Abstract 5067: Identifying exosomal mRNA, microRNA and protein signatures in melanoma cells

Abstract

Abstract Background: Exosomes are tiny membrane bound vesicles released by all cell types, with increased secretion associated with disease states such as cancer. Current research reveals an active role of the exosomal mRNA, miRNA, and protein contents in malignant progression. The emission of exosomes offers a unique opportunity to access, in a non-invasive manner, the wealth of biological information related to the melanoma cells that produce these exosomes. mRNA, miRNA, and protein signatures in exosomes represent an exciting potential biomarker. Materials and Methods: Exosome samples were isolated from two human melanoma cell lines (A375 and SK-MEL-28), with one normal melanocyte cell line (HEMa-LP) serving as a control. The exosome purity was confirmed using electronic microscope. The total RNA content was isolated from both exosomal and cellular samples. Two completely independent experiments were conducted in order to prepare duplicate samples for all microarray experiments on both exosomal and cellular RNA. Microarray experiments were performed using Affymetrix GeneChip Human HG-U133 Plus 2.0 array and miRNA array chips. For proteomics studies, proteins from the cells and the exosomes were extracted and analyzed by 2-D DIGE (two-dimensional difference in gel electrophoresis). Protein spots were chosen based on their differential expression and identified by mass spectrometry. Results: miRNA and mRNA array revealed distinctive exosomal expression signatures, which differed significantly from their corresponding cellular samples. Significant differences were also observed between exosomes from normal and melanoma cell lines. A subset of nine miRNAs showing significant variation was chosen, many of which have been shown to be involved in cancer or melanoma progression. Of these, seven were confirmed using real time PCR (let-7i, miR-15b, miR-30d, miR-106b, miR-181a, miR-182, and miR-221). Nine mRNAs were chosen based on these results, with consideration of known cancer and melanoma gene associations and links to the identified miRNA subset. Of these nine mRNAs, four were confirmed using real time PCR (CCND1, CTHRC1, EPS8, and NCOA3). Proteomic analysis showed that the protein profiles of normal melanocyte derived exosomes was strikingly different from those of the melanoma cell derived exosomes. We have identified some of the exosomal proteins associated with melanoma progression, such as syntenin-1, annexin A2, proteoglycan, and hyaluronan. Conclusion: To our knowledge, this is the first in-depth screening of the whole genome/miRNome/proteome in melanoma exosomes. These results provide a starting point for the identification of mRNA, miRNA, and protein signatures of melanoma-derived exosomes with great potential for translation into clinical application as non-invasive diagnostic and prognostic biomarkers. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 102nd Annual Meeting of the American Association for Cancer Research; 2011 Apr 2-6; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2011;71(8 Suppl):Abstract nr 5067. doi:10.1158/1538-7445.AM2011-5067