Date of Award

Spring 3-31-2025

Embargo Period

3-31-2030

Document Type

Dissertation - MUSC Only

Degree Name

Doctor of Philosophy (PhD)

Department

Biochemistry and Molecular Biology

College

College of Graduate Studies

First Advisor

Besim Ogretmen

Second Advisor

Michael Ostrowski

Third Advisor

Shikhar Mehrotra

Fourth Advisor

Ozgur Sahin

Fifth Advisor

Denis Guttridge

Abstract

Metastasis is the leading cause of cancer-related mortality. Immunotherapy, like immune checkpoint blockade (ICB), has revolutionized how we treat metastatic cancer, but ICB resistance substantially limits efficacy, especially in solid cancers like triple-negative breast cancer (TNBC). Understanding the molecular drivers underlying metastasis and immunotherapy resistance is crucial to improve clinical outcomes. This dissertation investigates ceramide metabolism, specifically Ceramide Synthase 4 (CerS4), in regulating metastasis and immunotherapy resistance through intracellular programmed death ligand 1 (PD-L1) signaling.

Using molecular in vitro and transcriptomic approaches, we established that CerS4 downregulation promotes PD-L1 internalization from the cell surface into an intracellular complex with the RNA-binding protein Caprin-1. This PD-L1/Caprin-1 complex stabilizes oncogenic mRNAs critical for promoting tumor migration/metastasis via activating Sonic hedgehog (Shh) and TGF-β signaling pathways. Genetic ablation of CerS4 in transgenic and orthotopic TNBC models significantly increased metastasis and generated an immunosuppressive tumor microenvironment (TME) characterized by elevated regulatory T cells and CD8+ T cell dysfunction. Importantly, pharmacologic targeting of the PD-L1/Caprin-1 complex with Shh pathway inhibitors combined with ICB synergistically enhanced antitumor immunity and significantly reduced metastasis.

Next, we developed a transplantable, orthotopic TNBC model of ICB resistance (E0771-2RA) via serial anti-PD-L1 therapy and interrogated this model with comprehensive lipidomics, transcriptomic analyses, and immune profiling to understand the role of CerS4 in immunotherapy resistance. The E0771-2RA model exhibited strong CerS4/ceramide suppression, enhanced intracellular PD-L1 accumulation, increased Caprin-1 interaction, and heightened prostaglandin E2 (PGE2) synthesis via prostaglandin-endoperoxide synthase 2 (Ptgs2) mRNA stabilization by the PD-L1/Caprin-1 complex. We identified that ceramide, regardless of chain length, directly binds PD-L1 and prevents Caprin-1 binding. Resistant tumors displayed significant immune remodeling within the TME with prominent CD8+ T cell dysfunction. After ICB, dynamic expression of PGE2receptors, EP2 and EP4, was observed among CD8+ T cells and impaired response in PGE2-rich tumors. CerS4/ceramide restoration or targeting the PD-L1/Caprin-1 complex via TGF-β inhibition reversed ICB resistance and reactivated CD8+ T cell response.

Collectively, these results identify the CerS4/PD-L1/Caprin-1 axis as a critical determinant of metastasis and immunotherapy resistance in cancer, providing new therapeutic strategies for targeting metastasis and improving ICB response rates.

Rights

Copyright is held by the author. All rights reserved.

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