Abstract LB-361: Radiosensitization and decreased neurosphere-forming capacity with TGFβ inhibition in glioma cells

Abstract

Abstract Transforming growth factor-β (TGFβ) is a pleotropic cytokine in the tumor microenvironment that can promote malignant behaviors, including invasion and motility, at late stages of tumorigenesis. Glioblastomas produce abundant TGFβ, are routinely treated with radiation, and have a very poor prognosis (median survival of only 14.6 months). An unexpected role for TGFβ in the DNA damage response has recently been discovered in which TGFβ inhibition in vitro and in vivo compromises ATM-kinase activity induced by ionizing radiation (Cancer Research 66:10861–68; 62:5627–31). These data suggest that TGFβ could actually protect cancer cells from the cytotoxic effects of radiation by promoting ATM dependent responses; if so, TGFβ inhibitors, which are in clinical trials, might increase therapeutic response to radiation. To test this idea, we asked whether TGFβ inhibition could radiosensitize glioblastoma cells. We used the murine glioblastoma cell line, GL261, to test the effects of TGFβ inhibition by LY364947 (a small molecule inhibitor of the TGFβ type I receptor kinase) on proliferation, radiosensitivity, and neurosphere-forming capacity. GL261 cells were found to produce 0.9 ng/mL per 106 cells of total TGFβ in media conditioned for 24 hr, the majority of which is latent TGFβ2. They also respond to exogenous TGFβ1 with an increase in Smad2 phosphorylation by Western blot. Despite intact TGFβ receptor kinase activity, GL261 cells displayed no growth modulation response to exogenous TGFβ1 (0.5–2ng/mL) treatment or to inhibition by LY364947 (400nM). Nonetheless, inhibition of TGFβ with LY364947 for 24 hours prior to radiation treatment significantly increased GL261 radiosensitivity in the clonogenic assay, with a 1.25 (p<0.001, ANOVA) dose enhancement ratio at 10% surviving fraction; in other words, an increase of 25% in clonogenic cell death. The increase in radiosensitivity correlated with a significant 55% decrease in γH2AX foci, which is a substrate of ATM, following radiation treatment with 2Gy (p<0.0001, ANOVA). We then assessed the response of glioma initiating cells (GIC) in a neurosphere assay. Irradiation of GL261 cells with 2Gy decreased neurosphere-forming capacity by 28% (p<0.001, ANOVA). Treatment with LY364947 alone had no effect on neurosphere formation, but treatment for 24 hours prior to irradiation decreased the neurosphere-forming capacity of irradiated GL261 cells by an additional 47% (p<0.001, ANOVA). Further studies using an orthotopic GL261 intracranial glioblastoma model are underway to test how these radiation effects mediated by TGFβ affect tumor growth in vivo. Given the radiosensitization and specifically the response of the GIC population, our results suggest that inhibition of TGFβ in combination with radiation represents promising therapeutic strategy in glioblastoma to improve poor response rates seen with the standard regimen of chemotherapy and radiotherapy. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 102nd Annual Meeting of the American Association for Cancer Research; 2011 Apr 2-6; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2011;71(8 Suppl):Abstract nr LB-361. doi:10.1158/1538-7445.AM2011-LB-361