Wnt3 Augments Adhesion Mediated Drug Resistance of Myeloma Cells Via Wnt/RhoA Pathway.

Despite the tremendous effort in developing of conventional chemotherapy and molecular targeting drugs for patients with multiple myeloma (MM), it has been proven difficult to completely abrogate neoplastic cells from bone marrow (BM). Hence, patients with refractory disease still experience poor outcome due to disease progression. Principle obstacle in the treatment of this disease is a chemo-resistance which is mainly caused by the interaction of myeloma cells with BM stromal cells. 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, HS-sultan and MM1S) by reverse transcription (RT)-polymerase chain reaction (PCR) 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. RPMI8226, ARH77 and KMS-5 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. This cell adhesion was augmented by addition of Wnt3 containing conditioned medium (CM) and suppressed by Wnt-receptor competitor, secreted Frizzled related protein (sFRP)-1, but not by specific inhibitor of canonical pathway (DKK-1). These results suggest that adhesion of myeloma cells was regulated by non-canonical pathway of Wnt signaling. We further examined whether the Wnt3 mediated adhesion to stromal cells involved in CAM-DR. The drug resistance of ARH-77 for doxorubicin was 1.8 folds enhanced by adhesion to stromal cells in comparison with stroma-free condition. This CAM-DR for doxorubicin was further augmented (2.6 folds) by addition of Wnt3 CM via enhancement of adhesion to stromal cells. Moreover, although the doxorubicin sensitivity of ARH-77 in coculture with stromal cells was significantly reduced by sFRP-1, this effect was not observed in stroma-free culture, indicating that Wnt3-mediated CAM-DR is dependent on attachment with stromal cells. Additionally, CAM-DR was completely restrained by addition of Rho kinase inhibitor Y27632. These results indicate that Wnt3 augments myeloma CAM-DR by enhancement of adhesion to human BM stromal cells via Wnt/RhoA signaling. Thus, Wnt/RhoA signaling pathway could be a promising molecular target to overcome CAM-DR.