PRH/HHEX in intrahepatic cholangiocarcinoma, disease progression and response to chemotherapeutics.

Cholangiocarcinoma is cancer of the bile ducts that carry bile from the liver to the gall bladder and the small intestine. The number of cases of Cholangiocarcinoma is increasing year by year as is the number of deaths caused by this disease. There are several established risk factors for Cholangiocarcinoma including chronic Hepatitis B and C infection, autoimmune-related diseases such as Primary Sclerosing Cholangitis (PSC), and, in some countries, liver fluke infection. In most cases the disease is very advanced at the time of diagnosis and only around 30% of patients are suitable for surgery. New treatment options and biomarkers that allow early detection and/or inform personalised cancer treatment for individual patients are urgently needed. Under normal circumstances cell proliferation, cell-cell contacts and cell movement are carefully controlled and a large part this control is brought about by proteins called transcription factors that determine which genes are turned on or off in our cells and thereby determine how each cell behaves. We are studying a transcription factor called PRH/HHEX that is essential for liver and bile duct formation because this factor can inhibit many important pathways in the cell that are known to be required for cancer cell division and movement. In this project we will investigate the importance of PRH in cholangiocarcinoma. Using samples taken from patients with cholangiocarcinoma we have already shown that PRH levels are reduced in cholangiocarcinoma cells. The aim of this project is to investigate this in more detail using a larger number of samples. We will also identify genes that are regulated by PRH in cholangiocarcinoma cells and we will determine whether the products of these genes, or PRH itself, can be used as biomarkers to predict how quickly the cancer will progress or even allow us to identify people at risk of developing cholangiocarcinoma before they show symptoms of the disease. We will also determine whether changes in PRH activity alter the ability cholangiocarcinoma cells to form tumours in mice and we will examine whether changes in PRH these cells makes them more or less sensitive to drugs that are used to treat this disease. Finally we think that the re-establishment of PRH activity in cholangiocarcinoma could be a useful strategy for cancer treatment. We will test this using drugs that prevent protein modifications that we have previously shown to result in PRH inactivation and PRH degradation.

Our central hypothesis is that depletion of PRH protein or PRH inactivation by increased protein kinase CK2-dependent phosphorylation is associated with ICC and progression to ICC and that changes in cell behaviour brought about by the loss of PRH activity are of critical importance in the response of ICC to chemotherapeutics. Moreover, we propose that restoration of PRH through pharmacological interventions that decrease PRH inactivation through phosphorylation such as the CK2 inhibitor CX-4945, will be of value in the treatment of ICC patients with elevated levels of phosphorylated PRH. We will use immunohistochemistry to test our hypothesis that PRH levels and/or phosphorylated PRH levels are associated with ICC and to determine whether they are an indicator of poor prognosis in primary sclerosing cholangitis. We will also determine PRH and pPRH protein levels in ICC cell lines and knockdown PRH in these cells using lentiviral constructs expressing PRH shRNA. This will allow us to determine whether PRH knockdown increases the sensitivity of these cells to Cisplatin and the role that PRH plays in mediating the effects of the CX-4945. We will determine whether increased PRH activity in ICC cells results in decreased tumour growth and metastasis in xenograft mouse models. To achieve this we will implant luciferase-labelled PRH over-expressing ICC cells in nude mice and measure tumour growth by repeated in vivo imaging. To examine changes in metastasis we will inject luciferase-labelled PRH knockdown and PRH over-expression ICC cells intravenously into nude mice and monitor the appearance of metastases in internal organs (lymph nodes, lungs, and liver). Finally, we will identify PRH target genes in cholangiocytes and compare the expression of these target genes in cholangiocytes and ICC cells using a genome-wide microarray screen. We will then examine whether the products of these genes are potential serum biomarkers for early diagnosis of ICC.

The incidence of Cholangiocarcinoma is increasing worldwide and early diagnosis is still not available. This contributes to a high mortality rate in Thailand and the UK and more widely. In the face of the massive disease burden of Cholangiocarcinoma worldwide, this UK-Thailand fund offers us a unique opportunity to conduct relevant research to alleviate this problem, and as well as allowing us to bring together experts in several fields within the UK and within Thailand i.e. molecular biologists, clinicians, and pathologists, to share their knowledge and experience in this area. This work will also allow our Thai collaborators to expand their tissue and cell line banks allowing both UK and Thai collaborators a synergistic output as well as the opportunity to forge new collaborations with Professor G. Gores at the Mayo Inst. one of the premier laboratories in the USA working on mouse models of ICC. It will provide an opportunity for face to face meetings and discussions, both physical and via the internet, on how best to collaborate to shed light on this disease.
The outcomes of the previous work from the Jayaraman and Gaston laboratories are expected to have a major impact in the development of new potential combinatorial treatments. This is reflected in the publication of this work in high impact factor Journals as well as the press coverage of these publications. Most recently their work has been highlighted in Genetic Engineering and Biotechnology News (www.genengnews.com/gen-news-highlights) as well as in About Biology and Science Daily and featuring as highlighted reports on the Breast Cancer Campaign website and the University of Bristol website. The insights from this work and from the collaboration with Dr Afford for the current project are likewise expected to have a major impact in the advancement of new combinatorial therapeutic approaches to Cholangiocarcinoma.
PRH also has great potential as a novel biomarker for several conditions and we are working with Bioscience Ventures (BSVL), a joint venture between University of Birmingham and Abingdon Health, to commercialise PRH antibodies for biomarker development. This is another route that is expected to have a significant impact on clinical practice. Our current studies and the ones outlined in this proposal will help to characterise PRH as a potential biomarker and a target molecule for future translational projects. In conjunction with Bioscience Ventures (BSVL), a joint venture between University of Birmingham and Abingdon Health, we have produced PRH monoclonal antibodies and we are investigating the use of these antibodies in cancer diagnosis and prognosis. Further collaborations with Industry are planned to explore the potential of PRH and PRH targets as biomarkers and drug targets. These projects indicate the broader applications of our work and its relevance to patients and to Industry.