Figure 6. Noggin silencing limits growth of PC-3/Fluc cells in bone xenografts. Bioluminescent signal (photons +/2 SD) emitted from the cancer cell-xenografted tibiae was quantified at day 7, 14, 21 and 25 after intra-osseous inoculation of tumor cells; n = 6–7 animals for each experimental group. ***P,0.001, Nog-KD clones versus PC-3/Fluc at day 25; *P,0.05, Nog-KD 14 versus PC-3/Fluc at day 21. doi:10.1371/journal.pone.0016078.g006 inhibition of bone formation. We also demonstrate that shRNA- mediated suppression of the constitutive expression of the BMP antagonist noggin in PC-3 cells, without interfering with the host microenvironment–derived noggin, restores the osteoblast number and bone formation in bone lesions induced by these cells. Accordingly, the bone response is converted from a purely osteolytic to a mixed osteoblastic/osteolytic one. Collectively, these results provide novel evidence strongly suggesting that noggin secretion by CaP cells mediates the inhibition of the osteoblast recruitment/activity. The resulting inhibition of bone formation prevents the repair of the osteolytic lesion generated by cytokine-stimulated, osteoclast-mediated bone resorption. Consis- tently with this notion, noggin may represent a potential therapeutic target in osteolytic CaP bone metastasis. The molecular mechanisms governing the osteoblastic and the osteolytic response in bone metastases by solid cancers are subject of intensive investigation. In the osteolytic response, the attention has been predominantly focused on extracellular factors and signaling pathways that mediate the crosstalk between tumor cells and the bone microenvironment leading to the vicious cycle of tumor proliferation and bone resorption [25]. This hypothesis postulates that factors such as PTHrP [26], RANKL [27] and IL-8 [28] are secreted by cancer cells and stimulate osteoclast recruitment and activity. The consequent increase in bone resorption releases matrix-embedded growth factors, such as insulin-like growth factor (IGF) and transforming growth factor beta (TGF-b), which, in turn, promote further cancer cell growth. The influence of cancer cells on osteoblast recruitment and activity has been studied almost exclusively in MM. Paradigmatic molecules inducing directly osteoblast recruitment and, conse- quently, bone formation, are members of the BMP and Wnt protein families. Extracellular antagonists are crucial for the modulation of their activities [29]. In a seminal study it has been demonstrated that in MM secretion of the Wnt antagonist Dkk-1 by the neoplastic cells inhibits osteoblast recruitment and activity. Accordingly, the osteolytic lesion in MM is not only the result of increased bone resorption, but also of repressed bone formation [30]. Furthermore, down-regulation of Dkk-1 expression seems to Role of Noggin in Osteolytic Bone Metastasis mediate the osteoinductive activity of endothelin-1 (ET-1) [31]. Another extracellular antagonist of the Wnt pathway, sFRP-2, may also contribute to this mechanism of inhibition of bone formation [32]. The modulation of the osteoblast recruitment/activity and the possible contribution of inhibition of bone formation in osteolytic bone metastasis by solid cancers have received little attention. A limited number of quantitative histomorphometric studies in osteolytic metastases by a variety of epithelial cancers have shown that, besides the increase in bone resorption, there is also impairment in bone formation, especially in advanced lesions [33,34,35]. It has been suggested that in these lesions bone resorption is uncoupled from the subsequent bone formation phase, which usually follows in normal bone remodeling [36]. This phenomenon may be mediated by a direct, negative influence of cancer cells on osteoblast recruitment, survival and activity, as shown in vitro for osteolytic CaM and CaP cell lines [37,38,39]. The present investigation, showing that indexes of bone formation are impaired in tibiae xenografted with PC-3 cells, further supports the clinical findings above and strongly suggests that a mechanism of uncoupling bone formation from resorption is also operating in this model. However, the identity of the molecules mediating this inhibitory effect on osteoblasts is yet unknown. Antagonism of BMP activity by noggin is critical for embryonic chondro-osteogenesis and joint formation [40]. Osteoblast-target- ed over-expression of noggin results in osteopenia as the result of impaired osteoblast recruitment [41,42], indicating that the extracellular modulation of the BMP concentration is also essential in adult life for the control of bone formation during bone remodeling. BMPs and noggin reciprocally induce their expression in osteoblasts [23], indicating that a positive feedback is necessary for maintaining an optimal balance between BMP and noggin concentrations in the bone microenvironment. Bone metastatic cancer cells may interfere with this balance by secreting an excess of either BMPs or noggin. It has been reported that BMP-6 expression positively correlates with CaP progression [43,44] and that BMP-6 is the BMP primarily responsible for inducing an osteoblast response in mouse models of CaP bone metastasis [17,45]. However, in one of these reports we demonstrated that, in addition, osteoinductive cancer cells lack secretion of the BMP inhibitor noggin and that noggin forced expression in these cells abolishes their osteoinductive activity in vivo. Therefore, an unopposed effect of an excess of BMP-6 locally released by cancer cells is also a determinant of the osteoblast response both in CaP and CaM bone metastasis [17]. Likewise, low expression of the Wnt antagonist Dkk-1 seems to favor the Wnt-induced osteoblast response in CaP bone metastasis [46]. These two studies proved for the first time that in osteoblastic bone metastases the physiological, tight balance between osteoinductive BMP-6 and/ or Wnt proteins and their antagonists is tilted toward the first and, thus, favors an abnormal osteoblast response. In contrast, osteolytic CaM cell lines express BMP-2 and -4, while the osteolytic CaP cell line PC-3 expresses BMP-3 [17,47]. However, both CaP and CaM osteolytic cell lines express low or no BMP-6 [17,45] and BMP-7 [48,49] and, most importantly, secrete constitutively relative high amounts of noggin [17]. Thus, in osteolytic bone metastases the balance between BMPs and noggin seems to be altered in a direction opposite to that of osteoblastic bone metastases. The present study proves that this noggin-tilted balance is responsible for the suppression of bone formation and, thus, prevents repair of the osteolytic lesion. Furthermore, the demonstration that specific targeting of the cancer cell-derived noggin preserves bone formation/repair strongly suggests noggin as a major ‘‘un-coupling factor’’ in osteolytic bone metastasis and PLoS ONE | www.plosone.org 5 January 2011 | Volume 6 | Issue 1 | e16078