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 ≃ 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 ⁴⁸⁰NGGNGTVEDRKPSE⁴⁹⁴L, 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-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 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.