title: CD44 enhances invasion of basal-like breast cancer cells by upregulating serine protease and collagen-degrading enzymatic expression and activity.
abstract: BACKGROUND
  Basal-like breast cancers (BL-BCa) have the worst prognosis of all subgroups of this disease. Hyaluronan (HA) and the HA receptor CD44 have a long-standing association with cell invasion and metastasis of breast cancer. The purpose of this study was to establish the relation of CD44 to BL-BCa and to characterize how HA/CD44 signaling promotes a protease-dependent invasion of breast cancer (BrCa) cells.
  
  
  METHODS
  CD44 expression was determined with immunohistochemistry (IHC) analysis of a breast cancer tissue microarray (TMA). In vitro experiments were performed on a panel of invasive BL-BCa cell lines, by using quantitative polymerase chain reaction (PCR), immunoblotting, protease activity assays, and invasion assays to characterize the basis of HA-induced, CD44-mediated invasion.
  
  
  RESULTS
  Expression of the hyaluronan (HA) receptor CD44 associated with the basal-like subgroup in a cohort of 141 breast tumor specimens (P = 0.018). Highly invasive cells of the representative BL-BCa cell line, MDA-MB-231 (MDA-MB-231Hi) exhibited increased invasion through a basement membrane matrix (Matrigel) and collagen. In further experiments, HA-induced promotion of CD44 signaling potentiated expression of urokinase plasminogen activator (uPA) and its receptor uPAR, and underpinned an increased cell-associated activity of this serine protease in MDA-MB-231Hi and a further BL-BCa cell line, Hs578T cells. Knockdown of CD44 attenuated both basal and HA-stimulated uPA and uPAR gene expression and uPA activity. Inhibition of uPA activity by using (a) a gene-targeted RNAi or (b) a small-molecule inhibitor of uPA attenuated HA-induced invasion of MDA-MB-231Hi cells through Matrigel. HA/CD44 signaling also was shown to increase invasion of MDA-MB-231 cells through collagen and to potentiate the collagen-degrading activity of MDA-MB-231Hi cells. CD44 signaling was subsequently shown to upregulate expression of two potent collagen-degrading enzymes, the cysteine protease cathepsin K and the matrix metalloprotease MT1-MMP. RNAi- or shRNA-mediated depletion of CD44 in MDA-MB-231Hi cells decreased basal and HA-induced cathepsin K and MT1-MMP expression, reduced the collagen-degrading activity of the cell, and attenuated cell invasion through collagen. Pharmacologic inhibition of cathepsin K or RNAi-mediated depletion of MT1-MMP also attenuated MDA-MB-231Hi cell invasion through collagen.
  
  
  CONCLUSIONS
  HA-induced CD44 signaling increases a diverse spectrum of protease activity to facilitate the invasion associated with BL-BCa cells, providing new insights into the molecular basis of CD44-promoted invasion.
authors.affiliation: Centre for Cancer Research and Cell Biology, Queens University Belfast, 97 Lisburn Road, Belfast, BT9 7BL, Northern Ireland.
authors.forename: Nicola
authors.initials: N
authors.lastname: Montgomery
authors.forename: Ashleigh
authors.initials: A
authors.lastname: Hill
authors.forename: Suzanne
authors.initials: S
authors.lastname: McFarlane
authors.forename: Jessica
authors.initials: J
authors.lastname: Neisen
authors.forename: Anthony
authors.initials: A
authors.lastname: O'Grady
authors.forename: Susie
authors.initials: S
authors.lastname: Conlon
authors.forename: Karin
authors.initials: K
authors.lastname: Jirstrom
authors.forename: Elaine W
authors.initials: EW
authors.lastname: Kay
authors.forename: David J J
authors.initials: DJ
authors.lastname: Waugh
mesh_terms: D001943:Breast Neoplasms / Q000201:enzymology / Q000378:metabolism* / Q000473:pathology; D056657:Cathepsin K / Q000378:metabolism; D045744:Cell Line, Tumor; D003094:Collagen / Q000378:metabolism*; D005260:Female; D006801:Humans; D018960:Hyaluronan Receptors / Q000235:genetics / Q000378:metabolism*; D006820:Hyaluronic Acid / Q000378:metabolism*; D053511:Matrix Metalloproteinase 14 / Q000235:genetics / Q000378:metabolism; D009361:Neoplasm Invasiveness; D009362:Neoplasm Metastasis; D018295:Neoplasms, Basal Cell / Q000378:metabolism*; D034622:RNA Interference; D034741:RNA, Small Interfering; D055293:Receptors, Urokinase Plasminogen Activator / Q000378:metabolism; D057057:Serine Proteases / Q000378:metabolism*; D015398:Signal Transduction; D015854:Up-Regulation; D014568:Urokinase-Type Plasminogen Activator / Q000235:genetics / Q000378:metabolism
chemical_list: D018960:Hyaluronan Receptors; D034741:RNA, Small Interfering; D055293:Receptors, Urokinase Plasminogen Activator; D006820:Hyaluronic Acid; D003094:Collagen; D057057:Serine Proteases; D014568:Urokinase-Type Plasminogen Activator; D056657:Cathepsin K; D053511:Matrix Metalloproteinase 14