Abstract
Background: Exosomes from cancer cells promote tumor growth and metastasis through intercellular communication. However, the exosomal bioactive molecules involved and the mechanism of action remain elusive. Carboxypeptidase E (CPE) is known to drive tumor progression in different cancers, including hepatocellular carcinoma (HCC), which is associated with high mortality rate. Here, we investigated if CPE is present within cancer cell exosomes and contributes to the molecular pathogenesis of HCC and other cancers by regulating tumor growth and invasion.Methods: Exosomes isolated from the culture media of cancer cells or human serum were analyzed for CPE mRNA and protein using quantitative PCR/ RT-PCR and western blot respectively. Low-metastatic HCC97L cells were incubated with exosomes derived from high-metastatic HCC97H cells. In other experiments, HCC97H cells were incubated with CPE-shRNA loaded exosomes isolated from HEK293T cells. The recipient HCC cells were assessed for proliferation and invasion using MTT cell proliferation, colony formation and matrigel invasion assays. Results: CPE mRNA and protein were found to be packaged within exosomes released from cancer cells. We observed elevated CPE mRNA levels in secreted exosomes from high versus low-malignant cells, from various cancer types including HCC, breast cancer and glioblastoma. In a pilot study, significantly higher CPE copy numbers were found in serum exosomes from cancer patients compared to healthy donors, suggesting that exosomal CPE mRNA could be a potential diagnostic biomarker. Low-malignant HCC97L cells treated with exosomes derived from HCC97H cells, displayed enhanced proliferation and invasion; however exosomes from HCC97H cells pre-treated with CPE-shRNA failed to promote proliferation. When HEK293T exosomes loaded with CPE-shRNA, were incubated with HCC97H cells, expression of CPE, Cyclin D1, a cell-cycle regulatory protein and c-MYC, a proto-oncogene, were suppressed, resulting in diminished proliferation of HCC97H cells. Conclusions: Our results identified CPE as a bioactive molecule in exosomes driving the growth and invasion of low-malignant HCC cells, and showed that CPE-shRNA loaded exosomes can inhibit malignant tumor cell proliferation via Cyclin D1 and c-MYC suppression. Thus, CPE is a key player in exosome transmission of tumorigenesis, and exosome-based delivery of CPE-shRNA offers a potential treatment for tumor progression.