title: Restoration of full-length APC protein in SW480 colon cancer cells induces exosome-mediated secretion of DKK-4.
abstract: Mutations in the adenomatous polyposis coli (APC) tumor suppressor gene are common in both inherited and sporadic forms of colorectal cancer (CRC), and are associated with dysregulated Wnt signaling. Colon carcinoma SW480 cells restored with stable expression of wild-type APC (SW480APC cells) exhibit attenuated Wnt signaling, and reduced tumorigenicity, including increased cell adhesion. We performed a comparative proteomic analysis of exosomes isolated from SW480 and SW480APC cells to examine the effects of restored APC on exosome protein expression. A salient finding of our study was the unique expression of the Wnt antagonist Dickkopf-related protein 4 (DKK4) in SW480APC, but not parental SW480 cell-derived exosomes. Upregulation of DKK4 in SW480APC cells was confirmed by semiquantitative RT-PCR, immunoblotting, and immunogold electron microscopy. Analysis of the DKK4 gene promoter by methylation-specific PCR revealed reduced methylation in SW480APC cells, while RT-PCR demonstrated the downregulation of DNMT-3a, compared to the parental cell line. Our discovery of exosome-mediated secretion of DKK4 opens up the possibility that exosomal DKK4 may be a mechanism used by epithelial colon cells to regulate Wnt signaling which is lost during CRC progression.
authors: Joint Proteomics Research Laboratory, Ludwig Institute for Cancer Research and The Walter and Eliza Hall Institute of Medical Research, Parkville, Victoria, Australia.
authors: Justin W E
authors: JW
authors: Lim
authors: Rommel A
authors: RA
authors: Mathias
authors: Eugene A
authors: EA
authors: Kapp
authors: Meredith J
authors: MJ
authors: Layton
authors: Maree C
authors: MC
authors: Faux
authors: Antony W
authors: AW
authors: Burgess
authors: Hong
authors: H
authors: Ji
authors: Richard J
authors: RJ
authors: Simpson
mesh_terms: D025601:Adenomatous Polyposis Coli Protein / Q000235:genetics / Q000378:metabolism*; D045744:Cell Line, Tumor; D003110:Colonic Neoplasms / Q000378:metabolism*; D004248:DNA (Cytosine-5-)-Methyltransferases / Q000235:genetics / Q000378:metabolism; D000090205:DNA Methyltransferase 3A; D055354:Exosomes / Q000737:chemistry / Q000378:metabolism*; D006801:Humans; D036341:Intercellular Signaling Peptides and Proteins / Q000235:genetics / Q000378:metabolism*; D008745:Methylation; D016133:Polymerase Chain Reaction; D011401:Promoter Regions, Genetic; D020543:Proteome / Q000032:analysis / Q000235:genetics / Q000378:metabolism; D040901:Proteomics; D015854:Up-Regulation; D060449:Wnt Signaling Pathway
chemical_list: D025601:Adenomatous Polyposis Coli Protein; C442333:DKK4 protein, human; C000717967:DNMT3A protein, human; D036341:Intercellular Signaling Peptides and Proteins; D020543:Proteome; D004248:DNA (Cytosine-5-)-Methyltransferases; D000090205:DNA Methyltransferase 3A