Abstract 1061: Glutathione S-transferase P1 is required for growth and survival of human glioma cells

Abstract

Abstract Overexpression of glutathione S-transferase p1 (GSTP1), a protein involved in Phase II metabolism and cell signaling, is a hallmark of diverse human malignancies, including gliomas, and is associated with resistance to therapy, aggressive tumor growth and poor patient survival. Although, the function of GSTP1 in drug metabolism and detoxification is well documented, its role in regulating tumor cell growth remains unclear. Here, we report that in human glioma characterized by high GSTP1 expression, GSTP1 is obligately required for the growth and survival of the tumor cells. For this, we stably infected cells of the MGR3 glioblastoma multiforme (GBM) cell line that expresses high de novo levels of GSTP1, with a lentiviral vector that either expresses a short hairpin RNA (shRNA) specifically targeting to GSTP1 or a control shRNA. We isolated two clones, 1 and 2 and showed by western blotting that the GSTP1 specific shRNA mediated decreases in GSTP1 expression of 92% and 100%, respectively, relative to controls while infected with eGFP shRNA. Growth kinetic analysis showed the in vitro growth rates of both clones 1 and 2 to be significantly lower than that of the parental or control clone, with population doubling times of 108.9 ± 17.6 hr for clone 1 and 94.8 ± 4.3 hr for clone 2, compared with 40.6 ± 2.6 hr for the parental cells and 44.7 ± 2.0 hr for the control transfected cells. Cell cycle analysis showed that the GSTP1 knockdown induced a significant almost 2-fold increase in the sub-G1 (apoptotic) population that correlated with an increase in caspase 3/7 activation. Western blotting showed no significant changes in total protein levels of ERK, JNK and p38 MAPK following GSTP1 knockdown, however, phosphorylation levels of both ERK1/2 and JNK1/2 increased significantly, while phospho- p38 MAPK levels did not change. JNK downstream signaling, viz., phosphorylation of c-Jun, increased dramatically in cells of the two clones. These alterations in MAPK signaling in the tumor cells associated with GSTP1 silencing correlated with the decreased survival and growth kinetics and corresponding changes in apoptosis/survival signals, including, decreased bcl2 and increased bax levels in the tumors. Our results show that GSTP1 is required for cell survival and growth of human GBM cells that naturally over-express high GSTP1 levels. This is consistent with the aggressive tumor growth and poor survival of patients whose tumors contain high levels of GSTP1 and provide a rationale for targeting GSTP1 as a potential therapeutic strategy for malignant gliomas. Supported by grants RO1CA112519, RO1CA127872, RO1CA91438, P30CA114236 and P5OCA108786 from the National Institutes of Health (USA). Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 101st Annual Meeting of the American Association for Cancer Research; 2010 Apr 17-21; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2010;70(8 Suppl):Abstract nr 1061.