CD44v(3,8-10) is involved in cytoskeleton-mediated tumor cell migration and matrix metalloproteinase (MMP-9) association in metastatic breast cancer cells

J Cell Physiol. 1998 Jul;176(1):206-15. doi: 10.1002/(SICI)1097-4652(199807)176:1<206::AID-JCP22>3.0.CO;2-3.

Abstract

In the present study, we have employed a unique breast cancer cell line (Met-1, which was derived from a high metastatic potential tumor in transgenic mice expressing polyomavirus middle T oncogene) to study the role of CD44 variant isoform(s) in the regulation of metastatic breast tumor cell behavior. The results of reverse transcriptase-polymerase chain reaction, Southern blot, nucleotide sequencing, immunoprecipitation, and immunoblot analyses indicated that these cells express a major CD44 isoform (molecular weight approximately 260 kDa) containing a v3,8-10 exon insertion (designated as CD44v3,8-10). In addition, we have determined that CD44v3,8-10 binds specifically to the cytoskeletal proteins such as ankyrin. Biochemical analyses, using competition binding assays and a synthetic peptide identical to NGGNGTVEDRKPSEL (a sequence located between aa480 and aa494 of CD44v3,8-10) indicate that this 15-amino acid peptide binds specifically to the cytoskeletal protein ankyrin (but not to fodrin or spectrin). This peptide competes effectively for ankyrin binding to CD44v3,8-10. Therefore, we believe that the sequence 480NGGNGTVEDRKPSE494L, located at the cytoplasmic domain of CD44v3,8-10, is required for the ankyrin binding. We have also detected that CD44v3,8-10-containing Met-1 cells are capable of forming membrane spikes or "invadopodia" structures and undergo active migration processes. Treatments of Met-1 cells with certain agents including anti-CD44v3 antibody, cytochalasin D (a microfilament inhibitor), and W-7 (a calmodulin antagonist), but not colchicine (a microtubule disrupting agent) effectively inhibit "invadopodia" formation and subsequent tumor cell migration. Further analyses using zymography assays and double immunofluorescence staining indicated that CD44v3,8-10 is closely associated with the active form of matrix metalloproteinase, MMP-9, in a complex within "invadopodia" structures. These findings suggest that CD44v3,8-10 plays an important role in linking ankyrin to the membrane-associated actomyosin contractile system required for "invadopodia" formation (coupled with matrix degradation activities) and tumor cell migration during breast cancer progression.

Publication types

  • Research Support, U.S. Gov't, Non-P.H.S.
  • Research Support, U.S. Gov't, P.H.S.

MeSH terms

  • Amino Acid Sequence
  • Animals
  • Ankyrins / metabolism
  • Binding, Competitive
  • Cell Movement / drug effects
  • Cell Movement / physiology*
  • Collagenases / metabolism*
  • Cytochalasin D / pharmacology
  • Cytoskeleton / physiology*
  • Gene Expression Regulation, Neoplastic / genetics
  • Hyaluronan Receptors / genetics*
  • Hyaluronan Receptors / immunology
  • Immunohistochemistry
  • Matrix Metalloproteinase 9
  • Mice
  • Molecular Sequence Data
  • Neoplasm Metastasis / physiopathology*
  • Neoplasms, Experimental / metabolism*
  • Peptide Fragments / metabolism
  • Protein Binding / physiology
  • Sulfonamides / pharmacology
  • Tumor Cells, Cultured

Substances

  • Ankyrins
  • Hyaluronan Receptors
  • Peptide Fragments
  • Sulfonamides
  • Cytochalasin D
  • W 7
  • Collagenases
  • Matrix Metalloproteinase 9