Proteomic analysis of low- and high-grade human colon adenocarcinoma tissues and tissue-derived primary cell lines reveals unique biological functions of tumours and new protein biomarker candidates

Abstract

Background: Colon cancer is the third most common cancer and second highest cause of cancer deaths worldwide. The aim of the study was to find new biomarkers for diagnosis, prognosis and therapeutic drug targets for this disease.Methods: Four low-grade and four high-grade human colon adenocarcinoma tumours with patient-matched normal colon tissues were analysed. Additionally, tissue-derived primary cell lines were established from each tumour tissue. The cell lines were validated using DNA sequencing to confirm that they are a suitable in vitro model for colon adenocarcinoma based on conserved gene mutations. Label-free quantitation proteomics was performed to compare the proteomes of colon adenocarcinoma samples to normal colon samples, and of colon adenocarcinoma tissues to tissue-derived cell lines to find significantly differentially abundant proteins. The functions enriched within the differentially expressed proteins were assessed using STRING. Proteomics data was validated by Western blotting. Results: A total of 4767 proteins were identified across all tissues, and 4711 across primary tissue-derived cell lines. Of these, 3302 proteins were detected in both the tissues and the cell lines. On average, primary cell lines shared about 70% of proteins with their parent tissue, and they retained mutations to key colon adenocarcinoma-related genes and did not diverge far genetically from their parent tissues. Colon adenocarcinoma tissues displayed upregulation of RNA processing, steroid biosynthesis during tumour initiation and immune system responses and detoxification during tumour progression, downregulation of cytoskeletal organisation and loss of normal muscle function. Tissue-derived cell lines exhibited increased interferon-gamma signalling and aberrant ferroptosis. Overall, 318 proteins were significantly up-regulated and 362 proteins down-regulated by comparisons of high-grade with low-grade tumours and low-grade tumour with normal colon tissues from both sample types. Conclusions: The differences exhibited between tissues and cell lines highlight the additional information that can be obtained from patient-derived primary cell lines. DNA sequencing and proteomics confirmed that these cell lines can be considered suitable in vitro models of the parent tumours. Various potential biomarkers for colon adenocarcinoma initiation and progression and drug targets were identified and discussed, including ten novel markers ACSL4, ANK2, AMER3, CAV3, EXOSC6, EXOSC6, GCLM, LDB3, MUC5B and TRFC.