showing that blocking Dkk-1 activity rescues bone formation in animal models of MM [16]. Previously, we have reported that the osteoinductive and osteolytic potential in vivo of CaP and CaM cell lines can be defined in vitro by their differential expression not only of osteolytic cytokines, but also of the BMP antagonist noggin. Osteoinductive cancer cell lines lack noggin expression, and the functional relevance of this finding was emphasized by showing that noggin forced expression in an osteoinductive CaP cell line abolishes the osteoblast response induced by its bone metastases in vivo. Conversely, constitutive noggin expression in vitro seems to be the hallmark of osteolytic CaP and CaM cell lines [17]. We then argued that, in analogy to what has been found for Dkk-1 in MM, constitutive noggin release by osteolytic cancer cells might contribute, through inhibition of bone formation, to the osteolytic lesion in bone metastases of solid cancers. To test this hypothesis, we first explored whether inhibition of bone formation is a constituent of osteolytic lesions induced in bones xenografted with the human CaP cell line PC-3. We then investigated whether short hairpin RNA (shRNA)-mediated noggin suppression in PC-3 cells, constitutively secreting this protein [17], could preserve bone formation in the osteolytic lesions. The osteolytic lesions in bones xenografted with PC-3 cells showed morphological and histomorphometric parameters of enhanced osteoclast-mediated bone resorption and decreased osteoblast-mediated bone formation. In contrast, bones xenograft- ed with noggin-silenced PC-3 cells were characterized by structural and histological modifications indicative of bone formation/repair activity. Therefore, noggin suppression in the osteolytic cell line PC-3 seems to preserve the bone formation that normally follows bone resorption, as an effect of the ‘‘coupling phenomenon’’. Conversely, cancer cell-secreted noggin prevents the repair of osteolytic lesions by uncoupling bone formation from the osteoclast-mediated bone resorption, which is stimulated by cancer cell-derived osteolytic cytokines. Results Silencing of Noggin mRNA and protein expression in the PC-3/Fluc Cell clone by shRNA The human osteolytic CaP cell line PC-3, constitutively expressing the BMP antagonist noggin [17], was first transfected with a luciferase-encoding vector to generate luc-positive clones. A cell clone, PC-3/Fluc, was selected based on stable luc expression and on gene expression profile in vitro, tumor take and bone reaction after intra-osseous inoculation in vivo equivalent to those of the parental PC-3 cells (Figure S1). The PC-3/Fluc clone was subsequently transfected with a combination of three different noggin-specific shRNA or with a non-targeting shRNA to derive noggin knock-down (Nog-KD) and negative control (mock) clones, respectively. Two Nog KD clones (Nog-KD 14 and Nog-KD 17) were selected based on the substantial reduction in noggin mRNA expression, as compared to PC-3/Fluc cells (Figure 1A). Immunoblot analysis of noggin protein secretion in the culture supernatant from the Nog-KD clones 14 and 17 showed a reduction of 93% and 98%, respectively (Figure 1B). Mock 5 clone showed an increase in noggin mRNA expression. However, noggin protein expression in this clone and in mock 4 was not affected. Cell proliferation and expression of osteotropic factors of the Noggin-silenced PC-3/Fluc cells in vitro Cell proliferation in vitro of the mock and Nog-KD clones was not affected, as compared to parental PC-3/Fluc (Figure 2A). Role of Noggin in Osteolytic Bone Metastasis Figure 1. Noggin silencing mediated by shRNA inhibits noggin mRNA and protein expression. A. Noggin mRNA expression in PC- 3/Fluc cells, non targeting-vector-transfected clones (mock 4 and 5) and Noggin-KD clones (Nog-KD 14 and 17). The mRNA expression levels (+/ 2 SD) are quantified by real-time RT-PCR and normalized to b-actin as endogenous control; mRNA level in PC-3/Fluc cells is set as 100%; the mean of 2 to 3 independent experiments is shown. ***P,0.001, Nog-KD clones versus PC-3/Fluc and mock clones. B. Noggin protein secreted in the conditioned medium (CM) from PC-3/Fluc, mock and Nog-KD clones. Equivalent amounts of proteins from concentrated CM were immunoblotted with anti-noggin antibody. Equivalent protein loading was verified by staining with Coomassie blue the total protein content in the CM. doi:10.1371/journal.pone.0016078.g001 In order to verify whether noggin silencing would affect mRNA expression of relevant osteotropic factors, the expression of Dkk-1, PTHrP, RANKL, CSF-1 and IL-8 was also investigated (Figure 2B). The expression of Dkk-1 and PTHrP was not modified in all transfected clones, as compared to the PC-3/Fluc. Consistently with previous investigations in parental PC-3 cells [17], RANKL expression was undetectable in PC-3/Fluc, mock and Nog-KD clones. CSF-1 was moderately up-regulated in all mock and Nog-KD clones, although the elevation did not reach significance. IL-8 expression was significantly higher in the mock clones and, especially, in Nog-KD 17. It has been shown in osteoblasts in vitro that expression of noggin is BMP-dependent, indicating that a probable feedback mecha- nism is necessary to maintain a BMP/noggin balance, thereby limiting the BMP action on these cells [23]. A similar feedback has also been reported in some prostate cancer-derived cell lines [17,24]. The mRNA expression of BMP-2,-3,-6 and -7 was not modified in both Nog-KD clones as compared to parental PC-3, while BMP-4 mRNA was undetectable in both parental and Nog- KD cells (not shown). Therefore, noggin silencing in PC-3 cells does not alter either the level or the spectrum of their BMP expression. Effect on bone in vivo of Noggin-silenced PC-3/Fluc cells A first experiment was terminated at day 25 after injection for all groups of animals, while in a following experiment the observation period for the Nog-KD clones was prolonged to 33 days. In both experiments the radiographic analysis detected the onset of an equivalent osteolytic reaction in all groups of cancer cell-bearing bones as early as day 14 after tumor cell xenografting (not shown). At day 21 all xenografted tibiae still showed a similar number and extension of osteolytic lesions, and an enlargement of the bone shaft, as compared to sham-operated tibiae. However, in PLoS ONE | www.plosone.org 2 January 2011 | Volume 6 | Issue 1 | e16078