(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.

Public Health Relevance

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.

National Institute of Health (NIH)
National Cancer Institute (NCI)
Research Program--Cooperative Agreements (U19)
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Special Emphasis Panel (ZRG1-OBT-S (50))
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Vanderbilt University Medical Center
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Zhang, Qin; Jeppesen, Dennis K; Higginbotham, James N et al. (2018) Mutant KRAS Exosomes Alter the Metabolic State of Recipient Colonic Epithelial Cells. Cell Mol Gastroenterol Hepatol 5:627-629.e6
Hinger, Scott A; Cha, Diana J; Franklin, Jeffrey L et al. (2018) Diverse Long RNAs Are Differentially Sorted into Extracellular Vesicles Secreted by Colorectal Cancer Cells. Cell Rep 25:715-725.e4
Sung, Bong Hwan; Weaver, Alissa M (2018) Directed migration: Cells navigate by extracellular vesicles. J Cell Biol 217:2613-2614
Maas, Sybren L N; Breakefield, Xandra O; Weaver, Alissa M (2017) Extracellular Vesicles: Unique Intercellular Delivery Vehicles. Trends Cell Biol 27:172-188
McKenzie, Andrew J; Hoshino, Daisuke; Hong, Nan Hyung et al. (2016) KRAS-MEK Signaling Controls Ago2 Sorting into Exosomes. Cell Rep 15:978-987
Higginbotham, James N; Zhang, Qin; Jeppesen, Dennis K et al. (2016) Identification and characterization of EGF receptor in individual exosomes by fluorescence-activated vesicle sorting. J Extracell Vesicles 5:29254
Dou, Yongchao; Cha, Diana J; Franklin, Jeffrey L et al. (2016) Circular RNAs are down-regulated in KRAS mutant colon cancer cells and can be transferred to exosomes. Sci Rep 6:37982
Cha, Diana J; Franklin, Jeffrey L; Dou, Yongchao et al. (2015) KRAS-dependent sorting of miRNA to exosomes. Elife 4:e07197
Laurent, Louise C; Abdel-Mageed, Asim B; Adelson, P David et al. (2015) Meeting report: discussions and preliminary findings on extracellular RNA measurement methods from laboratories in the NIH Extracellular RNA Communication Consortium. J Extracell Vesicles 4:26533
Sung, Bong Hwan; Ketova, Tatiana; Hoshino, Daisuke et al. (2015) Directional cell movement through tissues is controlled by exosome secretion. Nat Commun 6:7164

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