Role of Par-3 in E-cadherin recycling and signalling

The mechanism that attaches neighbouring units, or cells, in our body to each other is known as cell-cell adhesion. Recent work has demonstrated that cell-cell adhesion is also important for communication between neighbouring cells to decide when to divide, migrate or die. Specific cell adhesion proteins ensure cell-cell adhesion: the proteins on the surface of one cell bind directly to similar proteins on the surface of adjacent cell. One of the major cell adhesion proteins is called E-cadherin. E-cadherin provides cell-cell adhesion between epithelial cells: the cells that outline all cavities and surface structures of the body. E-cadherin is vital for proper development of the body from very early stages, whereas faulty E-cadherin adhesion contributes to cancer progression by increasing growth and metastasis.
Two reasons make it important for a cell to be able to control amounts of E-cadherin at its surface. First, more E-cadherin results in stronger adhesion between neighbouring cells, and reverse. Increasing strength of adhesion is required in response to mechanical forces to prevent rapture of epithelia, whereas reducing strength of adhesion is needed when cells decide to exchange neighbours. Both mechanical stretching and neighbour exchange participate in normal development of an organism and its maintenance during adult life, and occur in disease. The second reason is that the amount of E-cadherin at the cell surface determines the number of E-cadherin molecules available to interact with other proteins that are involved in communication between cells. To be able to rapidly adjust the amount of E-cadherin at the cell surface according to the current needs of a cell, a portion of E-cadherin constantly circulates between cell surface and cell's interior, a process called recycling. To date, little is known about how E-cadherin recycling is regulated, for example why after E-cadherin moves inside the cell, it is then returned back to the cell surface instead of being destroyed inside the cell. I have chosen a simple animal to study this problem, the fruit fly Drosophila. Fruit flies use E-cadherin in the same way as we do. For example, if fruit fly embryos lack E-cadherin they die early in development because epithelial cells cannot maintain contacts to each other and tissues fall apart. I have recently discovered that recycled E-cadherin is specifically associated with the protein called Bazooka/Par-3 in Drosophila embryos. Bazooka/Par-3 is a large protein that has many parts, which bind other proteins. During my past research I obtained a list of all proteins that interact with Bazooka/Par-3, and found that it includes several proteins that are known to either participate in transport of proteins between cell's surface and interior, or in communication between cells. Therefore, Bazooka/Par-3 is a good candidate to link E-cadherin to recycling and cell-cell communication machineries, and it is the focus of this proposal to discover how Bazooka/Par-3 does this. The knowledge of how Bazooka/Par-3 regulates E-cadherin recycling and links it to communication between cells may be used to regulate the levels, distribution or action of E-cadherin.
I anticipate to discover basic mechanisms that are shared between all animals. In future, I will be able to apply this knowledge to treatment of medical conditions arising from defects in E-cadherin function such as epithelia-derived tumours. For example, if I find that Bazooka/Par-3 binds a particular protein that allows E-cadherin to be re-delivered to the cell surface instead of being destroyed inside cells, absence of this protein might be used to mark cells that are likely to break down cell-cell adhesion and start invading other tissues, or this protein might be used as a drug target to prevent cells from re-building adhesion and forming secondary tumours in other tissues.

Cell-cell adhesion is vital for attachment between cells and cell-cell communication. E-cadherin is one of the major transmembrane proteins to provide cell-cell adhesion in epithelial cells. E-cadherin amounts at the cell surface determine adhesion strength and signalling. Cells regulate amounts of E-cadherin at their surfaces by constantly recycling a substantial portion of E-cadherin molecules. Despite vast knowledge of general recycling machinery, it is unknown how the decision between recycling and degradation is made in the case of E-cadherin, and how E-cadherin recycling is coupled with signalling pathways.
The proposal follows on from my discovery that recycled E-cadherin associates with the scaffolding protein Bazooka/Par-3 (Par-3), and E-cadherin amounts depend on amounts of Par-3 in epidermal cells of Drosophila embryos. The mass-spectrometry data after Par-3 immunoprecipitation revealed that Par-3 interacts with regulators of intracellular trafficking and components of signalling pathways.
The goals of this research are to identify how Par-3 regulates recycling of E-cadherin and links it to signalling pathways. By manipulating four recycling regulators, discovered to interact with Par-3, a research associate, supported by this grant, and I will confirm their roles in E-cadherin turnover using Fluorescence Recovery After Photobleaching and quantitative imaging. Then we will determine stages of the E-cadherin trafficking that they regulate, how they are linked to E-cadherin--Par-3, and how they facilitate recycling of the E-cadherin--Par-3 complex. In parallel, we will characterise how E-cadherin recycling regulates signalling pathways by manipulating E-cadherin recycling and monitoring activities of the JAK/STAT and EGF signalling pathways, selected based on mass-spectrometry and published data. Then, we will describe mechanisms that link the signalling pathways to recycled E-cadherin--Par-3, and the roles of these links in development of an organism.

Beneficiaries:
1. Future patients suffering from cancer: The proposed study is most relevant to carcinomas, which derive from epithelial cells. Carcinomas include breast, prostate, lung and bowel cancers: the four most common cancers in UK (53% of all new cancer cases and 46% of all cancer deaths in UK in 2012). Loss or reduction of E-cadherin is correlated with carcinomas spread, e.g. 85% of invasive lobular breast carcinomas are completely E-cadherin negative, and reciprocally, E-cadherin is often elevated in secondary tumours, e.g. 62% of secondary tumours from primary breast cancers have elevated E-cadherin. This research could benefit patients in two ways:
A) by providing biomarkers that specifically label cells that are likely to form metastases following loss of E-cadherin, thus permitting earlier and better diagnoses. For example, if a specific protein, e.g. deubiquitinase Usp7, is required for decision between recycling and degradation, reduced levels of this protein might correlate with increased degradation of E-cadherin before changes in overall E-cadherin levels can be detected. Timescale:
-identify potential biomarkers - year 1,
-characterise how they regulate E-cadherin recycling - years 2-3,
-validate potential biomarkers in mammalian cells - years 2-3,
-communicate the discovery to clinical researchers to initiate collaboration - years 2-3.
B) by providing drug targets for treating secondary tumours through discovery of proteins, which promote E-cadherin recycling or couples E-cadherin recycling to pro-proliferative signalling pathways. For example, if we find that E-cadherin recycling activates JAK/STAT pathway, drug inhibition of discovered recycling regulators might be used to prevent proliferation of tumour cells. Timescale:
-identify potential drug targets - year 1,
-characterise how they regulate E-cadherin recycling or link it to signalling pathways - years 2-3,
-validate potential drug targets in mammalian cells - years 2-3,
-engage with pharmaceutical and biotech companies - year 3.
Carcinomas are predominantly diagnosed in older adults, for example 80% of breast cancers occur in women aged over 50 years. Therefore, earlier and better diagnostics and treatment could contribute to healthy ageing of affected individuals and allow sufferers to live longer and remain active longer contributing to society as a whole.
2. Biotechnology and pharmaceutical industries: The companies licensed to use and develop drugs targeting the above drug targets will benefit from their successful identification. Development of such products will be beneficial for the UK economy and attract investment from global business. Timescale: see 1B.
3. Health services: would benefit from increasing efficiency in diagnosis and treatment of E-cadherin-associated diseases following discovery of new biomarkers and drug targets. Timescale: see 1.
4. Those recruiting scientifically trained staff, including business, industrial and public sector: RA who will be supported by this grant will improve their training, including transferable skills, e.g. project management and leadership skills. Additionally, I will supervise undergraduate students, contributing to their training in scientific experimentation, experimental design, data analysis, and transferable skills. Thus, this grant will contribute towards health of UK science and higher education through developing expertise and training highly skilled researchers. Timescale: years 1-3.
5. The public: RA supported by this grant and I will communicate the importance, excitement and beauty of scientific research to public, e.g. by presenting our research during departmental open days and working with the Public Engagement team at the Research and Innovation Services, University of Sheffield. Examining molecular and cellular behaviour in living animals provides beautiful images that effectively capture and communicate the concepts of biomedicine. Timescale: years 1-3.