Saliva and stool proteomic profiling revealed potential biomarkers for inflammatory bowel diseases and colorectal carcinoma

Inflammatory Bowel Diseases (IBDs) are a heterogeneous group of chronic disorders, with Crohn"s disease (CD) and ulcerative colitis (UC) being the most frequent. Patients diagnosed with IBDs are at high risk of developing several neoplasia, including colorectal carcinoma (CRC), as a result of the inflammation. IBDs and CRC often exhibit shared pathogenetic mechanisms and display common non-specific symptoms, making the diagnostic process challenging. Moreover, traditional clinical practice based on blood laboratory tests and endoscopy can hardly detect such diseases at early onset. Hence, there is an urgent need for predictive novel biomarkers for both IBDs and CRC. Although serum is the preferred biological matrix, stools and saliva are also promising matrices to screen for biomarkers, considering their ease and non-invasive collection methods.

Mass spectrometry-based proteomics is a high-throughput technology to study potential biomarkers on a large scale. In this study, we aimed to investigate the presence of biomarkers for IBDs and CRC in saliva and stool samples belonging to 20 healthy controls (CS), 12 CD, 13 UC, and 37 CRC, using a proteomic approach. Saliva samples were pooled and proteins were separated by SDS-PAGE before digestion, whereas endogenous peptides were extracted from pooled fecal samples using 10 kDa cut-off filters. LC-MS/MS analyses were performed with an LTQ Orbitrap XL coupled to a nano-HPLC Ultimate 3000 (Thermo Fisher Scientific), using a linear gradient of ACN / 0.1% FA to separate the peptide mixtures.

A total of 152 proteins were identified in saliva samples, of which 73 were commonly found in CRC, CD, and UC, but not in CS. According to a Gene Ontology-based enrichment analysis, these proteins are involved in cell adhesion, regulator activity of peptidases, and glucose and nucleic acid metabolism. Of the remaining 79 proteins, those highly represented in CRC and CD, are related to inflammation, DNA stability, immunity, and oxidation. In stool samples, peptides belonging to 30 different proteins were identified, of which 4 were specific for CD. Among these, Basic Salivary Proline-Rich Protein 1 (PRB1) was highly abundant, whereas its isoform PRB2 was reduced in abundance in IBDs and CRC saliva samples. Moreover, two PRB1 peptides were shown to stimulate in vitro CRC cells" growth and the pro-proliferative signaling pathways Erk1/2, Akt, and p38.

These results demonstrated that CRC and CD share common salivary proteins, and PRB1-derived peptides stimulate cancer cell growth, suggesting a potential role of such proteins in IBDs and cancer pathogenesis.

Grant: PNRR-POC-2022-12375750 "Diagnostic innovation for inflammatory bowel diseases in children and adults: non invasive stool and salivary testing with ecologic new methods and biomaterials", funded by the European Union - NextGenerationEU – NRRP M6C2 - Investment: 2.1 "Enhancement and strengthening of biomedical research within the NHS. CUP I93C22000590006