Abstract 4315: System Xc- in glioma alters redox regulation, energetics and morphology

Glioblastoma multiforme is the most common malignant primary brain tumor with a median life expectancy of 12 months from diagnosis. Due to the metabolic demands of rapidly proliferating cancer cells and higher free radical generation, we show that human glioma cells, compared to primary normal human astrocytes, express higher levels of system XC-, a sodium-independent electroneutral transporter composed of a catalytic subunit, xCT, and a heavy chain subunit, 4F2hc. System XC- is responsible for maintaining redox homeostasis by importing cystine, where it is then reduced to cysteine and used to synthesize the major antioxidant glutathione (GSH). We demonstrate that inhibition of system XC-, accomplished using sulfasalazine (SAS), increased intracellular reactive oxygen species (ROS) and decreased survival of U251 glioma cells in a dose-dependent manner. To further delineate the role of system XC- in glioma survival and minimize the off-target effects of SAS, we generated stable xCT-over-expressing and xCT-knockdown U251 glioma cell lines. The xCT-over-expressing glioma cells were more resistant to oxidative stress and chemotherapy treatment. Interestingly, the xCT-over-expressing U251 cells exhibit increased basal ATP generation compared to xCT-knockdown and control cells, despite no difference in viability or proliferation. The higher ATP levels correlated with increased basal levels of ROS in the xCT-over-expressing cells. Since mitochondria generate much of the cellular energy and produce most of the cellular ROS, we performed transmission electron microscopy (TEM) for morphological examination of the mitochondria. TEM revealed no alteration in mitochondrial morphology but there was a significant increase in the number of mitochondria in the xCT-over-expressing cells compared to xCT-knockdown and control cells. This indicates that system XC- may not only play a role in redox regulation but also in altered energetics. Further studies to assess the mitochondrial biogenesis and metabolism are currently under investigation. Additionally, the xCT-over-expressing U251 cells exhibited an altered morphology compared to the typical elongated morphology of parental and empty vector controls. This cuboidal morphology was associated with increased cortical F-actin staining compared to the elongated F-actin stress fibers in control cells. Since actin stress fibers promote cell migration, we assessed the migratory and invasive characteristics of the xCT-modified cell lines. The xCT-over-expressing U251 cells exhibited decreased migration and invasion while the xCT-knock-down U251 cells exhibited increased invasion. These findings indicate that system XC- over-expression in glioma not only promotes survival under oxidative stress and chemotherapy treatment, but may also modulate mitochondrial function and the migratory/ invasive properties to promote integration into host tissue. Citation Format: Monika D. Polewski, Rosyli F. Reveron, Karen S. Aboody. System Xc- in glioma alters redox regulation, energetics and morphology. [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 4315. doi:10.1158/1538-7445.AM2014-4315