Career Development Program
Project Eight: Investigating the Role of EGFR Degradation in Targeting Tumor and Tumor Endotyhelial Cells
Aarif Ahsan, Ph.D.
Specific Aims are:
The overall success of a treatment depends on multiple factors influencing both tumor cells and the tumor microenvironment including tumor-associated blood vessels and stroma. Epidermal growth factor receptor (EGFR) expression plays a central role in the survival of not only the head and neck tumor cells but also their microenvironment. Treatment with EGFR inhibitors has improved the outcome of certain patients but has met with limited success. We have found that treatment that induces EGFR degradation increases the effectiveness of chemo and radiotherapy beyond that obtained by EGFR inhibition alone (1). Inhibition of HSP90-ATPase activity by geldanamycin or other agents is very effective in potentiating EGFR degradation and enhancing chemo- and radio-sensitization (2). However, such an approach is likely to be non-specific and toxic to normal tissue, since it would be anticipated to affect all the HSP90 client proteins. Our long term goal is to develop a specific approach to enhance EGFR degradation via blocking the interaction between EGFR and HSP90, without affecting HSP90-ATPase activity. Towards this end, we have identified an 8 amino acid motif of EGFR that is critical in the EGFRHSP90 interaction and in the EGFR protein stability. We show that a peptide which we have developed that competitively blocks protein-protein interaction between EGFR-HSP90 (named as Disruptin), promotes EGFR degradation and induces death of EGFR driven tumors and tumor-associated endothelial cells. As tumor cells are known to secrete pro-angiogenic factors such as VEGF, bFGF, IL-8 and EGF, loss of tumor cells is expected to compromise tumor- associated blood vessels as well. We expect this approach to be more effective than blocking the tyrosine kinase activity of EGFR and less toxic to normal tissues than current strategies of targeting HSP90 activity. The goal of this application is to determine the role of EGFR degradation in targeting tumor and tumor-associated endothelial cells. These goals will be achieved through 2 specific aims:
1. Determine the effects of Disruptin on survival of tumor and tumorassociated endothelial cells in vivo and establish a correlation with EGFR degradation. In this aim, we will investigate the efficacy and mechanism of Disruptin on EGFR-dependent or independent tumor xenografts. Our preliminary data show that EGFR is degraded in UMSCC1 tumor xenografts treated with Disruptin and Disruptin also caused significant reduction of micro-blood vessels within tumors. We hypothesize that Disruptin will be most effective in EGFR driven tumors where it will inhibit EGFR activity by promoting its degradation in both tumor and in the tumor- associated endothelial cells, and thus induce cell death in both the compartments. Aim 1A is to assess the efficacy of Disruptin and its correlation with EGFR degradation. We hypothesize that this approach will produce regression of tumors driven by EGFR, which will correlate with dose of Disruptin, EGFR degradation and reduced CD31 staining. Aim 1B is to understand the cross-talk between EGFR-dependent or independent tumor cells and endothelial cells upon treatment with Disruptin. We hypothesize that Disruptin will directly affect endothelial cells and therefore will cause reduction in CD31 staining in tumors, but this effect will be substantially higher in EGFR driven tumors than in EGFR independent tumors. These data will provide evidence for development of this novel approach to promote EGFR degradation via blocking the interaction between EGFR and HSP90 specifically.
2. Determine the precise role of EGFR-HSP90 interaction in the survival of tumor and associated endothelial cells and the mechanism of Disruptin action. We hypothesize that the head and neck cancer and tumor-associated endothelial cells depend on EGFR, and loss of EGFR would induce cell death in these cells. Specific Aim 2 A is to understand the effects of Disruptin on endothelial cell invasion, migration and capillary sprouting. Our preliminary data show that Disruptin significantly reduced capillary sprouting in human dermal microvascular endothelial cells (HDMEC). We hypothesize that these effects of Disruptin on capillary formation are due to EGFR degradation. We will analyze the changes in capillary sprouting and EGFR degradation in HDMEC treated with Disruptin and scrambled peptide. We will then perform mechanistic studies by over-expressing wild-type and degradation resistant (Y1045F) EGFR in HDMEC and assess the effects of Disruptin on capillary sprouting. Aim 2B is to understand the effects of Disruptin on the production and/or release of angiogenic growth factors, such as VEGF, bFGF, IL-8 and EGF by tumor or endothelial cells. We hypothesize that Disruptin will degrade EGFR in both tumor and endothelial cells, which will affect the overall production and/or release of pro-angiogenic factors as reported in case of other EGFR inhibitors (3). The effects of Disruptin on these angiogenic factors will be tested using established ELISA methods in culture media of tumor and endothelial cells. We expect Disruptin will block the production/release of angiogenic growth-factors which will correlate with the response. Finally, the effects of conditioned medium produced from scrambled peptide and Disruptin treated HNSCC tumor cells will be assessed 0n the migration of primary endothelial cells using standard migration assays. We expect Disruptin to cause a decrease in the release of angiogenic factors by tumor cells which will be reflected by a decrease in endothelial cell migration.
Results from this proposal would represent a proof-of-principle that disruption of HSP90 and EGFR complex would potentiate EGFR degradation, and the knowledge can be utilized further to develop a small molecule to treat EGFR driven tumors.