EGFR modulates microRNA maturation in response to hypoxia through phosphorylation of AGO2

Abstract

MicroRNAs (miRNAs) are generated by two-step processing to yield small RNAs that negatively regulate target gene expression at the post-transcriptional level. Deregulation of miRNAs has been linked to diverse pathological processes, including cancer. Recent studies have also implicated miRNAs in the regulation of cellular response to a spectrum of stresses, such as hypoxia, which is frequently encountered in the poorly angiogenic core of a solid tumour. However, the upstream regulators of miRNA biogenesis machineries remain obscure, raising the question of how tumour cells efficiently coordinate and impose specificity on miRNA expression and function in response to stresses. Here we show that epidermal growth factor receptor (EGFR), which is the product of a well-characterized oncogene in human cancers, suppresses the maturation of specific tumour-suppressor-like miRNAs in response to hypoxic stress through phosphorylation of argonaute 2 (AGO2) at Tyr 393. The association between EGFR and AGO2 is enhanced by hypoxia, leading to elevated AGO2-Y393 phosphorylation, which in turn reduces the binding of Dicer to AGO2 and inhibits miRNA processing from precursor miRNAs to mature miRNAs. We also identify a long-loop structure in precursor miRNAs as a critical regulatory element in phospho-Y393-AGO2-mediated miRNA maturation. Furthermore, AGO2-Y393 phosphorylation mediates EGFR-enhanced cell survival and invasiveness under hypoxia, and correlates with poorer overall survival in breast cancer patients. Our study reveals a previously unrecognized function of EGFR in miRNA maturation and demonstrates how EGFR is likely to function as a regulator of AGO2 through novel post-translational modification. These findings suggest that modulation of miRNA biogenesis is important for stress response in tumour cells and has potential clinical implications.

Figure 1. EGFR interacts with AGO2 in response to hypoxia

a, Split-half-YFP-fused EGFR and AGO2 were stably expressed in HTC-1080 cells to screen for upstream stimuli that might trigger EGFR–AGO2 interaction. E, EGFR; A, AGO2. b, Top, representative live-cell image. N, nuclear; C, cytoplasmic. Bottom, fluorescence-activated cell sorting (FACS) analysis of HTC-1080 stable transfectants as indicated. FITC, fluoresce in isothiocyanate. c, Immunoprecipitation and western blot analysis of HeLa cells in response to different stimuli. EGF, 20 ng ml⁻¹; SA (sodium arsenite), 500 μM; hypoxia, 1% O₂. d, Confocal microscopy analysis of live HeLa cells as indicated. Rab7, a marker for late endosomes. Regions 1 and 2 at top are shown in magnified view at bottom. NC, normoxia; H24, hypoxia 24 h. e, Immunoprecipitation and western blot analysis of HeLa stable transfectants expressing HIF1/2α shRNAs.

Figure 2. EGFR modulates miRNA maturation in response to hypoxia

a, Top, proposed role of EGFR in miRNA maturation. Bottom, hierarchical clustering analysis of R-Pre (log₂[precursor miR (scrambled)] − log₂[precursor miR (EGFR shRNA-E1)]) and R-Mature (log₂[mature miR (scrambled)] − log₂[mature miR (EGFR shRNA-E1)]) identified one distinct cluster of miRNAs whose maturation was suppressed by EGFR under hypoxia. We define this subcluster as mHESM. b, Top, Venn diagram highlighting the mRNAs that are regulated by EGFR and likely to be targeted by top-scoring mHESM (those for which R-Pre − R-Mature ≥0.8 and R-Mature ≤ −0.4). Bottom, EGFR-mediated suppression of top-scoring mHESM concurrent with the upregulation of targeting mRNAs in response to hypoxia. AE, average expression.

Figure 3. EGFR phosphorylates AGO2 at Tyr 393 to suppress the maturation of long-loop mHESM in response to hypoxia

a, In vitro kinase assay detected by 4G10 antibody. b, Immunoprecipitation and western blot analysis of HeLa Tet-Off-inducible AGO2 stable clones. c, Relative position of Tyr 393 in the structure of human AGO2 with bound guide RNA^,. The N (cyan), linker L1 (yellow), PAZ (violet), linker L2 (grey), MID (orange) and PIWI (green) domains are shown in ribbon representation, and the co-purifying guide RNA (red) is depicted in stick representation. Tyr 393 and the PIWI box are highlighted in blue. The expanded boxed segment highlights the electrostatic surface around Tyr 393, including a modelled phosphate attached to Tyr 393. d, Normalized expression of miRNA and corresponding luciferase activity of miR reporters (n = 4). e, RNA-binding protein immunoprecipitation (RIP) enrichment of precursor and mature miRNAs (n = 3). qPCR, quantitative PCR. f, g, Northern blot analysis as indicated. Dashed lines indicate different exposure times. RNA integrity was examined by ethidium bromide staining (Supplementary Figs 32 and 33). Data represent mean ± s.d.; *P <0.05, t-test.

Figure 4. p-Y393-AGO2 enhances cell survival and invasiveness under hypoxia and correlates with poorer overall survival in breast cancer patients

a, Cell apoptosis analysed by FACS (n = 3). b, In vitro migration assay. Result was calculated on the basis of five randomly selected fields per membrane in triplicate (n = 3). c, In vitro three-dimensional invasion assay (n = 5). d, Representative images of immunohistochemical staining of p-Y393-AGO2 in human breast tumour and its adjacent normal tissue. e, Correlation between p-Y393-AGO2 and overall survival in breast cancer patients (n = 125). P <0.0001, Kaplan–Meier survival analysis. All data represent mean ± s.d.; *P <0.05, t-test.