Uncovering H. Pylori associated and independent Gastric cancer signature by tissue phosphoproteomic analysis

Gastric cancer, the fifth most prevalent cancer worldwide, involves complex etiologies, including but not limited to H. pylori infection. While numerous genomic studies have found molecular signatures associated with pathogenic phenotypes, 40% patients, especially young male, do not associate with H. pylori that necessitates understanding of their underlying mechanism. To gain more insights into gastric cancer pathogenesis and therapeutic drug discovery, we performed a deep proteomic and phosphoproteomic profiling of paired tumors and adjacent normal tissues (NAT) from a prospectively collected gastric cancer cohort of 120 patients, predominantly male (65%), with an average age of 64. Global proteome and phosphoproteome profiles were conducted by TMT 11-plex-based proteomic workflow using pooled NAT and tumor as an internal standard to ensure long-term analysis reproducibility. A total of 11,498 proteins and 95,341 unique phosphosites from 110,454 phosphopeptides in 10,044 proteins were identified. The PCA analysis revealed separate patterns of normal and tumor profiles. Unsupervised hierarchical clustering of proteomic and phosphoproteomic profiles stratifies patients into three (PT1-PT3) and four (PS1-PS4) clusters, respectively. Proteomic cluster 2 (PT2, 37 patients) is enriched with male (84%), H. Pylori infection (73%) and distal location of the stomach (81%). Similarly, phosphoproteomic subtype 2 (PS2) is also enriched with H. Pylori infection (81%), including 17 common patients from the PT2 cluster with a higher proportion of male and H. Pylori infection. Further pathway enrichment and functional annotation indicate a strong association with bacterial invasion, cell adhesion, and aberrant ECM-receptor modulation. In addition, differential proteins in H. pylori negative and positive populations were compared in NATs and tumor proteome. The findings revealed distinctly differential proteins in tumor and NATs, showing enrichment of immune response and metabolic pathways regulation in the tumor proteome, while the NAT proteome is enriched with focal adhesion and PI3K-AKT signaling pathway. Taken together, the proteomic and phosphoproteomic profiling revealed new insight into the etiology of gastric cancer patients associated with H. Pylori infection, and may provide potential mechanisms for biomarker development and drug treatment.