Graduation Date

Spring 5-4-2024

Document Type

Dissertation

Degree Name

Doctor of Philosophy (PhD)

Programs

Biochemistry & Molecular Biology

First Advisor

Surinder K. Batra

Second Advisor

Sushil Kumar

Abstract

Mucins facilitate the pancreatic cancer (PC) initiation, progression, and metastasis. Among mucins, MUC4 has been reported to inhibit lymphokine-activated cell killing and induce the apoptosis of cytotoxic T-cells. Counterintuitively, MUC4 expression is upregulated by multiple T-cell-secreted cytokines, such as IFN-γ, IL-17, and stroma-secreted factors like retinoic acid. Previously, we have identified that nuclear receptor coactivator 3 (NCOA3) regulates the MUC1 and MUC4 expression by increasing chromatin accessibility and maintaining protein stability. However, the comprehensive crosstalk mediated by MUC4 in cancer cells and T-cells and how its upstream regulator, NCOA3, modifies the cancer cell-intrinsic behavior is still elusive. Here, we show that the activated T-cell conditioned media (CM), containing IL-2, IFN-γ, and TNF-α, induced MUC4 expression in PC cells, and the subsequent RNA-seq analysis also showed the activation of cell death-associated genes. Further investigations revealed that CRISPR-Cas9-mediated knockout (KO) of MUC4 increased PC cell death upon activated T-cell CM treatment. Concurrently, the subcutaneous implantation of MUC4 proficient and deficient cells on the contralateral flanks of C57BL/6 and athymic mice demonstrated a significant reduction in the tumor weight with increased infiltration of both CD3+ and CD8+ cells (C57BL/6) in MUC4 KO tumors. Validating these findings, PanCancer Immune cell type profiling of tumors from KrasG12D/+, Tp53R127H/+, Pdx-1-Cre (KPC), and after MUC4 depletion (PKMC) showed an increased infiltration of cytotoxic cells in KPCM tumors. Mechanistically, MUC4 glycosylation modifies the interactions between MUC4 and perforin, protecting cancer cells from perforin-mediated PC cell death. Next, we generated the NCOA3 conditional KO murine model and bred it with a KPC autochthonous murine model. NCOA3 depletion reduced PC cell growth both in vitro and in vivo. The RNA-seq. analysis from KPNC/KPC tumor tissue and cell lines derived from the murine models showed upregulation of UDP-glucose dehydrogenase (UGDH), which may lead to the accumulation of UDP-glucuronic acid (UDP-GA), a toxic metabolite, and glycosaminoglycans (GAGs) synthesis, suggesting that GAGs synthesis may be an alternate route to compensate for the loss of mucins, downregulated after NCOA3 depletion. In summary, MUC4 expression protects the PC cells from T-cell mediate apoptosis through glycosylation-dependent inhibition of perforin function. Further, NCOA3 depletion leads to UDP-GA accumulation and GAGs synthesis, the impact of which will be investigated in future studies.

Comments

2024 Copyright, the authors

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