Myoferlin is a novel exosomal protein and functional regulator of cancer-derived exosomes

Abstract

Exosomes are communication mediators participating in the intercellular exchange of proteins, metabolites and nucleic acids. Recent studies have demonstrated that exosomes are characterized by a unique proteomic composition that is distinct from the cellular one. The mechanisms responsible for determining the proteome content of the exosomes remain however obscure. In the current study we employ ultrastructural approach to validate a novel exosomal protein myoferlin. This is a multiple C2-domain containing protein, known for its conserved physiological function in endocytosis and vesicle fusion biology. Emerging studies demonstrate that myoferlin is frequently overexpressed in cancer, where it promotes cancer cell migration and invasion. Our data expand these findings by showing that myoferlin is a general component of cancer cell derived exosomes from different breast and pancreatic cancer cell lines. Using proteomic analysis, we demonstrate for the first time that myoferlin depletion in cancer cells leads to a significantly modulated exosomal protein load. Such myoferlin-depleted exosomes were also functionally deficient as shown by their reduced capacity to transfer nucleic acids to human endothelial cells (HUVEC). Beyond this, myoferlin-depleted cancer exosomes also had a significantly reduced ability to induce migration and proliferation of HUVEC. The present study highlights myoferlin as a new functional player in exosome biology, calling for novel strategies to target this emerging oncogene in human cancer.

Keywords: angiogenesis; endothelial cells; proteomics; vesicle trafficking.

Figure 1. Myoferlin is expressed in exosomes from breast and pancreas cancer cell lines

A. Western blot analysis of myoferlin expression in exosomes isolated from multiple breast and pancreas cancer cell lines. CD9 and FLOT1 were used as positive controls for exosomes whereas the GRP78 (ER) and COX1 (mitochondria) were employed as controls to exclude presence of contaminating organelles. B. Immunogold electron microscopy of exosome preparations from MDA-MB-231 cells stained for myoferlin. (A-B) Representative images of three independent experiments are shown.

Figure 2. Loss of myoferlin decreases exosome size but not quantity

A. Western blot validation of myoferlin depletion in cells (left) and exosome preparation (right) purified from breast (MDA-MB-231) and pancreas (BxPC-3) cancer cell supernatants. HSC70 (cell extracts) and CD9 (isolated exosomes) were used as loading controls. Representative images of three independent experiments are shown. B. Exosome quantity was assessed by protein quantification of the total amount of isolated exosomes relative to the number of cells. C. Measurement of average exosome size using DLS. (B-C) Data are averages of three independent experiments; error bars indicate standard error of means.

Figure 3. Myoferlin depletion induces major change in cancer exosome proteome content

A. Exosomal localization of proteins identified in MDA-MB-231 (left) and BxPC-3 (right) proteomic analysis of isolated exosomes, according to the STRING database (http://string-db.org/). B. Venn diagram showing the differential repartition of modulated proteins in MDA-MB-231 and BxPC-3 isolated exosomes. (A-B) The values are averages of two independent experiments. C. Enrichment pathway analysis of down-regulated proteins in absence of myoferlin. D. STRING Protein interaction network of proteins commonly down-regulated in breast and pancreatic myoferlin-deficient exosomes E. Western blot validation of vesicular makers modulated in response to myoferlin silencing (CD9 was used as control). Red Ponceau is used as a loading control. Shown are representative images of three independent experiments.

Figure 4. Myoferlin-deficient exosomes are impaired to enter target cells

A-B. Immunofluorescence of HUVEC incubated with PKH67 labelled cancer-derived exosomes. Shown are representative images of three independent experiments. Quantification was conducted on 10 randomly selected fields, error bars indicate standard error of means. C. Relative levels of the murine miRNA miR-298, measured by qRT-PCR, in HUVEC incubated with cancer-derived exosomes pre-loaded with miR-298. Ubiquitously expressed RNU44 was used for normalization. Data are averages of three independent experiments; error bars indicate standard error of means. D. Confocal analysis of cancer-derived exosomes localization in HUVEC. CD31 was used for membrane staining while nuclei were labelled with DAPI. The absence of cell surface adsorption was excluded using the Pearson correlation analysis.

Figure 5. Myoferlin-depleted cancer-derived exosomes decrease proliferation and migration of endothelial cells

A. Proliferation and B. migration of endothelial cells incubated with exosomes isolated from myoferlin-deficient or control cancer cells. (A-B) Data are averages of three independent experiments; error bars indicate standard error of means.