The long-term objective of this proposal is to uncover mechanisms involved in the regulation of HNSCCpathogenesis and therapy. Human (dihydro)ceramide syntheses 1-6, (dh)CerS1-6, identified as yeasthomologues of the longevity assurance gene 1-6 (LASS1-6) regulate the de novo generation of endogenousceramides with specific fatty acid chain lengths; for example, whereas (dh)CerS1/LASS1 is responsible forthe generation of C18-(dihydro)ceramide, (dh)CerS6/LASS6 generates C16-(dihydro)ceramide. Thesedihydroceramides are then desaturated to ceramides (with their distinct fatty acid chain lengths) bydihydroceramide desaturase (Des). In the cell, these two enzymatic steps (dihydroceramide synthesis anddesaturation) occur in the endoplasmic reticulum (ER). Remarkably, our ongoing mechanistic studies revealthat knock down of LASS6/C16-(dihydro)ceramide induces ER stress, which then triggers mitochondrialapoptosis in HNSCC cells. In addition, treatment of HNSCC cells with known ER stress inducers, such astunicamycin, results in a rapid degradation of LASS6 protein prior to apoptosis. More importantly, increasedlevels of C16-ceramide via induction of hl_ASS6 expression enhance resistance, and protect HNSCC cellsfrom ER stress and cell death. Collectively, these data suggest a novel hypothesis that LASSS-generatedC16-(dihydro)ceramide plays important roles in the regulation of ER homeostasis, such that down-regulationof this pathway mediates a significant component of the ER stress response, which then leads to apoptosisin HNSCC cells. To test this novel hypothesis, three Specific Aims are proposed: 1) Determine the roles ofLASS6/C16-(dihydro)ceramide in the regulation of ER stress in HNSCC cells; 2) Identify the mechanisms bywhich down-regulation of LASS6/C16-(dihydro)ceramide induces ER stress (or a component of the stress),and consequent apoptosis in HNSCC cells; and 3) Establish the in vivo roles and clinical relevance ofLASS6/C16-(dihydro)ceramide in HNSCC pathogenesis and/or response to therapy via the regulation of ERstress. Thus, these studies will help determine the roles and mechanisms of action of LASS6/C16-(dihydro)ceramide in the regulation of ER stress in HNSCC cells. Importantly, these results have tremendousimplications in unraveling the complexities of ceramide signaling and ER stress, in addition to cleartherapeutic implications that will be defined in this proposal.
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