Phosphoproteome Profiling Reveals Molecular Mechanisms of Growth-Factor-Mediated Kinase Inhibitor Resistance in EGFR-Overexpressing Cancer Cells

Although
substantial progress has been made regarding the use of
molecularly targeted cancer therapies, resistance almost invariably
develops and presents a major clinical challenge. The tumor microenvironment
can rescue cancer cells from kinase inhibitors by growth-factor-mediated
induction of pro-survival pathways. Here we show that epidermal growth
factor receptor (EGFR) inhibition by Gefitinib is counteracted by
growth factors, notably FGF2, and we assessed the global molecular
consequences of this resistance at the proteome and phosphoproteome
level in A431 cells. Tandem mass tag peptide labeling and quantitative
mass spectrometry allowed the identification and quantification of
22 000 phosphopeptides and 8800 proteins in biological triplicates
without missing values. The data show that FGF2 protects the cells
from the antiproliferative effect of Gefitinib and largely prevents
reprogramming of the proteome and phosphoproteome. Simultaneous EGFR/FGFR
or EGFR/GSG2 (Haspin) inhibition overcomes this resistance, and the
phosphoproteomic experiments further prioritized the RAS/MEK/ERK as
well as the PI3K/mTOR axis for combination treatment. Consequently,
the MEK inhibitor Trametinib prevented FGF2-mediated survival of EGFR
inhibitor-resistant cells when used in combination with Gefitinib.
Surprisingly, the PI3K/mTOR inhibitor Omipalisib reversed resistance
mediated by all four growth factors tested, making it an interesting
candidate for mitigating the effects of the tumor microenvironment.