title: APC/β-catenin-rich complexes at membrane protrusions regulate mammary tumor cell migration and mesenchymal morphology.
abstract: BACKGROUND
  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. 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.
  
  
  METHODS
  The 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.
  
  
  RESULTS
  APC/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.
  
  
  CONCLUSIONS
  These 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.
authors.affiliation: Department of Surgery, University of Chicago, Chicago, IL 60637, USA.
authors.forename: Matthew A
authors.initials: MA
authors.lastname: Odenwald
authors.forename: Jenifer R
authors.initials: JR
authors.lastname: Prosperi
authors.forename: Kathleen H
authors.initials: KH
authors.lastname: Goss
mesh_terms: D025601:Adenomatous Polyposis Coli Protein / Q000235:genetics / Q000378:metabolism*; D000818:Animals; D015153:Blotting, Western; D001943:Breast Neoplasms / Q000235:genetics / Q000378:metabolism* / Q000473:pathology; D002465:Cell Movement / Q000187:drug effects; D049109:Cell Proliferation; D048430:Cell Shape / Q000187:drug effects; D022081:Cell Surface Extensions / Q000187:drug effects / Q000378:metabolism* / Q000473:pathology; D003599:Cytoskeleton / Q000378:metabolism; D004285:Dogs; D058750:Epithelial-Mesenchymal Transition / Q000187:drug effects; D005260:Female; D005455:Fluorescent Antibody Technique; D017930:Genes, Reporter; D045325:HCT116 Cells; D006801:Humans; D061985:Madin Darby Canine Kidney Cells; D051379:Mice; D008858:Microscopy, Phase-Contrast; D046912:Multiprotein Complexes; D009361:Neoplasm Invasiveness; D046988:Proteasome Endopeptidase Complex / Q000187:drug effects / Q000378:metabolism; D061988:Proteasome Inhibitors / Q000494:pharmacology; D034622:RNA Interference; D020133:Reverse Transcriptase Polymerase Chain Reaction; D013997:Time Factors; D014162:Transfection; D016212:Transforming Growth Factor beta / Q000378:metabolism; D051176:beta Catenin / Q000378:metabolism*
chemical_list: C555337:APC protein, human; D025601:Adenomatous Polyposis Coli Protein; C495270:CTNNB1 protein, human; C495267:CTNNB1 protein, mouse; D046912:Multiprotein Complexes; D061988:Proteasome Inhibitors; D016212:Transforming Growth Factor beta; D051176:beta Catenin; D046988:Proteasome Endopeptidase Complex