ORCID
http://orcid.org/0000-0002-5098-4963
Date of Award
Spring 5-15-2023
Degree Name
Doctor of Philosophy (PhD)
Degree Type
Dissertation
Abstract
Innate immunity is a fundamental defense mechanism against non-self. Similar to its role in pathogenic infections, innate immunity is capable of eliminating cancerous cells by activating cytotoxic inflammatory signaling. Adenosine deaminase acting on RNA 1 (ADAR1) is a key regulator of cell-intrinsic innate immunity. ADAR1 converts adenosines to inosines on double-stranded RNAs (dsRNAs), altering dsRNA structure and its interactive properties. This suppresses cytoplasmic sensing of immunogenic dsRNA and activation of the type I interferon (IFN) signaling pathway. Frequently, suppressing endogenous dsRNA from triggering cytotoxic inflammatory signaling is essential for cancer cell survival. This is known as ADAR1 dependency, which has been proposed as a potential therapeutic target in cancer. In this dissertation, I aimed to analyze cellular mechanisms mediating ADAR1 dependency in breast cancer to identify novel molecular targets.As ADAR1 dependency is associated with elevated type I IFN signature, I investigated a potential interplay between ADAR1 and hypoxia-inducible factor 1 (HIF1) in the context of cell-intrinsic type I IFN signaling activated by co-depletion of tumor suppressors p53 and ARF. In the mouse embryonic fibroblast model and human breast cancer cell lines, knockdown of ADAR1 decreased the normoxic expression of HIF1 α subunit (HIF1α). Knockdown of HIF1α in breast cancer cell lines phenocopied ADAR1 knockdown, characterized by reduced proliferation and translational shutdown. However, unlike the ADAR1 dependency mechanism, the translational shutdown upon HIF1α knockdown was not mediated by protein kinase R (PKR). This suggests that HIF1α is not a downstream mediator of ADAR1 dependency. Therefore, in tumors with ADAR1-driven expression of HIF1α, targeting ADAR1 can potentially activate multiple stress response kinases to trigger translational shutdown. Additionally, tumor suppressor von Hippel-Lindau (VHL) was downregulated by ADAR1 post-translationally although it was not coupled with HIF1α expression. Next, I sought to determine the factors that establish ADAR1 dependency or ADAR1 independency. Using proximity labeling, putative ADAR1-interacting proteins were identified in breast cancer cell lines. Of the proteins identified, DExH box helicase 9 (DHX9) was of particular interest. Knockdown of DHX9 in ADAR1-dependent cell lines caused cell death and activation of PKR. In ADAR1-independent cell lines, combined knockdown of DHX9 and ADAR1, but neither alone, caused activation of multiple dsRNA sensing pathways leading to a viral mimicry phenotype. Furthermore, the dsRNA-binding domain of DHX9 was sufficient to prevent PKR activation upon combined knockdown. Taken together, DHX9 plays an important role in suppression of dsRNA sensing, and its functional coordination with ADAR1 distinguishes ADAR1-dependent from -independent cells. Another RNA helicase identified by proximity labeling, DDX54, was examined for its potential role in ADAR1 dependency. However, DDX54 did not suppress ADAR1 dependency. Unexpectedly, a short hairpin RNA targeting DDX54 was shown to directly activate PKR. Other dsRNA-sensing pathways were not activated, suggesting that the shRNA acts as a specific agonist of PKR rather than a broad immunostimulant. Collectively, this dissertation highlights pathway analyses surrounding ADAR1 function and identifies molecular determinants of ADAR1 dependency. It excludes HIF1α as a mechanistic component of ADAR1-driven cancer cells and proposes DHX9 as a novel target in ADAR1-associated viral mimicry. The findings establish dysregulation of RNA metabolism as a major driver of cancer cell-intrinsic inflammatory signaling. In the broader context of molecular cell biology, this work demonstrates the use of genetic and interactome-based approaches to uncover functional interplays between cellular pathways.
Language
English (en)
Chair and Committee
Jason D. Weber
Committee Members
Jennifer M. Heemstra, Jason C. Mills, Joshua B. Rubin, Jieya Shao,
Recommended Citation
Ryu, Sua, "Identifying Molecular Targets of ADAR1 Dependency in Breast Cancer" (2023). Arts & Sciences Electronic Theses and Dissertations. 2910.
https://openscholarship.wustl.edu/art_sci_etds/2910