Glioblastoma multiforme (GBM), a grade IV astrocytoma, is a very aggressive and prevalent primary brain tumor with a median survival rate of 12-15 months. Though the standard treatment relies on combinations of chemotherapy, radiation therapy and surgical resection, GBM remains radio-resistant with a high recurrence rate after resection. Understanding the molecular mechanisms involved in the therapeutic response could lead to the identification of novel targets to improve therapy. Radiation can trigger the activation of cytosolic phospholipase A2 (cPLA2), leading to production of lipid second-messengers such as lysophosphatidylcholine (LPC). Autotaxin (ATX), also known as lysophospholipase D (LysoPLD), is a secreted enzyme that catalyzes the production of lysophosphatidic acid (LPA) in the tumor microenvironment by cleaving the head-group of LPC. Specific G-protein coupled receptors (GPCRs) mediate the autocrine and paracrine effects of LPA such as migration, angiogenesis and proliferation in cancer. ATX is highly expressed in glioblastoma cells and is known to contribute to its invasive properties. We studied ATX as a potential target to enhance radiosensitivity in GBM by targeting ATX with PF-8380, a specific inhibitor developed by Pfizer Inc., and genetically by using shRNA. Pre-treatment with 1μM PF-8380 followed by irradiation with 4Gy resulted in decreased clonogenic survival, decreased migration and decreased invasion in mouse glioblastoma GL-261and human glioblastoma U87-MG cells. We confirmed these results by knocking down ATX with shRNA in GL-261 and U87-MG cells. Inhibition of ATX by PF-8380, or its knockdown by shRNA, lead to attenuation of Akt phosphorylation in GL-261 and U87-MG cells.

Inhibition or knockdown of ATX resulted in significantly reduced radiation-induced neovascularization as assayed with GL-261 tumor vascular window model. The effect of ATX inhibition by PF-8380 in-vivo was studied using a heterotopic mouse GL-261 tumor model. Tumor bearing mice were treated with DMSO or 1 mg/kg PF-8380 followed by irradiation with 1.8Gy for 5 consecutive days. Tumor growth delay was analyzed using a non-parametric (Kruskal-Wallis) test to determine the amount of days to reach a tumor volume of 0.7 cm3 and ANOVA to determine the difference between treatments. Tumors treated with DMSO or PF-8380 took 11.2 and 12.2 days respectively to reach a volume of 0.7 cm3. Tumors treated with radiation alone took 23.3 days while the combination of PF-8380 with radiation significantly delayed tumor growth to 32 days.

In this study, we found that inhibition of pro-survival cellular signaling pathways, migration, invasion and radiation-induced neovascularization can be achieved by targeting ATX. Thus, ATX could serve as a novel and effective molecular target for the development of radiosensitizers of GBM, and may provide a new approach to treating patients with this lethal form of brain cancer.

Citation Format: David Y.A. Dadey, Sandeep R. Bhave, Daniel J. Ferraro, Rowan M. Karvas, Dennis E. Hallahan, Dinesh Thotala. Inhibition of autotaxin enhances radiosensitivity in human and murine glioblastoma cell lines. [abstract]. In: Proceedings of the 104th Annual Meeting of the American Association for Cancer Research; 2013 Apr 6-10; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2013;73(8 Suppl):Abstract nr 2136. doi:10.1158/1538-7445.AM2013-2136