Plasticity of epithelial cell boundaries governed by EGF and actin remodelling

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.

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.

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.