eIF4E-mediated translational control of cancer incidence.

Mitogen activated translation initiation factor eIF4E mediates normal cell proliferation, yet induces tumorigenesis when deregulated and overexpressed. It remains unknown, how activated eIF4E directs such distinct biological outputs. Our experimental data provide evidence that distinct threshold levels of eIF4E govern its biological output in lactating mammary glands and that eIF4E overexpression in the context of cell population expansion can initiate malignant transformation by enabling cells to evade DNA damage checkpoints caused by hyperproliferative oncogenic stimuli. These findings point at the cellular level of eIF4E as an important sensor for normal or pro-neoplastic propagation of cells. Here, we describe a model that links the pro-neoplastic function of eIF4F to its ability to disable oncogene-activated tumor surveillance programs; and propose a novel therapeutic strategy for cancer prevention based upon targeting aberrant eIF4E with safe doses of small-molecule antagonists to ensure the maintenance of eIF4E levels below the pro-neoplastic threshold. This article is part of a Special Issue entitled: Translation and Cancer.

Activation of translation complex eIF4F is essential for the genesis and maintenance of the malignant phenotype in human mammary epithelial cells.

Common human malignancies acquire derangements of the translation initiation complex, eIF4F, but their functional significance is unknown. Hypophosphorylated 4E-BP proteins negatively regulate eIF4F assembly by sequestering its mRNA cap binding component eIF4E, whereas hyperphosphorylation abrogates this function. We found that breast carcinoma cells harbor increases in the eIF4F constituent eIF4GI and hyperphosphorylation of 4E-BP1 which are two alterations that activate eIF4F assembly. Ectopic expression of eIF4E in human mammary epithelial cells enabled clonal expansion and anchorage-independent growth. Transfer of 4E-BP1 phosphorylation site mutants into breast carcinoma cells suppressed their tumorigenicity, whereas loss of these 4E-BP1 phosphorylation site mutants accompanied spontaneous reversion to a malignant phenotype. Thus, eIF4F activation is an essential component of the malignant phenotype in breast carcinoma.

Eukaryotic translation initiation factor 4E induced progression of primary human mammary epithelial cells along the cancer pathway is associated with targeted translational deregulation of oncogenic drivers and inhibitors.

Pathologic redirection of translational control by constitutive activation of eukaryotic translation initiation factor 4F (eIF4F), the cap-dependent translation initiation apparatus, is an obligatory step in oncogenesis; however, its mechanism remains undefined. Here, we simulate this pro-oncogenic state by overexpressing eIF4E, the rate-limiting component of eIF4F, in primary human mammary epithelial cells (HMECs) and examine the resultant changes in cell biology and gene expression profiles of total and polyribosome-bound mRNA genome wide. Overexpressed eIF4E rescues primary HMECs from telomere-independent growth arrest and disables checkpoints governing S-phase entry as well as apoptosis in HMECs immortalized by telomerase, imparting cells with proliferative and survival autonomy. Although the transcriptional response to increased eIF4E was modest, the translational response was large, selective, and bidirectional. In addition to translational activation of known and novel eIF4E-responsive oncogenic drivers regulating cell growth and survival, our data unveil previously unrecognized cellular defenses including translational activation of tumor suppressors, translational repression of transcripts enriched with miRNA target sites, and translational modulation of genes governing translation itself. These findings provide insight into the proneoplastic and compensatory mechanisms embedded in the oncogenic translational program. They support a model whereby deregulated eIF4E moves human epithelial cells along the cancer pathway by profoundly altering ribosomal recruitment to cancer-related transcripts, and eIF4E-modified cells counter these potentially oncogenic alterations with a compensatory translational mechanism that mitigates acquisition of malignancy.

Expression, purification and characterization of recombinant mouse translation initiation factor eIF4E as a dihydrofolate reductase (DHFR) fusion protein.

One of the earliest steps in translation initiation is recognition of the mRNA cap structure (m7GpppX) by the initiation factor eIF4E. Studies of interactions between purified eIF4E and its binding partners provide important information for understanding mechanisms underlying translational control in normal and cancer cells. Numerous impediments of the available methods used for eIF4E purification led us to develop a novel methodology for obtaining fractions of eIF4E free from undesired by-products. Herein we report methods for bacterial expression of eIF4E tagged with mutant dihydrofolate reductase (DHFR) followed by isolation and purification of the DHFR-eIF4E protein by using affinity and anion exchange chromatography. Fluorescence quenching experiments indicated the cap-analog, 7MeGTP, bound to DHFR-eIF4E and eIF4E with a dissociation constant (K(d)) of 6+/-5 and 10+/-3 nM, respectively. Recombinant eIF4E and DHFR-eIF4E were both shown to significantly enhance in vitro translation in dose dependent manner by 75% at 0.5 microM. Nevertheless increased concentrations of eIF4E and DHFR-eIF4E significantly inhibited translation in a dose dependent manner by a maximum at 2 microM of 60% and 90%, respectively. Thus, we have demonstrated that we have developed an expression system for fully functional recombinant eIF4E. We have also shown that the fusion protein DHFR-eIF4E is functional and thus may be useful for cell based affinity tag studies with fluorescently labeled trimethoprim analogs.

Apoptosis resistance downstream of eIF4E: posttranscriptional activation of an anti-apoptotic transcript carrying a consensus hairpin structure.

Aberrant activation of the translation initiation machinery is a common property of malignant cells, and is essential for breast carcinoma cells to manifest a malignant phenotype. How does sustained activation of the rate limiting step in protein synthesis so fundamentally alter a cell? In this report, we test the post transcriptional operon theory as a possible mechanism, employing a model system in which apoptosis resistance is conferred on NIH 3T3 cells by ectopic expression of eIF4E. We show (i) there is a set of 255 transcripts that manifest an increase in translational efficiency during eIF4E-mediated escape from apoptosis; (ii) there is a novel prototype 55 nt RNA consensus hairpin structure that is overrepresented in the 5'-untranslated region of translationally activated transcripts; (iii) the identified consensus hairpin structure is sufficient to target a reporter mRNA for translational activation under pro-apoptotic stress, but only when eIF4E is deregulated; and (iv) that osteopontin, one of the translationally activated transcripts harboring the identified consensus hairpin structure functions as one mediator of the apoptosis resistance seen in our model. Our findings offer genome-wide insights into the mechanism of eIF4E-mediated apoptosis resistance and provide a paradigm for the systematic study of posttranscriptional control in normal biology and disease.

Repression of cap-dependent translation attenuates the transformed phenotype in non-small cell lung cancer both in vitro and in vivo.

Aberrant hyperactivation of the cap-dependent protein synthesis apparatus has been documented in a wide range of solid tumors, including epithelial carcinomas, but causal linkage has only been established in breast carcinoma. In this report, we sought to determine if targeted disruption of deregulated cap-dependent translation abrogates tumorigenicity and enhances cell death in non-small cell lung cancer (NSCLC). NSCLC cell lines were stably transfected with either wild-type 4E-BP1 (HA-4E-BP1) or the dominant-active mutant 4E-BP1(A37/A46) (HA-TTAA). Transfected NSCLC cells with enhanced translational repression showed pronounced cell death following treatment with gemcitabine. In addition, transfected HA-TTAA and HA-4E-BP1wt proteins suppressed growth in a cloning efficiency assay. NSCLC cells transduced with HA-TTAA also show decreased tumorigenicity in xenograft models. Xenograft tumors expressing HA-TTAA were significantly smaller than control tumors. This work shows that hyperactivation of the translational machinery is necessary for maintenance of the malignant phenotype in NSCLC, identifies the molecular strategy used to activate translation, and supports the development of lung cancer therapies that directly target the cap-dependent translation initiation complex.

Translational control of cell fate: availability of phosphorylation sites on translational repressor 4E-BP1 governs its proapoptotic potency.

Translational control has been recently added to well-recognized genomic, transcriptional, and posttranslational mechanisms regulating apoptosis. We previously found that overexpressed eukaryotic initiation factor 4E (eIF4E) rescues cells from apoptosis, while ectopic expression of wild-type eIF4E-binding protein 1 (4E-BP1), the most abundant member of the 4E-BP family of eIF4E repressor proteins, activates apoptosis--but only in transformed cells. To test the possibility that nontransformed cells require less cap-dependent translation to suppress apoptosis than do their transformed counterparts, we intensified the level of translational repression in nontransformed fibroblasts. Here, we show that inhibition of 4E-BP1 phosphorylation by rapamycin triggers apoptosis in cells ectopically expressing wild-type 4E-BP1 and that expression of 4E-BP1 phosphorylation site mutants potently activates apoptosis in a phosphorylation site-specific manner. In general, proapoptotic potency paralleled repression of cap-dependent translation. However, this relationship was not a simple monotone. As repression of cap-dependent translation intensified, apoptosis increased to a maximum value. Further repression resulted in less apoptosis--a state associated with activation of translation through internal ribosomal entry sites. These findings show: that phosphorylation events govern the proapoptotic potency of 4E-BP1, that 4E-BP1 is proapoptotic in normal as well as transformed fibroblasts, and that malignant transformation is associated with a higher requirement for cap-dependent translation to inhibit apoptosis. Our results suggest that 4E-BP1-mediated control of apoptosis occurs through qualitative rather than quantitative changes in protein synthesis, mediated by a dynamic interplay between cap-dependent and cap-independent processes.

Acquired Tamoxifen Resistance in MCF-7 Breast Cancer Cells Requires Hyperactivation of eIF4F-Mediated Translation.

While selective estrogen receptor modulators, such as tamoxifen, have contributed to increased survival in patients with hormone receptor-positive breast cancer, the development of resistance to these therapies has led to the need to investigate other targetable pathways involved in oncogenic signaling. Approval of the mTOR inhibitor everolimus in the therapy of secondary endocrine resistance demonstrates the validity of this approach. Importantly, mTOR activation regulates eukaryotic messenger RNA translation. Eukaryotic translation initiation factor 4E (eIF4E), a component of the cap-dependent translation complex eIF4F, confers resistance to drug-induced apoptosis when overexpressed in multiple cell types. The eIF4F complex is downstream of multiple oncogenic pathways, including mTOR, making it an appealing drug target. Here, we show that the eIF4F translation pathway was hyperactive in tamoxifen-resistant (TamR) MCF-7L breast cancer cells. While overexpression of eIF4E was not sufficient to confer resistance to tamoxifen in MCF-7L cells, its function was necessary to maintain resistance in TamR cells. Targeting the eIF4E subunit of the eIF4F complex through its degradation using an antisense oligonucleotide (ASO) or via sequestration using a mutant 4E-BP1 inhibited the proliferation and colony formation of TamR cells and partially restored sensitivity to tamoxifen. Further, the use of these agents also resulted in cell cycle arrest and induction of apoptosis in TamR cells. Finally, the use of a pharmacologic agent which inhibited the eIF4E-eIF4G interaction also decreased the proliferation and anchorage-dependent colony formation in TamR cells. These results highlight the eIF4F complex as a promising target for patients with acquired resistance to tamoxifen and, potentially, other endocrine therapies.

The cap-dependent translation apparatus integrates and amplifies cancer pathways.

Deregulation of a plethora of cancer genes causes pathological changes in only a small set of pathways that confer a cell with malignant properties. This principle of convergence of oncogenic signaling-the ability of several hundred oncogenes to focus their effects on a few critical regulatory nodes that impart autonomy to the cell-motivates the search for putative focal points. Genomic, transcriptional and posttranslational mechanisms that regulate the function of cancer gene pathways are all well established. It has recently become evident that cancer is also subject to translational control. Here we discuss cancer-related regulatory events that are mediated by the cap-dependent mRNA binding stage of translation initiation. This information implicates the cap-dependent protein synthesis pathway as a pleotropic integrator and amplifier of many essential oncogenic signals, and the translational control network as a bona fide molecular target for anti-cancer therapeutic interventions.

eIF4E threshold levels differ in governing normal and neoplastic expansion of mammary stem and luminal progenitor cells.

Translation initiation factor eIF4E mediates normal cell proliferation, yet induces tumorigenesis when overexpressed. The mechanisms by which eIF4E directs such distinct biologic outputs remain unknown. We found that mouse mammary morphogenesis during pregnancy and lactation is accompanied by increased cap-binding capability of eIF4E and activation of the eIF4E-dependent translational apparatus, but only subtle oscillations in eIF4E abundance. Using a transgenic mouse model engineered so that lactogenic hormones stimulate a sustained increase in eIF4E abundance in stem/progenitor cells of lactogenic mammary epithelium during successive pregnancy/lactation cycles, eIF4E overexpression increased self-renewal, triggered DNA replication stress, and induced formation of premalignant and malignant lesions. Using complementary in vivo and ex vivo approaches, we found that increasing eIF4E levels rescued cells harboring oncogenic c-Myc or H-RasV12 from DNA replication stress and oncogene-induced replication catastrophe. Our findings indicate that distinct threshold levels of eIF4E govern its biologic output in lactating mammary glands and that eIF4E overexpression in the context of stem/progenitor cell population expansion can initiate malignant transformation by enabling cells to evade DNA damage checkpoints activated by oncogenic stimuli. Maintaining eIF4E levels below its proneoplastic threshold is an important anticancer defense in normal cells, with important implications for understanding pregnancy-associated breast cancer.

Introduction to Translation.

We introduce here the inaugural issue of the new scientific journal Translation. The overarching aim of this endeavor is to establish a new forum for a broad spectrum of research in the area of protein synthesis in living systems ranging from structural biochemical, evolutionary and regulatory aspects of translation to the fundamental questions related to post-translational control of somatic phenomena in multicellular organisms including human behavior and health. The journal will publish high quality research articles, provide novel insights, ask provocative questions and discuss new hypothesis in this emerging field. Launching a new journal is always challenging. We hope that strong criteria for the peer-review process, transparency of the editorial policy and the scientific reputation of its founders, editors and editorial board assure the success of Translation; and we rely on continuing support of the scientific community in all aspects of the journal's activity.

Attacking a nexus of the oncogenic circuitry by reversing aberrant eIF4F-mediated translation.

Notwithstanding their genetic complexity, different cancers share a core group of perturbed pathways converging upon a few regulatory nodes that link the intracellular-signaling network with the basic metabolic machinery. The clear implication of this view for cancer therapy is that instead of targeting individual genetic alterations one by one, the next generation of cancer therapeutics will target critical hubs in the cancer network. One such hub is the translation-initiation complex eIF4F, which integrates several cancer-related pathways into a self-amplifying signaling system. When hyperactivated by apical oncogenic signals, the eIF4F-driven translational apparatus selectively switches the translational repertoire of a cell toward malignancy. This central integrative role of pathologically activated eIF4F has motivated the development of small-molecule inhibitors to correct its function. A genome-wide, systems-level means to objectively evaluate the pharmacologic response to therapeutics targeting eIF4F remains an unmet challenge.

Translational control of cell fate: from integration of environmental signals to breaching anticancer defense.

Despite their genetic diversity, different cancers manifest common features at the protein pathway level. They share a core group of perturbed pathways that converge upon a few regulatory hubs linking the cellular signaling network with the basic metabolic machinery. Available evidence indicates that one such hub is the eIF4F-mediated cap-dependent mRNA translation initiation apparatus, whose integrity is required for physiological control of growth, proliferation and viability. However, when hyperactivated by upstream oncogenic signaling, eIF4F selectively stimulates the translation of a group of mRNAs required for cancer genesis and progression. Here, we describe a model that links the pro-neoplastic function of eIF4F to its ability to disable oncogene-activated tumor surveillance programs and propose a novel therapeutic strategy for cancer based upon targeting aberrant eIF4F with small-molecule antagonists.

Eukaryotic initiation factor 4E binding protein family of proteins: sentinels at a translational control checkpoint in lung tumor defense.

The usurping of translational control by sustained activation of translation initiation factors is oncogenic. Here, we show that the primary negative regulators of these oncogenic initiation factors--the 4E-BP protein family--operate as guardians of a translational control checkpoint in lung tumor defense. When challenged with the tobacco carcinogen 4-(methylnitrosamino)-I-(3-pyridyl)-1-butanone (NNK), 4ebp1(-/-)/4ebp2(-/-) mice showed increased sensitivity to tumorigenesis compared with their wild-type counterparts. The 4E-BP-deficient state per se creates pro-oncogenic, genome-wide skewing of the molecular landscape, with translational activation of genes governing angiogenesis, growth, and proliferation, and translational activation of the precise cytochrome p450 enzyme isoform (CYP2A5) that bioactivates NNK into mutagenic metabolites. Our study provides in vivo proof for a translational control checkpoint in lung tumor defense.

Nontoxic chemical interdiction of the epithelial-to-mesenchymal transition by targeting cap-dependent translation.

Normal growth and development depends upon high fidelity regulation of cap-dependent translation initiation, a process that is usurped and redirected in cancer to mediate acquisition of malignant properties. The epithelial-to-mesenchymal transition (EMT) is a key translationally regulated step in the development of epithelial cancers and pathological tissue fibrosis. To date, no compounds targeting EMT have been developed. Here we report the synthesis of a novel class of histidine triad nucleotide binding protein (HINT)-dependent pronucleotides that interdict EMT by negatively regulating the association of eIF4E with the mRNA cap. Compound eIF4E inhibitor-1 potently inhibited cap-dependent translation in a dose-dependent manner in zebrafish embryos without causing developmental abnormalities and prevented eIF4E from triggering EMT in zebrafish ectoderm explants without toxicity. Metabolism studies with whole cell lysates demonstrated that the prodrug was rapidly converted into 7-BnGMP. Thus we have successfully developed the first nontoxic small molecule able to inhibit EMT, a key process in the development of epithelial cancer and tissue fibrosis, by targeting the interaction of eIF4E with the mRNA cap and demonstrated the tractability of zebrafish as a model organism for studying agents that modulate EMT. Our work provides strong motivation for the continued development of compounds designed to normalize cap-dependent translation as novel chemo-preventive agents and therapeutics for cancer and fibrosis.

Synthesis and evaluation of potential inhibitors of eIF4E cap binding to 7-methyl GTP.

Cap-dependent translation is initiated by the binding of eIF4E to capped mRNA (m(7)GpppN). We have prepared a small library of 7-methyl guanosine nucleoside and nucleotide analogs and evaluated their ability to inhibit eIF4E binding to 7-methyl GTP with a competitive eIF4E binding immunoassay. 5'-H-Phosphonate derivatives in which the 2'- and 3'-riboside hydroxyls were tethered together by an isopropylidene group were shown to be a new class of inhibitors of eIF4E binding to capped mRNA.

Translation initiation factor 4E blocks endoplasmic reticulum-mediated apoptosis.

Eukaryotic translation initiation factor 4E (eIF4E) is the mRNA cap-binding protein required for translation of cellular mRNAs utilizing the 5' cap structure. The rate-limiting factor for mRNA recruitment to ribosomes, eIF4E is a major target for regulation of translation by growth factors, hormones, and other extracellular stimuli. When overexpressed, eIF4E exerts profound effects on cell growth and survival, leading to suppression of oncogene-dependent apoptosis, causing malignant transformation and conferring tumors with multiple drug resistance. We found previously that overexpressed eIF4E interdicts the apoptotic pathway induced by growth factor withdrawal and cytotoxic drugs by selectively activating the expression of Bcl-X(L), thus preventing mitochondrial release of cytochrome c. In this study, we examined the impact of ectopic eIF4E expression on apoptosis mediated by the endoplasmic reticulum (ER). Here we show that eIF4E rescued cells from the ER stressors brefeldin A, tunicamycin, thapsigargin, and the Ca(2+) ionophore A23187. In addition, we found that cells rescued from Ca(2+) ionophore-triggered apoptosis did not release calcium from their ER nor did they translocate caspase-12 from the ER to the cytoplasm. These data lend strong support to the concept that eIF4E functions as a pleiotropic regulator of cell viability and that integration of critical organelle-mediated checkpoints for apoptosis can be controlled by the cap-dependent translation apparatus.

Translation factor eIF4E rescues cells from Myc-dependent apoptosis by inhibiting cytochrome c release.

Eukaryotic translation initiation factor 4E (eIF4E) markedly reduces cellular susceptibility to apoptosis. However, the mechanism by which the translation apparatus operates on the cellular apoptotic machinery remains uncertain. Here we show that eIF4E-mediated rescue from Myc-dependent apoptosis is accompanied by inhibition of mitochondrial cytochrome c release. Experiments achieving gain and loss of function demonstrate that eIF4E-mediated rescue is governed by pretranslational and translational activation of bcl-x as well as by additional intermediates acting directly on, or upstream of, the mitochondria. Thus, our data trace a pathway controlling apoptotic susceptibility that begins with the activity state of the protein synthesis machinery and leads to interdiction of the apoptotic program at the mitochondrial checkpoint.