Abstract B075: eIF4A regulates ERK activation by controlling the translation of DUSP6

Abstract Background: Dysregulation of ERK activation is a common occurrence in human cancers. Under physiological conditions, cellular ERK activity is tightly controlled by the regulation of activation of upstream kinases and the inhibitory phosphatases. However, the detailed mechanisms by which the ERK phosphatases are regulated are not well categorized. Here, we identified a novel mechanism by which the ERK phosphorylation is regulated by the eIF4A-dependent translation of DUSP6. Material and Methods: Three different eIF4A inhibitors (silvestrol, hippuristanol and pateamine A) were used to treat 3 different cell lines harboring the BRAF V600E mutant. Silvestrol treatment was also extended to a panel of cell lines, which harbor BRAF V600E, KRAS or NRAS mutant or RAS/RAF WT. We studied the change of MAPK signaling by detecting phospho-MEK, phospho-ERK, DUSP6 by Western blot, and the RNA expression of downstream targets of ERK (including DUSP6, SPRY2, ETV4, ETV5) by qRT-PCR. To test the necessity of proteins of interest, eIF4A1 and eIF4A2 was knocked down by siRNA, DUSP6 was knocked out by CRISPR-Cas9 technology. The translation rate of DUSP6 was determined by polysome profiling. Lastly the effect of eIF4A inhibition on autophagy was investigated by 1) measuring LC3-II levels by Western blot and 2) measuring the GFP-LC3/RFP-LC3ΔG ratio by flow cytometry using a recently reported system. Results: Here, we show that ERK activation is regulated by eIF4A-dependent translation of DUSP6, the ERK1/2 phosphatase. We found that, in a panel of cell lines (BRAF V600E mutant, KRAS or NRAS mutant or RAS/RAF WT), treatment with silvestrol, an inhibitor of eIF4A, caused increased phosphorylation of ERK (pERK) as well as ERK signaling outputs without affecting pMEK. The induction of pERK by silverstrol was highly associated with decreased expression of DUSP6, but not of other DUSP proteins. We confirmed that the decrease in DUSP6 is required for induction of pERK by silverstrol. In cells in which DUSP6 was knocked out, pERK is not affected by silverstrol. ERK activation is known to increase DUSP6 expression and induction of pERK by silvestrol increased DUSP6 mRNA, while decreasing DUSP6 protein. The results suggest that translation of DUSP6 might be dependent on eIF4A and that silvestrol decreases DUSP6 protein expression by inhibiting its translation. This hypothesis is supported by data that shows a) expression of an eIF4A mutant that cannot bind silvestrol abrogates its effect on DUSP6, b) two other inhibitors of eIF4A (hippuristanol and pateamine A) and c) knocking down eIF4A also reduce DUSP6 protein expression. Moreover, the translation of DUSP6 is cap-independent and inhibited by silvestrol as determined by polysome profiling. Inhibition of DUSP6 translation by silvestrol results in a rapid, proteasome-dependent, reduction in DUSP6 expression and a concomitant marked increase in ERK phosphorylation. In multiple BRAF V600E cancer cell lines, silvestrol-induced ERK activation is not associated with increased cellular senescence or apoptosis, as suggested by previous studies. Instead, the ERK activation increases LC3-II abundance and decreases LC3/ LC3ΔG ratio. Conclusions: The data show that that DUSP6 is translated in an eIF4A-depenent cap-independent manner and is sensitive to eIF4A inhibitors. By decreasing the expression of DUSP6, eIF4A inhibitors induce ERK activation and promote autophagy, which may promote cell survival. Citation Format: Jianing Xu, Hans-Guido Wendel, Jerry Pelletier, Zhan Yao, Neal Rosen. eIF4A regulates ERK activation by controlling the translation of DUSP6 [abstract]. In: Proceedings of the AACR-NCI-EORTC International Conference on Molecular Targets and Cancer Therapeutics; 2019 Oct 26-30; Boston, MA. Philadelphia (PA): AACR; Mol Cancer Ther 2019;18(12 Suppl):Abstract nr B075. doi:10.1158/1535-7163.TARG-19-B075