Abstract
BackgroundThe APC tumor suppressor is mutated or downregulated in many tumor types, and is prominently localized to punctate clusters at protrusion tips in migratory cells, such as in astrocytes where it has been implicated in directed cell motility. Although APC loss is considered an initiating event in colorectal cancer, for example, it is less clear what role APC plays in tumor cell motility and whether loss of APC might be an important promoter of tumor progression in addition to initiation.MethodsThe localization of APC and β-catenin was analyzed in multiple cell lines, including non-transformed epithelial lines treated with a proteasome inhibitor or TGFβ to induce an epithelial-to-mesenchymal transition (EMT), as well as several breast cancer lines, by immunofluorescence. APC expression was knocked down in 4T07 mammary tumor cells using lentiviral-mediated delivery of APC-specific short-hairpin (sh) RNAs, and assessed using quantitative (q) reverse-transcriptase (RT)-PCR and western blotting. Tumor cell motility was analyzed by performing wound-filling assays, and morphology via immunofluorescence (IF) and phase-contrast microscopy. Additionally, proliferation was measured using BrdU incorporation, and TCF reporter assays were performed to determine β-catenin/TCF-mediated transcriptional activity.ResultsAPC/β-catenin-rich complexes were observed at protrusion ends of migratory epithelial cells treated with a proteasome inhibitor or when EMT has been induced and in tumor cells with a mesenchymal, spindle-like morphology. 4T07 tumor cells with reduced APC levels were significantly less motile and had a more rounded morphology; yet, they did not differ significantly in proliferation or β-catenin/TCF transcriptional activity. Furthermore, we found that APC/β-catenin-rich complexes at protrusion ends were dependent upon an intact microtubule cytoskeleton.ConclusionsThese findings indicate that membrane protrusions with APC/β-catenin-containing puncta control the migratory potential and mesenchymal morphology of mammary tumor cells and suggest that APC loss during later stages of tumor progression might impact tumor cell dissemination or colonization.
Highlights
The adenomatous polyposis coli (APC) tumor suppressor is mutated or downregulated in many tumor types, and is prominently localized to punctate clusters at protrusion tips in migratory cells, such as in astrocytes where it has been implicated in directed cell motility
Cell culture EpH4 mouse mammary epithelial cells [16] and MadinDarby Canine Kidney (MDCK) were obtained from Karl Matlin ([17]; University of Chicago) and maintained at 37°C with 5% CO2 in DMEM supplemented with 5% fetal bovine serum (FBS), 1% penicillin/streptomycin, and 1:5000 Plasmocin (Invivogen). 4T1, 4T07, and 67NR mouse mammary tumor cells obtained from Fred Miller ([18]; Karmanos Cancer Institute), and Hs578T human breast cancer and SW480 colon cancer cells obtained from the American Type Culture Collection (ATCC) were maintained in DMEM with 10% FBS and pen/strep as above
To explore whether there were other circumstances in which β-catenin colocalized with APC at protrusion ends in epithelial cells, we analyzed the well-characterized model of TGFβinduced epithelial-to-mesenchymal transition (EMT) in MDCK cells
Summary
The APC tumor suppressor is mutated or downregulated in many tumor types, and is prominently localized to punctate clusters at protrusion tips in migratory cells, such as in astrocytes where it has been implicated in directed cell motility. Identification of APC mutations and loss of heterozygosity (LOH) in primary mammary tumorigenesis. These studies have indicated that the effects of Apc mutation in both normal mammary homeostasis and mammary tumor development are not solely dependent on Wnt pathway regulation, which is APC’s best-characterized molecular activity. Through de-repression of the canonical Wnt pathway, APC loss results in β-catenin stabilization, accumulation and nuclear translocation where it associates with TCF/LEF transcription factors to regulate Wnt target genes. Emerging data like those in the mammary models described above indicate that the consequences of APC inactivation are not solely restricted to Wnt pathway activation
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