Effects of AKT inhibition on HGF-mediated erlotinib resistance in non-small cell lung cancer cell lines

Abstract

Purpose: Acquired resistance to erlotinib in patients with EGFR-mutant non-small cell lung cancer can result from aberrant activation of alternative receptor tyrosine kinases, such as the HGF-driven c-MET receptor. We sought to determine whether inhibition of AKT signaling could augment erlotinib activity and abrogate HGF-mediated resistance.

Methods: The effects of MK-2206, a selective AKT inhibitor, were evaluated in combination with erlotinib on a large panel of 13 lung cancer cell lines containing different EGFR or KRAS abnormalities. The activity of the combination was assessed using proliferation assays, flow cytometry and immunoblotting. The MEK inhibitor PD0325901 was used to determine the role of the MAP kinase pathway in erlotinib resistance.

Results: The combination of MK-2206 and erlotinib resulted in synergistic growth inhibition independent of EGFR mutation status. In cell lines where HGF blocked the anti-proliferative and cytotoxic effects of erlotinib, MK-2206 could restore cell cycle arrest, but MEK inhibition was required for erlotinib-dependent apoptosis. Both AKT and MEK inhibition contributed to cell death independent of erlotinib in the T790M-containing H1975 and the EGFR-WT cell lines tested.

Conclusions: These findings illustrate the potential advantages and challenges of combined signal transduction inhibition as a generalized strategy to circumvent acquired erlotinib resistance.

Fig. 1

a Growth curves of single-agent MK-2206 in NSCLC cell lines. Cells were treated for 72 h prior to MTT. Legend is in order of sensitivity as indicated by IC₅₀. Mutation status indicates the presence of an EGFR or KRAS mutation, with the specific substitution in parentheses; wt indicates no known abnormalities in either EGFR or KRAS. b Immunoblot of phospho-AKT (S473) following 3 h of treatment with MK-2206 at the indicated dose

Fig. 2

a Growth response of NSCLC cell lines following 72 h of treatment with erlotinib (E) at 0.05 μM (HCC827/PC-9) or 0.5 μM, MK-2206 (M) at 0.5 μM, HGF at 50 ng/mL, and/or PHA-665752 (PHA) at 0.5 μM using the CellTiter-Fluor Cell Viability Assay. Data are graphed as % growth relative to untreated cells. White columns have no added HGF or PHA; black columns are supplemented with HGF; hatched columns are supplemented with both HGF and PHA. b Immunoblot of the HCC827 cell line following 24 h of treatment with erlotinib, HGF, and/or PHA-665752 at similar dose levels

Fig. 3

Growth response of NSCLC cell lines models of HGF-dependent erlotinib resistance following treatment with erlotinib (E)* (0.5 μM), MK-2206 (M) (0.5 μM), PD0325901 (PD) (0.5 μM), PHA665752 (PHA) (0.5 μM), and HGF (50 ng/mL) as single-agents or in combination using the CellTiter-Fluor Cell Viability Assay. All cells were treated for 72 h prior to analysis. Data are graphed as % growth relative to untreated cells. White columns have no added HGF or PHA; black columns are supplemented with HGF; hatched columns are supplemented with both HGF and PHA. *HCC827 and PC-9 cells were treated with lower doses of erlotinib (0.05 μM)

Fig. 4

Immunoblotting analysis of phospho-EGFR, AKT, and ERK following 24 h of treatment in NSCLC cell lines. a Cells were treated with erlotinib* (0.5 μM), MK-2206 (0.5 μM), and PD0325901 (0.5 μM) as single-agents and in combination. b Cells were treated with erlotinib, MK-2206, PD0325901 with or without HGF (50 ng/mL) and PHA665752 (0.5 μM). *HCC827 and PC-9 cells were treated with lower doses of erlotinib (0.05 μM)

Fig. 5

Immunoblotting analysis of PARP cleavage and cleavage caspase-3 (CC3) following 24 h of treatment with erlotinib* (0.5 μM), MK-2206 (0.5 μM), PD0325901 (0.5 μM) as single-agents and in combination with NSCLC cell lines with or without HGF (50 ng/mL) and PHA665752 (0.5 μM). *HCC827 and PC-9 cells were treated with lower doses of erlotinib (0.05 μM)