Bourguignon L Y, Gunja-Smith Z, Iida N, Zhu H B, Young L J, Muller W J, Cardiff R D
Department of Cell Biology and Anatomy, University of Miami Medical School, Florida 33136, USA.
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.
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.
在本研究中，我们采用了一种独特的乳腺癌细胞系（Met-1，它源自表达多瘤病毒中T癌基因的转基因小鼠的高转移潜能肿瘤）来研究CD44变异体同工型在调节转移性乳腺肿瘤细胞行为中的作用。逆转录聚合酶链反应、Southern印迹、核苷酸测序、免疫沉淀和免疫印迹分析结果表明，这些细胞表达一种主要的CD44同工型（分子量约260 kDa），其含有v3,8 - 10外显子插入（命名为CD44v3,8 - 10）。此外，我们已确定CD44v3,8 - 10特异性结合细胞骨架蛋白，如锚蛋白。使用竞争结合试验和与NGGNGTVEDRKPSEL（CD44v3,8 - 10的aa480和aa494之间的序列）相同的合成肽进行的生化分析表明，这种15个氨基酸的肽特异性结合细胞骨架蛋白锚蛋白（但不结合血影蛋白或肌动蛋白）。该肽有效地竞争锚蛋白与CD44v3,8 - 10的结合。因此，我们认为位于CD44v3,8 - 10细胞质结构域的序列480NGGNGTVEDRKPSE494L是锚蛋白结合所必需的。我们还检测到含有CD44v3,8 - 10的Met-1细胞能够形成膜钉或“侵袭伪足”结构并经历活跃的迁移过程。用某些试剂处理Met-1细胞，包括抗CD44v3抗体、细胞松弛素D（一种微丝抑制剂）和W - 7（一种钙调蛋白拮抗剂），但不包括秋水仙碱（一种微管破坏剂），可有效抑制“侵袭伪足”形成及随后的肿瘤细胞迁移。使用酶谱分析和双重免疫荧光染色的进一步分析表明，CD44v3,8 - 10在“侵袭伪足”结构内的复合物中与基质金属蛋白酶MMP - 9的活性形式密切相关。这些发现表明，CD44v3,8 - 10在将锚蛋白与“侵袭伪足”形成（与基质降解活性相关）和乳腺癌进展过程中的肿瘤细胞迁移所需的膜相关肌动球蛋白收缩系统连接方面发挥重要作用。
