LRRC8A-containing anion channels promote glioblastoma proliferation via a WNK1/mTORC2-dependent mechanism

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 the present study, we investigated the role of LRRC8A and VRACs in the progression of glioblastoma (GBM), the most common and aggressive primary brain tumour. We found that, compared with healthy brain tissue, LRRC8A mRNA is significantly upregulated in surgical GBM specimens, patient-derived GBM cell lines and 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, RNA interference-mediated knockdown of LRRC8A or pharmacological blockade of VRAC with 4,4'-diisothiocyano-2,2'-stilbenedisulfonic acid (DIDS) reduced cell proliferation, lowered intracellular chloride levels and inhibited activity of mammalian target of rapamycin (mTOR) complex 2 (mTORC2). The anti-proliferative 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 signalling axis for VRAC-dependent control of cellular functions and highlight several potential molecular targets for limiting GBM proliferation. KEY POINTS: Volume-regulated anion channels (VRACs) are considered to contribute to the progression of several human cancers. The essential VRAC subunit LRRC8A is significantly overexpressed in clinical specimens of glioblastoma, the most common and aggressive primary brain malignancy. RNA interference-mediated downregulation of LRRC8A reduces proliferation in patient-derived GBM cell cultures, suggesting that VRACs promote cancer cell growth. LRRC8A/VRAC-mediated effects on cell proliferation are driven by a mechanism involving the chloride-sensitive protein kinase WNK1, mTOR complex 2 and activation of downstream kinases AKT and SGK.