BioProject

Never-smoker lung adenocarcinoma (NSLA) is prevalent in Asian populations, and is even more in women. EGFR mutations and ALK fusions are major alterations observed in NSLA. We have focused on NSLA without EGFR and ALK alteration (NENA) rather than NSLA with EGFR and ALK (EA). First, we selected 141 NSLA tissues, and performed proteogenomic analyses including the whole-genome sequencing (WGS), transcriptome, methylation EPIC array, total proteome and phosphoproteome. We then excluded 40 patients with EA and 7 patients with NENA microsatellite instability. Genome analysis revealed that TP53 (25%), KRAS (22%), ROS1 fusion (14%), and SETD2 (11%) were the most frequently mutated genes in NENA patients. Proteogenomic impact analysis revealed that STK11 and ERBB2 somatic mutations had broader effects on cancer-associated genes in NENA. Through DNA copy-number alteration analysis, we identified 22 prognostic proteins, influencing transcriptomic and proteomic changes. Gene set enrichment analysis revealed that the estrogen signaling emerged as the key pathway activated in NENA. A lot of proteogenomic evidence supported the increased estrogen signaling, such as copy-number deletions in chromosomes 14 and 21, STK11 mutation, and DNA hypomethylation of LLGL2 and ST14. Finally, saracatinib, an Src inhibitor, was suggested as a potential drug for targeting activated estrogen signaling in NENA, and was experimentally validated in vitro using cell line model. In this study, we enhanced our understanding of the etiology of NENA NSLA through the proteogenomic landscape, based on which we proposed saracatinib as an effective drug Overall design: The Infinium MethylationEPIC BeadChip (Illumina) was employed for methylation array experiments following the manufacturer’s instructions. Briefly, 500 ng of gDNA from lung tissues was treated with 20 µL of sodium bisulfite solution using an EZ DNA Methylation-Gold µKit (Zymo Research). Bisulfite-converted DNA (4 µL) was amplified using the Infinium Methylation Assay Kit (Illumina). The amplified DNA was hybridized to the Infinium MethylationEPIC BeadChip and scanned using an Illumina iSCAN system. CpG methylation values were calculated as average-values using Minfi (v3.11), with the following equation: average = M/(M + U + 100), where M and U are the methylated and unmethylated signal intensities, respectively. Measurements with a p-value <0.05 were considered significant above the background. Additionally, redundant probes, including multi-hit probes and SNPs, which could affect the analysis results, were filtered using the ChAMP package filter function.