Kobune Masayoshi, Chiba Hiroki, Kato Junji, Kato Kazunori, Nakamura Kiminori, Kawano Yutaka, Takada Kohichi, Takimoto Rishu, Takayama Tetsuji, Hamada Hirofumi, Niitsu Yoshiro
Fourth Department of Internal Medicine, Sapporo Medical University School of Medicine, Sapporo, Japan.
Mol Cancer Ther. 2007 Jun;6(6):1774-84. doi: 10.1158/1535-7163.MCT-06-0684.
Adhesion of myeloma cells to bone marrow stromal cells is now considered to play a critical role in chemoresistance. However, little is known about the molecular mechanism governing cell adhesion-mediated drug resistance (CAM-DR) of myeloma cells. In this study, we focused our interests on the implication of the Wnt signal in CAM-DR. We first screened the expression of Wnt family in myeloma cell lines and found that Wnt3 was overexpressed in all the myeloma cells examined. KMS-5 and ARH77, which highly expressed Wnt3 protein, tightly adhered to human bone marrow stromal cells, and accumulation of beta-catenin and GTP-bounded RhoA was observed in these myeloma cell lines. Conversely, RPMI8226 and MM1S, which modestly expressed Wnt3 protein, rather weakly adhered to human bone marrow stromal. We then examined the relevance of Wnt3 expression to adhesive property to stromal cells and to CAM-DR of myeloma cells. KMS-5 and ARH-77 exhibited apparent CAM-DR against doxorubicin. This CAM-DR was significantly reduced by anti-integrin beta(1) antibody, anti-integrin alpha(6) antibody and a Wnt-receptor competitor, secreted Frizzled-related protein-1, and Rho kinase inhibitor Y27632, but not by the specific inhibitor of canonical signaling (Dickkopf-1), indicating that Wnt-mediated CAM-DR that is dependent on integrin alpha(6)/beta(1) (VLA-6)-mediated attachment to stromal cells is induced by the Wnt/RhoA/Rho kinase pathway signal. This CAM-DR was also significantly reduced by Wnt3 small interfering RNA transfer to KMS-5. These results indicate that Wnt3 contributes to VLA-6-mediated CAM-DR via the Wnt/RhoA/ROCK pathway of myeloma cells in an autocrine manner. Thus, the Wnt3 signaling pathway could be a promising molecular target to overcome CAM-DR of myeloma cells.
骨髓瘤细胞与骨髓基质细胞的黏附目前被认为在化疗耐药中起关键作用。然而，关于骨髓瘤细胞的细胞黏附介导的耐药性（CAM-DR）的分子机制却知之甚少。在本研究中，我们将兴趣集中在Wnt信号在CAM-DR中的作用。我们首先筛选了骨髓瘤细胞系中Wnt家族的表达，发现Wnt3在所有检测的骨髓瘤细胞中均过表达。高表达Wnt3蛋白的KMS-5和ARH77细胞与人骨髓基质细胞紧密黏附，并且在这些骨髓瘤细胞系中观察到β-连环蛋白和GTP结合的RhoA的积累。相反，适度表达Wnt3蛋白的RPMI8226和MM1S细胞与人骨髓基质细胞的黏附较弱。然后，我们研究了Wnt3表达与骨髓瘤细胞对基质细胞的黏附特性以及CAM-DR的相关性。KMS-5和ARH-77对阿霉素表现出明显的CAM-DR。抗整合素β(1)抗体、抗整合素α(6)抗体以及Wnt受体竞争者分泌型卷曲相关蛋白-1和Rho激酶抑制剂Y27632可显著降低这种CAM-DR，但经典信号通路的特异性抑制剂（Dickkopf-1）则不能，这表明Wnt介导的依赖整合素α(6)/β(1)（VLA-6）介导的与基质细胞黏附的CAM-DR是由Wnt/RhoA/Rho激酶途径信号诱导的。将Wnt3小干扰RNA转染到KMS-5细胞中也可显著降低这种CAM-DR。这些结果表明，Wnt3以自分泌方式通过骨髓瘤细胞的Wnt/RhoA/ROCK途径促进VLA-6介导的CAM-DR。因此，Wnt3信号通路可能是克服骨髓瘤细胞CAM-DR的一个有前景的分子靶点。
