Targeting glucosylceramide generation induces cancer cell death through lysosomal dysfunction independent of ceramide accumulation

The bioactive lipid ceramide is usually implicated in mediating anti-proliferative signaling. Therefore, preventing its conversion to complex sphingolipids was proposed as a valid target to induce cancer cell death. Higher expression of the sphingolipid metabolic enzyme glucosylceramide synthase (GCS), which converts ceramide to glucosylceramide (GlcCer), correlates with poor prognosis in cancers. Pre-clinical and cellular models targeting GCS to treat cancer achieves increases in ceramide levels with subsequent apoptosis in tumor cells. However, the underpinning mechanisms and the possible roles of elevated ceramide versus loss of GlcCer or downstream glycosphingolipids are not established. We found that inhibition of GCS by eliglustat, an FDA-approved GCS inhibitor, or other pharmacological inhibitors (Genz123346 and miglustat), or genetic silencing of GCS induced cell death in several cancer cell lines, which was not rescued by prevention of ceramide generation, suggesting that the cell death may be independent of ceramide accumulation. In addition, genetic or pharmacological inhibition of GCS resulted in defective lysosomal turnover and stalling of cytoprotective autophagy independent of ceramide levels. GlcCer loss also caused changes in lysosomal morphology and distribution and induced accumulation of protein, lipid, and organellar degradation products within the lysosomes. Importantly, restoration of lysosomal function through lysosomal biogenesis and functional autophagy by the mTOR inhibitor Torin1 rescued cell death and lysosomal dysfunction, suggesting a previously unknown role for GCS in the maintenance of lysosomal function. In summary, our study demonstrates a previously unappreciated function for GlcCer and glycosphingolipids, probably independent of ceramide, in maintaining lysosome function and a new mechanistic view in targeting GCS for cancer therapy.