Understanding the biology of transforming growth factor-β in colorectal cancers

Colorectal cancer (CRC affects millions of people every year globally. Abnormal cells utilise several mutated proteins and perturbed pathways to progress from a benign tumour to malignant cancer. Expression of proteins such as uPAR, integrin β6 and TGFβ have been extensively implicated by us and others in CRC. The primary aim of this thesis was to contribute additional knowledge regarding the role of TGFβ in CRC through investigation of the proposed hypothetical uPAR·αvβ6·TGFβ1 interactome. This was achieved by using six CRC cell lines as model systems where expression levels of two known activator systems of TGFβ, namely integrin β6 (SW480Mock, SW480β6OE, HT29Mock, HT29β6AS) and the uPA protease receptor uPAR (HCT116WT and HT29uPARAS) have been artificially expressed or down regulated. The changes in these model systems following active TGFβ1 treatment were investigated using state-of-the-art proteomics and a cell signalling assay (i.e., AlphaScreen® SureFire® Assay) technologies in conjunction with sophisticated bioinformatics. The cells expressing β6 (SW480β6OE, HT29Mock, HT29β6AS) exhibited increased proliferation, invasion and wound healing upon treatment with TGFβ1. The cells with higher uPAR expression did not respond to (HCT116WT) TGFβ treatments. These results determined that malignancy was attained in a TGFβ-dependent manner when β6 was expressed or in a TGFβ-independent manner when uPAR was expressed. Additionally, the proteomic data presented in this thesis identified several perturbed proteins and biomolecular pathways that could be associated with CRC and has given important clues to understanding the role of TGFβ and the proposed hypothetical uPAR•αvβ6•TGFβ1 interactome. Additionally, an Olink Proseek study using Dukes’ stage A-D CRC patient plasma samples (1μL of plasma) identified CEA, IL-8 and prolactin were determined to differentiate unaffected controls from non-malignant (Dukes’ A + B) and malignant (Dukes’ C + D) stages and were published as potential plasma Dukes’-stage CRC biomarkers. This thesis has demonstrated the immense power of high-throughput modern proteomic and multiplexing technologies to gain insights into the TGFβ associated CRC pathogenesis at detailed molecular level and to identify avenues for disease biomarker exploration.