Abstract
BackgroundExtracellular vesicles (EVs) are secreted from many cells, carrying cargoes including proteins and nucleic acids. Research has shown that EVs play a role in a variety of biological processes including immunity, bone formation and recently they have been implicated in promotion of a metastatic phenotype.MethodsEVs were isolated from HCT116 colon cancer cells, 1459 non-malignant colon fibroblast cells, and tumor and normal colon tissue from a patient sample. Co-cultures were performed with 1459 cells and malignant vesicles, as well as HCT116 cells and non-malignant vesicles. Malignant phenotype was measured using soft agar colony formation assay. Co-cultures were also analyzed for protein levels using mass spectrometry. The importance of 14-3-3 zeta/delta in transfer of malignant phenotype was explored using siRNA. Additionally, luciferase reporter assay was used to measure the transcriptional activity of NF-κB.ResultsThis study demonstrates the ability of EVs derived from malignant colon cancer cell line and malignant patient tissue to induce the malignant phenotype in non-malignant colon cells. Similarly, EVs derived from non-malignant colon cell lines and normal patient tissue reversed the malignant phenotype of HCT116 cells. Cells expressing an EV-induced malignant phenotype showed increased transcriptional activity of NF-κB which was inhibited by the NF--κB inhibitor, BAY117082. We also demonstrate that knock down of 14-3-3 zeta/delta reduced anchorage-independent growth of HCT116 cells and 1459 cells co-cultured with HCT derived EVs.ConclusionsEvidence of EV-mediated induction of malignant phenotype, and reversal of malignant phenotype, provides rational basis for further study of the role of EVs in tumorigenesis. Identification of 14-3-3 zeta/delta as up-regulated in malignancy suggests its potential as a putative drug target for the treatment of colorectal cancer.
Highlights
Extracellular vesicles (EVs) are secreted from many cells, carrying cargoes including proteins and nucleic acids
We found an increased expression of the proteins 14-3-3 zeta/delta, prohibitin, phosphorylated Raf kinase Inhibitor Protein and Signal Transducer and Activator of Transcription1 (STAT1) in 1459 cells acquiring malignant phenotype after co-culture with malignant EVs
Our study shows that 14-3-3 zeta/delta is important in the EV mediated induction of malignant phenotype, suggesting it’s role as a potential target for therapeutic interventions
Summary
Extracellular vesicles (EVs) are secreted from many cells, carrying cargoes including proteins and nucleic acids. Even with medical advances, the average 5-year survival rate of patients is a moderate 64.9 % [3]. This statistic fails to emphasize the impact of disease presentation at diagnosis on the outcome. Anti-EGFR treatments, including panitumumab and cetuximab, have been shown to improve outcome for patients with tumors carrying wild-type KRAS gene. The role of EVs is especially important in regards to tumor progression, because cancer cells have been shown to secrete EVs at an elevated rate. Further understanding of the mechanisms underlying increased EV secretion in cancer, and the mechanisms behind phenotypic transfer, are crucial to the potential identification of many novel therapeutic targets
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