To determine if this feedback model explains the activation of phosphatidylinositol 3-kinase signaling in EGFR-mutant cancers, we used shRNA to knockdown endogenous EGFR (which carries an exon 19 deletion) in the HCC827 NSCLC cell line and replaced with either EGFR (exon 19del) wild-type at T669//669, or EGFR (exon 19del) carrying a T669A mutation.
Of note, this is the same EGFR-mutant cell line in which we observed that EGFRT669//669 is phosphorylated in MEK-dependent manner (Figure 5, Supplemental Figure 8A).
When endogenous EGFR was replaced with EGFR (exon19del) wild-type at T669//669, MEK inhibition led to significant feedback activation of ERBB3/phosphatidylinositol 3-kinase/AKT signaling (Figure 6C).
However, replacement with the EGFR (exon19 del) T669A mutant led to increased tyrosine phosphorylation of both EGFR and ERBB3, and activation of phosphatidylinositol 3-kinase/AKT signaling, mimicking the effect of MEK inhibition (Figure 6C).
As expected, addition of AZD6244 failed to further augment ERBB3 and AKT phosphorylation in cells expressing the 669A mutant.
These results demonstrate that EGFRT669//669 phosphorylation is necessary for MEK/ERK to suppress EGFR-mediated activation of ERBB3.
This supports the hypothesis that a dominant ERK feedback on ERBB3/phosphatidylinositol 3-kinase/AKT is mediated though phosphorylation of T669//669 on EGFR (or T677//677 HER2).
