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

Lead Research Organisation: University of Sheffield
Department Name: Biomedical Science

Abstract

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.

Technical Summary

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.

Planned Impact

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.

Publications

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Greig J (2020) Arf6 determines tissue architecture by stabilizing intercellular adhesion. in Philosophical transactions of the Royal Society of London. Series B, Biological sciences

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Ramirez Moreno M (2021) Interactions and Feedbacks in E-Cadherin Transcriptional Regulation. in Frontiers in cell and developmental biology

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Ramírez Moreno M (2021) The Cross-Talk Between EGFR and E-Cadherin. in Frontiers in cell and developmental biology

 
Description WithThis project aimed to decipher the mechanisms that control E-cadherin (E-cad) recycling and the consequences of those mechanisms on a tissue and organism level. Following the assessment of proposed candidate proteins, we focused on the Adaptor Protein Complex 1 (AP-1). We found that AP-1, which is known to be involved in intracellular recycling, regulates the surface levels of E-cad, as shown with the use of fluorescent-tagged versions of the protein. The study of the changes in E-cad dynamics led to two striking conclusions.
Firstly, we found that AP-1 regulates E-cad's membrane presentation by at least two mechanisms: a) by controlling the trafficking of E-cad within the cell (trackable using fluorescent probes and specific antibodies for compartments of interest) and b) by limiting the internalization of E-cad from the plasma membrane. The latter is an unexpected novel role of AP-1, discovered in vivo and matching its newly found localization at the vicinity of the E-cad at the cell surface.
Secondly, we determined that an excessive internalization of E-cad at the membrane, in conditions such as the removal of AP-1, increases the expression of the E-cad gene. This cellular response supports the existence of the feedback loop regulating E-cad dynamics and promoting epithelial robustness. Indeed, we observed opposite compensatory changes in E-cad expression upon forced overexpression of E-cad.
At the same time, we found that the loss of AP-1 leads to a complex phenotype in our tissue model - the Drosophila larval wing discs. This phenotype includes ectopic tissue folding and the triggering of programmed cell death (apoptosis). In particular, cell death is activated by defects in E-cad internalisation and/or recycling as we found that preventing E-cad from being internalised rescues cell death. Upon blocking cell death, the tissue undergoes hyperplastic overgrowth. These findings suggest that a tumour-suppressing mechanism keeps E-cad in check, consistent with recent views regarding the importance of the regulation of E-cad in cancer development.
Exploitation Route Several types of stakeholders will benefit from the output of this project. Our publications provide new knowledge about the regulation of E-cadherin recycling and expression, therefore, promoting the progress of other research groups, particularly those involved in the study of cellular trafficking using tissue models, Drosophila researchers, and scientists and clinicians with interest in E-cad regulation. These and other researchers will be able to use new data analysis approaches we created during the project - our scripts for image analysis are openly available online. Lastly, this project has served as one of the first research experiences for four undergraduate and master students. Two of these students have since proceeded to PhD studies, thus highlighting the contribution of this research grant to the higher education landscape in the UK.
Sectors Healthcare,Pharmaceuticals and Medical Biotechnology

URL https://www.biorxiv.org/content/10.1101/2021.11.08.467679v1
 
Description How do the cadherins Fat and Dachsous control polarised cell behaviours during development?
Amount £400,340 (GBP)
Funding ID BB/S007342/1 
Organisation Biotechnology and Biological Sciences Research Council (BBSRC) 
Sector Public
Country United Kingdom
Start 05/2019 
End 05/2022
 
Description Research Project Grants
Amount £234,000 (GBP)
Funding ID RPG-2017-249 
Organisation The Leverhulme Trust 
Sector Charity/Non Profit
Country United Kingdom
Start 02/2018 
End 01/2020
 
Description Modelling intracellular transport 
Organisation University of Edinburgh
Department School of Mathematics
Country United Kingdom 
Sector Academic/University 
PI Contribution My expertise in intracellular trafficking of E-cadherin, together with the discovery of several regulators of it allows producing biological data for developing the mathematical model of intracellular transport aimed to understand how particular distributions of intracellular components are achieved and maintained. Additionally, I test model predictions therefore providing guidance for further developments.
Collaborator Contribution The model already generated several predictions that were then tested in cells and proved to be correct, altogether helping understanding how E-cadherin is transported inside cells and how its distribution is maintained.
Impact This collaboration is multi-disciplinary and is between a biology (our) and mathematics teams. The main outputs so far is future funding - the research grant from the Leverhulme Trust, and a collaborative involvement in the Science Festival at the University of Edinburgh.
Start Year 2016
 
Description Discovery night 
Form Of Engagement Activity Participation in an open day or visit at my research institution
Part Of Official Scheme? No
Geographic Reach Local
Primary Audience Public/other audiences
Results and Impact Discovery night is an annual event when the university is open to public. In particular, we explain to the public the idea behind our research and how flies help us to discover new medical treatments.
Year(s) Of Engagement Activity 2017,2018
URL https://www.sheffield.ac.uk/discoverynight
 
Description Edinburgh International Science Festival 2018 
Form Of Engagement Activity Participation in an activity, workshop or similar
Part Of Official Scheme? No
Geographic Reach Local
Primary Audience Public/other audiences
Results and Impact We (both PI and Research Associate) prepared the outreach material (badges and two games) for our collaborator's team to be displayed during Edinburgh International Science Festival, 2018. The aim was to increase awareness and interest in STEM disciplines in both children and adults. The stand was popular with attendees of the Festival, and visited by about 100 people/day. The feedback was very positive, which led to it being currently reused by the University fo Edinburgh mathematics outreach team during the regularly occurring "maths circle" event for children.
Year(s) Of Engagement Activity 2018
URL https://www.sciencefestival.co.uk/festival