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. 2009 May 15;4(5):367-77.
doi: 10.1021/cb9000475.

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

Brahma Ghosh  1 , Alexey O Benyumov, Phalguni Ghosh, Yan Jia, Svetlana Avdulov, Peter S Dahlberg, Mark Peterson, Karen Smith, Vitaly A Polunovsky, Peter B Bitterman, Carston R Wagner
Affiliations

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

Brahma Ghosh et al. ACS Chem Biol. .

Abstract

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.

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Figures

Figure 1
Figure 1. Chemical structure of cap analogs
Figure 2
Figure 2. Inhibition of cap-dependent translation by cap analogs in vitro
A) Cap-dependent translation quantified as the expression of Renilla luciferase reporter. B) IRES-mediated translation quantified as the expression of firefly luciferase reporter. Shown is the impact of compounds (100 and 500 µM) on reporter luminescence (arbitrary units) with all values normalized to the luminescence of a compound-free control reaction. Data are presented as mean ± SEM for 3 independent experiments; * indicates a significant difference at p < 0.01 compared to the calibration control set at 100.
Figure 3
Figure 3. Inhibition of cap-dependent translation by cap analogs in vivo
A) Cap-dependent translation quantified as the expression of Renilla luciferase reporter. B) IRES-mediated translation quantified as the expression of firefly luciferase reporter. Shown is reporter translation for test compounds microinjected in doses of 5 and 25 pmols with all values normalized to the luminescence in embryos injected with HBSS. Data are presented as mean ± SEM for 3 to 5 independent experiments conducted with 20 embryos each; * indicates a significant difference at p < 0.01 compared to the HBSS calibration control set at 100.
Figure 4
Figure 4. Manipulating cap-dependent translation in zebrafish embryos
A) Luciferase reporter mRNA in vitro-transcribed from the construct pcDNA3-rLuc-polIRES-fLuc, B) mRNA encoding murine eIF4E WT or the cap binding mutant eIF4E W56A from the constructs pcDNA3-3HA-eIF4E -wt or -W56A, C) Experimental approach to analyze cap-dependent translation following genetic or pharmacological intervention. Luciferase reporter mRNA is microinjected at the one-cell stage. Effectors - i) mRNA encoding the protein of interest (wt or mutant); ii) translation inhibitor 4Ei-1; or iii) both HA-eIF4E WT + 4Ei-1 are microinjected at the 2-cell stage. Half of the embryos are analyzed for expression of the HA-tagged exogenous proteins at the late blastula stage; the remaining embryos are analyzed at mid-gastrula for luminescence. D) Representative immunoblot demonstrating expression of exogenous wild-type and mutant eIF4E proteins in the injected embryos. Negative controls: lysates from xEF-1α- (1), buffer-injected (2) and non-injected embryos (3); Positive control: lysates from human mammary epithelial cells ectopically over expressing eIF4E (HMEC/HA-4E). E) Impact of each treatment on Renilla luciferase reporter expression as a measure of cap-dependent translation, with all values normalized to the luminescence of embryos injected with xEF-1α (left bar). Data are presented as mean ± SEM for 3 independent experiments conducted with 20 embryos each; * indicates a significant difference at p < 0.01 compared to the xEF-1α calibration control set at 100.
Figure 5
Figure 5. Inhibition of the epithelial-to-mesenchymal transition by 4Ei-1
A) Experimental setup to analyze the impact of cap-dependent translation alteration in ectoderm blastula explants. Buffer (HBSS); mRNA encoding Xenopus elongation factor (xEF-1α; negative control), eIF4E WT or the cap binding mutant eIF4E W56A, or a mixture of mRNA and 4Ei-1 (as indicated) are injected at the one-cell stage. Ectoderm blastula explants are excised prior to germ layer restriction and cultured individually. B) Explant morphology at 1, 24 and 48 h post excision. Within 1h, explants changed their shape from rectangular to spheroid. Within 24h, 79% of the explants derived from eIF4E WT-injected embryos (98 of 124) became elongated and developed distinct compartments consisting of two cell types: i) visually transparent and ii) opaque. By 48 h, the opaque cells migrated from the explants, assuming positions randomly throughout the culture dish. In contrast, explants derived from embryos injected with buffer or the control xEF-1α mRNA remained spherical for 48h. Of note, explants derived from embryos injected with either eIF4E W56A or eIF4E WT + 4Ei-1 did not differ from control, showing that the ability of eIF4E to bind the mRNA cap is required for development of the motile cell phenotype. C) Stage and lineage specific markers. RT-PCR amplified fragments resolved on agarose gel, standards on the left (STD). C1) goosecoid (gsc) expression. Maternally expressed mesodermal gsc, which demarcates future dorsal cells at the lateral portion of the embryo (59) was detected only in the intact embryos at the time point corresponding to 1 h post explant excision and in none of the explants, confirming that there were no mesoderm-destined cells present in the explants. C2). The ectodermal marker cytokeratin type 1 (cyt1) and a ventral cell marker gta3 (58,60) were observed in all samples, establishing the integrity of the RNA being analyzed – and the ectodermal origin of the explants. The notochord mesoderm marker no tail (ntl), and the paraxial mesoderm marker myogenic D (myoD) were detected only in explants injected with eIF4E WT mRNA 24h post excision and in the intact embryos at the expected times.
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