Date of Award

2018

Document Type

Dissertation

Degree Name

Doctor of Philosophy (PhD)

Department

Biochemistry and Molecular Biology

College

College of Graduate Studies

First Advisor

Besim Ogretmen

Second Advisor

Alan Diehl

Third Advisor

Robert Gemmil

Fourth Advisor

Christina Voelkel-Johnson

Fifth Advisor

Vamsi Gangaraju

Abstract

Sphingolipids have over the years, been explored as cancer therapy targets. FTY720 (Fingolimod, Gilenya) is a sphingosine analogue drug used for the treatment of multiple sclerosis. FTY720 is phosphorylated by sphingosine kinase 2, to generate P- FTY720 to exert its immunosuppressive properties through binding to sphingosine -1 phosphate receptors (S1PRs). FTY720 also exhibits anti-cancer properties. Our previous studies indicated that FTY720’s anti-cancer property is through induction of necroptosis. FTY720 directly binds to I2PP2A/SET (Inhibitor 2 of PP2A), consequently activating the tumor suppressor protein phosphatase 2A (PP2A). The activated PP2A then induces cell death by activating Receptor-Interacting Protein kinase-1 (RIPK1), involved in necroptosis signaling. Little is known about the roles of FTY720 in ceramide signaling and regulation of necroptosis in lung cancer. We therefore, sought to investigate the mechanisms of FTY720 in inducing necroptosis with regard to ceramide signaling. Our data indicate that C16-ceramide is crucial for FTY720-induced cell death. Interestingly, FTY720 does not induce ceramide generation but leads to the formation of specific ceramide-multi-protein complexes at the plasma membrane involved in plasma membrane disruption and necroptosis. Here we show D147, N148 and N169 residues in RIPK1 are important for RIPK1- ceramide binding and cell death. In addition, we show a novel role for non-muscle Myosin IIA (NMIIA) in trafficking these complexes to the plasma membrane to promote membrane rupture. We identify RIPK1 L112 and C256 residues as critical for RIPK1-NMIIA interaction. We hereby propose that these novel ceramide complexes form non-selective pores supported by RIPK1 and NMIIA. Our data suggest that these structures are very specific and are important in the execution of necroptosis independent of RIPK3 and MLKL. Additionally, preliminary data suggest that these novel structures can also form in-vivo, without external stimulus, indicating a possible role in non-pathological conditions.

Rights

All rights reserved. Copyright is held by the author.

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