Wnt3 Signaling Augments integrinβ1-Dependent Adhesion-Mediated Drug Resistance of Multiple Myeloma in Autocrine and Paracrine Manner.

Adhesion of myeloma cells to BM stromal cells is now considered to play a critical role in chemo-resistance. However, little is known about the molecular mechanism of cell adhesion mediated drug resistance (CAM-DR) in MM. In this study, we focused on relationship between drug resistance and expression of Wnts, the factor regulating the cell adhesion and proliferation, in myeloma cells. To gain insight into involvement of Wnt signaling in CAM-DR, we first screened the expression of Wnt family in myeloma cell lines (RPMI8226, ARH77, KMS-5, and MM1S) by reverse transcription-polymerase chain reaction analysis. Although the mRNAs of Wnt2b, Wnt7a and Wnt10b were variably expressed in some of myeloma cell lines, Wnt3 mRNA was detected in all the myeloma cells examined. KMS-5 and ARH77, which highly expressed Wnt3 protein, tightly adhered to human BM 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 BM stromal cells. 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-integrinβ1 antibody, a Wnt-receptor competitor, secreted Frizzled related protein-1 and Rho kinase inhibitor (Y27632 and OH-fasudil), but not by the specific inhibitor of canonical signaling (DKK-1), indicating that Wnt-mediated CAM-DR which is dependent on integrinβ1- mediated attachment to stromal cells is induced by Wnt/Rho pathway signal. This CAM-DR for doxorubicin was also significantly reduced by Wnt3 siRNA transfer to KMS-5. On the other hand, the cell adhesion of MM1S was dramatically augmented by addition of Wnt3 containing conditioned medium (CM) and suppressed by integrinα4 or β1 antibody (VLA4)(Fig 1). Furthermore, CAM-DR of MM1S was significantly augmented by Wnt3 CM or adhesion of mesenchymal stem cells which expressed Wnt3, but not BM stromal cells which did not express Wnt3 mRNA. These results suggest that adhesion of myeloma cells on stromal cells was regulated by Wnt signaling in autocrine or paracrine manner. The Wnt3 signaling pathway could be a promising molecular target to overcome CAM-DR.