The Regulation of Ceramide Generation and Its Downstream Metabolites as Targets for Cancer Therapy

Sphingolipids, a class of bioactive lipid signaling molecules, have been shown to play vital roles in cancer and other human diseases. The central sphingolipid molecule, ceramide, is known to play primarily growth-inhibitory roles in tumors, while its downstream metabolites, such as sphingomyelin, glycosphingolipids, and sphingosine-1-phosphate, are known to play growth-promoting roles. Their metabolism, then, plays a pivotal role in tumor progression and metastasis. Although much work has been done in understanding the mechanistic underpinnings of sphingolipid metabolism in cancer, the various ways in which ceramide and its downstream metabolites control tumor growth remain elusive. The works presented here reveal novel regulatory mechanisms of sphingolipid generation as well as the biological consequences of dysregulated sphingolipid metabolism. The first chapter provides an overview of sphingolipid metabolism as well as the established roles of sphingolipids in human health and disease. The second chapter investigates the role of post-translational modifications on ceramide synthase 6 (CerS6) activity in HCT116 colon cancer cells. The data presented demonstrates that glycosylation of CerS6 at N18 is essential for its activity, and ablated glycosylation results in modulation of cellular signaling pathways. Work presented in the third chapter shows a novel role for glycosphingolipids in lysosomal turnover and autophagy, and proposes a mechanism by which glucosylceramide synthase (GCS) inhibition induces tumor cell death in colon, cervical, and ovarian cancers. Further, these data provide critical mechanistic insights into the use of the GCS inhibitor, eliglustat, in combination with immunotherapies in cancer patients. The fourth chapter provides preliminary characterizations of subcellular pools of sphingolipids using mitochondrial and lysosomal rapid-immunoprecipitation and LC/MS-MS lipidomics analysis in HCT116 colon cancer cells. These results serve as a proof-of-concept for the use of rapid-immunoprecipitation in studying the regulation and biological impacts of subcellular pools of sphingolipids species. The final chapter offers analysis of the data presented and future directions. Together, the work presented in this dissertation shows novel regulatory mechanisms of sphingolipid generation, and the biological consequences of sphingolipid dysregulation. These mechanistic insights may be critical for developing novel chemotherapeutics to be used for the treatment of patients suffering from various cancers.