Abstract 6064: ADAR1-dependent RNA editing of GLI1 drives hepatocellular carcinoma stem cell self-renewal by initiating mitophagy
Authors: Luo Jie , Gong Lanqi , Yang Yuma , Huang Jiao , Fang Xiaona , Zhang Baifeng , Tang Ying , Liu Beilei , Liu Ming , Bai Lu , Lee Victor Ho-Fun , Guan Xin-Yuan 
Published: June 2022

Background: Hepatocellular carcinoma (HCC) is one of the most common and most difficult to cure malignancies worldwide. Identifying new effective therapeutic targets is of great significance for the management of HCC. Cumulative evidence suggests cancer stem cells (CSCs) are key drivers of tumor growth and heterogeneity. Post-transcriptional RNA editing of adenosine to inosine (A-to-I) catalyzed by ADAR1 dramatically alters cellular transcriptome in cancers. However, ADAR1 editase-dependent mechanisms governing hepatic CSC generation have not been elucidated. Therefore, we have systematically and comprehensively investigated ADAR1’s effect on CSC self-renewal in HCC.

Methods: An in vitro human hepatocyte differentiation model along with relevant transcriptomic data form GEO and TCGA were analyzed to characterize the oncofetal role of ADAR1. Using genetic approaches, RNA sequencing was performed to identify putative ADAR1-mediated recoding edited genes in HCC cell lines. Clinical implication of GLI1 editing was studied in a cohort of 88 HCC patients. GLI1 editing-mediated change in its promoter activity and protein stability was investigated by dual reporter assay, CHIP, Co-IP, and PLA assays. Functional difference in stemness properties, including abilities of self-renewal, differentiation, tumorigenesis, chemo-resistance and metastasis between wild-type and edited GLI1 (GLI1wt vs. GLI1R701G) and the exact mechanisms were also studied in cell models and mice.

Results: Lentiviral ADAR1 wild-type but not editing-defective ADAR1E912A mutant, editing at nt2101 of GLI1 transcript caused arginine-to-glycine substitution at the residue 701. Importantly, increased editing of GLI1 was implicated in the pathogenesis of HCC. Upon editing, C-terminal half of GLI1 harbored a lower susceptibility to the inhibition of SUFU, thus promoting its nuclear translocation and activation. Moreover, GLI1R701G appeared more stable than GLI1wt due to reduced formation of specific K63-GLI1 substrate and β-TrCP-GLI1 complex. Edited GLI1 was found to strongly enhance targeted activation of NANOG and SOX9, resulting in accumulation of hepatic CSCs population, hepatocarcinogenesis, sorafenib-resistance and metastasis. Additionally, GLI1 editing initiated mitophagy via PINK1/Parkin-dependent pathway. Impaired mitophagy effectively antagonized the functions of GLI1R701G on hepatic CSC self-renewal and overcome chemoresistance.

Conclusion: The critical advance of this study is that ADAR1 editase activity drives GLI1-dependent maintenance of the hepatic CSC population. Our discovery of a pivotal ADAR1-GLI1-PINK1 self-renewal axis provides the first mechanistic link between RNA-editing-driven malignant progression and mitochondrial homeostasis. Thus, ADAR1 represents a unique therapeutic vulnerability in liver CSCs with active mitophagy mediated by edited GLI1.

Citation Format: Jie Luo, Lanqi Gong, Yuma Yang, Jiao Huang, Xiaona Fang, Baifeng Zhang, Ying Tang, Beilei Liu, Ming Liu, Lu Bai, Victor Ho-Fun Lee, Xin-Yuan Guan. ADAR1-dependent RNA editing of GLI1 drives hepatocellular carcinoma stem cell self-renewal by initiating mitophagy [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2022; 2022 Apr 8-13. Philadelphia (PA): AACR; Cancer Res 2022;82(12_Suppl):Abstract nr 6064.