Targeting Wnt Signaling in Myeloma In Vivo; Differential Effects on Tumor Burden and Myeloma Bone Disease.

Multiple myeloma is characterized by uncontrolled proliferation of myeloma cells within the bone marrow and the development of a severe osteolytic bone disease. In addition to a well characterized increase in osteoclastic bone resorption, myeloma bone disease is associated with a reduction in bone formation. Osteoblast differentiation and bone formation are regulated in vivo by canonical Wnt signaling and activation of β-catenin. Therefore increasing Wnt signaling in the bone microenvironment in multiple myeloma may prevent the development of myeloma bone disease. In support of this, we have previously demonstrated that activation of Wnt signaling with lithium chloride (LiCl) in the 5TGM1 murine model of myeloma reduces tumor burden and osteolytic bone disease. However, we also found that LiCl treatment increased subcutaneous (s.c.) tumor growth. This suggests that the reduction in tumor burden within the bone microenvironment may be an indirect effect mediated through the effects of LiCl to prevent myeloma bone disease. The aim of the current study was to determine the effect of specific molecular blockade of Wnt signaling in myeloma cells in vivo. 5TGM1-GFP myeloma cells were transfected by electroporation with either myc-tagged dominant negative TCF4 (DNTCF4) or pcDNA. Following stable selection by culture in G418, expression of DNTCF4 was confirmed by western blot for myc. No difference was found in the growth rates of 5TGM1-pcDNA or 5TGM1-DNTCF4 in vitro. Treatment with LiCl or Wnt3A had no significant effect on cell viability in vitro, but significantly increased β-catenin activity, as measured by TOPFLASH activity in 5TGM1-pcDNA cells. This increase was not observed in 5TGM1-DNTCF4, confirming that expression of DNTCF4 blocked Wnt signaling induced by LiCl in 5TGM1 myeloma cells. C57Bl/KaLwRij mice were inoculated with 5TGM1-pcDNA or 5TGM1-DNTCF4 cells by either intravenous (i.v.) or s.c. injection. Mice were treated from time of tumor cell inoculation with 200mg/kg/day LiCl or vehicle control (d.H₂0) by oral gavage for 28 days. I.v. inoculation of myeloma cells resulted in a significant increase in serum IgG2bκ concentrations and the proportion of GFP-positive cells in the bone marrow. A significant reduction in trabecular bone volume was also observed. MicroCT analysis of the tibia demonstrated that LiCl significantly increased trabecular bone volume in both 5TGM1-pcDNA and 5TGM1-DNTCF4 myeloma-bearing mice. LiCl significantly decreased serum IgG2bκ concentrations in both 5TGM1-pcDNA and 5TGM1-DNTCF4 myeloma-bearing mice, with a greater effect in 5TGM1-DNTCF4 myeloma-bearing mice. FACS analysis of GFP-positive cells demonstrated that LiCl significantly reduced tumor burden in the bone marrow in both 5TGM1-pcDNA and 5TGM1-DNTCF4 myeloma-bearing mice. However, following s.c inoculation, LiCl significantly increased s.c. tumor volume of 5TGM1-pcDNA tumors, but had no effect on 5TGM1-DNTCF4 s.c. tumor volume. Taken together these results demonstrate that the effect of increasing Wnt signaling in myeloma is dependent upon the microenvironment. By specific inhibition of β-catenin activity in myeloma cells combined with systemic stimulation of the Wnt signaling pathway, our results suggest that increasing Wnt signaling in myeloma in vivo has dual effects; firstly to enhance myeloma growth directly, and secondly to enhance osteoblast differentiation and thus indirectly reduce tumor burden in bone, highlighting the importance of the bone marrow microenvironment in regulating myeloma growth and survival.