(Project Leader: Alissa Weaver) Exosomes are late endosomal-derived secretory vesicles that are frequently secreted by cancer cells and have been linked to tumor aggressiveness and metastasis. In addition to carrying proteins, exosomes are enriched in a variety of extracellular RNAs (eRNAs), including miRNAs and mRNAs. A major unanswered question is whether and how cellular signaling affects the export of eRNAs into exosomes and other described RNA transport vehicles. Recent advances in sequencing technologies combined with advanced intracellular microscopy and flow cytometry vesicle sorting approaches make it possible to perform in-depth analysis of the specificity and regulation of eRNA export. These studies should have a profound impact on our understanding of eRNA secretion mechanisms and tumor biology. Using an isogenically matched colon cancer cell line model of KRAS signaling, we recently found that an oncogenic mutant of KRAS selectively affects both the protein and RNA content of colon cancer cell exosomes. In addition, we find that mutant KRAS-expressing cells have differential import of a model miRNA into late endosomes. Finally, we find that KRAS mutant cell exosomes have increased content of lipid raft-associated proteins, suggesting differential control of exosome biogenesis and cholesterol trafficking. Based on these data, we hypothesize that KRAS signaling selectively controls the sorting of specific RNAs and proteins into exosomes. To test this hypothesis, we propose to comprehensively analyze the eRNA content from the isogenically matched KRAS colon cancer cell lines and determine whether specific sorting mechanisms exist to control eRNA export. We will also identify how K-RAS signaling controls two critical regulatory points in RNA secretion: subcellular localization of RNA machineries to late endosomes and exosome biogenesis pathways.
The discovery of extracellular RNA (eRNA) has raised the possibility that intercellular communication might be mediated by RNAs delivered in exosomes or lipoprotein particles. We seek to determine how eRNAs are packaged for export in these particles and how they signal between cells using a colon cancer model dependent on expression of oncogenic K-Ras. Transfer of eRNAs between cells in the tumor microenvironment could constitute a novel mechanism to promote tumorigenesis.