Molecular and regional characterization of colorectal polyps: insights from proteomics, phosphoproteomics, and immune profiling.

BACKGROUND: Familial adenomatous polyposis (FAP) is an inherited predisposition to colorectal cancer and characterized by profuse colorectal adenomas starting from the second decade of life. Regional (left vs. right) differences in the colonic microbiologic and immunologic microenvironment may impact adenoma evolution but are poorly understood. We aimed to characterize regional molecular, microbial, DNA damage, and immune differences in pediatric FAP polyps to test the hypothesis that polyps in pediatric FAP exhibit distinct regional and molecular features that contribute to differential growth and genomic instability. METHODS: Colonic polyps and adjacent non-polyp mucosa were harvested from pediatric FAP patients undergoing colonoscopy. Tandem mass tag-based proteomic and phosphoproteomic profiling was performed and were followed by functional assays including colony formation, spheroid growth, and patient-derived organoid culture. γH2AX staining was used to quantify induction of DNA double-strand breaks (DSBs) in HCT116 colon cancer cells cultured in Fusobacterium nucleatum conditioned media (FnCM). Immunohistochemistry and immunofluorescence were used to assess ATR, CDK4, γH2AX, and oxidative damage (8-OxoG). Immune profiling was performed by flow cytometry, focusing on CD103(+) tissue-resident memory T cells (TRMs). RESULTS: Right-sided polyps exhibited increased ATR and CDK4 expression compared with left-sided lesions and adjacent mucosa. FnCM exposure induced a marked increase in γH2AX staining in HCT116 cells, consistent with our in vivo findings of elevated DSB burden in proximal versus distal FAP polyps. Biofilm enrichment and higher microbial staining were observed in right-sided lesions, whereas distal polyps were enriched with CD103(+) TRM populations. Pharmacologic inhibition of ATR or CDK4 significantly suppressed both colony formation and spheroid growth. Organoids derived from proximal colon polyps exhibited accelerated growth and crypt budding, with higher expression of stemness markers (CD44, CD133, Lgr5, BMI-1) compared with distal polyps. CONCLUSIONS: Integrated proteomic, phosphoproteomic, and immune-microbiome profiling reveals regional heterogeneity of adenomas in pediatric FAP. Right compared to left sided polyps harbor greater DNA damage, elevated ATR/CDK4 kinase activity, reduced immune surveillance, and increased stem-like growth. These findings identify ATR and CDK4 as potential therapeutic targets and suggest that regional microenvironmental differences can impact chemoprevention strategies in pediatric FAP.