title: The cell surface mucin podocalyxin regulates collective breast tumor budding.
abstract: BACKGROUND
  Overexpression of the transmembrane sialomucin podocalyxin, which is known to play a role in lumen formation during polarized epithelial morphogenesis, is an independent indicator of poor prognosis in a number of epithelial cancers, including those that arise in the breast. Therefore, we set out to determine if podocalyxin plays a functional role in breast tumor progression.
  
  
  METHODS
  MCF-7 breast cancer cells, which express little endogenous podocalyxin, were stably transfected with wild type podocalyxin for forced overexpression. 4T1 mammary tumor cells, which express considerable endogenous podocalyxin, were retrovirally transduced with a short hairpin ribonucleic acid (shRNA) targeting podocalyxin for stable knockdown. In vitro, the effects of podocalyxin on collective cellular migration and invasion were assessed in two-dimensional monolayer and three-dimensional basement membrane/collagen gel culture, respectively. In vivo, local invasion was assessed after orthotopic transplantation in immunocompromised mice.
  
  
  RESULTS
  Forced overexpression of podocalyxin caused cohesive clusters of epithelial MCF-7 breast tumor cells to bud off from the primary tumor and collectively invade the stroma of the mouse mammary gland in vivo. This budding was not associated with any obvious changes in histoarchitecture, matrix deposition or proliferation in the primary tumour. In vitro, podocalyxin overexpression induced a collective migration of MCF-7 tumor cells in two-dimensional (2-D) monolayer culture that was dependent on the activity of the actin scaffolding protein ezrin, a cytoplasmic binding partner of podocalyxin. In three-dimensional (3-D) culture, podocalyxin overexpression induced a collective budding and invasion that was dependent on actomyosin contractility. Interestingly, the collectively invasive cell aggregates often contained expanded microlumens that were also observed in vivo. Conversely, when endogenous podocalyxin was removed from highly metastatic, but cohesive, 4T1 mammary tumor cells there was a decrease in collective invasion in three-dimensional culture.
  
  
  CONCLUSIONS
  Podocalyxin is a tumor cell-intrinsic regulator of experimental collective tumor cell invasion and tumor budding.
authors.affiliation: Department of Cellular and Physiological Sciences, University of British Columbia, 2350 Health Sciences Mall, Vancouver, BC, V6T 1Z3, Canada. marcia.l.graves@gmail.com.
authors.forename: Marcia L
authors.initials: ML
authors.lastname: Graves
authors.affiliation: Department of Cellular and Physiological Sciences, University of British Columbia, 2350 Health Sciences Mall, Vancouver, BC, V6T 1Z3, Canada. jane.cipollone@mail.utoronto.ca.
authors.forename: Jane A
authors.initials: JA
authors.lastname: Cipollone
authors.affiliation: Department of Cellular and Physiological Sciences, University of British Columbia, 2350 Health Sciences Mall, Vancouver, BC, V6T 1Z3, Canada. paaustin@mail.ubc.ca.
authors.forename: Pamela
authors.initials: P
authors.lastname: Austin
authors.affiliation: Department of Cellular and Physiological Sciences, University of British Columbia, 2350 Health Sciences Mall, Vancouver, BC, V6T 1Z3, Canada. ebell750@gmail.com.
authors.forename: Erin M
authors.initials: EM
authors.lastname: Bell
authors.affiliation: Deeley Cancer Research Center, 2410 Lee Avenue, Victoria, V8R 6V5, BC, Canada. jnielsen@bccrc.ca.
authors.forename: Julie S
authors.initials: JS
authors.lastname: Nielsen
authors.affiliation: Genetic Pathology Evaluation Center, Vancouver General Hospital, 2660 Oak Street, Vancouver, V6H 3Z6, BC, Canada. Blake.Gilks@vch.ca.
authors.forename: C Blake
authors.initials: CB
authors.lastname: Gilks
authors.affiliation: Biomedical Research Center, University of British Columbia, Vancouver, BC, V6T 1Z3, Canada. kelly@brc.ubc.ca.
authors.forename: Kelly M
authors.initials: KM
authors.lastname: McNagny
authors.affiliation: Department of Cellular and Physiological Sciences, University of British Columbia, 2350 Health Sciences Mall, Vancouver, BC, V6T 1Z3, Canada. roskelly@mail.ubc.ca.
authors.forename: Calvin D
authors.initials: CD
authors.lastname: Roskelley
mesh_terms: D000818:Animals; D001943:Breast Neoplasms / Q000235:genetics* / Q000473:pathology; D002465:Cell Movement / Q000235:genetics*; D005260:Female; D015972:Gene Expression Regulation, Neoplastic; D055785:Gene Knockdown Techniques; D006801:Humans; D061986:MCF-7 Cells; D008321:Mammary Glands, Animal / Q000378:metabolism / Q000473:pathology; D051379:Mice; D009361:Neoplasm Invasiveness / Q000235:genetics*; D012795:Sialoglycoproteins / Q000096:biosynthesis* / Q000235:genetics
chemical_list: D012795:Sialoglycoproteins; C001643:podocalyxin