Abstract PO1-24-01: ADAR1 drives ribosomal biogenesis by regulating NOP14 and stabilizing FBL-NOP58 complex in breast cancer

Abstract

Abstract Background Breast cancer (BC) has been widely accepted as a highly heterogeneous disease. Transcriptional and translational dysregulation is commonly found in BC patients. Adenosine-to-inosine (A-to-I) editing on RNA is the most abundant RNA editing form in human, mediated mainly by ADARs. Though the role of ADAR1 in RNA editing and immune response has been deeply investigated, the change of the translational profile and the biology sequalae after base editing are still unclear. In this study, we uncovered a previously unknown role of ADAR1 in the regulation of ribosome biosynthesis and translational dysregulation in BC cells. Method RNA-seq was used for gene set enrichment analysis (GSEA) and A-to-I editing analysis. Transmission electron microscopy (TEM) was conducted to visualize the nucleolar architecture. Nuclear rRNA abundances of 18S and 28S rRNAs were monitored using Qsep100. Ribosomal fractions were isolated with a Beckman SW41Ti rotor and quantified on a 2100 Bioanalyzer. 3' Untranslated region (3′UTR) reporter construction and dual-luciferase reporter assay were used to validate the correlation. In-vitro transcription, RNA pull-down assay and sanger sequencing were used to directly confirm the direct interaction and A-to-I editing status. Cell proliferation, migration assay, apoptosis assay in vitro and xenograft mouse model in vivo were performed for phenotype study. Co-immunoprecipitation (Co-IP) was used to identify the interactive protein. Immunofluorescence (IF) assay was applied to indicate the co-localization. Immunohistochemistry (IHC) staining was applied to tissue microarrays and assess of expression. Survival analysis was made by Kaplan-Meier method. RNA polymerase I inhibitor CX-5461 was used for treatment. Result Through bioinformatic analysis with RNA-seq data from cell lines and clinical samples, we found the expression of ADAR1 was correlated with signatures of ribosomes. Consistently, BC cells with overexpression of ADAR1 showed larger nucleoli and contained more 18S and 28S rRNAs. To uncover the potential ribosomal genes involved in this process, we analyzed the transcriptional profile of 88 BC samples in combination with ADAR1 editing profiles identified by comparing ADAR1-null and control cells. Nucleolar Protein 14 (NOP14), a nucleolar ribosome biogenesis gene, was found to be hyper-edited. Phenotype experiments revealed that NOP14 promotes tumor growth and invasion in vitro and in vivo. The RNA and protein level of NOP14 is regulated by ADAR1 through A-to-I editase activity. Mechanically, ADAR1 binds directly with NOP14 transcripts in the 3’UTR region through Sanger sequencing and RNA-pull down. The binding region of NOP14 with ADAR1 was also a target of miR-939-3p, while the ADAR1 mediated-editing led to less binding of miR-939-3p with NOP14 and resulted in NOP14 up-regulation. Notably, NOP14 facilitates the assembly of the box C/D small nucleolar ribonucleoprotein (snoRNP) and stabilize the interaction of fibrillarin (FBL)-nucleolar Protein 58 (NOP58) with this complex. Loss of NOP14 impaired the nucleolar distribution of FBL and the abundance of ribosomes. CX-5461, an RNA polymerase I inhibitor, suppressed the overexpression of ribosomes in ADAR1 high cells and displayed a strong inhibition efficacy on tumor growth both in vitro and in vivo. Conclusion Taken together, these results shed light on the role of ADAR1 in ribosomal biogenesis in breast cancer. The promising results also provide potential targets in BCs with high ADAR1 by suppressing translational dysregulation. Citation Format: Liren Wangxu, Hengyu Ren, Zhibo Shao, Jiong Wu, Yayun Chi. ADAR1 drives ribosomal biogenesis by regulating NOP14 and stabilizing FBL-NOP58 complex in breast cancer [abstract]. In: Proceedings of the 2023 San Antonio Breast Cancer Symposium; 2023 Dec 5-9; San Antonio, TX. Philadelphia (PA): AACR; Cancer Res 2024;84(9 Suppl):Abstract nr PO1-24-01.