Proteomic Identification and Immunological Validation of Protein Biomarkers for Early Detection and Prognosis of Colorectal Cancer

Abstract

Colorectal cancer (CRC) is an important public health problem both internationally and in New Zealand. Currently available systems for early cancer detection (Faecal Occult Blood Test) and prediction of prognosis (pathological staging) are widely regarded as being imperfect, although no alternative tests are available. Identification and validation of sensitive and accurate biomarkers for the management of CRC is the goal of the work undertaken and described in the present PhD thesis.

Recent developments in proteomic technologies have allowed comparison of multiple proteins from different tissues to be accomplished with good reproducibility and sensitivity. This has led to identification of many putative serological and tissue biomarkers for cancer. The vast majority of these early proteomic studies used gross tumour tissues for analysis. High intratumoural tissue heterogeneity such as differences in the amount of stroma and body fluid content within the gross tissue mass makes the crude tissue lysate less representative of the epithelial cancer cells under study and increases the technical variability. The development of laser capture microdissection (LCM) has allowed isolation of pure populations of cancer cells for analysis, significantly increasing the accuracy of quantitative tumour proteomics. In this thesis, a combined use of LCM and two-dimensional difference gel electrophoresis (2D-DIGE) in profiling CRC tissues is reported. This approach greatly improved the profiling accuracy as evidenced by a significant reduction in inter-patient variation as well as the discovery and validation of six previously unrecognised CRC–associated proteins. By performing immunohistochemistry on tumour tissue microarrays, two candidate markers — ACY1 and TUFM have been further evaluated as potential novel adverse prognostic factors for CRC. The study also developed a novel strategy to identify tumour-specific secreted proteins by incorporating secretome samples (total proteins released from cultured CRC cells) into the same 2D-DIGE analysis of the tumour tissue proteome as mentioned above. Fifteen proteins were identified to be both secreted from cancer cells and overexpressed in the tumour tissues. These tumour-specific secreted proteins have potential as specific serum/plasma markers for early cancer detection.Using an ELISA kit, developed in-house, one candidate marker — RUVBL1 was confirmed to be present in the cancer patients’ plasma at a concentration 4 fold greater on average than that found in the normal donor plasma, suggesting the potential utility of RUVBL1 as a non-invasive blood test for early detection of CRC. These results prove that the strategy used in this study is valid in discovering potentially novel biomarkers for CRC. Studies with more participants are required to confirm the clinical utility of these findings. The biological implications of these novel candidates also need to be further explored in the future.