Abstract 511: EGFR-initated NADPH oxidase activity regulates Fyn expression in glioblastoma multiforme

Glioblastoma multiforme (GBM) constitute the most aggressive and common form of primary brain tumors, conferring the worst clinical prognosis. Epidermal growth factor receptor (EGFR) amplification, mutation and re-arrangement are commonly observed genetic lesions in GBM. The most frequently occurring EGFR variant in GBM, EGFRvIII, is characterized by a truncated extracellular domain, leading to a receptor which is unable to bind ligand yet rendered constitutively active. In addition to increasing proliferation and survival signaling, EGFRvIII increases the production of reactive oxygen species (ROS); redox signaling in GBM, however, remains an understudied oncogenic feature. Consistent with published findings, our studies indicate that EGFRvIII elevates ROS levels in comparison to wild-type EGFR or vector-transfected control in U87-MG and the observed increase in ROS levels is alleviated by pharmacological and genetic means of EGFR inhibition. ROS increases were determined to occur independent of augmented spare mitochondrial respiratory capacity, but did coincide with elevated basal levels of non-mitochondrial oxygen consumption as measured by Seahorse extracellular flux analysis. Given this finding, our studies focused on the NADPH oxidase (NOX), where chemical inhibition of NOX by apocynin effectively reduced ROS by greater than 50% in EGFRvIII expressing cells. Likewise, EGFRvIII increased the consumption of NADPH greater than 2.0-fold relative to vector control, as measured by cellular NADP/NADPH ratios, and the resulting increases in NADPH consumption were diminished by chemical inhibition of EGFRvIII as well as apocynin inhibition of NOX. These findings indicated that EGFRvIII-induced ROS increases might be related to increased activity of the NOX complex. To this end, siRNA knockdown of p47phox, an essential component of the NOX-2 complex, markedly reduced ROS content as well as cellular proliferation in these cells, thus reinforcing a possible link between aberrant EGFR signaling and ROS production through NOX. Our studies additionally indicate that EGFRvIII-mediated elevations in ROS up-regulate mRNA and protein level expression (>3.0-fold) of the SRC family kinase member Fyn, as Fyn expression is up-regulated in an EGFRvIII-dependent manner and concordantly down-regulated via chemical and genetic means of NOX inhibition. Moreover, siRNA knockdown of Fyn effectively lowers cellular proliferation, survival and increases sensitivity to EGFR kinase inhibition in EGFRvIII, thus indicating that redox-initiated signaling through Fyn, in part, mediates GBM proliferation. Taken together, our results suggest that EGFRvIII increases NOX-2 generated ROS through p47phox, enhancing the proliferative capacity of GBM, in part, via increased Fyn expression, suggesting that targeting redox alterations may prove beneficial in improving clinical outcomes in GBM. Citation Format: Blake Johnson, Joya Chandra. EGFR-initated NADPH oxidase activity regulates Fyn expression in glioblastoma multiforme. [abstract]. In: Proceedings of the 105th Annual Meeting of the American Association for Cancer Research; 2014 Apr 5-9; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2014;74(19 Suppl):Abstract nr 511. doi:10.1158/1538-7445.AM2014-511