Ceramide Signaling in the Regulation of Head & Neck Cancer Cell Death and Therapy

SUMMARY Head and neck squamous cell carcinoma (HNSCC) includes mucosal squamous cell carcinomas of the oral cavity, pharynx, and larynx. It represents over 65,000 new cancer cases, with 14,500 cancer-related deaths in the United States annually. Previous studies demonstrated that ~75% of HNSCC tumors contain decreased ceramide synthase 1 (CerS1)-generated C18-ceramide, a bioactive sphingolipid with anti-proliferative signaling functions. Lower ceramide levels are associated with advanced disease stage and poor survival in HNSCC patients with defective mitophagy. Our preliminary data showed that the reconstitution of C18-ceramide synthesis in mitochondria by CerS1 restored mitophagy and HNSCC tumor suppression. Recently, we discovered that instead of trafficking C18-ceramide, its metabolic enzyme, CerS1, is transported from the endoplasmic reticulum (ER) to damaged mitochondria, a paradigm-shifting mechanism, to induce mitophagy in response to cisplatin, rapamycin or sodium selenite (SoSe)-mediated stress signaling. A previously unidentified 17 kDA protein catalyzes this process. We coined its name as p17/PERMIT (protein ER-mitochondrial transporter). However, mechanisms that regulate CerS1-p17/PERMIT and subsequent ceramide-dependent mitophagy and cell death in HNSCC remain unknown. Accordingly, to restore ceramide accumulation in mitochondria, we developed a novel ceramide analog drug, LCL768. This ceramide analog contains C18-ceramide, conjugated with selenium and pyridinium moieties, which target C18-ceramide selectively to HNSCC cell mitochondria, leading to mitophagy induction. Thus, our preliminary data suggested to us the novel hypothesis that mitochondrial trafficking of CerS1 by p17/PERMIT mediates ceramide-dependent mitophagy, leading to HNSCC tumor suppression in response to cancer therapeutics and stress signaling. As a corollary, we also hypothesize that LCL768 reconstitutes mitochondrial ceramide and mitophagy in HNSCC with defective mitochondrial CerS1- p17/PERMIT trafficking. To test these hypotheses, we propose three Specific Aims: 1) Determine the mechanism whereby p17/PERMIT-mediated trafficking of CerS1 to mitochondria induces C18-ceramide generation and mitophagy. 2) Define the mechanisms by which mitochondrial CerS1/C18-ceramide mediates mitophagy via Drp1 activation. 3) Identify the therapeutic mechanisms and efficacy of mitochondria-targeted ceramide-analog drug, LCL768, in HNSCC tumor suppression. Our research team, which includes both basic and clinical scientists, is uniquely positioned to execute studies proposed in this application based on our complementary expertise in the fields of sphingolipid metabolism, mitochondrial damage signaling, mechanisms of cancer cell death, and HNSCC therapy. In addition to ceramide analogs (such as LCL768), we propose experiments here using clinically available therapeutic drugs, such as SoSe, rapamycin and cisplatin in patient-derived 2D HNSCC organoids and PDX tumors, which can readily be translated into the clinic to induce ceramide-dependent mitophagy and tumor suppression in HNSCC.