In this study, we demonstrate that bortezomib is capable of increasing bone mass in myelomatous and nonmyelomatous bones in vivo and that increased bone formation stimulated by this agent is associated with a concomitant reduction in primary myeloma tumor burden in our experimental model. Using stroma-dependent BN myeloma cells and live-animal imaging in SCID-rab mice, we further validate our findings and demonstrate that the antimyeloma effects of bortezomib, but not melphalan, are associated with increased BMD. Bortezomib significantly reduced the number of osteoclasts and increased the number of differentiating osteoblasts in implanted bones in SCID-rab mice. The in vitro data further indicate that bortezomib affects bone remodeling by simultaneous inhibition of osteoclastogenesis and stimulation of osteoblastogenesis. The results of this study support our and others’ previous findings demonstrating that increased bone mass through exogenous MSC cytotherapy [29
] or treatment with DKK1-neutralizing antibody [29
], Wnt3a [35
], or lithium chloride [36
] have inhibitory effects on myeloma tumor burden in bone.
The mechanisms by which proteasome inhibitors promote bone formation are gradually being revealed. In our work, bortezomib acted directly on osteoclast and osteoblast precursors, confirming previous in vitro findings [16
]. The ubiquitin-proteasome pathway may modulate osteoblast differentiation through upregulating bone morphogenetic protein 2 (BMP-2) expression [11
] and preventing proteolytic degradation of the osteoblast transcription factor Runx2/Cbfa1 [16
]. Bortezomib reduces circulating levels of DKK1 and RANKL in patients with MM [15
]. DKK1 plays a critical role in regulating osteoblastogenesis in physiological and pathological conditions, including MM [9
]. The inhibitory effects of bortezomib on DKK1 levels in patients with MM is probably a consequence of reduced DKK1
expression in osteoblast precursors [18
] and myeloma cells themselves [9
]. RANKL expression is upregulated in myelomatous bones and is critically involved in stimulating osteoclast activity [19
]. In addition to inhibiting RANKL, bortezomib inhibits osteoclast differentiation through a mechanism associated with inhibition of NF-κB activity in osteoclast precursors [17
]. Collectively, these studies suggest that bortezomib increases bone mass by simultaneously stimulating bone formation and suppressing bone resorption.
Despite direct effects on bone cells, bortezomib seems to have more profound bone-anabolic efficacy in myelomatous bones of SCID-rab mice and patients with MM who also show an antimyeloma response to the drug [13
]. Although other clinical studies showed that bortezomib promotes osteoblast activity in patients with MM irrespective of response to treatment, the effects are significantly higher in responding patients than nonresponding patients [14
]. It should also be noted that these previous studies were based on retrospective observations in patients who were previously treated with a bisphosphonate. The antimyeloma responses of other potent drugs, such as melphalan, were not associated with bone anabolism, suggesting that bortezomib’s effect on skeletal homeostasis is not a consequence of reduced tumor burden. The failure of bortezomib to efficiently prevent bone loss in nonresponding patients, as well as an understanding of its transient effects on bone formation markers in certain responding patients [13
], requires further investigation.
The majority of patients with MM are elderly individuals whose bone remodeling is impaired by age and MM. In these patients, bortezomib may initially effectively lower MM burden, inhibit osteoclast differentiation, and reduce levels of restraint factors, such as DKK1, thus accelerating osteogenic differentiation of the existing pool of MSCs. However, this effect is transient, perhaps due to the limited pool of MSCs or an inability of these cells to retain their proliferative and differentiation potential over time. In contrast, MSCs in implanted rabbit bones in SCID-rab mice may retain their potential to differentiate into osteoblasts for lengthy periods of time, resulting in marked increases in bone formation.
As in the clinical setting, bortezomib heterogeneously affected myeloma tumor growth in our animal model. Although an exact comparison cannot be done, paraprotein levels were reduced >50% from pretreatment levels in 7 (43%) of 16 experiments (). Similar effects were observed in various clinical trials with bortezomib as a single agent [10
]. Our study also revealed that the BMD of uninvolved murine bones was not affected by growth of MM in SCID-rab mice. This observation supports the notion that, in contrast to bone manifestations, such as breast cancer, clinical and experimental MM bone disease mainly occurs in bone areas adjacent to tumor cells [2
Bortezomib also slightly increases BMD of nonmyelomatous rabbit bones; however, interestingly, bortezomib had no effect on BMD of mouse femurs in our experimental model.
In previous studies, Garrett et al. [11
] used 5-week-old Swiss ICR white mice and Mukherjee et al. [12
] used 7-week-old C57BL/6 mice to demonstrate the bone-anabolic response of proteasome inhibitors PS1, epoxomicin, and bortezomib in mouse bones in vivo. In the current study, we used CB.17/Icr-SCID mice to construct the SCID-rab model; treatment of SCID-rab mice with bortezomib was initiated at least at 15 weeks of age. Our conflicting results may suggest that regulation of mouse bone remodeling by the ubiquitin-proteasome pathway is influenced by age, strain, and/or immune function.
Interestingly, recent studies indicated that restoring or increasing Wnt signaling in myelomatous bones by using DKK1-neutralizing antibody [25
], Wnt3a [35
], or lithium chloride [36
] prevented bone disease, stimulated bone formation, and inhibited myeloma growth. Furthermore, in their in vitro study, Oyajobi et al. [18
] demonstrated that bortezomib downregulated DKK1
expression in calvarial cultures and stromal cells and that DKK1 blocked bortezomib’s stimulatory effects on bone formation. These studies suggest that proteasome inhibitors also promote bone formation through regulation of Wnt signaling, including reduction in DKK1 levels and upregulation of BMP-2 in bone.
Bortezomib has been shown to directly inhibit myeloma cell growth at low nanomolar concentrations [39
]. Although we were unable to prove that the antimyeloma response of bortezomib could be entirely mediated through effects on bone remodeling, previous experimental studies showing that prevention of osteolysis [19
] or promotion of bone formation [25
] has a negative impact on progression of MM. Our results are also supported by the in vitro findings that culture of osteoclasts alone supports longterm survival of primary myeloma cells [34
] and that mature osteoblasts attenuate the growth of myeloma cells in a subset of patients [29
]. Intriguingly, we have recently discovered that decorin, a main small leucine-rich proteoglycan highly expressed and produced by osteoblasts, is involved in the anti-MM effects of osteoblasts and that bortezomib had a tendency to increase decorin expression in osteoblasts, particularly those generated from patients with MM [40
Collectively, we have shown that bortezomib can induce osteoblastogenesis and suppress osteoclastogenesis in vivo and in vitro. The increased BMD of myelomatous bones by bortezomib is accompanied by a reduction in tumor burden. Our study suggests that in addition to direct effect on MM cells bortezomib controls MM also through regulation of osteoblast and osteoclast activity, resulting in an inhospitable BM environment for tumor cells.