title: Proteins, which are upregulated at early time points following Apc deletion, are involved in intestinal tumourigenesis and represent potential colorectal cancer biomarkers
Creators: name: Ibrahim, Shahram
Publisher: name: University of Liverpool Repository
Year: 2015

description: Colorectal cancer is a potentially curable disease if diagnosed at its earliest stages. However, as the current tools for early diagnosis of colorectal cancer are suboptimal, this condition is still a major health issue in the UK and the whole world. Each year nearly 40,000 new cases are diagnosed in the UK. Approximately half of these patients have advanced disease at the time of diagnosis and this is associated with a less favourable prognosis. Therefore, there is still an urgent need for better colorectal cancer screening tools. Moreover, it has long been suggested that APC deletion is an early and major event in the initiation of more than 80% of all colorectal cancers. However, the molecular events that occur following APC deletion are yet to be fully understood. Working on an acute Apc deletion animal model, our group has identified several candidate biomarker proteins. It is postulated that studying these proteins will reveal new aspects about early colorectal tumourigenesis. We hypothesised that these proteins are upregulated very early following Apc deletion and are involved in various aspects of colorectal cancer development, therefore they are potential biomarkers and/or therapeutic targets for this disease. Most of the project’s aims were addressed using immunohistochemical assessment of the expression of several candidate biomarkers in a novel in vivo model of acute Apc deletion (AhCre+Apcfl/fl mouse), a model of established early intestinal neoplasia (ApcMin/+ mouse) and a model of invasive disease (AhCreERT+Apcfl/+Ptenfl/fl mouse) as well as clinical samples from patients with early and advanced colorectal cancer. Mechanistic studies were carried out using the human colonic adenocarcinoma cell lines, HCT116 and HT29. In the animal models, deletion of Apc resulted in an early activation of the Wnt signalling pathway as indicated by nuclear localisation of Beta catenin. Six (NAP1L1, RPL6, SFRS2, PHB, FABP6 and NCL) out of the nine candidate biomarker proteins tested and two proposed partner molecules (Cyclin E and CDC5L), demonstrated obvious differential patterns of expression in areas where Apc loss had induced Wnt pathway activation. Specific knockdown studies of selected members of this protein list in human colon adenocarcinoma cell lines using siRNA identified important effects of these proteins on critical cellular functions such as proliferation and apoptosis. NAP1L1 and RPL6 knockdown had an inhibitory effect on cell proliferation and survival and caused a simultaneous increase in apoptosis. SFRS2 knockdown caused increased abundance of nuclear CDC5L and vice versa. SFRS2 down regulation had marginal effects on cell proliferation and cell survival and resulted in a small reduction in the amount of apoptosis. In contrast, CDC5L knockdown caused a dramatic inhibition of cellular proliferation, loss of the G2 cell cycle peak and a significant increase in the amount of apoptosis and caspase 8 activity. HCT116 cells with a p53 deletion were more sensitive to toxicity of experimental (transfection) reagents and SFRS2 knockdown in these cells caused obvious inhibition of cell proliferation with increased apoptosis. Immunohistochemical staining of the candidate biomarker proteins in human samples of colorectal cancer generally supported the results shown in animal studies for early stages of the disease. Moreover, it demonstrated reduced nuclear expression of Beta catenin, nuclear relocalisation of CDC5L and cytoplasmic displacement of SFRS2 in the more advanced stages of colorectal cancer. PHB showed increased cytoplasmic staining in Dukes’ stage A and B cancers. These results were backed up by appropriate scoring systems. Our results concerning these proteins in colorectal cancer are novel and agree with several studies by other research groups describing their roles in other cancers or cancer models. Due to the relatively late detection of lesions in humans, the early changes which were observed in animals might have been missed in humans. NAP1L1, RPL6, SFRS2 and NCL showed early overexpression during colorectal tumourigenesis. These proteins therefore have the potential as screening biomarkers. Due to their role in regulating cellular proliferation, NAP1L1, RPL6 and CDC5L are potential therapeutic targets for colorectal cancer. PHB is also a potential marker for Dukes’ stage A and B cancers. Mechanistically, the interplay between SFRS2 and CDC5L during colorectal tumourigenesis is a promising case to follow. Based on this study and other studies conducted in our group, the above proteins are promising screening, predictive or prognostic markers as well as potential therapeutic targets for colorectal cancer.