Targeting the Wnt/β-Catenin Signaling Pathway in Multiple Myeloma: A Possible New Therapeutic Approach to Overcome Bortezomib-Resistance

Multiple myeloma (MM) is a neoplasm of plasma cells that is often fatal, despite the use of high dose chemotherapy and hematopoietic stem cell transplantation. Although new therapeutic approaches, including novel agents such as thalidomide, lenalidomide, and bortezomib (a proteasome inhibitor), are now used clinically and have improved the outcome of patients with MM, most patients eventually relapse, and it remains an incurable disease.

Wnt/β-catenin signaling plays a critical role in both cell proliferation and differentiation in normal tissue. β-catenin, a key player and downstream effector in canonical Wnt signaling, is involved in the regulation of cell fate, proliferation, and self renewal of stem cells. The activation of Wnt signaling is aberrantduring the pathogenesis of various malignant neoplasms, and it has been suggested that Wnt/β-catenin signaling is involved in the regulation of cancer stem cells. When the Wnt signaling pathway is activated, stabilized β-catenin translocates to the nucleus, where it interacts with T-cell factor, followed by transcription of target genes such as c-myc, cyclin D1 and survivin. Nuclear β-catenin also recruits the co-activator, cyclic AMP response element-binding protein (CBP), which is essential for hematopoietic cell proliferation and hematopoietic stem cell self renewal, or its related homolog p300, which is involved in hematopoietic cell differentiation. Recent studies have shown that CBP and p300 have distinct functions in the regulation of β-catenin expression: CBP promotes β-catenin expression, whereas p300 inhibits β-catenin expression. In the context of hematological malignancies, β-catenin is overexpressed in myeloma-derived cell lines and primary myeloma cells, whereas expression is very low in normal plasma cells. Myeloma cells are maintained by several growth factors and cytokines, including Wnt ligands secreted by stromal cells in the bone marrow. These data suggest that Wnt/β-catenin signaling contributes to the pathogenesis of MM and thus might be a promising target for the treatment of this incurable hematological malignancy. Moreover, it has been reported that CBP, rather than p300, plays an important role in the expression of apurinic endonuclease/redox factor-1, an important regulator of multidrug resistance, in retinoic acid-induced chemo-resistant myeloma cells, suggesting that CBP is involved in the acquisition of drug resistance. Taken together with the previous data, Wnt/β-catenin signaling, especially CBP, might be an attractive target for new therapeutic agents against MM. ICG-001, small-molecule inhibitor of the canonical Wnt signaling pathway, specifically binds to CBP, thereby disrupting CBP/β-catenin interaction. In the present study, we investigated the role of Wnt/β-catenin signaling in myeloma cells using ICG-001. MTS and trypan blue dye exclusion assays showed that ICG-001 inhibits the proliferation of U266, RPMI8226, and KMS myeloma cell lines in a dose- (0-15 μM) and time- (0-72 h) dependent manner. Assays for apoptotic cell death were performed to determine the cause of growth inhibition by ICG-001 and demonstrated that ICG-001 induced both early and late apoptosis in myeloma cells. To investigate the molecular mechanisms of ICG-001-induced cell death in myeloma cells, the expression of various cell-death associated proteins and down-stream molecules of Wnt/β-catenin signaling was examined. Western blotting analysis showed that ICG-001 arrested cell growth and induced apoptotic cell death in myeloma cells by reducing the expression of three β-catenin target molecules: survivin, cyclin D1, and c-Myc. We next examined the effects of ICG-001 on bortezomib (BTZ)-resistant MM cells. BTZ resistance is an urgent issue in clinics, and therapeutic approaches for overcoming BTZ resistance are important. Interestingly, ICG-001 inhibited the proliferation of both BTZ-sensitive wild-type KMS and BTZ-resistant KMS cells, suggesting that targeting CBP may overcome BTZ-resistance. Furthermore, when combined with cyclosporine, which inhibits non-canonical Wnt/β-catenin signaling, ICG-001 synergistically induced the growth arrest of myeloma cells by inducing apoptotic cell death. These results indicate that inhibition of Wnt/β-catenin signaling may be an attractive therapeutic option both for patients with newly diagnosed MM and for those in a refractory or relapsed state.