With-No-Lysine Kinase 3 (WNK3) stimulates glioma invasion by regulating cell volume

Among the most prevalent and deadly primary brain tumors, high-grade gliomas evade complete surgical resection by diffuse invasion into surrounding brain parenchyma. Navigating through tight extracellular spaces requires invading glioma cells to alter their shape and volume. Cell volume changes are achieved through transmembrane transport of osmolytes along with obligated water. The sodium-potassium-chloride cotransporter isoform-1 (NKCC1) plays a pivotal role in this process, and previous work has demonstrated that NKCC1 inhibition compromises glioma invasion in vitro and in vivo by interfering with the required cell volume changes. In this study, we show that NKCC1 activity in gliomas requires the With-No-Lysine Kinase-3 (WNK3) kinase. Western blots of patient biopsies and patient-derived cell lines shows prominent expression of Ste-20-related, proline-alanine-rich kinase (SPAK), oxidative stress response kinase (OSR1), and WNK family members 1, 3, and 4. Of these, only WNK3 colocalized and coimmunoprecipitated with NKCC1 upon changes in cell volume. Stable knockdown of WNK3 using specific short hairpin RNA constructs completely abolished NKCC1 activity, as measured by the loss of bumetanide-sensitive cell volume regulation. Consequently, WNK3 knockdown cells showed a reduced ability to invade across Transwell barriers and lacked bumetanide-sensitive migration. This data indicates that WNK3 is an essential regulator of NKCC1 and that WNK3 activates NKCC1-mediated ion transport necessary for cell volume changes associated with cell invasion.