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Updated: Jul 3, 2026
Detection of Targetable Alterations in Non-small Cell Lung Cancer using Next-generation Sequencing
Published on: October 10, 2025
Laser capture microdissection and protein microarray analysis of human non-small cell lung cancer: differential
Amy J VanMeter1, Adrianna S Rodriguez, Elise D Bowman
1Center for Applied Proteomics and Molecular Medicine, George Mason University, Manassas, Virginia 20110, USA.
Abstract:
Little is known about lung carcinoma epidermal growth factor (EGF) kinase pathway signaling within the context of the tissue microenvironment. We quantitatively profiled the phosphorylation and abundance of signal pathway proteins relevant to the EGF receptor within laser capture microdissected untreated, human non-small cell lung cancer (NSCLC) (n = 25) of known epidermal growth factor receptor (EGFR) tyrosine kinase domain mutation status. We measured six phosphorylation sites on EGFR to evaluate whether EGFR mutation status in vivo was associated with the coordinated phosphorylation of specific multiple phosphorylation sites on the EGFR and downstream proteins. Reverse phase protein array quantitation of NSCLC revealed simultaneous increased phosphorylation of EGFR residues Tyr-1148 (p < 0.044) and Tyr-1068 (p < 0.026) and decreased phosphorylation of EGFR Tyr-1045 (p < 0.002), HER2 Tyr-1248 (p < 0.015), IRS-1 Ser-612 (p < 0.001), and SMAD Ser-465/467 (p < 0.011) across all classes of mutated EGFR patient samples compared with wild type. To explore which subset of correlations was influenced by ligand induction versus an intrinsic phenotype of the EGFR mutants, we profiled the time course of 115 cellular signal proteins for EGF ligand-stimulated (three dosages) NSCLC mutant and wild type cultured cell lines. EGFR mutant cell lines (H1975 L858R) displayed a pattern of EGFR Tyr-1045 and HER2 Tyr-1248 phosphorylation similar to that found in tissue. Persistence of phosphorylation for AKT Ser-473 following ligand stimulation was found for the mutant. These data suggest that a higher proportion of the EGFR mutant carcinoma cells may exhibit activation of the phosphatidylinositol 3-kinase/protein kinase B (AKT)/mammalian target of rapamycin (MTOR) pathway through Tyr-1148 and Tyr-1068 and suppression of IRS-1 Ser-612, altered heterodimerization with ERBB2, reduced response to transforming growth factor beta suppression, and reduced ubiquitination/degradation of the EGFR through EGFR Tyr-1045, thus providing a survival advantage. This is the first comparison of multiple, site-specific phosphoproteins with the EGFR tyrosine kinase domain mutation status in vivo.
Insights
This study reveals distinct epidermal growth factor receptor (EGFR) signaling patterns in non-small cell lung cancer (NSCLC) with EGFR mutations. These differences in phosphorylation suggest mechanisms driving cancer cell survival and offer new therapeutic targets.
Area of Science:
- Oncology
- Molecular Biology
- Biochemistry
Background:
- Signaling pathways of epidermal growth factor (EGF) receptor (EGFR) in lung carcinoma tissue microenvironment are not well understood.
- EGFR tyrosine kinase domain mutations are key drivers in non-small cell lung cancer (NSCLC).
- Understanding site-specific phosphorylation patterns in relation to EGFR mutation status is crucial for targeted therapies.
Purpose of the Study:
- To quantitatively profile phosphorylation and abundance of EGFR-related signaling proteins in NSCLC tissues based on EGFR mutation status.
- To investigate the association between EGFR mutation status and coordinated phosphorylation of EGFR and downstream proteins in vivo.
- To explore ligand-induced versus intrinsic signaling phenotypes in EGFR-mutated NSCLC cell lines.
Main Methods:
- Laser capture microdissection of 25 untreated human NSCLC tissues with known EGFR mutation status.
- Reverse-phase protein array (RPPA) to quantify phosphorylation sites on EGFR, HER2, IRS-1, and SMAD.
- Time-course profiling of 115 cellular signaling proteins in EGF ligand-stimulated NSCLC cell lines (mutant and wild-type).
Main Results:
- Mutated EGFR NSCLC samples showed increased phosphorylation at EGFR Tyr-1148 and Tyr-1068, and decreased phosphorylation at EGFR Tyr-1045, HER2 Tyr-1248, IRS-1 Ser-612, and SMAD Ser-465/467 compared to wild-type.
- EGFR mutant cell lines exhibited phosphorylation patterns (EGFR Tyr-1045, HER2 Tyr-1248) mirroring tissue findings.
- Mutant cell lines showed persistent AKT Ser-473 phosphorylation post-ligand stimulation, suggesting sustained PI3K/AKT/mTOR pathway activation.
Conclusions:
- EGFR mutant NSCLC cells may activate the PI3K/AKT/mTOR pathway and suppress IRS-1 signaling.
- Altered HER2 heterodimerization, reduced TGF-beta suppression response, and impaired EGFR ubiquitination/degradation contribute to a survival advantage in EGFR-mutant cells.
- This study provides the first comprehensive comparison of site-specific phosphoproteins with EGFR tyrosine kinase domain mutation status in vivo.