Abstract
Background: Exosomes promote tumor growth and metastasis through intercellular communication, although the mechanism remains elusive. Carboxypeptidase E (CPE) supports the progression of different cancers, including hepatocellular carcinoma (HCC). Here, we investigated whether CPE is the bioactive cargo within exosomes, and whether it contributes to tumorigenesis, using HCC cell lines as a cancer model. Methods: Exosomes were isolated from supernatant media of cancer cells, or human sera. mRNA and protein expression were analyzed using PCR and Western blot. 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. Cell proliferation and invasion were assessed using MTT, colony formation, and matrigel invasion assays. Results: Exosomes released from cancer cells contain CPE mRNA and protein. CPE mRNA levels are enriched in exosomes secreted from high- versus low-metastastic cells, across various cancer types. In a pilot study, significantly higher CPE copy numbers were found in serum exosomes from cancer patients compared to healthy subjects. 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, the expression of CPE, Cyclin D1, a cell-cycle regulatory protein and c-myc, a proto-oncogene, were suppressed, resulting in the diminished proliferation of HCC97H cells. Conclusions: We identified CPE as an exosomal bioactive molecule driving the growth and invasion of low-metastatic HCC cells. CPE-shRNA loaded exosomes can inhibit malignant tumor cell proliferation via Cyclin D1 and c-MYC suppression. Thus, CPE is a key player in the exosome transmission of tumorigenesis, and the exosome-based delivery of CPE-shRNA offers a potential treatment for tumor progression. Notably, measuring CPE transcript levels in serum exosomes from cancer patients could have potential liquid biopsy applications.
Highlights
Exosomes are nano-sized extracellular vesicles (30–140 nm in diameter), which facilitate critical intercellular communication by way of transferring bioactive molecules
To determine if Carboxypeptidase E (CPE) mRNA and its splice variant, CPE-∆N, are present within exosomes derived from three different cancer cell lines, we used a specific primer set ∆F/∆R which flanks the region of deletion in exon1 to differentiate CPE-∆N mRNA sequence, in addition to primers flanking the rest of the CPE mRNA
Our results indicate that exosomes derived from hepatocellular carcinoma (HCC), breast, and ovarian cancer cell lines contain CPE mRNA
Summary
Exosomes are nano-sized extracellular vesicles (30–140 nm in diameter), which facilitate critical intercellular communication by way of transferring bioactive molecules. Using orthotopic pancreatic cancer mouse models, it was demonstrated that exosomes carrying KRAS specific siRNA can suppress tumor growth, inhibit metastasis, and improve overall survival [13]. It remains to be determined what exosomal factors induce tumor growth and metastasis in HCC and other cancers, and whether exosomes can be exploited for targeted cancer therapy. When HEK293T exosomes loaded with CPE-shRNA were incubated with HCC97H cells, the expression of CPE, Cyclin D1, a cell-cycle regulatory protein and c-myc, a proto-oncogene, were suppressed, resulting in the diminished proliferation of HCC97H cells. Conclusions: We identified CPE as an exosomal bioactive molecule driving the growth and invasion of low-metastatic HCC cells. CPE-shRNA loaded exosomes can inhibit malignant tumor cell proliferation via Cyclin D1 and c-MYC suppression. Measuring CPE transcript levels in serum exosomes from cancer patients could have potential liquid biopsy applications
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