Background: The APC tumor suppressor is mutated or downregulated in many tumor types, and is prominently\r\nlocalized to punctate clusters at protrusion tips in migratory cells, such as in astrocytes where it has been\r\nimplicated in directed cell motility. Although APC loss is considered an initiating event in colorectal cancer, for\r\nexample, it is less clear what role APC plays in tumor cell motility and whether loss of APC might be an important\r\npromoter of tumor progression in addition to initiation.\r\nMethods: The localization of APC and �Ÿ-catenin was analyzed in multiple cell lines, including non-transformed\r\nepithelial lines treated with a proteasome inhibitor or TGF�Ÿ to induce an epithelial-to-mesenchymal transition\r\n(EMT), as well as several breast cancer lines, by immunofluorescence. APC expression was knocked down in 4T07\r\nmammary tumor cells using lentiviral-mediated delivery of APC-specific short-hairpin (sh) RNAs, and assessed using\r\nquantitative (q) reverse-transcriptase (RT)-PCR and western blotting. Tumor cell motility was analyzed by performing\r\nwound-filling assays, and morphology via immunofluorescence (IF) and phase-contrast microscopy. Additionally,\r\nproliferation was measured using BrdU incorporation, and TCF reporter assays were performed to determine\r\n�Ÿ-catenin/TCF-mediated transcriptional activity.\r\nResults: APC/�Ÿ-catenin-rich complexes were observed at protrusion ends of migratory epithelial cells treated with a\r\nproteasome inhibitor or when EMT has been induced and in tumor cells with a mesenchymal, spindle-like\r\nmorphology. 4T07 tumor cells with reduced APC levels were significantly less motile and had a more rounded\r\nmorphology; yet, they did not differ significantly in proliferation or �Ÿ-catenin/TCF transcriptional activity.\r\nFurthermore, we found that APC/�Ÿ-catenin-rich complexes at protrusion ends were dependent upon an intact\r\nmicrotubule cytoskeleton.\r\nConclusions: These findings indicate that membrane protrusions with APC/�Ÿ-catenin-containing puncta control the\r\nmigratory potential and mesenchymal morphology of mammary tumor cells and suggest that APC loss during later\r\nstages of tumor progression might impact tumor cell dissemination or colonization.
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