Regulatory Role of Sphingolipid Metabolites in Immunogenic Cancer Cell Death
Restricted (Penn State Only)
- Author:
- Nduwumwami, Asvelt
- Graduate Program:
- Biomedical Sciences
- Degree:
- Doctor of Philosophy
- Document Type:
- Dissertation
- Date of Defense:
- June 21, 2022
- Committee Members:
- Jong Yun, Chair & Dissertation Advisor
Nadine Hempel, Special Member
Victor Ruiz-Velasco, Outside Unit & Field Member
Jeffrey Neighbors, Major Field Member
Ralph Keil, Program Head/Chair
Arati Sharma, Major Field Member
Jeremy Andrew Hengst, Special Member - Keywords:
- Immunogenic cell death
sphingosine kinase
ceramide
sphingolipid metabolism
calreticulin - Abstract:
- The ability of cancer cells to elicit an antitumor immunity relies on two determining features of immunogenicity: antigenicity and adjuvanticity. Antigenicity, which arises mainly from somatic mutations that form neoantigens, allows endogenous T cells to recognize tumors. Adjuvanticity on the other hand is a consequence of endogenous alarm signals emitted by malignant cells succumbing to immunogenic cell death (ICD) induced by chemotherapeutic or physical agents. These alarm entities include various immunostimulatory molecules such as extracellular ATP and cell surface exposure of the endoplasmic reticulum resident protein calreticulin. They communicate damage or stress and are therefore termed damage associated molecular patterns (DAMPs). DAMPs interact with their cognate receptors on antigen presenting cells (APCs) to drive the maturation and recruitment of APCs to tumor microenvironment, and to promote phagocytosis of dying cancer cells and antigen cross-presentation to naïve T cells, thereby paving the way for adaptive antitumor immunity. The findings presented in this dissertation demonstrate that bioactive sphingolipid metabolites including sphingosine-1-phosphate (S1P) and ceramide regulate processes that are integral to the emission of ICD-associated DAMPs. We show that inhibition of the oncogenic sphingosine kinase (SphK) enhances mitoxantrone-induced ceramide production and results in increased ATP secretion and cell surface exposure of calreticulin. Examination of the isolated plasma membrane lipid raft fraction demonstrated that cell surface calreticulin exists as a disulfide linked dimer in response to ICD induction, suggesting that this structural modification of calreticulin may underlie its ICD-associated immunological function. Pharmacological inhibition and genetic manipulation of enzymes in the sphingolipid metabolic pathway coupled with LC/MS-based sphingolipidomic analyses identified ceramide synthase 6-derived C16 ceramide to be required for the translocation of dimerized calreticulin to the lipid raft microdomain at the plasma membrane. Importantly, we show that agents that induce intracellular ceramide accumulation convert cancer cells into a “vaccine” that elicits an antitumor protection against inoculation of live cancer cells in syngeneic immunocompetent murine models. This ability of ceramide to induce ICD provides an attractive complement to its established direct cytotoxic effects on tumors. While intracellular ceramide accumulation is required for the ICD-associated production of the cell surface calreticulin, we show that SphK1-derived S1P is a stress-induced pro-survival lipid that suppresses ICD. Mechanistically, under ER stress conditions, S1P stabilizes the cellular FADD-like IL-1β-converting enzyme-inhibitory protein (c-FLIP), which suppresses caspase 8-mediated initiation of ICD signaling pathways. These findings indicate that inhibition of sphingosine kinase may represent a means to enhance the therapeutic efficacy of immunogenic cell death-inducing agents, such as mitoxantrone, while reducing their overt toxicity and immunosuppressive effects, leading to better therapeutic outcomes for patients.