Brian P. Ceresa - US grants

This subproject is one of many research subprojects utilizing the resources provided by a Center grant funded by NIH/NCRR. The subproject and investigator (PI) may have received primary funding from another NIH source, and thus could be represented in other CRISP entries. The institution listed is for the Center, which is not necessarily the institution for the investigator. Corneal epithelial wound healing and homeostasis is regulated by EGFR activity. This process is mediated by three cellular changes: cell migration, proliferation, and stratification. Inhibition of ligand-stimulated EGFR activation, either through small molecule inhibitors or blocking antibodies, results in a decrease in all three cellular changes as well as impaired wound healing. Based on our own findings and the work of others, we hypothesize that corneal wound healing can be enhanced by promoting EGFR-mediated signals that enhance cell migration. To test this hypothesis, we must understand EGFR-mediated signaling on the molecular and cellular level. This information will be used to target molecules and mechanisms that promote epithelial cell migration, which can ultimately accelerate coverage of the damaged area and healing of the wounded cornea. In the first aim of this proposal, we will study other endogenous EGFR ligands that have been reported to be more potent activators of corneal wound healing. We hypothesize that these ligands promote different endocytic trafficking itineraries of the EGFR than EGF (i.e. recycling rather than degradation) and this is the basis of their enhance wound healing. In the second aim, we will disrupt EGFR endocytic trafficking at discrete stages along the endocytic pathway and assess the magnitude and duration of the EGFR phosphorylation, effector signaling, and cell physiology. These findings will indicate whether enrichment of the activated receptor at discrete endocytic location will specifically promote the molecular and cellular events associated with corneal wound healing.

DESCRIPTION (provided by applicant): The long-term goal of our laboratory is to understand how signal transduction by cell surface receptors is regulated. We would like to develop strategies for selectively activating or inhibiting these cellular activities and bypass limitations of the receptor (i.e. low receptor number or receptor desensitization). Extracellular ligands (growth factor, hormone, neurotransmitter, etc.) bind to unique cell surface receptors that induce intracellular, biochemical changes that are integrated to invoke specific changes in cell physiology. While the exquisite specificity of this system has been long appreciated, the molecular mechanism by which it occurs is poorly understood. Understanding how an overlapping set of biochemical responses produces a specific physiology is the key to this problem. To understand this process, we are using the prototypical receptor tyrosine kinase, the epidermal growth factor receptor (EGFR), as a model. The EGFR is critical for many developmental and homeostatic processes; stimulation of the EGFR leads to a variety of cellular changes including cell proliferation, differentiation, migration, and viability. Overexpression of the EGFR is associated with many cancers. The magnitude and duration of signaling to these biochemical intermediates dictates how cell physiology is altered. One way this occurs is through the internalization and degradation of the receptor following ligand binding. In addition to activating intracellular signaling pathways, ligand binding also causes most cells surface receptors to internalize via clathrin-coated pits. Once inside the cell, the ligand:receptor complex moves through a series of well-defined endocytic stages until it ultimately reaches the lysosome where it undergoes degradation. It has been shown previously that disrupting EGFR endocytosis can alter the response of the cell. However, these studies have been limited to distinguishing between cell surface and intracellular receptors. The overarching hypothesis of our research is that the endocytic pathway is a key positive and negative regulator of cell surface receptor signaling. In Aim 1, we will selectively disrupt EGFR trafficking through the endocytic pathway. We will assess EGFR signaling at several endocytic stages and determine whether differences in signaling occur due to changes in the receptor itself, receptor:effector interactions, or the duration/magnitude of signaling. In Aim 2 we will build on our recent findings that spatially restricting the EGFR in cancer cells (MDA-MB-468 cells) dramatically changes cell growth and viability properties. We will determine how the intracellular EGFRs produce apoptotic signals and whether changing the trafficking and signaling of EGFRs in other cell lines can cause them to undergo EGFR-mediated apoptosis. Finally, in Aim 3, we will explore how the endocytic pathway negatively regulates EGFR signaling. In these studies, we will determine the mechanism of signal inactivation and determine if altering the rate of inactivation is sufficient to change EGFR signaling. PUBLIC HEALTH RELEVANCE: The epidermal growth factor receptor (EGFR) is a fundamental cell surface protein that functions by detecting the presence of growth factors outside the cell and converting the information into biochemical changes within the cell. Proper function of the EGFR is necessary for development and homeostasis of the whole organism; overexpression/hyperactivation of the EGFR is associated with many cancers. The immediate goal of this research is to better understand the molecular mechanisms that regulate the biological and pathological functions of the EGFR and use that information for treatment of diseases associated with the EGFR.

DESCRIPTION (provided by applicant): The long-term goal of our laboratory is to develop strategies to restore and maintain the integrity of the corneal epithelium. Activation of the epidermal growth factor receptor (EGFR) is necessary and sufficient for the homeostasis and repair of the corneal epithelium. Despite evidence that the EGFR is a viable target for corneal epithelial wound healing, its use therapeutically has been limited. Understanding the molecular details of how signaling by the receptor is regulated will provide clues for utilizing EGFR-targeted therapies. One such regulatory mechanism is ligand-stimulated EGFR endocytosis. The internalization and subsequent endocytic trafficking of the receptor negatively controls EGFR signaling by targeting the active receptor to lysosomes for degradation. Our studies will determine how endocytosis affects EGFR signaling in the corneal epithelium. Based on our recent findings and reports in the literature, we propose the following hypothesis: slowing the endocytic trafficking of the liganded EGFR will prolong EGFR signaling and enhance corneal wound healing. This overall hypothesis will be tested with the following Aims. In Aim 1, we will test the hypothesis that different endogenous EGFR ligands possess different routes and/or kinetics of endocytic trafficking. Secondarily, we will determine if those ligands that promote EGFR recycling will enhance corneal epithelial cell migration and wound healing. Using immortalized and primary human corneal epithelial cells, we will examine the kinetics and routes of EGFR endocytic trafficking in response to endogenous EGFR ligands. These ligands have been reported to have varying kinetics and/or routes of EGFR endocytic trafficking in other cell lines. The effect of these ligands on EGFR endocytic trafficking in corneal epithelial cells is unknown due to differences in the level of EGFR expression and intrinsic properties of the corneal epithelial cells. Once we determine how these ligands impact EGFR endocytic trafficking in the corneal epithelium, we will determine how they affect the cell biology of corneal epithelial cells (cell migration) and corneal wound healing using an in vivo model (Sprague- Dawley rats). In Aim 2, we will test the hypothesis that the inhibition of EGFR endocytic trafficking will prolong receptor activity and enhance the rate of corneal epithelial cell migration and wound healing. Using the tissue culture cells described above, we will disrupt endocytic trafficking at selected stages in the endocytic pathway (at the plasma membrane, pre-early endosome, and in the late endosome/multivesicular body) and assess whether the phosphorylation of the EGFR and downstream effectors is prolonged. Next, we will determine if such changes are reflected in EGFR-mediated corneal epithelial cell migration and wound healing. In Aim 3, we will determine if other EGFR family members (ErbB2, 3, and 4) play a role in promoting corneal wound healing, if they do so more efficaciously that EGFR. If we are able to develop strategies for enhancing EGFR signaling, we have the potential for therapeutically accelerating the rate of corneal wound healing and minimize patient discomfort, the risk of infection, and blindness.

PROJECT SUMMARY/ABSTRACT An intact and fully differentiated corneal epithelium is critical for proper vision and to keep foreign objects (bacteria, viruses, small particles) out of the eye. However, damage to the corneal epithelium is one of the most common ocular problems presented in primary care facilities and arises from a variety of factors, including trauma, disease, and a side-effect of drugs. Despite the prevalence, discomfort, and potential for blindness associated with perturbation of the corneal epithelium, there are no FDA-approved agents that promote the restoration and homeostasis of this tissue. The long-term goal of our research is to identify novel tools to modulate the molecular mechanisms that regulate and promote corneal epithelial wound healing and homeostasis. The overall objective of this application is to identify novel compounds that prolong EGFR signaling. To accomplish this, we will test the central hypothesis that compounds that block c-Cbl?s ability to ubiquitylate the EGFR, will divert the activated EGFR from the lysosome for degradation. We believe inhibition of this interaction will lead to greater EGFR activity and increase corneal epithelial cell migration, proliferation, and differentiation, three cell biological responses that are central to the restoration and maintenance of corneal epithelial homeostasis. The rationale for these studies is based on our previous findings that knockdown of c-Cbl and inhibition of ubiquitylation enhances EGFR-dependent corneal epithelial wound healing. This research has the following specific aims: 1) Identify candidate compounds that bind with the highest affinity for c-Cbl and 2) Determine whether the highest affinity compounds are the most efficacious antagonists of EGFR ubiquitylation and best prolong EGFR signaling. We have completed an in silico screen of 28,000,000 compounds for their ability to disrupt EGFR:c-Cbl interactions. In Aim 1, we will test the top 50 candidates for their ability to bind recombinant, purified, c-Cbl using a Thermofluor assay and Isothermal Titration Calorimetry. In Aim 2, we will use immortalized corneal epithelial cells to test whether our highest affinity compounds are most effective in preventing ligand-dependent EGFR ubiquitylation, slowing the rate of receptor degradation, and enhancing the rate of corneal epithelial wound healing. Our proposed studies are innovative, in our opinion, because we will modulate novel, ligand-independent molecular mechanisms to increase the magnitude and duration of EGFR activity as a means of accelerating restoration of damaged corneal epithelium. These studies are significant because accelerating re-epithelialization and homeostasis of compromised corneas will decrease the duration of patient distress, minimize the likelihood of infection, and reduce the incidence of blindness. Corneal epithelial homeostasis is a significant public health issue with limited treatment options. This research has the potential to identify new ways of treating damaged corneas following trauma, disease, or as a side-effect of drug treatment as well as provide a better understanding of the molecular mechanisms that regulate corneal epithelial homeostasis.

Project Summary/Abstract This application is a resubmission for a T35 summer medical student training program in vision research with two Specific Aims: 1) To provide hands-on training in basic and clinical vision research to medical students in a structured mentored environment and 2) To provide an interactive, educational experience that introduces medical students the fundamental skills necessary for basic, translational, and clinical research in vision biology. The co-directors of this program request support for 6 second-year medical students each summer in vision related research laboratories that focus on topics such as visual circuitry, retinal degeneration, corneal epithelial homeostasis, uveitis, and development of the visual system. Trainees will be selected from the first year class at University of Louisville School of Medicine. Students will review research projects submitted by 19 faculty funded by the NEI or vision related foundations. The directors will make a special effort to recruit students from ethnic minorities and disadvantaged backgrounds. Students will review the available projects and enter their 1st-3rd choices, allowing matching of trainees with mentors. Final decisions will be made based on interviews between mentors and Trainees. During the 10-week summer training, students work with mentors on their research project in clinical or laboratory settings, complete training in the Responsible Conduct of Research including topics such as fabrication and falsification of data, plagiarism, managing scientific data, publication practices and responsible authorship, mentorship, stewardship, and conflict of interest. Trainees will complete human subject, IRB training, and animal care and handling as required by their specific research projects. All Trainees will attend a 2-hour weekly ?Vision Sciences Research Training Seminar? designed to introduce clinical/translational research in Ophthalmology led by mentors in this T35 program. The seminar series will culminate with each Trainee presenting his/her research project to peers and mentors. Trainees present the results of their research as posters with peers at a School of Medicine-wide, week-long celebration of research that includes nationally recognized physician- scientists as keynote speakers. Trainees will be encouraged to continue research as part of the Distinction in Research track that is an enrichment program focusing on providing meaningful and productive research experiences throughout second through fourth years of medical school, with ongoing presentations by students with mentors and 4+ weeks of research time in the fourth year toward the goal of developing clinician-researchers. Trainees will be tracked to assess the impact of this T35 program on careers as physician-scientists.

Project Summary An intact and fully differentiated corneal epithelium is critical for proper vision and to keep foreign objects (bacteria, viruses, small particles) out of the eye. However, damage to the corneal epithelium is one of the most common ocular problems presented in primary care facilities and arises from a variety of factors, including trauma, disease, and a side-effect of drugs. Despite the prevalence, discomfort, and potential for blindness associated with perturbation of the corneal epithelium, there are no FDA-approved agents that promote the restoration and homeostasis of this tissue. The long-term goal of our research is to develop new compounds that promote corneal epithelial wound healing and homeostasis. The overall objective of this application is to chemically optimize a recently identified c-Cbl antagonist to increase its affinity and potency, while decreasing cytotoxicity. To accomplish this, we will test the central hypothesis that compounds that disrupt EGFR:c-Cbl interactions will prevent EGFR ubiquitylation, divert the activated EGFR from the lysosome, and prolong EGFR signaling. Based on preliminary studies, this prolongs EGFR activity and increases the restoration and maintenance of corneal epithelium via cell migration, proliferation, and differentiation. The rationale for these studies is that knockdown of c-Cbl and inhibits EGFR ubiquitylation, prolongs EGFR activity, and enhances corneal epithelial wound healing. Following an in silico screen of 25,000,000 compounds, we have identified several lead compounds that bind c-Cbl and inhibit ligand-mediated EGFR ubiquitylation. We seek to develop the top compound into a therapeutic agent with the following specific aims. In Aim 1, we will structurally optimize our lead compound to increase its affinity for c-Cbl and disrupt EGFR:c-Cbl binding. Multiple rounds of chemical modifications to our lead compound will be used to identify new derivatives that bind recombinant c-Cbl with high affinity and disrupt EGFR:c-Cbl interactions. In Aim 2, we will test the highest affinity compounds for disruption of c-Cbl-mediated ubiquitylation and trafficking of the EGFR. The top candidates will be tested for their ability to block ligand-mediated EGFR ubiquitylation and lysosomal degradation, and if they prolong EGFR signaling in corneal epithelial cells. In Aim 3, we will determine whether inhibitors of EGFR ubiquitylation promote corneal epithelial wound healing. The top compounds will be assayed for corneal epithelial wound healing using in vitro, ex vivo, and in vivo assays. Our proposed studies are innovative, in our opinion, because we will by-pass the limitation of EGFR occupancy and target a novel protein (c-Cbl) and molecular mechanism (receptor desensitization). These studies are significant because accelerating re-epithelialization and homeostasis of compromised corneas will decrease patient distress, minimize infections, and reduce the incidence of blindness. Corneal epithelial homeostasis is a major public health issue with limited treatments. Our goal is to develop our lead compounds for the topical treatment of compromised corneal epithelium to restore tissue homeostasis.