Nkcc1 Regulates Migration Of Glioblastoma Tumor Initiating Cells By Interacting With The Actin Cytoskeleton

Proceedings: AACR 106th Annual Meeting 2015; April 18-22, 2015; Philadelphia, PAGlioblastoma (GBM) is the most aggressive and deadliest primary human brain tumor in adults due to the extensive tumor cell migration throughout surrounding brain parenchyma. The capacity to form new tumors, distant from the original location, resides in a specific cell subpopulation called brain tumor initiating cells (BTICs). Combined local and systemic therapies have been ineffective at targeting these invasive cells; furthermore, the mechanisms that confer GBM cells their invasive behavior have not been fully elucidated. The electroneutral Na+-K+-Cl- cotransporter 1 (NKCC1) is an important cell volume regulator that is implicated in cell migration and overexpressed in GBM compared to non-cancer brain tissue. We have shown that both downregulation and pharmacological inhibition (using Bumetanide, BMT) of NKCC1 in BTICs lead to decreased cell migration, in vitro and in vivo. In addition, we reported that NKCC1 knockdown cells (NKCC1 KD) show significantly larger focal adhesions, suggesting a potential role of NKCC1 in cell adhesion. We now report the role of NKCC1 on cytoskeletal dynamics. We found that glioma cells display a significant decrease in cell spreading capacity upon NKCC1 KD or inhibition by BMT. Further, F-actin organization (observed with phalloidin staining) showed dramatic changes upon NKCC1 KD in BTICs. These changes include concentration of actin filaments on the membrane periphery in a ring shaped form and decreased bundled actin content. To analyze actin changes in a dynamic model, we transduced BTICs with the Lifeact-RFP construct for live imaging of F-actin. We observed that upon EGF stimulation, control cells responded by uniform spreading, as reflected by an increased cell circularity within seconds after stimulation, while NKCC1 KD cells did not respond. This suggests that the dynamic response of the actin cytoskeleton to external stimuli is decreased when NKCC1 is down-regulated. To determine the potential actin-regulatory mechanisms affected by NKCC1 inhibition we studied the small Rho-GTPases, RhoA and Rac1. We observed that the levels of active (GTP-bound) forms of RhoA and Rac1 were decreased in NKCC1 KD cells. Moreover, when BTICs were treated with the ROCK1 inhibitor, Y-27632, they showed a dose-dependent decrease in cell migration and activity of MLC (ROCK substrate). These effects were significantly weaker when the ROCK inhibitor was applied on NKCC1 KD or BMT-treated cells, suggesting that decreased RhoA and ROCK1 activity mediates the effects of NKCC1 inhibition. In summary, NKCC1 regulates BTIC migration beyond cell volume changes, by modulating the cytoskeleton through multiple targets that include F-actin regulation and RhoA and Rac1 activity. Due to its essential role in cell migration and high expression in GBM, NKCC1 may serve as a specific therapeutic target to decrease BTIC cell invasion and increase survival in patients with primary brain cancer.Citation Format: Paula V. Schiapparelli, Roxana Magana-Maldonado, Susan Hamilla, Eric Goulin Lippi Fernandes, Sara Ganaha, Chuan-Hsiang Huang, Hugo Guerrero-Cazares, Helim Aranda-Espinoza, Peter N. Devreotes, Alfredo Quinones-Hinojosa. NKCC1 regulates migration of glioblastoma tumor initiating cells by interacting with the actin cytoskeleton. [abstract]. In: Proceedings of the 106th Annual Meeting of the American Association for Cancer Research; 2015 Apr 18-22; Philadelphia, PA. Philadelphia (PA): AACR; Cancer Res 2015;75(15 Suppl):Abstract nr 4152. doi:10.1158/1538-7445.AM2015-4152