Abstract
BackgroundThe epidermal growth factor receptor (EGFR) is closely implicated in cancer, and sequencing analyses have revealed a high mutation rate of EGFR in lung cancer. Recent advances have provided novel insights into the endocytic regulation of wild-type EGFR, but that of mutated EGFR remains elusive. In the present study, we aim to investigate the endocytic degradation of a frequently occurred exon 19-deleted mutant in lung cancer.MethodsThe EGF-induced endocytic degradation of EGFR was examined in a panel of lung cancer cells using immunoblotting. The subcellular distribution of internalized EGFR was investigated using immunofluorescence and confocal microscopy. The effects of dynamin were assessed using its small molecule inhibitors, while the influence of RTN3 was tested using shRNA-mediated knockdown. Finally the ubiquitylation status of EGFR mutant was studied using immunoprecipitation under steady state and tyrosine kinase inhibitor-treated conditions.ResultsEGF induced various rates of EGFR endocytic degradation in lung cancer cells. Interestingly, the exon 19 deletion mutant is constantly internalized and sorted to lysosome for degradation, and this process is independent of dynamin activity. EGF stimulation and HSP90 inhibition further enhance the endocytic degradation of the exon 19 deletion mutant, in a dynamin activity-dependent and -independent manner, respectively. Albeit with different modes of internalization, the uptake of the exon 19-deleted EGFR is mediated through receptor ubiquitylation.ConclusionsThe internalized EGFR mutant is constantly routed through endosome to lysosome for degradation. The endocytosis of EGFR mutant occurs through both dynamin activity-dependent and -independent mechanisms. Our findings gain novel insights into the endocytic regulation of mutated EGFR and may have potential clinical implications.
Highlights
The epidermal growth factor receptor (EGFR) is closely implicated in cancer, and sequencing analyses have revealed a high mutation rate of EGFR in lung cancer
As exon 19 deletion is the most prevalent EGFR mutation detected from non-small cell lung cancer (NSCLC) patients, the current study focused on this EGFR mutant and investigated its endocytosis [12]
When serum-starved cells were exposed to EGF at 20 ng/ml, downregulation of EGFR was observed in all cell types, with the fastest degradation happened in A549, H460, and SK-MES-1 cells that bear wild-type EGFR (Fig. 1)
Summary
The epidermal growth factor receptor (EGFR) is closely implicated in cancer, and sequencing analyses have revealed a high mutation rate of EGFR in lung cancer. Wang et al Cell Communication and Signaling (2018) 16:40 is internalized through clathrin-mediated endocytosis (CME) or non-clathrin endocytosis (NCE), with the former mode favors signal propagation while the latter one promotes receptor degradation to attenuate signaling. Such balance between receptor signaling and degradation is prudently maintained in normal cells but frequently decoupled in cancer cells overexpressing EGFR [7]. The exon 19-deletion mutation of EGFR is recurrently observed in non-small cell lung cancer (NSCLC) patients, which accounts for nearly 50% of all EGFR abnormalities [10, 12, 13]. The in-frame deletion of exon 19 confers enhanced kinase activity on mutated EGFR and leads to the overstimulation of downstream signaling cascades that promotes tumorigenesis
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