The role of EGFR* signaling pathway for tumor maintenance and alternative pathways in glioblastoma which enhance tumor growth

A74

Epidermal growth factor receptor (EGFR) gene amplification occurs in approx. 40% of all glioblastomas. EGFR* (deletion of exons 2-7), which is found in approximately 20-30 % of human glioblastomas, is the most commonly occurring mutant form and is shown to confer enhanced tumorigenecity. EGFR has been used as a tumor-specific drug target in kinase inhibitor, antibody-based immunotherapy and immunotoxin approaches, although EGFR* has not been established as being required for tumor maintenance. Moreover, recent experience with kinase inhibitor therapy indicates that tumors will eventually grow through the therapy, either because of desensitizing mutations in the kinase itself or through the activation of alternate oncogenic pathways. This study seeks to address both of these issues by using glioma cells that conditionally express EGFR* to test the requirement of EGFR* for tumor maintenance and to uncover alternate pathways that gliomas employ to bypass EGFR* inhibition.For these purposes, we developed a tetracycline-regulatable EGFR* expression system, in which EGFR* expression levels in tumor xenografts are repressed by administering doxycycline-containing drinking water to engrafted mice. The cell line chosen for this approach is the human glioblastoma cell line, U373MG, which we have previously shown requires EGFR* for xenograft establishment in mice. Mice fed doxycycline (dox) in their drinking water and injected subcutaneously with tet-regulatable EGFR*- expressing U373 cells failed to form tumors after 50 days, however when dox was omitted from their water, EGFR* was expressed and this resulted in tumor formation. Tumors that were allowed to establish and then switched to dox conditions, resulting in the silencing of EGFR* expression, underwent an extended period of stasis. The growth rate of tumors before and after suppression of EGFR* were dramatically different, and a significant reductions in proliferation rate (Ki-67) was observed in EGFR*-negative tumors. After a period of stasis for EGFR*-negative tumors, some tumors eventually regained aggressive growth, with kinetics similar to EGFR*-expressing tumors. .We conclude that EGFR* is required only for tumor establishment and that gliomas have selective pressure in vivo to employ alternative compensatory pathways to acquire aggressiveness. Currently we are aiming to define these alternate pathways and to validate their activation in primary human gliomas as a prelude for preclinical testing. We believe that this will provide novel therapeutic targets that serve as alternatives to EGFR* expression and which can be used in combination targeted therapies.