Role of heparin binding growth factors in O.viverrini induced Cholangiocarcinoma (O-CCA) development, progression and angiogenesis

Cancer of the bile duct (cholangiocarcinoma, or CCA) is a severe, often fatal cancer, rare in the western world. However, it is a hundred times more common in areas of Thailand, Laos and Vietnam than anywhere else in the world. This is mainly because it may be caused by a liver fluke called Opisthorchis. This is a parasite that lives in fish found in the rivers of that area. The exact type of cancer caused by this parasite is different from other cancers of the bile duct. This means that we can think of CCA as being two (or more) different types of cancer - Opisthorchis-induced CCA (or O-CCA), and CCA cause by other causes (e.g. hepatitis, inflammatory bowel disease, smoking, etc - which we have termed NonO-CCA. In Thailand this disease is devastating, particularly in poor areas, where both men and women suffering from this disease are unable to provide for their families and leave children as orphans when they die.
There is a known class of proteins that contribute to cancer growth called the heparin binding factor growth factors (HB-GF). Heparin is a carbohydrate (starch like molecule) that is found in the space between cells, including in cancer. These HB-GFs include epidermal growth factor (EGF), vascular endothelial growth factor (VEGF), and hepatocyte growth factor (HGF). Drugs that inhibit these growth factors have all been made and are used for various cancers (e.g. Herceptin for breast cancer), but not yet shown to be useful for CCA. We have recently found out that a molecule that mimics heparin, isolated from a seaweed commonly used in Thai food, can interfere with how HB-GFs help CCAs grow in the laboratory. This seaweed extract, called sulphated galactan (or SG) can stop CCA cells from growing moving, and making new blood vessels. However, we do not know which HB-GFs it sticks to, or how I stops them working. In this project we intend to develop a panel of O-CCA cells by using parts of the cancers surgically removed from patients with the disease, and compare them with NonO-CCA patients in the UK and find out whether there are notable differences in the way their heparin binding growth factors are made or work, find out whether growth of these Thailand specific cancers can be halted with drugs that target HB-GFs and whether the Thai seaweed extract can be used to tell us what is different about these types of cancer cell.
Ultimately we would like this work to pave the way for clinical trials of specific, already approved, drugs in CCA in Thailand. We think that by understanding the biology of these cancer cells we will be able to design trials that use these drugs in combination to target Thai CCA patients. Moreover, we would like to investigate whether the Thai seaweed extract can be used to help research into cancer of the bile duct in Thailand and the rest of the world.

Technical Summary.
We aim to test four hypotheses.
a) O-CCA tumours produce growth factor combinations that lend themselves to heparin binding mediated tumour growth.
This will be done by combining tissue collection of samples and processing them into a tissue microarray for screening of tumours for expression of growth factors and their receptors. We will also generate a bank of CCA tumour cell lines from fresh tissue collected in Thailand and Nottingham. We will investigate growth factor expression by multiplex ELISA, HB-GF receptor expression and signalling by RNA expression by digital droplet PCR and western blot using phosphospecific antibodies, and by pharmacologically and molecular interference with the signalling pathways. We will develop patient derived tumour cell lines and xenografts for in vivo modelling of these tumours. We will compare NonO with O-CCA to determine differences in signalling.
b) O-CCA mediated angiogenic growth factor production is a consequence of that growth factor signalling
Production of angiogenic cytokines, in particular VEGF but others too will by these cells will be examined in response to pharmacological inhibition and molecular knockdown of the HN-GFs and their receptors.
c) that preventing that upregulation in O-CCA cells can slow O-CCA in animal models
These will then be followed up in vivo by determining cacner growth rates in heterotopic (subcutaneous, measured by calliper) and orthotopic (bile duct, measured by bioluminescent imaging) tumour growth in immunosuppressed animals with human stromal cells.
d) that this can be done using sulphated galactans from G. fisherii.
O-CCA and NonO-CCA cells will be treated with heparin binding inhibition with sulphated galactan and the identify of the HBGFs discovered, the mechanism of action determined, and the role of those HBGFs in mediating cell migration, proliferation and angiogenesis investigated.

The involvement of individual heparin binding growth factors in cholangiocarcinoma (CCA) has been demonstrated by numerous studies. These are now beginning to be translated into individual clinical trials using anti-growth factor antibodies such as bevacizumab and trastuzimab, and growth factor receptor inhibitors such as sunitinib and erlotinib. However, it is unlikely that inhibition of a single HB-GF will provide long lasting efficacy in patients, as numerous HB-GF are expressed in various CCA. In contrast understanding how multiple HB-GF interact, and in particular the effect of inhibition of multiple HB-GF by inhibitors that target common pathways, such as the sulphated galactans described here, could provide proof of principle of a novel treatment for a disease that affects up to 85 patients per 100000 people in areas of Thailand. The impact of this would be stimulate drug discovery for competitive heparin binding agents, either synthetic or from natural sources, and potentially stimulate both Thai medical research, and investment in Thai aquaculture. The ability to understand the difference between CCA derived from fluke infection and CCA derived from viral or non infectious disease at a growth factor signalling level will also impact on our understanding of cancer diversity - already partially indicated by the findings of significant genetic discrepancy between the two cancer types.
The establishment of tissue arrays for tumour types has had a significant impact on understanding of the involvement of specific signalling pathways of disease. There is one other tissue array published from Thailand, by Khon Kaen University, the results of which would benefit from validated by a second independent array. Ramathibodi is a tertiary care hospital, one of the best in Thailand, with one of the highest research activities. A tissue array generated from cases of CCA, which can be used under the MRC data-sharing package - allowing other researchers to access the data, will significantly impact on research into O-CCA. Generation of data from these types of array becomes synergistic once the number of case gets above 200, and validation of two arrays can confirm expression patterns with confidence that allows high profile publications for both teams. The establishment of primary tumour lines from multiple patients would also be a huge benefit, as would the establishment of patient derived xenografts in Thailand and the UK. The impact of these sorts of experiments on additional drug trials that can be done using avatars becomes possible once established, further supporting Thai and UK science.
This type of joint grant will also have a significant impact on the collaboration between the two institutions, and on CCA research within Thailand. Both the training and experiences of the younger research assistants, and the establishment of the Nottingham-Bangkok network will have significant impact on cross fertilisation of ideas, the development of new training programs and additional international collaboration. In addition we have set aside £4.5K (270K baht) in Impact Funding to support networking meetings of CCA scientists both within and outide this collaboration. This will be used to host meetings of CCA researchers in Thailand, with external advisers, and access for PhD students and postdocs from sites around Thailand to get together annually for three years to present data, attend workshops, and plan projects. It will promote the sharing of resources, tissue samples, gene expression and protein expression profiles and enhance the quality of sceince on CCA in Thailand.