Abstract 1076: Identifying biomarkers of metastasis through biosynthetic tagging

Introduction: Short, noncoding RNAs called microRNAs (miRNAs) have demonstrated huge potential as diagnostic and therapeutic agents. One hurdle to their application as biomarkers is that they are not labeled by cell of origin, making it impossible to distinguish host- from disease-specific species. The current proposal is a novel combination of chemical and genetic tools to identify the tumor-specific miRNA secretome. We leverage murine models of the extremes of neuroblastoma and lung adenocarcinoma metastatic behavior, a protozoan enzyme that can selectively incorporate a modified base into RNA, and expression profiling by next-generation sequencing to identify miRNA markers of metastasis. Methods: To biosynthetically label RNA in vivo by cell of origin (tumor versus host), we exploit a nucleotide salvage enzyme used by the protozoan parasite Toxoplasma gondii to yield uridine monophosphate (UMP). When the uracil analog 4-thiouracil (4TU) is provided as a substrate, UPRT couples ribose-5-phosphate to the 4TU N1 nitrogen, resulting in 4-thiouridine-monophosphate (4TUMP). 4TUMP is then incorporated into RNA, providing a molecular handle by which RNA species from UPRT-expressing cells can be manipulated. The thiol-labeled (“tagged”) RNA can be separated from untagged RNA by thiol-specific biotinylation followed by affinity purification on streptavidin-coated magnetic beads. Importantly: (1) thiol-containing nucleotides do not naturally occur in eukaryotic mRNAs, (2) multicellular eukaryotes lack UPRT activity, and (3) expression of UPRT has little effect on cellular physiology. Targeted expression of UPRT therefore allows cell-specific tagging of miRNAs with 4TU. This approach is highly efficient; nearly all human and murine miRNAs are taggable. Results: Cell lines derived from metastatic and nonmetastatic models of adult and pediatric cancers were stably transfected to express the UPRT enzyme and injected into syngeneic mice to induce tumor formation. The mice were fed a diet supplemented with 4TU. Since the tumor cells expressed UPRT, they synthesized TU-tagged miRNAs, while host cells, which did not express UPRT, did not. miRNAs released by the tumor into the blood or adjacent tissue were TU-tagged. Separation of the miRNAs present in tumor, normal adjacent tissue and serum into labeled and unlabeled fractions followed by small RNA sequencing enabled the controlled comparison of the profile of miRNAs released into the blood specifically by the tumor relative to the profile of miRNAs from the host. Conclusions: Preliminary experiments have allowed us to identify miRNAs that are biomarkers of cancer metastasis that we are investigating as therapeutic targets in the same mouse models, extending to models based on human cell lines, evaluating in patient populations, and developing as clinical tools. Citation Format: Yiqiang Zhang, Xiaojie Yu, Shaimar Gonzalez, Sarah Almasri, Leonidas Bleris, Alexander Pertsemlidis. Identifying biomarkers of metastasis through biosynthetic tagging [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2018; 2018 Apr 14-18; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2018;78(13 Suppl):Abstract nr 1076.