CD44v(3,8-10) is involved in cytoskeleton-mediated tumor cell migration and matrix metalloproteinase (MMP-9) association in metastatic breast cancer cells.
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.
Bourguignon
L Y
LY
Department of Cell Biology and Anatomy, University of Miami Medical School, Florida 33136, USA.
Gunja-Smith
Z
Z
Iida
N
N
Zhu
H B
HB
Young
L J
LJ
Muller
W J
WJ
Cardiff
R D
RD
D000595:Amino Acid Sequence; D000818:Animals; D017487:Ankyrins / Q000378:metabolism; D001667:Binding, Competitive; D002465:Cell Movement / Q000187:drug effects / Q000502:physiology*; D017364:Collagenases / Q000378:metabolism*; D015638:Cytochalasin D / Q000494:pharmacology; D003599:Cytoskeleton / Q000502:physiology*; D015972:Gene Expression Regulation, Neoplastic / Q000235:genetics; D018960:Hyaluronan Receptors / Q000235:genetics* / Q000276:immunology; D007150:Immunohistochemistry; D020780:Matrix Metalloproteinase 9; D051379:Mice; D008969:Molecular Sequence Data; D009362:Neoplasm Metastasis / Q000503:physiopathology*; D009374:Neoplasms, Experimental / Q000378:metabolism*; D010446:Peptide Fragments / Q000378:metabolism; D011485:Protein Binding / Q000502:physiology; D013449:Sulfonamides / Q000494:pharmacology; D014407:Tumor Cells, Cultured
D017487:Ankyrins; D018960:Hyaluronan Receptors; D010446:Peptide Fragments; D013449:Sulfonamides; D015638:Cytochalasin D; C017967:W 7; D017364:Collagenases; D020780:Matrix Metalloproteinase 9