Abstract B31: NOX2 generated reactive oxygen species promotes glioblastoma growth through inhibition of PTEN

The tumor suppressor protein Phosphatase and Tensin Homolog (PTEN) is a negative regulator of the Akt pathway which promotes proliferation, migration, and self-renewal. PTEN is mutated or deleted in ~40% of glioblastoma (GBM) resulting in increased Akt activity; however almost all GBM9s have increased Akt activity, regardless of PTEN status. This raises the possibility that GBM may inactivate PTEN through means other than mutation or deletion. Treatment with exogenous reactive oxygen species (ROS), like hydrogen peroxide, inactivates PTEN through oxidation in the phosphatase domain. The low oxygen environment of tumors (1%O2) increases ROS levels as compared to normal body tissue (4% O2), and tissue culture conditions (20% O2). Recent data indicate that low to moderate levels of ROS are required for cell maintenance and stem cell renewal while moderate increases in ROS may promote migration, invasion, and proliferation. Indeed, our lab recently found that normal neural stem cells have higher ROS levels, which is required for stem cell renewal. Moreover, data from our lab and others, have found that growing cells under physiological oxygen levels increases ROS and promotes renewal and proliferation. This suggests that the low-oxygen environment of tumors may promote proliferation and tumorigenesis through post-translational modification of proteins, like PTEN. NADPH oxidase (NOX) is a membrane bound complex of proteins that produces a significant amount of endogenous intracellular ROS. While this protein was originally identified in macrophages as being important for immune surveillance, new data suggests that the family of NOX proteins (1-4) has many other biological functions in a variety of different cell types. New data has found that the constitutively active NOX4 acts as an oncogene in several different cancer types by promoting migration, invasion, and proliferation. Within the brain, NOX2 promotes ROS-induced stem cell renewal in normal neural stem cells. Additionally, increased NOX2 expression in GBM patients correlates with shorter survival, while patients with low NOX2 expression generally live longer. Together these data suggest that, through NOX2, the low-oxygen environment of GBM increases ROS levels that oxidize and inhibit PTEN. This novel post-translational modification may increase proliferation and promote tumorigenicity through activation of Akt in cells with intact PTEN. We examined endogenous ROS levels in patient-derived GBM samples and found that increased ROS correlated with faster proliferating cells. Furthermore, low levels of O2 (1%) increased proliferation and Akt activation in a PTEN dependent manner. Both PTEN null and knockdown cells showed no increase in either proliferation or Akt activation over cells grown at room air (20%). Moreover, pharmacological inhibition of Akt abrogated ROS-induced proliferation, suggesting this mechanism functions through the PTEN/Akt pathway. This response was due directly to ROS production, as treatment with antioxidants decreased both ROS production and proliferation at low oxygen and room air. Additionally, we found that growth at 1% O2 led to the oxidation and inhibition of PTEN as seen by Akt phosphorylation. Treatment with apocynin, a NOX2 inhibitor decreased ROS production, proliferation, and Akt phosphorylation, suggesting that ROS-induced proliferation may be mediated through the NOX2 protein. This data not only show PTEN oxidation and inhibition under physiological conditions for the first time, it suggests that the low-oxygen tumor microenvironment may alter PTEN positive GBM to up-regulate the Akt pathway and promote tumorigenesis. Citation Format: Kirsten Ludwig, Harley Kornblum. NOX2 generated reactive oxygen species promotes glioblastoma growth through inhibition of PTEN. [abstract]. In: Proceedings of the AACR Special Conference: Advances in Brain Cancer Research; May 27-30, 2015; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2015;75(23 Suppl):Abstract nr B31.