MEK inhibition leads to PI3K/AKT activation by relieving a negative feedback on ERBB receptors

Abstract

The phosphoinositide 3-kinase (PI3K)/AKT and RAF/MEK/ERK signaling pathways are activated in a wide range of human cancers. In many cases, concomitant inhibition of both pathways is necessary to block proliferation and induce cell death and tumor shrinkage. Several feedback systems have been described in which inhibition of one intracellular pathway leads to activation of a parallel signaling pathway, thereby decreasing the effectiveness of single-agent targeted therapies. In this study, we describe a feedback mechanism in which MEK inhibition leads to activation of PI3K/AKT signaling in EGFR and HER2-driven cancers. We found that MEK inhibitor-induced activation of PI3K/AKT resulted from hyperactivation of ERBB3 as a result of the loss of an inhibitory threonine phosphorylation in the conserved juxtamembrane domains of EGFR and HER2. Mutation of this amino acid led to increased ERBB receptor activation and upregulation of the ERBB3/PI3K/AKT signaling pathway, which was no longer responsive to MEK inhibition. Taken together, these results elucidate an important, dominant feedback network regulating central oncogenic pathways in human cancer.

Figure 1. MEK inhibition leads to feedback activation of ERBB3/PI3K/AKT signaling

(A) EGFR-addicted (HCC827, H4006, H1975) or HER2-addicted (BT-474, NCI-N87, MDA-MB-453) cell lines were treated with 2µM AZD6244 for 6 hours. Cell lysates were immunoblotted to detect the indicated proteins. (B) H1975 (EGFR L858R/T790M) xenografts were treated with AZD6244 25mg/kg BID. Mice were sacrificed 6hrs following treatment and tumors were harvested. Tumor cell lysates were immunoblotted to detect the indicated proteins. (C) MDA-MB-453 and HCC827 cells were treated for 6hrs or 18 hours with 2µM AZD6244. Phospholipids were isolated from cell lysates and relative PIP₃ and PI(4,5)P₂ levels were quantified by ELISA. Each data point represents the mean ±SEM of two independent experiments performed in triplicate. For normalization, the PIP₃/PI(4,5)P₂ ratio was set to 100% for untreated cells. Asterisks represent p<0.05. (D) HCC827 cells were treated for 6hrs with 2µM AZD6244. Cell extracts were immunoprecipitated with an anti-p85 antibody followed by Western blot with anti-p-Tyr and anti-p85 antibodies. Blots were stripped and re-probed with antibodies specific to ERBB3, GAB1, and IRS-1.

Figure 2. Feedback activation and increased surface localization of ERBB3 occurs within one hour of treatment with AZD6244

(A) HCC827 cells were treated for the indicated number of hours with AZD6244 (2µM). Cell lysates were immunoblotted to detect the indicated proteins. (B) HCC827 cells were treated with DMSO (NoRx) or AZD6244 (2µM) for 1 hour at 4degC during biotin labeling of surface proteins. Following labeling, cells were returned to media (+/− AZD6244) and treated for the indicated number of hours before lysis. Biotin labeled surface proteins were immunoprecipitated with NeutrAvidin beads, separated by SDS page, and immunoblotted to detect the indicated proteins.

Figure 3. Suppression of ERBB3 expression abrogates MEK/ERK feedback on PI3K/AKT signaling

(A) HCC827 cells infected with a lentivirus expressing a Tet-inducible shERBB3 hairpin were induced with 100ng/mL doxycycline (+Dox) for 48hrs. Following knockdown, cells were treated with AZD6244 (2µM) for the indicated number of hours. Cell lysates were immunoblotted to detect the indicated proteins. (B) BT-474 cells were transfected with control or ERBB3 targeted siRNA for 48hrs, followed by treatment with AZD6244 for 6hrs. Cell lysates were immunoblotted with the indicated proteins. (C) BT-474 cells were transfected with control or ERBB3 targeted siRNA for 48hrs, followed by treatment with 2µM AZD6244, 1µM of the PI3K inhibitor GDC-0941, or the combination for 72hrs. Cells were collected and stained for propidium iodide and AnnexinV to determine the percentage of apoptotic cells. Each data point represents the mean ±SD of three independent experiments.

Figure 4. MEK/ERK feedback on ERBB3 occurs in KRAS-mutant cell lines and is distinct from TORC1 feedback on IRS-1

(A) KRAS-mutant cell lines were treated with AZD6244 (2µM) or Rapamycin (50 nM) for 6 or 24hrs. Cell lysates were immunoblotted to detect the indicated proteins. (B) SW1463 cells were treated with 1µM of the EGFR inhibitor gefitinib or 1µM of the IGF-IR inhibitor NVP-AEW541, alone or in combination with 2µM AZD6244 or 50nM Rapamycin for 24 hours. Cell lysates were immunoblotted to detect the indicated proteins. (C) Model of MEK feedback on ERBB3 in EGFR/HER2-addicted cancers (top) and KRAS-mutant cancers with low phospho-ERBB3 (bottom).

Figure 5. MEK inhibition blocks phosphorylation of a direct ERK target site in the conserved JM domains of EGFR and HER2

(A) HCC827 and BT-474 cells were treated with AZD6244 (2µM) for 6 or 24hrs. Cell lysates were immunoblotted to detect the indicated proteins. Phospho-HER2 (677) runs at 185kDa and is recognized by the anti-phospho-EGFR (669) antibody. (B) HCC827 or BT-474 cells were treated with AZD6244 (2µM) for 6hrs. EGFR or HER2 was immunoprecipitated, and coomassie blue bands were excised for quantitative mass spectrometry analysis of peptide phosphorylation. Each data point represents the mean ±SD of three independent experiments.

Figure 6. T669A mutation of EGFR or T677A mutation of HER2 blocks MEK feedback activation of ERBB receptors

(A, B) CHO-KI cells were transiently transfected to express wild-type ERBB3 in combination with a GFP control, or wild-type or mutant (A) EGFR or (B) HER2. 48hrs post transfection cells were treated with AZD6244 (2µM) for 90 minutes. Cell lysates were immunoblotted to detect indicated proteins. Cells expressing EGFR T669A were also treated with 50ng/mL HRG ligand for 30 minutes to achieve maximal ERBB3 phsophorylation. (C) HCC827 cells were infected with a control or shEGFR hairpin, followed by infection with lentiviral vectors expressing GFP, T669 wild-type EGFR (exon 19del), or EGFR T669A (exon 19del). Following knockdown and puro selection for 72hrs, cells were treated with AZD6244 (2µM) for 6hrs. Cell lysates were immunoblotted to detect the indicated proteins.

Figure 7. Model of MEK inhibitor-induced feedback on ERBB receptor signaling pathways

In untreated cells EGFR is phosphorylated at T669 by MEK/ERK, which inhibits activation of EGFR and ERBB3. In the presence of AZD6244, ERK is inhibited and T669 phosphorylation is blocked, increasing EGFR and ERBB3 tyrosine phosphorylation and up-regulating downstream signaling.