Abstract
Emerging advances in cancer therapy have transformed the landscape towards cancer immunotherapy regimens. Recent discoveries have resulted in the development of clinical immune checkpoint inhibitors that are 'game-changers' for cancer immunotherapy. Here we show that eEF2K, an atypical protein kinase that negatively modulates the elongation stage of protein synthesis, promotes the synthesis of PD-L1, an immune checkpoint protein which helps cancer cells to escape from immunosurveillance. Ablation of eEF2K in prostate and lung cancer cells markedly reduced the expression levels of the PD-L1 protein. We show that eEF2K promotes the association of PD-L1 mRNAs with translationally active polyribosomes and that translation of the PD-L1 mRNA is regulated by a uORF (upstream open reading-frame) within its 5'-UTR (5'-untranslated region) which starts with a non-canonical CUG as the initiation codon. This inhibitory effect is attenuated by eEF2K thereby allowing higher levels of translation of the PD-L1 coding region and enhanced expression of the PD-L1 protein. Moreover, eEF2K-depleted cancer cells are more vulnerable to immune attack by natural killer cells. Therefore, control of translation elongation can modulate the translation of this specific mRNA, one which contains an uORF that starts with CUG, and perhaps others that contain a similar feature. Taken together, our data reveal that eEF2K regulates PD-L1 expression at the level of the translation of its mRNA by virtue of a uORF in its 5'-region. This, and other roles of eEF2K in cancer cell biology (e.g. in cell survival and migration), may be exploited for the design of future therapeutic strategies.
Highlights
It is clear that immunotherapy is a promising and effective approach to tackling a range of cancers
IFNγ did increase the levels of the PD-ligand 1 (PD-L1) protein in prostate cancer PC3 cells (Figure 1A, data quantified in Figure 1B; note that PD-L1 generally appears as a rather fuzzy band on immunoblots, likely reflecting the fact it is subject to multiple post-translational modifications [24])
We used CRISPR–Cas9 genome editing to disrupt the EEF2K gene in PC3 cells (Supplementary Figure S1); as expected, since eEF2 kinase (eEF2K) is the only kinase that phosphorylates the regulatory site, Thr56, in eukaryotic elongation factor 2 (eEF2) [26], no phosphorylation of eEF2 at this site was seen in eEF2K-KO PC3 cells (Figure 1A). eEF2K was undetectable in the knockout (KO) cells. eEF2K-KO showed lower levels of PD-L1 than the corresponding control cells (Figure 1A,B)
Summary
It is clear that immunotherapy is a promising and effective approach to tackling a range of cancers (reviewed [1]). PD-1, which is expressed on the surface of T lymphocytes, binds ligands expressed on other cells which, through PD-1, repress the activity of T cells. One such ligand is the protein PD-ligand 1 (PD-L1; [1], termed CD274) which can be induced by signals such as interferon-γ (IFNγ), a cytokine. It is increasingly clear that countering these so-called immune checkpoints promotes immune responses, including against cancer cells. Antibodies that block either PD-1 or PD-L1 have shown striking efficacy in tackling a number of cancers (see, e.g. [1])
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