Full Western Blot dataset for the manuscript by AM Fidaleo et al.

Leucine-rich repeat-containing protein 8A (LRRC8A) is an essential subunit of the ubiquitously expressed volume-regulated anion channels (VRACs). Previous work has shown that LRRC8A is overexpressed in several cancers and is associated with poor survival outcomes. However, the underlying mechanisms remain obscure. In this study, we investigated the role of LRRC8A and VRACs in the progression of glioblastoma (GBM), the most common and aggressive primary brain tumor. We found that, as compared to healthy brain tissue, LRRC8A mRNA is significantly upregulated in surgical GBM specimens, patient-derived GBM cell lines, and in GBM datasets from The Cancer Genome Atlas. GBM patients in the lowest quartile of LRRC8A expression exhibited a trend toward longer survival. In patient-derived GBM cultures, RNAi-mediated knockdown of LRRC8A or pharmacological blockade of VRAC with DIDS reduced cell proliferation, lowered intracellular chloride levels, and inhibited activity of mTOR complex 2 (mTORC2). The antiproliferative effects of LRRC8A knockdown and DIDS were non-additive, suggesting a shared mechanism. Biochemical and molecular analyses revealed that LRRC8A-containing VRACs promote GBM cell proliferation through a new non-enzymatic function of the chloride-sensitive protein kinase WNK1. Specifically, VRAC activity facilitates WNK1-dependent activation of mTORC2 and its downstream kinases AKT and SGK. In support of this model, either downregulation of WNK1 or pharmacological inhibition of mTOR or SGK/AKT suppressed GBM cell proliferation and mimicked the effect of LRRC8A knockdown. Together, these findings establish a new mTORC2-centric signaling axis for VRAC-dependent control of cellular functions and highlight several potential molecular targets for limiting GBM proliferation.