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 lin e (Met-1, which was derived from a high metastatic potential tumor in transgenic mise expressing polyomavirus middle T oncogene) to study th e role of CD44 variant isoform(s) in the regulation of metastatic brea st tumor cell behavior. The results of reverse transcriptase-polymeras e chain reaction, Southern blot, nucleotide sequencing, immunoprecipit ation, and immunoblot analyses indicated that these cells express a ma jor CD44 isoform (molecular weight approximate to 260 kDa) containing a v3,8-10 exon insertion (designated as CD44v(3,8-10)). In addition, w e have determined that CD44v(3,8-10) binds specifically to the cytoske letal proteins such as ankyrin. Biochemical analyses, using competitio n 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 cytoskeleta l protein ankyrin (but not to fodrin or spectrin). This peptide compet es effectively for ankyrin binding to CD44v(3,8-10). Therefore, we bel ieve that the sequence (480)NGGNGTVEDRKPSE(494)L, located at the cytop lasmic domain of CD44v(3,8-10), is required for the ankyrin binding. W e have also detected that CD44v(3,8-10)-containing Met-1 cells are cap able of forming membrane spikes or ''invadopodia'' structures and unde rgo active migration processes. Treatments of Met-1 cells with certain agents including anti-CD44v(3) antibody, cytochalasin D (a microfilam ent inhibitor), and W-7 (a calmodulin antagonist), but not colchicine (a microtubule disrupting agent) effectively inhibit ''invadopodia'' f ormation and subsequent tumor cell migration. Further analyses using z ymography assays and double immunofluorescence staining indicated that CD44v(3,8-10) is closely associated with the active form of matrix me talloproteinase, 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 sys tem required for ''invadopodia'' formation (coupled with matrix degrad ation activities) and tumor cell migration during breast cancer progre ssion. (C) 1998 Wiley-Liss, Inc.