Plasticity of epithelial cell boundaries governed by EGF and actin remodelling

Lead Research Organisation: University of Liverpool
Department Name: Institute of Translational Medicine

Abstract

The tissue cells in our body respond to circulating factors, amongst them so-called growth factors, by undergoing changes in shape, movement or division rates. One of the most important growth factors is called EGF and it binds to its own receptor to convey the instructions necessary for these changes to the interior of the cell. In some cancer types, this receptor can be altered such that it continuously demands these types of changes from the cell; i.e. it forces the cell to grow and divide abnormally, extract itself from the tissue boundaries and move to another site. It follows that drugs, which inhibit this receptor are of widespread interest and a major focus of pharmaceutical companies.

We have studied normal, non-cancerous breast cells that can routinely be grown in petri dishes in the presence of growth factors including EGF. These breast cells form a continuous sheet, but within this sheet, cells remain able to roam around. However when we remove the EGF, these cells project long, finger-like protrusions into each other, which we call "interdigitations". In these interdigitated sheets, cell positions are fixed like the pieces of a completed jigsaw puzzle. If we add fresh EGF to this mesh of interdigitated cells, the finger-like protrusions disappear and the cells start to move around again. Such interdigitations are in fact common in tissues that have to endure mechanical stress. They may help to give the tissue superior flexibility and strength to withstand, for example, changes in volume in breast tissue due to lactation.

Our work has defined an experimental set-up in which we can study the proteins that respond to the EGF receptor and mediate this reversible remodeling process. New insights that will be gained from this work may help to inform both on the mechanism by which normal tissues increase their strength but also on disease states in which tissue boundaries are compromised due to a deregulation of the proteins involved.

Technical Summary

Epidermal growth factor (EGF) plays a critical role in tissue development, including mammary gland growth and differentiation. Deregulation of its receptor, EGFR, and other members of the ErbB family of receptor tyrosine kinases (RTK), is commonly associated with malignancy. MCF10A cells are "near-normal", untransformed mammary epithelial cells that recapitulate key features of mammary acini formation and are commonly used as a model system to study epithelial cell biology. These cells are routinely grown in EGF-containing, complex media in which they form a smooth contact inhibited cobblestone monolayer.
We have discovered a dramatic morphological change that these cells undergo upon long-term EGF withdrawal: This results in a highly interdigitated, desmosome-rich monolayer, wherein adjacent cells project actin containing protrusions deep into each other, causing a near-complete arrest of cell mobility within the monolayer. This phenotype is tonically suppressed by EGF (or activated EGFR) and rapidly reversed by acute EGF application. This is a novel, highly specific manifestation of EGFR-modulated signaling, which is insensitive to Neuregulin, HGF or Insulin. We note that this interdigitated, desmosome-rich phenotype recapitulates ultrastructural observations of mammary glands during lactation and post-weaning breast-fluid derived cells. Such interdigitations have been proposed to contribute to the maintenance of epithelial cell integrity in particular in tissues under mechanical stress, e.g. whilst undergoing dramatic volume changes.
We propose to use this unique, tractable and reversible in vitro assay system to study the mechanism and function of cellular interdigitation. We will explore the interplay between EGFR, membrane dynamics and the actin cytoskeleton and aim to identify novel mediators of relevant signaling networks using a combination of 'Omics and candidate based approaches with a focus on Rho GTPase family members and their regulators.

Planned Impact

This is primarily a basic research project addressing fundamental questions of growth factor signaling, cellular behavior and epithelial cell integrity. The work described in this proposal offers the opportunity for the elucidation of novel signalling circuitry with profound influence upon cellular behaviour. Our work will offer new screening opportunities, insights into physiological effects of EGFR inhibition and fundamental knowledge of the EGFR pathway. This will be of interest to both the academic and industrial communities.

Industrial impact:
EGFR is commonly deregulated in cancer and is a well-established drug target for treatment of metastatic disease. EGFR belongs to the ErbB family of receptors, which have spawned a multi-billion pound industry as validated drug targets. The assay we describe in this project is unique as it offers the opportunity to develop a novel, potentially high throughput phenotypic screening approach for EGFR activity. Importantly, in contrast to most other read-outs, this is based on near-normal cells grown in a confluent monolayer as opposed to already transformed cells grown under subconfluent conditions. We will continuously monitor opportunities for patenting our findings as well as forging collaborations with industrial partners based on our findings.
We already have existing links with industry, which have potential interests in this subject area, e.g. Horizon Technologies, a UK Biotech who engineered isogenic MCF10A cell line panels, and Forma Therapeutics, a major US Biotech company who is concerned with development of new drugs in a variety of medically invested areas. We are also engaged in a broad EU FP7 network aimed at understanding the mechanisms of Cetuximab resistance and improving clinical outcomes for patients with colon cancer (COLTHERES), through which we have access to several industrial partners.

Economic & Social Impact:
Generation, testing and production of drugs generates business and job opportunities within the UK. Drugs successfully targeting growth factor signalling impact upon both patient treatment and prognosis. Cetuximab, a monoclonal antibody targeting EGFR is used for treating metastatic colon cancer and squamous cancer of the head and neck in combination with chemotherapy or radiotherapy. Small molecule inhibitors (Iressa/Gefitinib) are used in the treatment of lung cancer. Unfortunately, treatment with these drugs often causes unpleasant dermatological reactions: The majority of people receiving Cetuximab develop papulopustular skin rash, which affects quality of life and can disrupt treatment regimens. Desmosomal proteins, the constituents of adhesive structures that maintain mechanical integrity of epithelial and other stress bearing tissues, are often mutated or silenced in breast cancer. Auto-antibodies against desmosomal components (Desmoglein 1,3) lead to a debilitating skin condition known as pemphigus. Our research into the modulation of epithelial cell plasticity and cell adhesion by EGFR may help with the development of new therapies or treatments to alleviate these side effects.
As our project has implications for our understanding of mammalian epithelial integrity and resistance to mechanical stress, another impact area, albeit far removed from our natural arena may lie in the development of protocols in regenerative medicine, tissue engineering and milk production.

Public/Education:
We will communicate our findings to the public via the University web-site and press releases when appropriate. Our laboratory hosts tours for visitors (both adults and local schoolchildren) interested in the work of the Liverpool Cancer Center. An immediate impact of the research lies in specialized skills training of staff associated with the project, who will gain experience handling large datasets and acquire technical prowess on a variety of state of the art imaging equipment, which will be beneficial for their future career development.

Publications

10 25 50
 
Description We have published a paper in Cell Reports (find url below) describing the initial findings associated with this project.
We now have
-undertaken a comprehensive morphometric and morphological characterisation of the actin-based interdigitations using a combination of fluorescence and electron microscopy based techniques and associated 3D-reconstruction. We are currently assessing a variety of EM-protocols to allow us to analyse the cytoskeletal elements and adhesion complexes in further detail.
-monitored actin dynamics within the interdigitations
- undertaken a morphometric analysis of the Golgi complex in MCF10A monolayers grown ± EGF.
-prepared samples for transcriptomic (RNA-seq) analysis, three experiments have been processed and analysed using a variety of bioinformatic databases and toolsets. We have identified and validated specific transcripts that response to EGF withdrawal and are now assessing their relevance for the process of Actin-finger induction upon EGF removal from these cells using siRNA-mediated depletion experiments.
-prepared and analysed phospho-proteomic samples, further proteomic analysis (total proteome) and validation experiments are in process.
- We have assembled a comprehensive siRNA library covering all annotated Rho-GAP and GEFs. This is now being used for an siRNA-depletion screen to identify specific GAP and GEFs that are required either for the formation or resolution of actin interdigitations.
Exploitation Route Our data provide a framework for future assay development to test EGFR inhibitors and modulators of epithelial cell integrity within a confluent monolayer cell system.
Sectors Education,Pharmaceuticals and Medical Biotechnology

URL http://dx.doi.org/10.1016/j.celrep.2014.08.026
 
Description Yvonne Tang, the post-doctoral researcher associated with this project has been communicating her findings and her understanding of epithelial cell biology to groups of school kids visiting ur laboratory in the past year. She has demonstrated several imaging methodologies to them, thus contributing to the education of the next generation of young scientists. Yvonne herself has gained considerable new skills including the ability to analyse complex transcriptomic and proteomic datasets, navigating databases like David and supervising MRes students in the lab.
First Year Of Impact 2015
Sector Education
Impact Types Societal

 
Title Generation of transcriptomic datasets associated with MCF10A model epithelial breast cell line cultured as a function of culture conditions 
Description We have generated a series of transcriptomic datasets which we aim to publish shortly. 
Type Of Material Database/Collection of data 
Provided To Others? No  
Impact These datasets will be a useful resource for other researchers who are either interested in this particular cell model, in breast cancer, and in the response of mammary cell lines to epidermal growth factor. 
 
Title Phospho-proteomic datasets 
Description We have generated a series of phospho-proteomic datasets that describe the response of a model mammary epithelial cell line grown in a monolayer in the absence of EGF to acuute stimulation with epidermal growth factor. We aim to publish this dataset shortly. 
Type Of Material Database/Collection of data 
Provided To Others? No  
Impact These datasets will be useful to other researchers studying mammary epithelial cell behaviour in response to growth factors. 
 
Description Compare transcriptomics data to published dataset 
Organisation University of Liverpool
Department Institute of Translational Medicine
Country United Kingdom 
Sector Academic/University 
PI Contribution Yvonne Tang and Michael Clague discussed published transcriptomic datasets of breast cancer cells with Dean Hammond. Yvonne Tang followed up on comparison work after discussions and training with Dean Hammond.
Collaborator Contribution Dean Hammond provided advice and provided aided the merging of our transcriptomics dataset to the published transcriptomics dataset (Marcotte et al 2016).
Impact Our transcriptomics data for MCF10A cells was found to be clustered near the published transcriptome of MCF10A cells.
Start Year 2016
 
Description Use of Ingenuity Pathway Analyses (IPA) software to analyse transcriptomics data 
Organisation University of Liverpool
Country United Kingdom 
Sector Academic/University 
PI Contribution Yvonne Tang was trained to use the IPA for transcriptomics data analyses.
Collaborator Contribution Olga Vasieva provided training and use of IPA software license, on behalf of the department on a collaborative basis
Impact Summary of trasncriptomics analyses were incorporated into graphs, to build hypotheses and for follow up validation work.
Start Year 2016
 
Description Laboratory visit of School Children to the lab 
Form Of Engagement Activity Participation in an open day or visit at my research institution
Part Of Official Scheme? No
Geographic Reach Regional
Primary Audience Schools
Results and Impact Approximately 22 school children from deprived areas visited our laboratory - Yvonne Tang the PDR employed on this project organised and led a series of demonstrations directly related to the project. This clearly increased the interest of the school children in Biological Sciences and it is anticipated that this will provoke this group to consider a Science related degree at the end of their school time.
Year(s) Of Engagement Activity 2015