Graduation Year

2025

Document Type

Open Access Senior Thesis

Degree Name

Bachelor of Arts

Department

Biology

Reader 1

Dr. Jeffrey N. Myers

Reader 2

Dr. Aditi Vyas

Abstract

Background: Gain-of-function TP53 mutations have previously been shown to induce chromosomal instability (CIN) in cancer cells, triggering the cGAS-STING innate immune response and subsequently activating the non-canonical NF-κB (NC-NF-κB) signaling pathway. This signaling cascade promotes cancer cell metastasis and an immunosuppressive tumor microenvironment (TME), significantly impacting tumor development and progression. However, the precise downstream mechanisms by which the mutp53-CIN-cGAS-STING-NC-NF-κB signaling pathway facilitates tumor development and progression remain poorly understood. Here, our objective was to identify direct downstream targets of mutp53-CIN-cGAS-STING-induced NC-NF-κB signaling that potentially impact the TME to promote the development and progression of oral squamous cell carcinoma.

Methods: RNA-seq results from human oral cancer UMSCC-1 stable cell lines expressing the R273H mutant p53, with or without RelB (a component of NC-NF-κB) knockdown, were analyzed. Potential direct transcriptional targets of NC-NF-κB were characterized and evaluated by data mining of chromatin immunoprecipitation sequencing (ChIP-seq) databases. Both western blot and RT-qPCR assays were used to validate target protein and mRNA expression in mouse cancer 4T1 and MOC2 cells, as well as in their isogenic RelB-knockout cells.

Results: Two potential target genes of interest, TMED2 and Sec13, involved in the endoplasmic reticulum (ER)-Golgi secretory pathway, were identified through Reactome/KEGG pathway analysis of the "gene signatures" modulated by mutp53 and NC-NF-κB, as well as through data mining of RelB ChIP-seq databases. RT-qPCR and western blot analyses revealed that when RelB is knocked out in NC-NF-κB-activated 4T1 and MOC2 cells, the mRNA and protein expression levels of both TMED2 and Sec13 increase. This strongly suggests that RelB transcriptionally suppresses TMED2 and Sec13 expression.

Conclusion: Our results indicate that within the mutp53-CIN-cGAS-STING-NC-NF-κB pathway, RelB potentially acts as a transcriptional repressor of TMED2 and Sec13 gene expression. Given that TMED2 and Sec13 are important molecules in the cellular secretory pathway and play a crucial role in the immunomodulation of the TME through mechanisms such as the ER stress response, cytokine secretion, and major histocompatibility complex class I (MHC I) suppression, our findings provide significant insight into how p53 mutations and CIN can lead to an immunosuppressive tumor microenvironment. This knowledge not only enhances our understanding of the cellular mechanisms of tumor cell immune evasion but also identifies potential molecular targets and strategies for the treatment of tumors with p53 mutations and CIN.

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