Dissecting the pathogenesis of ovine pulmonary adenocarcinoma with RNA-Seq

This proposal concerns a disease in sheep known as ovine pulmonary adenocarcinoma (OPA), which is caused by a virus known as JSRV. JSRV is spread through the air similarly to many other viruses (e.g., flu) and infects cells in the lung where it triggers the growth of cancer. OPA is an important disease affecting sheep flocks in the UK and many other countries, causing significant animal suffering and severe economic losses to affected farms.

OPA was first described in South Africa in 1825. Almost 200 years later, we still do not know how to prevent the disease, or even how to reliably diagnose it in the early stages. This is because the infection is silent for many months while the cancer grows; only becoming evident when the tumours become large enough to restrict breathing. Importantly, sheep have no immunity to JSRV and we cannot use the presence of antibodies to diagnose infection. More research is needed to increase knowledge about the interaction of JSRV and the sheep lung, and how that interaction gives rise to cancer. We believe that understanding that process in more detail will provide information that can be used to develop new diagnostic tests or vaccines to control the spread of the disease in farmed sheep.

The purpose of this research project is therefore to increase our understanding of the mechanisms by which JSRV causes OPA. The project has 3 main objectives. First, we wish to determine how infection by JSRV alters the physiology of sheep lung cells. We will do this by measuring the activity of sheep genes in infected lung tissue and comparing to that of healthy lungs. Secondly, we will examine how the lungs respond to a modified version of JSRV that can infect sheep but that does not cause tumours. Collectively, these first two experiments will generate a great deal about information on which genes are switched on or off during early infection and during later stages of disease. Our third objective is to study the function of a small number of genes in greater detail to determine their role in cancer or in blocking the immune response to JSRV. The selection of genes for study will be decided by the results from the first two aims.

Together, these 3 strands of work will allow us to dissect out the molecular changes that are important in the development of OPA. This information will allow us to design new strategies for controlling this disease. Better disease control will improve the economic sustainability of sheep farming, which is under threat from infectious diseases such as OPA. In turn, this will benefit rural communities and the wider public through strengthening the supply of safe, nutritious food. Importantly, we have already performed a small-scale project that has shown our planned experimental approach will deliver valuable results. In those pilot experiments we identified a number of proteins that are much more abundant in cancer cells than in normal lung. We are currently investigating the role of some of these proteins in OPA. The planned experiments will allow us to repeat this on a more comprehensive scale, so that we fully understand the interaction of JSRV and lung tissue in infected sheep. In addition, there are many unusual aspects to OPA and a deeper understanding of how this disease develops will also provide valuable information on normal lung function. Therefore, the results of this project will also be of great interest to researchers studying lung biology in humans.

Ovine pulmonary adenocarcinoma (OPA) caused by jaagsiekte sheep retrovirus (JSRV) is an important disease of sheep that causes significant losses to the sheep industry and is an important animal welfare issue. Our aim in this project is to dissect the molecular nature of pathogenesis of OPA using high-throughput transcriptome sequencing (RNA-Seq) and to examine the function of selected dysregulated pathways in an ovine lung culture system.

We will perform RNA-Seq and microRNA-Seq on tumour cells and infiltrating macrophages from microdissected tumour tissue from experimentally infected lambs. We will also obtain sequence from epithelial cells and macrophages from uninfected control lung. Differentially regulated genes and gene networks that are likely to be important in pathogenesis will be identified. Preliminary data has confirmed that this approach can identify differentially regulated genes in these tissues. Next, we will determine the host transcriptional response to infection by a non-transforming JSRV mutant. This will allow differentiation of host gene expression pathways that are altered in response to infection alone from those altered due to tumour growth and will determine whether oncogenesis has a role in the lack of adaptive immunity to JSRV in sheep.

The differential expression of selected target genes will be validated on a panel of natural and experimental cases of OPA using RT-qPCR and immunohistochemistry. Subsequently, we will test the function of selected pathways in JSRV pathogenesis in vitro using an ovine lung tissue culture system. This system allows the manipulation of the expression of genes of interest to predict and directly test the function of selected targets in vitro. Collectively, these objectives will allow us to define the molecular events occurring during JSRV pathogenesis. This information will underpin the design of new control strategies for OPA, e.g., by identifying new diagnostic targets and vaccination strategies.

Ovine pulmonary adenocarcinoma (OPA, also known as jaagsiekte) is a common infectious disease of sheep that is widespread in the UK and in many countries where sheep are farmed. OPA causes significant economic losses to producers and the severe respiratory distress it produces is a serious animal welfare concern. Our aim in this project is to dissect the molecular nature of pathogenesis of OPA using state of the art genetic technologies. Our vision is that the outputs of this research will stimulate new avenues of research in the area of pulmonary biology and deliver information that will underpin the design of novel strategies to control OPA.

WHO WILL BENEFIT FROM THIS RESEARCH?
A variety of stakeholder groups will be beneficiaries of the proposed research in the UK and elsewhere, including research scientists, academic and government research organisations, farmers, veterinarians, policymakers, government and the public.

HOW WILL THEY BENEFIT?
Research scientists and research organisations will benefit from the increased understanding of gene expression and lung function in sheep. While this has obvious impact for researchers studying ruminant disease, it will also have much broader relevance for anyone studying pulmonary biology or pathology. The knowledge gained will support research into other lung diseases, including infectious disease, inflammatory disease and other disorders of immune function. We also expect that the genetic information generated in this project will provide the intellectual framework for greater exploitation of sheep as an improved animal model of human pulmonary biology. The identification of pathways activated during tumour growth in OPA will be valuable for scientists studying human lung cancer or other epithelial tumours, while tumour biomarkers identified in OPA could have utility in diagnosing in human lung cancer. Furthermore, the use of RNA-Seq in sheep will demonstrate the utility of this technology for investigating other species with relatively uncharacterised genomes. This will also support the study of related species that are not target species for genome sequencing.

Sheep farmers and associated veterinary industries will benefit from the increased understanding of the pathogenesis of OPA because this information is required to design novel strategies for controlling this disease. The cost of OPA to individual farmers can be significant, particularly if breeding rams are affected, and the disease can have disastrous effects on the financial sustainability of sheep farming in some geographical areas, with consequent negative effects on associated industries and local communities. More broadly, respiratory disease in ruminant livestock species has enormous economic impact and the detailed characterisation of lung function at the transcriptome level, including microRNAs, will also contribute to knowledge underpinning efforts to control those diseases.

Government, policymakers and the wider public will benefit from this research in a number of ways. Improved animal health and welfare will result in more efficient animal production, contributing to greater food security and safety. In addition, improving efficiency in farming reduces its impact on the environment, leading to reduced usage of natural resources and reduced generation of greenhouse gases. Furthermore, the enhanced financial security of sheep producers will help to improve the sustainability of rural communities. While this has obvious advantages for those living in those communities, it also helps to sustain an inviting rural landscape that supports wider industries such as tourism and leisure.