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 mise 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 approximate to 260 kDa) containing a v3,8-10 exon insertion (designated as CD44v(3,8-10)). In addition, we have determined that CD44v(3,8-10) 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 CD44v(3,8-10)) 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 CD44v(3,8-10). Therefore, we believe that the sequence (480)NGGNGTVEDRKPSE(494)L, located at the cytoplasmic domain of CD44v(3,8-10), is required for the ankyrin binding. We have also detected that CD44v(3,8-10)-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-CD44v(3) 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 CD44v(3,8-10) is closely associated with the active form of matrix metalloproteinase, MMP-9, in a complex within "invadopodia" structures. These findings suggest that CD44v(3,8-10) 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. (C) 1998 Wiley-Liss, Inc.
