CD44 enhances invasion of basal-like breast cancer cells by upregulating serine protease and collagen-degrading enzymatic expression and activity

Breast Cancer Res. 2012 May 23;14(3):R84. doi: 10.1186/bcr3199.

Abstract

Introduction: 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.

Conclusion: 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.

Publication types

  • Research Support, Non-U.S. Gov't

MeSH terms

  • Breast Neoplasms / enzymology
  • Breast Neoplasms / metabolism*
  • Breast Neoplasms / pathology
  • Cathepsin K / metabolism
  • Cell Line, Tumor
  • Collagen / metabolism*
  • Female
  • Humans
  • Hyaluronan Receptors / genetics
  • Hyaluronan Receptors / metabolism*
  • Hyaluronic Acid / metabolism*
  • Matrix Metalloproteinase 14 / genetics
  • Matrix Metalloproteinase 14 / metabolism
  • Neoplasm Invasiveness
  • Neoplasm Metastasis
  • Neoplasms, Basal Cell / metabolism*
  • RNA Interference
  • RNA, Small Interfering
  • Receptors, Urokinase Plasminogen Activator / metabolism
  • Serine Proteases / metabolism*
  • Signal Transduction
  • Up-Regulation
  • Urokinase-Type Plasminogen Activator / genetics
  • Urokinase-Type Plasminogen Activator / metabolism

Substances

  • Hyaluronan Receptors
  • RNA, Small Interfering
  • Receptors, Urokinase Plasminogen Activator
  • Hyaluronic Acid
  • Collagen
  • Serine Proteases
  • Urokinase-Type Plasminogen Activator
  • Cathepsin K
  • Matrix Metalloproteinase 14