Abstract 2786: Investigation on the role of tumor suppressive microRNAs in angiosarcoma

Abstract Angiosarcoma (AS) is an aggressive tumor resulting in a very poor prognosis for patients. MicroRNAs (miRNAs) regulate gene expression and play important roles in a variety of diseases including cancer. Our previous studies demonstrate miRNA loss leads to AS in mice. To further understand the role of miRNAs that function as critical tumor suppressors in AS, we performed a miRNA-focused CRIPSR-Cas9 screen. The gRNA library was transduced into a human AS cell line expressing doxycycline (dox)-inducible Cas9. After passaging the cells for 28 days, sgRNA amplicon sequencing was performed to determine the change in the frequency of gRNAs. We anticipated that the loss of function of tumor suppressing miRNAs would lead to gRNA enrichment in the cells. Indeed, three miRNAs were identified as hits with significant enrichment of multiple gRNAs, including miR-200b, miR-181b, and miR-410. We hypothesize these miRNAs act as tumor suppressors and reduce proliferation and cell viability in AS. Based on our RNA-seq data, all three miRNAs are expressed in normal endothelial cells. Additionally, miR-410, miR-181b and a miR-200 family member are significantly downregulated in a human AS cell line and miR-410 is frequently deleted in AS patient samples. We are currently conducting functional validation studies by overexpressing these miRNAs in a panel of AS cell lines by lentiviral based expressing the pre-miRNAs. In preliminary results, the overexpression of miR-410 consistently inhibits cell proliferation and colony formation ability in both human and mouse AS cell lines. Additionally, to gain mechanistic insights for these miRNAs, a novel technique called AgoTRIBE was used to identify important mRNA targets regulated by miRNAs. In AgoTRIBE, the miRNA effector Ago2 is fused to ADAR's RNA editing domain, directing ADAR activity to miRNA natural targets. The ADAR editing events on miRNA targets can be detected by RNA seq. We used a dox-inducible lentiviral vector expressing human ADAR (hADAR) or hADAR-Ago in a mouse AS cell line. In preliminary data, we validated the protein expression of ADAR and ADAR-Ago upon dox treatment and the cellular localization of these complexes in cytoplasm. We are working on validating the function of AgoTRIBE by RNA seq and utilize this tool to identify targets regulated by our screen hits. This project represents the first global screen on miRNAs in AS. Functional validation of the hits from the screen in combination with the mechanistic insights from AgoTRIBE can provide fundamental evidence for the role of tumor suppressive miRNAs in AS. Citation Format: Bozhi Liu, Jillian Stachon, Annaleigh Powell-Benton, Sagar Utturkar, Nimod Janson, Anthony Murphy, Nadia Lanman, Jason Hanna. Investigation on the role of tumor suppressive microRNAs in angiosarcoma [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2025; Part 1 (Regular Abstracts); 2025 Apr 25-30; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2025;85(8_Suppl_1):Abstract nr 2786.