4F-targeting agents are in clinical trials, we propose that a broader understanding of their effect on gene expression in the TME will reveal unappreciated therapeutic vulnerabilities that could be used to improve the efficacy of existing cancer therapies.

Inhibition of MYC translation through targeting of the newly identified PHB-eIF4F complex as a therapeutic strategy in CLL.

Dysregulation of messenger RNA (mRNA) translation, including preferential translation of mRNA with complex 5' untranslated regions such as the MYC oncogene, is recognized as an important mechanism in cancer. Here, we show that both human and murine chronic lymphocytic leukemia (CLL) cells display a high translation rate, which is inhibited by the synthetic flavagline FL3, a prohibitin (PHB)-binding drug. A multiomics analysis performed in samples from patients with CLL and cell lines treated with FL3 revealed the decreased translation of the MYC oncogene and of proteins involved in cell cycle and metabolism. Furthermore, inhibiting translation induced a proliferation arrest and a rewiring of MYC-driven metabolism. Interestingly, contrary to other models, the RAS-RAF-(PHBs)-MAPK pathway is neither impaired by FL3 nor implicated in translation regulation in CLL cells. Here, we rather show that PHBs are directly associated with the eukaryotic initiation factor (eIF)4F translation complex and are targeted by FL3. Knockdown of PHBs resembled FL3 treatment. Importantly, inhibition of translation controlled CLL development in vivo, either alone or combined with immunotherapy. Finally, high expression of translation initiation-related genes and PHBs genes correlated with poor survival and unfavorable clinical parameters in patients with CLL. Overall, we demonstrated that translation inhibition is a valuable strategy to control CLL development by blocking the translation of several oncogenic pathways including MYC. We also unraveled a new and direct role of PHBs in translation initiation, thus creating new therapeutic opportunities for patients with CLL.

CDKAL1 Drives the Maintenance of Cancer Stem-Like Cells by Assembling the eIF4F Translation Initiation Complex.

Cancer stem-like cells (CSCs) have a unique translation mode, but little is understood about the process of elongation, especially the contribution of tRNA modifications to the maintenance of CSCs properties. Here, it is reported that, contrary to the initial aim, a tRNA-modifying methylthiotransferase CDKAL1 promotes CSC-factor SALL2 synthesis by assembling the eIF4F translation initiation complex. CDKAL1 expression is upregulated in patients with worse prognoses and is essential for maintaining CSCs in rhabdomyosarcoma (RMS) and common cancers. Translatome analysis reveals that a group of mRNAs whose translation is CDKAL1-dependent contains cytosine-rich sequences in the 5' untranslated region (5'UTR). Mechanistically, CDKAL1 promotes the translation of such mRNAs by organizing the eIF4F translation initiation complex. This complex formation does not require the enzyme activity of CDKAL1 but requires only the NH2 -terminus domain of CDKAL1. Furthermore, sites in CDKAL1 essential for forming the eIF4F complex are identified and discovered candidate inhibitors of CDKAL1-dependent translation.

Ferritin Iron Responsive Elements (IREs) mRNA Interacts with eIF4G and Activates In Vitro Translation.

BACKGROUND: Eukaryotic initiation factor (eIF) 4G plays an important role in assembling the initiation complex required for ribosome binding to mRNA and promote translation. Translation of ferritin IRE mRNAs is regulated by iron through iron responsive elements (IREs) and iron regulatory protein (IRP). The noncoding IRE stem-loop (30-nt) structure control synthesis of proteins in iron trafficking, cell cycling, and nervous system function. High cellular iron concentrations promote IRE RNA binding to ribosome and initiation factors, and allow synthesis of ferritin. METHODS: In vitro translation assay was performed in depleted wheat germ lysate with supplementation of initiation factors. Fluorescence spectroscopy was used to characterize eIF4F/IRE binding. RESULTS: Eukaryotic initiation factor eIF4G increases the translation of ferritin through binding to stem loop structure of iron responsive elements mRNA in the 5'-untranslated region. Our translation experiment demonstrated that exogenous addition of eIF4G selectively enhanced the translation of ferritin IRE RNA in depleted WG lysate. However, eIF4G facilitates capped IRE RNA translation significantly higher than uncapped IRE RNA translation. Addition of iron with eIF4G to depleted WG lysate significantly enhanced translation for both IRE mRNA (capped and uncapped), confirming the contribution of eIF4G and iron as a potent enhancer of ferritin IRE mRNA translation. Fluorescence data revealed that ferritin IRE strongly interacts to eIF4G (Kd = 63 nM), but not eIF4E. Further equilibrium studies showed that iron enhanced (~4-fold) the ferritin IRE binding to eIF4G. The equilibrium binding effects of iron on ferritin IRE RNA/eIFs interaction and the temperature dependence of this reaction were measured and compared. The Kd values for the IRE binding to eIF4G ranging from 18.2 nM to 63.0 nM as temperature elevated from 5 °C to 25 °C, while the presence of iron showed much stronger affinity over the same range of temperatures. Thermodynamic parameter revealed that IRE RNA binds to eIF4G with ΔH = -42.6 ± 3.3 kJ. mole-1, ΔS = -11.5 ± 0.4 J. mole-1K-1, and ΔG = -39.2 ± 2.7 kJ. mole-1, respectively. Furthermore, addition of iron significantly changed the values of thermodynamic parameters, favoring stable complex formation, thus favoring efficient protein synthesis. This study first time demonstrate the participation of eIF4G in ferritin IRE mRNA translation. CONCLUSIONS: eIF4G specifically interacts with ferritin IRE RNA and promotes eIF4G-dependent translation.

The domains of yeast eIF4G, eIF4E and the cap fine-tune eIF4A activities through an intricate network of stimulatory and inhibitory effects.

Translation initiation in eukaryotes starts with the recognition of the mRNA 5'-cap by eIF4F, a hetero-trimeric complex of eIF4E, the cap-binding protein, eIF4A, a DEAD-box helicase, and eIF4G, a scaffold protein. eIF4G comprises eIF4E- and eIF4A-binding domains (4E-BD, 4A-BD) and three RNA-binding regions (RNA1-RNA3), and interacts with eIF4A, eIF4E, and with the mRNA. Within the eIF4F complex, the helicase activity of eIF4A is increased. We showed previously that RNA3 of eIF4G is important for the stimulation of the eIF4A conformational cycle and its ATPase and helicase activities. Here, we dissect the interplay between the eIF4G domains and the role of the eIF4E/cap interaction in eIF4A activation. We show that RNA2 leads to an increase in the fraction of eIF4A in the closed state, an increased RNA affinity, and faster RNA unwinding. This stimulatory effect is partially reduced when the 4E-BD is present. eIF4E binding to the 4E-BD then further inhibits the helicase activity and closing of eIF4A, but does not affect the RNA-stimulated ATPase activity of eIF4A. The 5'-cap renders the functional interaction of mRNA with eIF4A less efficient. Overall, the activity of eIF4A at the 5'-cap is thus fine-tuned by a delicately balanced network of stimulatory and inhibitory interactions.

RNA-binding protein FXR1 drives cMYC translation by recruiting eIF4F complex to the translation start site.

Fragile X-related protein-1 (FXR1) gene is highly amplified in patients with ovarian cancer, and this amplification is associated with increased expression of both FXR1 mRNA and protein. FXR1 expression directly associates with the survival and proliferation of cancer cells. Surface sensing of translation (SUnSET) assay demonstrates that FXR1 enhances the overall translation in cancer cells. Reverse-phase protein array (RPPA) reveals that cMYC is the key target of FXR1. Mechanistically, FXR1 binds to the AU-rich elements (ARE) present within the 3' untranslated region (3'UTR) of cMYC and stabilizes its expression. In addition, the RGG domain in FXR1 interacts with eIF4A1 and eIF4E proteins. These two interactions of FXR1 result in the circularization of cMYC mRNA and facilitate the recruitment of eukaryotic translation initiation factors to the translation start site. In brief, we uncover a mechanism by which FXR1 promotes cMYC levels in cancer cells.

Assessing eukaryotic initiation factor 4F subunit essentiality by CRISPR-induced gene ablation in the mouse.

Eukaryotic initiation factor (eIF) 4F plays a central role in the ribosome recruitment phase of cap-dependent translation. This heterotrimeric complex consists of a cap binding subunit (eIF4E), a DEAD-box RNA helicase (eIF4A), and a large bridging protein (eIF4G). In mammalian cells, there are two genes encoding eIF4A (eIF4A1 and eIF4A2) and eIF4G (eIF4G1 and eIF4G3) paralogs that can assemble into eIF4F complexes. To query the essential nature of the eIF4F subunits in normal development, we used CRISPR/Cas9 to generate mouse strains with targeted ablation of each gene encoding the different eIF4F subunits. We find that Eif4e, Eif4g1, and Eif4a1 are essential for viability in the mouse, whereas Eif4g3 and Eif4a2 are not. However, Eif4g3 and Eif4a2 do play essential roles in spermatogenesis. Crossing of these strains to the lymphoma-prone Eµ-Myc mouse model revealed that heterozygosity at the Eif4e or Eif4a1 loci significantly delayed tumor onset. Lastly, tumors derived from Eif4e∆38 fs/+/Eµ-Myc or Eif4a1∆5 fs/+/Eµ-Myc mice show increased sensitivity to the chemotherapeutic agent doxorubicin, in vivo. Our study reveals that eIF4A2 and eIF4G3 play non-essential roles in gene expression regulation during embryogenesis; whereas reductions in eIF4E or eIF4A1 levels are protective against tumor development in a murine Myc-driven lymphoma setting.

Deep computational analysis details dysregulation of eukaryotic translation initiation complex eIF4F in human cancers.

eIF4F plays diverse roles in human cancers, which complicate the development of an overarching understanding of its functional and regulatory impacts across tumor types. Typically, eIF4F drives initiation from the mRNA 5' end (cap) and is composed of eIF4G1, eIF4A1, and cap-binding eIF4E. Cap-independent initiation is possible without eIF4E, from internal ribosomal entry sites (IRESs). By analyzing large public datasets, we found that cancers selectively overexpress EIF4G1 more than EIF4E. That expression imbalance supports EIF4G1 as a prognostic indicator in patients with cancer. It also attenuates "housekeeping" pathways that are usually regulated in a tissue-specific manner via cap-dependent initiation in healthy tissues and reinforce regulation of cancer-preferred pathways in cap-independent contexts. Cap-independent initiation is mechanistically attributable to eIF4G1 hyperphosphorylation that promotes binding to eIF4A1 and reduced eIF4E availability. Collectively, these findings reveal a novel model of dysregulated eIF4F function and highlight the clinical relevance of cap-(in)dependent initiation in cancer.

Translation Initiation Regulated by RNA-Binding Protein in Mammals: The Modulation of Translation Initiation Complex by Trans-Acting Factors.

Protein synthesis is tightly regulated at each step of translation. In particular, the formation of the basic cap-binding complex, eukaryotic initiation factor 4F (eIF4F) complex, on the 5' cap structure of mRNA is positioned as the rate-limiting step, and various cis-elements on mRNA contribute to fine-tune spatiotemporal protein expression. The cis-element on mRNAs is recognized and bound to the trans-acting factors, which enable the regulation of the translation rate or mRNA stability. In this review, we focus on the molecular mechanism of how the assembly of the eIF4F complex is regulated on the cap structure of mRNAs. We also summarize the fine-tuned regulation of translation initiation by various trans-acting factors through cis-elements on mRNAs.

KRASG12C Can Either Promote or Impair Cap-Dependent Translation in Two Different Lung Adenocarcinoma Cell Lines.

KRASG12C is among the most common oncogenic mutations in lung adenocarcinoma and a promising target for treatment by small-molecule inhibitors. KRAS oncogenic signaling is responsible for modulation of tumor microenvironment, with translation factors being among the most prominent deregulated targets. In the present study, we used TALENs to edit EGFRWT CL1-5 and A549 cells for integration of a Tet-inducible KRASG12C expression system. Subsequent analysis of both cell lines showed that cap-dependent translation was impaired in CL1-5 cells via involvement of mTORC2 and NF-κB. In contrast, in A549 cells, which additionally harbor the KRASG12S mutation, cap-dependent translation was favored via recruitment of mTORC1, c-MYC and the positive regulation of eIF4F complex. Downregulation of eIF1, eIF5 and eIF5B in the same cell line suggested a stringency loss of start codon selection during scanning of mRNAs. Puromycin staining and polysome profile analysis validated the enhanced translation rates in A549 cells and the impaired cap-dependent translation in CL1-5 cells. Interestingly, elevated translation rates were restored in CL1-5 cells after prolonged induction of KRASG12C through an mTORC1/p70S6K-independent way. Collectively, our results suggest that KRASG12C signaling differentially affects the regulation of the translational machinery. These differences could provide additional insights and facilitate current efforts to effectively target KRAS.

Cancer Plasticity: The Role of mRNA Translation.

Tumor progression is associated with dedifferentiated histopathologies concomitant with cancer cell survival within a changing, and often hostile, tumor microenvironment. These processes are enabled by cellular plasticity, whereby intracellular cues and extracellular signals are integrated to enable rapid shifts in cancer cell phenotypes. Cancer cell plasticity, at least in part, fuels tumor heterogeneity and facilitates metastasis and drug resistance. Protein synthesis is frequently dysregulated in cancer, and emerging data suggest that translational reprograming collaborates with epigenetic and metabolic programs to effectuate phenotypic plasticity of neoplasia. Herein, we discuss the potential role of mRNA translation in cancer cell plasticity, highlight emerging histopathological correlates, and deliberate on how this is related to efforts to improve understanding of the complex tumor ecology.

Rocaglates Induce Gain-of-Function Alterations to eIF4A and eIF4F.

Rocaglates are a diverse family of biologically active molecules that have gained tremendous interest in recent years due to their promising activities in pre-clinical cancer studies. As a result, this family of compounds has been significantly expanded through the development of efficient synthetic schemes. However, it is unknown whether all of the members of the rocaglate family act through similar mechanisms of action. Here, we present a comprehensive study comparing the biological activities of >200 rocaglates to better understand how the presence of different chemical entities influences their biological activities. Through this, we find that most rocaglates preferentially repress the translation of mRNAs containing purine-rich 5' leaders, but certain rocaglates lack this bias in translation repression. We also uncover an aspect of rocaglate mechanism of action in which the pool of translationally active eIF4F is diminished due to the sequestration of the complex onto RNA.

Repression of oncogenic cap-mediated translation by 4Ei-10 diminishes proliferation, enhances chemosensitivity and alters expression of malignancy-related proteins in mesothelioma.

Activated cap-dependent translation promotes cancer by stimulating translation of mRNAs encoding malignancy-promoting proteins. The nucleoside monophosphate Protide, 4Ei-10, undergoes intracellular uptake and conversion by Hint1 to form 7-Cl-Ph-Ethyl-GMP. 7-Cl-Ph-Ethyl-GMP is an analog of cap and inhibits protein translation by binding and sequestering eIF4E thus blocking eIF4E from binding to the mRNA cap. The effects of inhibiting translation initiation by disruption of the eIF4F complex with 4Ei-10 were examined in malignant mesothelioma (MM). In a cell-free assay system, formation of the eIF4F complex was disabled in response to exposure to 4Ei-10. Treatment of MM with 4Ei-10 resulted in decreased cell proliferation, increased sensitivity to pemetrexed and altered expression of malignancy-related proteins. In light of these findings, suppression of translation initiation by small molecule inhibitors like 4Ei-10 alone or in combination with pemetrexed represents an encouraging strategy meriting further evaluation in the treatment of MM.

Single-stranded regions modulate conformational dynamics and ATPase activity of eIF4A to optimize 5'-UTR unwinding.

Eukaryotic translation initiation requires unwinding of secondary structures in the 5'-untranslated region of mRNA. The DEAD-box helicase eIF4A is thought to unwind structural elements in the 5'-UTR in conjunction with eIF4G and eIF4B. Both factors jointly stimulate eIF4A activities by modulation of eIF4A conformational cycling between open and closed states. Here we examine how RNA substrates modulate eIF4A activities. The RNAs fall into two classes: Short RNAs only partially stimulate the eIF4A ATPase activity, and closing is rate-limiting for the conformational cycle. By contrast, longer RNAs maximally stimulate ATP hydrolysis and promote closing of eIF4A. Strikingly, the rate constants of unwinding do not correlate with the length of a single-stranded region preceding a duplex, but reach a maximum for RNA with a single-stranded region of six nucleotides. We propose a model in which RNA substrates affect eIF4A activities by modulating the kinetic partitioning of eIF4A between futile, unproductive, and productive cycles.

Targetome analysis of chaperone-mediated autophagy in cancer cells.

Chaperone-mediated autophagy (CMA) is a lysosomal degradation pathway of select soluble proteins. Nearly one-third of the soluble proteins are predicted to be recognized by this pathway, yet only a minor fraction of this proteome has been identified as CMA substrates in cancer cells. Here, we undertook a quantitative multiplex mass spectrometry approach to study the proteome of isolated lysosomes in cancer cells during CMA-activated conditions. By integrating bioinformatics analyses, we identified and categorized proteins of multiple cellular pathways that were specifically targeted by CMA. Beyond verifying metabolic pathways, we show that multiple components involved in select biological processes, including cellular translation, was specifically targeted for degradation by CMA. In particular, several proteins of the translation initiation complex were identified as bona fide CMA substrates in multiple cancer cell lines of distinct origin and we show that CMA suppresses cellular translation. We further show that the identified CMA substrates display high expression in multiple primary cancers compared to their normal counterparts. Combined, these findings uncover cellular processes affected by CMA and reveal a new role for CMA in the control of translation in cancer cells. Abbreviations: 6-AN: 6-aminonicotinamide; ACTB: actin beta; AR7: atypical retinoid 7; CHX: cycloheximide; CMA: chaperone-mediated autophagy; CQ: chloroquine; CTS: cathepsins; DDX3X: DEAD-box helicase 3 X-linked; EEF2: eukaryotic translation elongation factor 2; EIF4A1: eukaryotic translation initiation factor 4A1; EIF4H: eukaryotic translation initiation factor 4H; GEO: Gene Expression Omnibus; GO: Gene Ontology; GSEA: gene set enrichment analysis; HK2: hexokinase 2; HSPA8/HSC70: heat shock protein family A (Hsp70) member 8; LAMP: lysosomal-associated membrane protein; LDHA: lactate dehydrogenase A; NES: normalized enrichment score; NFKBIA: NFKB inhibitor alpha; PCA: principle component analysis; PQ: paraquat; S.D.: standard deviation; SUnSET: surface sensing of translation; TMT: tandem mass tags; TOMM40/TOM40: translocase of outer mitochondrial membrane 40.

Nitric oxide triggers the assembly of "type II" stress granules linked to decreased cell viability.

We show that 3-morpholinosydnonimine (SIN-1)-induced nitric oxide (NO) triggers the formation of SGs. Whereas the composition of NO-induced SGs is initially similar to sodium arsenite (SA)-induced type I (cytoprotective) SGs, the progressive loss of eIF3 over time converts them into pro-death (type II) SGs. NO-induced SG assembly requires the phosphorylation of eIF2α, but the transition to type II SGs is temporally linked to the mTOR-regulated displacement of eIF4F complexes from the m7 guanine cap. Whereas SA does not affect mitochondrial morphology or function, NO alters mitochondrial integrity and function, resulting in increased ROS production, decreased cytoplasmic ATP, and plasma membrane permeabilization, all of which are supported by type II SG assembly. Thus, cellular energy balance is linked to the composition and function of NO-induced SGs in ways that determine whether cells live or die.

Translational control of tumor immune escape via the eIF4F-STAT1-PD-L1 axis in melanoma.

Preventing the immune escape of tumor cells by blocking inhibitory checkpoints, such as the interaction between programmed death ligand-1 (PD-L1) and programmed death-1 (PD-1) receptor, is a powerful anticancer approach. However, many patients do not respond to checkpoint blockade. Tumor PD-L1 expression is a potential efficacy biomarker, but the complex mechanisms underlying its regulation are not completely understood. Here, we show that the eukaryotic translation initiation complex, eIF4F, which binds the 5' cap of mRNAs, regulates the surface expression of interferon-γ-induced PD-L1 on cancer cells by regulating translation of the mRNA encoding the signal transducer and activator of transcription 1 (STAT1) transcription factor. eIF4F complex formation correlates with response to immunotherapy in human melanoma. Pharmacological inhibition of eIF4A, the RNA helicase component of eIF4F, elicits powerful antitumor immune-mediated effects via PD-L1 downregulation. Thus, eIF4A inhibitors, in development as anticancer drugs, may also act as cancer immunotherapies.

Inhibition of eIF4F complex loading inhibits the survival of malignant glioma.

The eukaryotic initiation factor (eIF)4E­binding proteins (4E­BPs) regulate cap­dependent protein translation and control the assembly of the eIF4F complex. In the present study, a phosphorylation­deficient truncated 4E­BP2 (eIF4FD) was constructed into the eukaryotic expression vector pSecTag2, and the in vitro and in vivo effects on malignant glioma survival were determined through inhibiting eIF4F complex assembly. Cell cycle distribution analysis and TUNEL staining show that overexpression of eIF4FD suppressed cell proliferation and induced apoptosis in U251 cells. Western blotting showed that the cell cycle­related genes cyclin D1 and C­myc, and anti­apoptotic genes B­cell lymphoma 2 (Bcl­2), Bcl­extra large and survivin were reduced following the overexpression of eIF4FD. Furthermore, eIF4FD suppressed glioma vascularization via reductions in the expression of ß­catenin and vascular endothelial growth factor. In the orthotopic xenograft model, the stable expression of eIF4FD in U251 cells attenuated cell growth and increased the rate of apoptosis. Accordingly, pSecTag2­PTD­eIF4FD injection via the tail vein of mice also lead to cell growth inhibition and the induction of apoptosis. Therefore, the study showed that phosphorylation­deficient truncated 4E­BP2 efficiently inhibited eIF4E and prevented the formation of the eIF4F complex, which further contributed to the inhibition of cell proliferation and vascularization, and the induction of apoptosis. Therefore, the 4E­BP2­based phosphorylation­deficient truncation designed in the present study may represent a novel approach for the targeted therapy of human malignant glioma though inhibition of the translation initiation complex.