Ribavirin suppresses eIF4E-mediated oncogenic transformation by physical mimicry of the 7-methyl guanosine mRNA cap

Abstract

The eukaryotic translation initiation factor eIF4E is deregulated in many human cancers, and its overexpression in cells leads to malignant transformation. Oncogenic properties of eIF4E are directly linked to its ability to bind 7-methyl guanosine of the 5' mRNA. Here, we observe that the antiviral guanosine analogue ribavirin binds to eIF4E with micromolar affinity at the functional site used by 7-methyl guanosine mRNA cap, competes with eIF4E:mRNA binding, and, at low micromolar concentrations, selectively disrupts eIF4E subcellular organization and transport and translation of mRNAs posttranscriptionally regulated by eIF4E, thereby reducing levels of oncogenes such as cyclin D1. Ribavirin potently suppresses eIF4E-mediated oncogenic transformation of murine cells in vitro, of tumor growth of a mouse model of eIF4E-dependent human squamous cell carcinoma in vivo, and of colony formation of eIF4E-dependent acute myelogenous leukemia cells derived from human patients. These findings describe a specific, potent, and unforeseen mechanism of action of ribavirin. Quantum mechanical and NMR structural studies offer directions for the development of derivatives with improved cytostatic and antiviral properties. In all, ribavirin's association with eIF4E may provide a pharmacologic means for the interruption of posttranscriptional networks of oncogenes that maintain and enhance neoplasia and malignancy in human cancer.

Figures

Fig. 1.

Ribavirin, but not Rib4C, binds to the functional m7G cap-binding site of eIF4E with the same affinity as m7G mRNA cap. (a) Normalized corrected tryptophan fluorescence intensity quenching and their fits for binding to ribavirin to eIF4E wild-type (▪), W73A (□), W56A (stars), Rib4C to wild-type eIF4E (▴), RTP to wild-type eIF4E (♦), and m7GTP to wild-type eIF4E (⋄). (b) Apparent dissociation constants in micromolar for nucleoside/nucleotide:eIF4E binding. (c) Western blot of eIF4E remaining bound to m7G-Sepharose upon competition with various concentrations of m7GTP or RTP. Both m7GTP and RTP lead to 50% reduction of binding at a concentration of ≈1 μM. (d) Chemical structures of the keto forms of m7G, ribavirin, and Rib4C nucleosides. +, positive charge; R, ribose.

Fig. 2.

Ribavirin specifically inhibits eIF4E:mRNA binding, inhibits nucleocytoplasmic mRNA transport, and depletes levels of transport-regulated proteins. (a) Northern blots of RNA extracts of nuclear and cytoplasmic fractions of ribavirin-treated NIH 3T3 cells, which were probed as indicated. U6 small nuclear RNA and tRNALys serve as controls for quality of the fractionation. Ribavirin inhibits nucleocytoplasmic mRNA transport of cyclin D1, but not β-actin, with an apparent EC50 of ≈1 μM, as judged from the bar graph quantification (top row). N, nuclear; C, cytoplasmic. This effect was confirmed by using quantitative real-time PCR (Fig. 6b). (b) Northern and Western blots of total extracts of ribavirin-treated cells, exhibiting depletion of cyclin D1, without affecting transcription, mRNA stability, and protein synthesis. (c) Western blot of total protein extract of Rib4C-treated cells that were probed for cyclin D1. (d) Semiquantitative RT-PCR of cyclin D1 mRNA contained in eIF4E purified from the nuclei of ribavirin-treated cells. Control samples were purified by using IgG antibody (–) instead of antibody specific for eIF4E (+). Semiquantitative PCR of VEGF from cytoplasmic extracts was immunopurified as above.

Fig. 3.

Ribavirin suppresses eIF4E-mediated oncogenic transformation. (a) (Left) Anchorage-dependent foci formation of NIH 3T3 cells treated with ribavirin and transfected with empty vector (black dashed line), eIF4E WT (blue line), eIF4E W56A (red line), and cells treated with Rib4C and transfected with eIF4E WT (black solid line). Error bars represent ± 1σ of three independent experiments. Probability of focus formation (Pfocus) is defined as the number of foci formed divided by the number of cells plated. (Right) Photograph of Giemsa-stained dishes of ribavirin-treated cells transformed by eIF4E. (b) Colony formation of primary human CD34+ myeloid progenitors isolated from patients with AML (M1, solid circles; M5, solid squares) and normal bone marrow (BM, open squares), as a function of ribavirin concentration. Ribavirin reduces colony formation of eIF4E-dependent AML-M5 with an apparent IC50 of ≈1μM, and with no effect on M1 and normal bone marrow myeloid progenitor cells at this concentration. Note that data are internally normalized and that absolute colony formation efficiencies of AML myeloid progenitors are greater than that of BM (data not shown). Error bars represent ± 1σ of four independent experiments. (c)(Left) Mean tumor volume in nude mice engrafted with cells derived from a hypopharyngeal eIF4E-dependent tumor, as a function of treatment with daily 1 μM ribavirin orally at a dose of 40μg per kg per day (solid squares). Error bars represent ± 1σ of 10 mice. (Right) Photograph of tumors resected after 20 days of treatment.

Fig. 4.

Ribavirin and m7G mRNA cap are recognized similarly by eIF4E. (a) 1H, 15N HSQC NMR spectra of eIF4E in the absence (black) and presence (red) of saturating concentrations of m7G nucleoside. Note that of the 273 residues of the construct, 207 resonances are observed. (b) 1H, 15N HSQC NMR spectra of eIF4E in the presence of saturating concentrations of m7G (red) and ribavirin nucleosides (blue). (c) eIF4E backbone residues that exhibit (red) and do not exhibit (blue) 1H, 15N HSQC NMR chemical shift perturbation upon binding of ribavirin and m7G mRNA cap. The difference between conformational rearrangements upon cap binding of mouse eIF4E observed here and those reported for yeast eIF4E (29) may be because of differences between mouse and yeast proteins as well as micelle binding to yeast eIF4E (29).