Restoration of full‐length APC protein in SW 480 colon cancer cells induces exosome‐mediated secretion of DKK ‐4

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 W nt signaling. Colon carcinoma SW 480 cells restored with stable expression of wild‐type APC ( SW 480 APC cells) exhibit attenuated W nt signaling, and reduced tumorigenicity, including increased cell adhesion. We performed a comparative proteomic analysis of exosomes isolated from SW 480 and SW 480 APC cells to examine the effects of restored APC on exosome protein expression. A salient finding of our study was the unique expression of the W nt antagonist Dickkopf‐related protein 4 ( DKK 4) in SW 480 APC , but not parental SW 480 cell‐derived exosomes. Upregulation of DKK 4 in SW 480 APC cells was confirmed by semiquantitative RT ‐ PCR , immunoblotting, and immunogold electron microscopy. Analysis of the DKK 4 gene promoter by methylation‐specific PCR revealed reduced methylation in SW 480 APC cells, while RT ‐ PCR demonstrated the downregulation of DNMT ‐3a, compared to the parental cell line. Our discovery of exosome‐mediated secretion of DKK 4 opens up the possibility that exosomal DKK 4 may be a mechanism used by epithelial colon cells to regulate W nt signaling which is lost during CRC progression.