Abstract

Exosomal Carboxypeptidase E Confers and CPE-shRNA Loaded Exosomes Inhibit Tumorigenesis

Y. Peng Loh, Chief and Senior Investigator, Section on Cellular Neurobiology, Eunice Kennedy Shriver National Institute of Child health and Human Development, National Institutes of Health (NIH)

Carboxypeptidase E (CPE) has many extracellular non-enzymatic functions including its ability to promote cancer growth and metastasis. Exosomes carry biomolecules (proteins, DNA, mRNA and miRNA) unique to their cell of origin and deliver them to recipient target cells thereby mediating cell-cell communication. We have explored the ability of exosomes from high metastatic HCCH (liver cancer) cells to confer growth and metastatic properties to HCCL (low metastatic). Exosomes were isolated from the supernatant culture media of cancer cells and a correlation was found between elevated CPE mRNA levels in exosomes from high vs low metastatic cell lines across various cancer types. Content analysis of exosomes derived from highly metastatic HCC97H cells revealed CPE-WT mRNA and protein. We showed that exosomes released from HCC97H cells were able to enhance invasion of HCC cells with poor metastatic ability (HCC97L) in Matrigel invasion assay and proliferation in MTT assay. However, when CPE expression was suppressed in the HCC97H cells before exosome isolation, the exosomes had no effect on proliferation and invasion. These data demonstrate the ability of exosomes to confer metastasis in cancer cells and the role of exosomal CPE in driving the process. We then utilized the inherent property of exosomes to act as efficient delivery tools to carry a therapeutic agent such as shRNA. Previously it was shown that down-regulation of CPE expression by shRNA can reverse tumor growth and metastasis in an HCC mouse model. We therefore loaded CPE-shRNA into exosomes by infecting HEK293 (Human Embryonic Kidney) cells with adenovirus carrying CPE shRNA-GFP. These modified exosomes were harvested from the cell medium, purified and then used to transfer CPE-shRNA to HCC97H cells. The exosomes taken up by the recipient cells resulted in significant reduction of CPE mRNA levels and decrease in colony formation of these cells. Thus, these studies demonstrate the ability of exosomal CPE to enhance invasion in low metastatic HCC cells and the potential to use shRNA loaded exosomes to target CPE as a therapeutic strategy to treat liver and other cancers.