further emphasizes the relevance of BMP antagonism in pathological bone remodeling. Here we clearly show that noggin silencing in osteolytic PC-3 cells, although not correcting the bone architecture, restores the bone mass of the xenografted tibiae, as assessed by radiography, m- CT and pQCT. This effect on the bone mass is the result of normalization or even enhancement of bone formation, as indicated by histomorphometric parameters of osteoblast number and activity. Noggin silencing in PC-3 cells does not alter either the level or the spectrum of their BMP expression. As a result, the physiological BMP/noggin balance in bone could be tilted in favor of first by an excess of cancer cell-derived BMPs, which may explain the trend toward increase in bone formation observed in bone xenografted with Nog-KD PC-3 cells. The bone repair is evident in advanced bone lesions, suggesting that a time interval is necessary to restore bone formation, consistently with the temporal organization of the physiological bone remodeling (bone coupling). A similar mechanism of restoration of bone coupling has been proposed to explain the reparative bone formation in bone metastasis by solid cancers as an effect of anti-cancer therapy [33,36,50]. The positive effect of noggin silencing on bone formation supports our original hypothesis and suggests noggin as one of the essential cancer cell-derived inhibitors of the osteoblast recruit- ment/activity contributing to the osteolytic lesions in CaP bone metastases. We have chosen a CaP model of osteolytic bone metastasis to be consistent with our previous study demonstrating that, conversely, lack of noggin has a relevant role in the osteoblastic response in CaP bone metastases [17]. However, a predominantly osteolytic phenotype is observed only in a small percentage of CaP bone metastases, while it represents the majority of the CaM metastases [5,6]. Therefore, in order to extend the relevance of noggin in cancer-mediated osteolysis, it will be important to confirm the present findings in a CaM model of osteolytic bone metastasis. Nevertheless, it has already been shown that inhibition of Dkk-1 by neutralizing antibodies in MM in vivo [51,52], or in CaM cells in vitro [53], or by shRNA-mediated interference in CaP cells in vitro [54] is also able to restore bone formation suppressed by neoplastic cells. Furthermore, Dkk-1 serum levels are elevated in CaM patients with bone metastases, but not in CaM patients with soft tissue metastases, and it has been suggested that Dkk-1-induced inhibition of bone formation may contribute to the osteolytic lesion in CaM [55]. Collectively, these and the present investigation support the view that cancer cell- derived antagonists of both BMP and Wnt signaling pathways contribute to the severity of the osteolytic process not only in MM, but also in various types of epithelial cancer. The BMP and Wnt signaling pathways clearly cooperate and regulate each other, but the exact hierarchy of these two pathways in stimulating osteoblast recruitment and activity is still debated [56,57,58,59]. In the late phase of the experimental observation, concomitantly to bone repair, noggin silencing in cancer cells also reduces tumor growth. The fact that the growth rate in vitro of noggin-silenced clones is not affected suggests that this effect in vivo may be exerted through an influence of the bone environment. Restoration of bone formation may physically limit the intramedullary space available for cancer cell expansion [60]. However, in this case cancer cells would most probably invade the surrounding soft tissue through trans-cortical, vascular channels, as it has been observed when bone resorption is inhibited [61,62]. Most likely, osteoblasts may secrete factors inhibiting tumor growth directly [63]. One of these factors could be BMP-7, which we have previously demonstrated to inhibit tumor growth in animal models of osteolytic bone metastasis [48,49]. Osteolytic cancer cell- Role of Noggin in Osteolytic Bone Metastasis derived noggin may antagonize endogenous, osteoblast-derived BMP-7 and, therefore, allow escape from its inhibitory effect on tumor growth. Alternatively, osteoblasts may affect tumor growth indirectly, acting through an inhibition of osteoclast recruitment [39,60]. The latter, indirect mechanism is also suggested by the decreased osteoclast number and activity observed in tibiae xenografted with noggin-silenced PC-3 cells. Taken together, these observations indicate that restoration of bone formation may limit growth progression in osteolytic bone metastasis. The establishment of systemic bone metastases induced by injection into the left cardiac ventricle of noggin-silenced PC-3 cells is not impaired. This suggests that interference with the constitutive secretion of noggin by cancer cells does not affect the early steps of the bone metastatic colonization (adhesion and extra-vasation) and, especially, the growth initiation by the osteolytic process. It has been previously reported that exogenous noggin inhibits BMP-2- and BMP-4-induced invasion and migration of PC-3 cells, in vitro, and that noggin over-expression in the same cells limits their expansion and osteolytic activity, in vivo [64]. This finding is apparently in contradiction with our present study, showing that noggin silencing, rather than over-expression, limits tumor growth and osteolysis in the same PC-3 bone xenograft model. Most likely, noggin over-expression in PC-3 cells antagonizes the overall BMPs secreted by both cancer cells and the bone microenvironment and, accordingly, interferes not only with their direct, stimulatory effect on the invasive capacity of PC-3 cells, but also with their normal, stimulatory role in bone formation. In contrast, noggin silencing in PC-3 cells targets exclusively the noggin of cancer cell origin, thereby eliminating its interference with the physiological BMP/ noggin balance within the bone microenvironment. Accordingly, noggin silencing seems to be a more targeted mean to understand the role of noggin in osteolytic bone metastasis. Based on the present results we postulate a revised model for the pathogenesis of osteolytic bone metastasis by solid cancers, where the osteolytic lesion is not only the result of excess of bone resorption induced by cancer cell-derived factors stimulating osteoclast recruitment and activity, but also of uncoupling bone formation through noggin constitutively secreted by cancer cells. Conversely, suppression of cancer cell-derived noggin restores the bone formation phase, which is physiologically coupled to the initial phase of bone resorption. As a result of this anabolic effect, tumor progression is contained by yet unidentified, osteoblast- derived factors (Figure 7). Thus therapeutic neutralization of noggin may not only favor bone repair, but also control bone metastatic growth. Targeting of the RANK/RANKL axis has been proven to successfully inhibit osteolysis in numerous animal models of bone metastasis [65] and this evidence has contributed significantly to the strategy to develop a humanized monoclonal antibody against RANKL for treating osteolytic bone disease [66]. The evidence presented in this investigation, suggesting that a dual mechanism of stimulated bone resorption and inhibited bone formation is responsible also for the osteolytic lesion by solid cancers, provides the rationale for a combinatorial targeting of both, the RANK/ RANKL axis and the BMP antagonist noggin for a more effective control of bone metastatic growth. Materials and Methods Ethics statement Experimental animal protocols, anesthesia, surgical procedure and post-surgical analgesia were approved by the Committee for Animal Experimentation and the Veterinary Authorities of the PLoS ONE | www.plosone.org 6 January 2011 | Volume 6 | Issue 1 | e16078