Proteomic and phosphoproteomic characterization of FFPE lung adenocarcinoma harboring ALK, EGFR or KRAS Mutations, and triple wild-type tissue sections

Lung cancer (LC) is the leading cause of cancer-related mortality. Lung adenocarcinoma subtype (LUAC) accounts for approximately 40% of all LC cases. Mutations in key oncogenes have been identified as important genetic alterations in LUAC, including genes encoding epidermal growth factor receptor (EGFR), Kirsten rat sarcoma viral oncogene homolog (KRAS), and anaplastic lymphoma kinase (ALK). Our aim was the characterization of the altered biological pathways and phosphorylation events in LUAC tumors harboring EGFR, KRAS, or ALK oncogenic mutation and triple wild-type (WT) LUAC tumors by MS-based quantitative proteomics and phosphoproteomics analyses of 84 formalin-fixed paraffin-embedded (FFPE) tissue sections. The on-surface tryptic digestion and subsequent phosphopeptide enrichment enabled the identification of differentially expressed proteins and phosphosites, respectively using label-free quantitation.

During the proteomic analysis, 1995 proteins were subjected to statistical analysis. To identify proteins differentially expressed between the four sample groups, two-group comparisons were performed. Altogether, the expression of 160 proteins was significantly altered in at least one of the six comparisons. Based on the functional enrichment analysis, most of these proteins were affiliated with intracellular organelle lumen, actin cytoskeleton, Golgi apparatus, vesicle, and focal adhesion terms. Samples with EGFR mutation showed the most differences. Moreover, 31 proteins had been identified as mutation-specific (significantly altered in one mutation compared to the others) serving as potential therapeutic targets.

In the phosphoproteomic analysis, 386 phosphosites were considered for statistical analysis. Principal component analysis of these phosphosites revealed considerable differences between the samples with different mutations. The expression of 202 sites was significantly altered between the four sample groups. Additionally, 61 sites were found to be significantly dysregulated specifically in one sample group. These 61 phosphosites derived from 48 phosphorylated proteins. Functional enrichment analysis revealed that most of these proteins were involved in cellular processes, like cytoskeleton organization, and metabolic processes, like regulation of primary metabolic process, regulation of cellular biosynthetic process, gene expression, and RNA processing including RNA splicing. Kinase-substrate enrichment analysis revealed altered activities of 10 kinases.

Our results could provide significant insights for developing therapeutic strategies for LUAC.

This project was supported by the Lendület (Momentum) Program of the Hungarian Academy of Sciences (HAS, MTA). Funding from the National Research, Development, and Innovation Fund of Hungary (FK 131603) is acknowledged. Samples were run at the ISTA LSF Mass Spectrometry Facility, part of the Scientific Service Units of ISTA. The Authors are grateful to Goran Mituloviĉ for the column.