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From the Urology Research Laboratory, Department of Urology and Department of Clinical Research,* University of Bern, Bern, Switzerland; Department of Endocrinology, Department of Urology, and Department of Molecular Cell Biology, Leiden University Medical Centre, Leiden, The Netherlands; Biochemical Cell Physiology,¶ German Cancer Research Center, Heidelberg, Germany; and the Academic Urology Unit,|| University of Sheffield, Royal Hallamshire Hospital, Sheffield, United Kingdom
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Abstract |
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Several factors stimulating osteoblast proliferation and differentiation in a paracrine manner have been shown to be released by CaP and CaM cells in the bone microenvironment and have been postulated to mediate osteoblast response in bone metastasis.3,4 Factors that modulate proliferation and differentiation can act directly on the osteoblast progenitors or indirectly by activation of factors involved in their generation.4 Paradigmatic molecules regulating directly osteoblast generation are the bone morphogenetic proteins (BMPs).5 BMPs were first identified by their ability to induce ectopic chondro-osteogenesis in vivo.6 They play a crucial role in skeletal and joint morphogenesis, bone remodeling, and fracture repair by inducing proliferation, lineage determination, differentiation, and apoptosis in chondrocyte and osteoblast precursors.7,8 Subsequently, they were shown to act as multifunctional regulators of embryonic patterning and organogenesis, tissue remodeling, and repair.9,10 A crucial regulatory mechanism is the modulation of BMP signaling by a class of secreted molecules, BMP antagonists, functioning through direct association with BMPs, thereby sequestering BMPs in the extracellular space and preventing binding to cell surface receptors. BMP antagonists have been divided in three subfamilies: the differential screening-selected gene aberrant in neuroblastoma (DAN), the twisted gastrulation, and the chordin/noggin.11
Antagonism of BMP activity by noggin and chordin is critical for embryonic chondro-osteogenesis and joint formation.12-14 Osteoblast-targeted overexpression of noggin15,16 and gremlin17 results in osteopenia attributable to impaired osteoblast recruitment and function, indicating that the extracellular control of the BMP concentration is also essential in adult life for maintaining the balance between bone resorption and bone formation during bone remodeling. Expression of BMP antagonists is BMP-dependent, indicating that a feedback mechanism maintaining an optimal ratio between BMPs and BMP antagonists is also necessary to achieve optimal bone mass.18 Further evidence that antagonism with molecules directly involved in osteoblast generation, such as the Wnt protein family,19-22 is critical for modulating bone mass has been given by the finding that the rare human disease sclerosteosis, characterized by excessive bone formation of the whole skeleton, is attributable to the loss of expression of the SOST gene,23 encoding the Wnt antagonist sclerostin.24,25 Conversely, bone-targeted overexpression of sclerostin in mice causes osteopenia.26 Furthermore, it has recently been shown that dickkopf-1 (DKK-1), another antagonist of Wnt signaling, modulates the osteoblast reaction in osteolytic foci of multiple myeloma.27
We hypothesized that loss of BMP and Wnt antagonist expression in bone metastatic CaP and CaM cells would unmask the osteoinductive effects of BMPs and Wnts released by the cancer cells at the bone metastatic site. To test this hypothesis, we first evaluated the expression in vitro of extracellular BMP and Wnt antagonists, as well as the expression of osteoinductive and osteolytic cytokines, in a variety of CaP and CaM cell lines, which possess either osteolytic or osteoinductive potential in vivo. The pattern of expression was verified also in vivo for CaP cell lines xenografted into bone and in clinical samples of bone metastasis. We further investigated whether forced expression of the BMP antagonist noggin in an osteoinductive CaP cell line would abolish the osteoblast response in its experimental bone metastasis in vivo.
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Materials and Methods |
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TopAbstractMaterials and MethodsResultsDiscussionReferences |
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Male CB17 SCID and BALB/c nude mice were purchased from Charles River France (L’Arbresle, France) and housed in individual ventilated cages according to the Swiss guidelines for the care and use of laboratory animals. Mice were 7 weeks old when used for the intraosseous implantation of tumor cells. For surgical manipulation, mice were anesthetized as described previously.28 Mice were sacrificed by CO2 euthanasia at the end of the observation period or at first signs of distress.
Clinical Samples of Bone Metastasis
Three CaP and one CaM clinical specimens of bone metastasis were obtained by radiography-guided biopsy, with prior informed consent of the patient and approval by the ethics committee of the University of Heidelberg–Mannheim, Germany. The CaM bone metastasis was classified as osteolytic whereas the three CaP bone metastases were classified as osteoblastic according to radiographical and histological criteria by a certified radiologist and a certified pathologist.
Bone specimens were snap-frozen in liquid nitrogen immediately after surgical excision and embedded in precooled RNase-free water. Twelve-µm-thick cryosections were obtained with a tungsten carbide blade (C profile; Leica Microsystems, Bensheim, Germany) and the aid of the CryoJane tape-transfer system (CTS; Instrumedics Inc., Hackensack, NJ) in a cryomicrotome (CM 3050; Leica Microsystems) set at –24°C. Bone cryosections were transferred onto 4x adhesive-coated slides (Instrumedics) as described recently,29 fixed in 70% ethanol, stained with Mayer’s hematoxylin (Sigma Diagnostics Inc., St. Louis, MO) and eosin Y (Merck, Darmstadt, Germany), and dehydrated by increasing grades of ethanol followed by xylene.
Laser Capture Microdissection
The P.A.L.M. Robot Micro-Beam (P.A.L.M. Instruments, Bernried, Germany) was used to excise by laser capture microdissection 
10,000 pure cancer cells from each clinical specimen of bone metastasis. The selection of purely neoplastic cells was in each case verified by a certified pathologist.
Cell Lines and Cell Culture
We studied a range of human CaP and CaM cell lines, with different tumorigenic and metastatic potential, shown to induce either osteolytic or osteosclerotic bone metastases, as shown by others28,30-35
and by us (A.W. and M.G.C., not shown) (Table 1)
. The osteolytic human CaP cell line PC-3 [American Type Culture Collection (ATCC)/LGC Promochem, Molsheim, France] and its isogenic clone PC-3M-Pro4, selected in vivo for enhanced metastatic potential32
(kindly provided by Dr. I.J. Fidler, Department of Cancer Biology, The University of Texas M. D. Anderson Cancer Center, Houston, TX), were grown in Dulbecco’s modified Eagle’s medium. The osteoinductive, androgen-dependent and non-bone metastatic human prostate cancer cell line LNCaP, and its isogenic variants C4-2 and C4-2B, androgen-independent and spontaneously metastasizing to bone after orthotopic implantation30
(kindly provided by Dr. L. Chung, Winship Cancer Center, Emory University, Atlanta, GA) were grown in T-medium. The osteolytic human mammary cancer cell line MDA-MB-231 (ATCC) and its isogenic clone MDA-231B, selected after sequential passaging in vivo for bone-restricted metastatic potential28
were grown in Dulbecco’s modified Eagle’s medium. The osteoinductive human mammary cancer cell lines T-47D and ZR-75-1 were purchased from ATCC and cultured in RPMI 1640 medium. The mouse osteoblast-like cell line KS48336
was cultured routinely in phenol red-free minimum essential medium-
. All media were supplemented with 10% fetal bovine serum (BioWittaker, Verviers, Belgium). Cell lines were stimulated either with 10 ng/ml of recombinant human transforming growth factor (TGF)-ß1 (R&D Systems Europe Ltd., Abingdon, UK) or 100 ng/ml of recombinant human BMP-2 (R&D Systems) or 100 ng/ml of recombinant human BMP-6 (kindly provided by Prof. S. Vukicevic, Genera, Croatia) for 6 or 24 hours.
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Cells were seeded at the density of 1.25 to 2.5 x 104 cells/cm2. After 1 day, the medium was replaced with serum-free medium, and the cells were cultured for a further 48 hours. The cell-conditioned media (CM) were centrifuged and stored in aliquots at –20°C for later use. The cell number was determined and, where necessary, serum-free medium was added to the CM to normalize for differences in cell density between samples.
Cell Proliferation Assay
Cells were seeded at the density of 104 cells/cm2 and cultured for a total of 8 days. Cell proliferation was determined daily with a tetrazolium salt-based method (MTT assay, Cell Proliferation Kit I; Roche Diagnostics, Rotkreuz, Switzerland) according to the manufacturer’s protocol.
Real-Time Polymerase Chain Reaction (PCR)
Total RNA extraction from subconfluent cultures of the various cell lines and from intraosseous xenografts of parental, noggin-, and mock-transfected C4-2B/luc+ cells, and of PC-3 and PC-3M-Pro4 cells, was performed with RNeasy (Qiagen, Hombrechtikon, Switzerland). Reverse transcription was performed with M-MLV-RT (Promega, Wallisellen, Switzerland) and random primers (Roche Diagnostics). Human-specific real-time PCR (TaqMan) primers and probes (Applied Biosystems, Rotkreuz, Switzerland) are listed in Table 2
. Extraction of total RNA from pure CaP or CaM cells, laser-microdissected from clinical specimens of bone metastasis, was performed according to the TriSpin method37
with several modifications, as previously described.29
cDNA was synthesized using SuperScript III reverse transcriptase (Invitrogen, Karlsruhe, Germany) and PCR amplified in Mx3000P (Stratagene, Amsterdam, The Netherlands) using QuantiTect SYBR Green PCR kit (Qiagen) according to the manufacturers’ protocols. Sense and anti-sense human-specific noggin primers used were 5'-TGTGCAAGCCGTCCAAGT-3' and 5'-GAGCACTTGCACTCGGAAAT-3'.
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Colony stimulating factor-1 (CSF-1) concentration in serum-free CM was determined by the Quantikine human CSF-1 immunoassay (R&D Systems), parathyroid hormone-related protein (PTHrP) by a two-site immunoradiometric assay (Nichols Institute Diagnostics, Bad Vilbel, Germany), and endothelin-1 (ET-1) by enzyme-linked immunosorbent assay (Biomedica, Wien, Austria). Protein secreted into the medium was normalized to the cell number at the end of the culture period.
Construction of the Noggin Expression Vector
The full-length human noggin cDNA was excised from the plasmid pBSII SK+.hNG (kindly provided by Regeneron Pharmaceuticals, Inc., Tarrytown, NY) with KpnI and NotI and ligated into the pcDNA3.1/Hygro expression vector (Invitrogen) (pcDNA3.1/Nog).
Stably Transfected Clones
The C4-2B cell line was transfected with the luciferase (luc) expression vector pCMVluc28 with SuperFect transfection reagent (Qiagen). A luc-positive clone was selected and transfected with pcDNA3.1/Nog to generate C4-2B-Nog clones and with the original plasmid pcDNA3.1/Hygro to generate C4-2B-mock clones.
Immunoblotting
Noggin expression in transfected C4-2B cells was determined in concentrated CM and in cell lysates prepared with radioimmunoprecipitation assay buffer supplemented with 1 mmol/L phenylmethyl sulfonyl fluoride (Sigma, Buchs, Switzerland). Protein concentration was determined by Dc protein assay (Bio-Rad, Reinach, Switzerland), and proteins were separated on 12% sodium dodecyl sulfate-polyacrylamide gels (30 µg of total protein per lane) and transferred on Hybond-P membranes (Amersham Biosciences, Otelfingen, Switzerland). Membranes were incubated with 40 ng/ml of a rat monoclonal antibody against the human native noggin protein38 (RP57-16; kindly provided by Dr. A.N. Economides, Regeneron Pharmaceuticals, Inc.) and detected with an horseradish peroxidase-labeled anti-rat secondary antibody (1:1000; Amersham Biosciences). A mouse monoclonal anti-actin antibody (1:5000; Chemicon International, Juro Supply GmbH, Lucerne, Switzerland) was used as loading control. Immunoreactivity was visualized with the ECL Advanced chemiluminescence substrate (Amersham Biosciences) using the VersaDoc imaging system and QuantityOne imaging software (Bio-Rad).
Secretion of Biologically Active Noggin by Stably Transfected Clones
For osteoblast differentiation assays, KS483 cells were seeded at a density of 1.5 x 104/cm2. At confluency (day 4), ascorbic acid (50 µg/ml; Merck Inc., Whitehouse Station, NJ) and CM of parental C4-2B, and of C4-2B-derived Nog and mock clones to a final concentration of 10% (v/v) or recombinant mouse noggin (R&D Systems) were added to the cultures. At day 7 alkaline phosphatase (ALP) activity in cell lysates was determined and corrected for the amount of DNA in the culture as previously described.36
Intraosseous Implantation of Parental, Noggin-, and Mock-Transfected C4-2B/luc+ Cells, and of PC-3 and PC-3M-Pro4 Cells
The cells were inoculated into the marrow cavity of the left tibia at the density of 5 to 8 x 105/10 µl of phosphate-buffered saline, as previously described.28 A group of five to seven animals was inoculated with each cell clone. A further group of five to seven animals, inoculated into the bone marrow cavity with phosphate-buffered saline alone (= sham), served as control. A first experiment with parental and three noggin- (Nog17, 19, and 20) and mock (mock1, 2, and 4)-transfected C4-2B/luc+ cells was terminated after 8 weeks. A second experiment with parental and only one noggin (Nog20)- and one mock-transfected (mock4) C4-2B/luc+ cells was terminated after 12 weeks. The experiments with PC-3 and PC-3M-Pro4 were terminated after 5 and 4 weeks, respectively.
Radiography
Radiographs of the mice were taken using X-Omat TL films (Kodak, Lausanne, Switzerland) as previously described.28
Whole Body Bioluminescent Imaging (BLI)
BLI was performed as previously described28 at 2-week intervals.
Peripheral Quantitative Computed Tomography (pQCT)
Bone mineral density and content of the tibia site of the tumor xenografts were determined with a small animal pQCT scanner (XCT Research SA; Norland Stratec, Pforzheim, Germany) at the end of experimental periods indicated above. Measurements were performed at 2.5 mm and 3 mm distal from the reference point (cleft of the knee joint).
High-Resolution Micro-CT
At the end of the experimental period indicated above, representative tibiae from a mouse implanted with C4-2B/luc+ cells and a sham-operated mouse were scanned with a micro-CT (Skyscan 1076; Gloor Instruments, Uster, Switzerland) at a voxel size resolution of 9 µm.
Histochemistry
The left tibia, site of intraosseous implantation of parental and stably Nog- and mock-transfected C4-2B/luc+ cells, were fixed and processed for paraffin embedding. Four-µm serial sections were stained either with Mayer’s hematoxylin and eosin Y or for tartrate-resistant acid phosphatase as described previously.39 The number of osteoclasts/bone surface was determined using the Scion Image software (Scion, Frederick, MD). Osteoclasts were counted as multinucleated tartrate-resistant acid phosphatase-positive cells on at least 5-mm total bone surface adjacent to tumor, on three mid-sagittal sections for each individual bone specimen.
Statistical Analysis
Rates of proliferation in vitro and growth in vivo were compared with the two-way analysis of variance test. The effect of cell-conditioned media on the ALP activity of KS483 cells and the bone architectural parameters obtained by pQCT were analyzed by the one-way analysis of variance. Osteoclast numbers were compared by the unpaired t-test.
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Results |
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To test whether differential expression of BMP and/or Wnt antagonists may explain the osteoblastic or osteolytic response in bone metastases, we investigated noggin, DAN, gremlin, SOST, and DKK-1 mRNA expression in vitro in a set of CaP and CaM cell lines ordered according to their osteoinductive or osteolytic potential in vivo (Table 1)
. All of the osteolytic cell lines tested, either of CaP or CaM origin, expressed considerable amounts of noggin mRNA, the CaP cell lines expressing approximately threefold higher mRNA levels than the CaM cell lines. In contrast, all of the osteoinductive CaP and CaM cell lines expressed noggin mRNA at extremely low levels (Figure 1A)
, which were in average almost 400-fold lower than those expressed by the osteolytic cell lines. This differential noggin mRNA expression was also reflected in different levels of secreted protein (Figure 1A)
. An equivalent pattern of distribution between osteoinductive and osteolytic cell lines was evident for DKK-1 mRNA expression. Although osteoinductive cell lines indifferently showed complete lack of its expression, all of the osteolytic cell lines showed considerable amounts of DKK-1 expression, the CaP cell lines expressing approximately fourfold higher mRNA levels than the CaM cell lines (Figure 1B)
. All of the osteolytic cell lines and osteoinductive CaM cell lines expressed DAN mRNA, whereas the osteoinductive CaP cell lines expressed it at very low or undetectable levels (Figure 1C)
. SOST mRNA expression was detectable at a very low level only in MDA-MB-231 cells, but it was absent in all remaining cell lines (not shown). Gremlin mRNA could not be detected in any of the cell lines tested (not shown).
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. Thus, the amount of constitutive or BMP-induced noggin expression in osteoinductive cell lines was negligible when compared with osteolytic cell lines and may still not have been sufficient for antagonizing the BMP locally released in vivo. Nevertheless, the increase in noggin expression induced in vitro by BMP-2 and BMP-6 seems to exclude that the lack of constitutive noggin expression in the osteoinductive cell lines is the consequence of an im-paired response to BMPs. TGF-ß1 did not stimulate noggin expression in any of the tested cell lines (not shown). Collectively, the results above suggest a strong correlation between lack of noggin and DKK-1 expression in vitro and the osteoinductive potential in vivo of all the CaP and CaM cell lines examined in this study. Expression of noggin in Pure CaP or CaM Cells, Laser-Microdissected from Clinical Specimens of Bone Metastasis
Noggin has been reported to be expressed in cells of the osteoblast lineage.41,42 Thus, to avoid the interference by noggin expressed by the bone stromal component in human bone metastasis biopsies, we analyzed noggin expression in 100% pure cancer cells isolated from metastatic bone lesions by laser capture microdissection. Relative noggin expression, normalized to ß-actin, was found to be 0.353 in an osteolytic CaM bone metastasis, and 0.182, 0.006, and 0.000093 in three osteoblastic CaP bone metastasis, respectively.
Expression of Members of the TGF-ß Superfamily in Osteolytic and Osteoinductive Cell Lines in Vitro
BMPs are considered major players in the osteoblast response to cancer bone metastasis.3,4
The osteoinductive cell lines, especially those of CaP origin, mainly expressed BMP-6 (Figure 2, A–D)
. In addition, the osteoinductive cell line T-47D expressed low levels of BMP-4 (Figure 2C)
. Conversely, osteolytic cell lines preferentially expressed BMP-2, -3, and -4, but very low amounts of BMP-6 (Figure 2, A–D)
. Discrete amounts of prostate-derived factor (PDF) were mainly expressed in the CaP cell lines, either osteoinductive or osteolytic (Figure 2E)
. Therefore, no pattern of BMP family member mRNA expression characteristic either of osteoinductive or of osteolytic cancer cell lines emerged from the cell lines analyzed in this study. In addition, expression of one or more members of the BMP family by the osteoinductive cell lines further suggested that their extremely low noggin expression is not attributable to lack of induction by endogenous BMP.
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. Thus, tumor cell-derived TGF-ß1 seems not to be related to the osteoinductive property of cancer cell lines. Expression in Vitro of Cytokines Influencing Bone Resorption
The relevance of bone-resorbing cytokines such as PTHrP, receptor activator of nuclear factor-
B ligand (RANKL), and its decoy receptor osteoprotegerin (OPG) in osteolytic bone metastasis is well documented.43,44
However, less attention has been devoted to a systematic investigation aiming at clarifying whether the absence of expression of bone-resorbing cytokines in cancer cells is a prerequisite for allowing full manifestation of their osteoinductive potential.
In the cell lines examined in this study, we found that only osteolytic cell lines expressed substantial amounts of PTHrP and CSF-1 mRNA and protein and interleukin (IL)-8 mRNA. Although osteolytic CaP cell lines expressed and secreted predominantly PTHrP and IL-8 (Figure 3, A and B
; and Supplemental Figure 1, see http://ajp.amjpathol.org), osteolytic CaM cell lines expressed and secreted mostly CSF-1 (Figure 3, C and D)
. These results are in agreement with previous studies reporting the expression of PTHrP,45,46
CSF-1,47-49
and IL-850,51
in both CaP and CaM cell lines.
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. OPG mRNA was expressed at detectable levels by both CaM or CaP osteolytic cell lines and by the osteoinductive CaM cell line ZR-75-1 (Figure 3F)
. OPG mRNA expression in intraosseous xenografts of the osteolytic cell line PC-3 was approximately threefold higher than in vitro, but it was reduced by 3.5-fold in intraosseous xenografts of the variant cell line PC-3M-Pro4. OPG mRNA was not detectable in intraosseous xenografts of the osteoinductive cell line C4-2B (not shown). Lack of RANKL expression by bone metastatic cancer cell lines has already been described.52,53
This is consistent with the view that in osteolytic bone metastasis RANKL is osteoblast-derived and is induced by cancer cell-derived PTHrP.44,53
Our finding showing OPG expression in vitro and in vivo prevalently in osteolytic, but not in osteoinductive, cancer cell lines is in agreement with previous reports.52,53
Osteolytic cell lines exhibit high tumorigenic and metastatic potential and their OPG expression is consistent with the postulated role of OPG in protecting cancer cells from tumor necrosis factor-related-apoptosis inducing ligand (TRAIL)-induced apoptosis.54
Taken together, the observations above, although limited to a small number of cancer cell lines, suggest that the osteolytic potential of cancer cell lines depends prevalently on their ability to stimulate osteoclast generation directly by secreting CSF-1 and IL-8, and, indirectly, via PTHrP-induced release of RANKL by bone stromal cells. Growth Characteristics and Bone Effects in Vivo of the C4-2B Cell Clone Stably Transfected with the pCMV-Luciferase Vector (C4-2B/luc+ Cells)
Based on the observation that low expression of noggin in vitro correlates with the osteoinductive potential in vivo of cancer cell lines, we tested whether forced noggin expression in an osteoinductive cell line, constitutively lacking noggin expression, could abolish the osteoblast response in vivo. We have previously reported on the sensitivity and reliability of noninvasive whole body BLI as a method to monitor and quantify the development and progression of bone metastasis in living animals.28
To apply this method, we first established C4-2B cell clones with stable expression of the luc gene. One clone was selected based on high luc expression, gene expression profile of osteotropic cytokines in vitro, tumor take, and bone reaction after intraosseous implantation in vivo equivalent to those of the parental C4-2B cells (not shown). After implantation of C4-2B/luc+ cells into the tibia marrow cavity of immunodeficient mice, tumor take was 100%, as illustrated in Figure 4A
. Tumor growth was monitored and quantified as bioluminescent emission, which was clearly detectable in all animals from day 28 on (Figure 4B)
. Signal intensity further increased in the following 8 weeks. Radiographs taken 12 weeks after intratibial inoculation of C4-2B cells, at which time animals were sacrificed, showed an enlargement in the upper half of the bone shaft, with cortical thinning and mixed areas of osteoblastic and osteolytic reaction in the upper one third of the bone shaft (Figure 4C)
. In contrast, radiographs taken 3 weeks after intratibial inoculation of PC-3 cells show only minor modifications of the bone shape and cortex but a well-defined area of osteolysis at the site of inoculation. Ex vivo micro-CT analysis of sham-operated and C4-2B-implanted bones demonstrates profound changes in the bone architecture of the tibia implanted with C4-2B cells, compared with the sham-operated tibia (Figure 4D)
. A marked enlargement of the bone shaft, associated with a thinning of the cortex, and an increased density of randomly oriented and thinner trabeculae were the principal modifications induced by the presence of tumor cells. The presence of an osteoblast reaction was confirmed by the histological examination showing thin and irregular bone trabeculae covered by active osteoblasts within bone marrow areas invaded by cancer cells (Figure 4E)
. These alterations are consistent with a mixed, osteosclerotic and osteolytic, reaction equivalent to that described for the C4-2 cell line, from which the C4-2B cells are derived.31,55
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C4-2B/luc+ cells were transfected with a noggin expression plasmid (Nog) or with the empty pcDNA3.1 vector (mock). Three noggin-expressing clones (Nog17, Nog19, and Nog20) and three mock clones (mock1, mock2, and mock4) were selected and tested for noggin protein expression (Figure 5A)
. No noggin was detected in the cell lysate of C4-2B/luc+ cells, which is consistent with the lack of noggin secretion by parental C4-2B cells (Figure 1A)
. All three Nog clones show detectable noggin protein, both in the cell lysate and in the culture supernatant. Nog19 and Nog20 secreted almost equivalent amounts of noggin protein, whereas Nog17 secreted a considerably lower amount, reflected by their noggin mRNA expression (not shown). In contrast, none of the three mock clones secreted detectable amounts of noggin (not shown).
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. No significant effect on ALP expression was induced by CM from mock clones. The difference for the levels of biologically active noggin between the Nog17 and Nog19/Nog20 cloneswas considerably less pronounced than for the amounts of the immunoreactive, secreted protein (Figure 5A)
. Secretion by Nog19/Nog20 clones of additional factors stimating ALP expression in KS483 cells, and thus interfering with the noggin inhibitory activity, may explain this discrepancy.
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It is widely recognized that the reciprocal interactions between cancer cells and the bone microenvironment in vivo may modify the phenotype and the pattern of cytokines expressed by cancer cells in vitro.35
To exclude this possibility, we analyzed noggin and PTHrP expression in vivo in intraosseous xenografts of either osteolytic or osteoinductive CaP cell lines. The osteolytic PC-3 and its derivative PC-3M-Pro4 cell line expressed both noggin and PTHrP, although to a lesser extent than in vitro. Instead, the osteoinductive C4-2B cell line, as well as the mock clones derived from the C4-2B/luc+ cells did not express either noggin or PTHrP (Figure 5, B and C)
. Thus, the differential expression of noggin and PTHrP between osteolytic and osteoinductive CaP cell lines shown in vitro could be confirmed in vivo. Noggin mRNA expression at the end of the in vivo experiment was high in both the C4-2B cell clones Nog17 and Nog20 (Figure 5B)
, thus confirming persistence of noggin expression in vivo. Equally to the parental C4-2B cells, Nog17/Nog20 and mock1/mock4 transfected clones did not express PTHrP (Figure 5C)
. DKK-1 mRNA expression in vivo was undetectable in parental and noggin- and mock-transfected C4-2B/luc+ cells (not shown).
Growth Characteristics in Vitro and in Vivo of C4-2B/luc+/Nog+ Clones
The growth rate in vitro was equivalent for Nog19, mock1, and mock2 cell clones and the parental C4-2B/luc+ cell line (Figure 5D)
. In contrast, Nog17, Nog20, and mock4 clones showed a slightly, but significantly (P < 0.05), lower growth than the parental C4-2B/luc+ cell line. Nog19 and Nog20 cells showed an intraosseous growth rate and an end-point tumor burden, as assessed by whole body BLI monitoring in the living animals, equivalent to that of parental cells, whereas Nog17 and all of the three mock-transfected clones grew to a lesser extent (P < 0.01 at day 56) (Figure 5E)
. Equivalent results were obtained in a second experiment in which the intraosseous growth rate of parental C4-2B/luc+, Nog20, and mock4 cells was monitored for 80 days (not shown). This marginal effect on tumor growth could be partially attributable to a difference in the original number of tumor cells inoculated. Taken together, the BLI monitoring of tumor growth indicates that noggin forced expression has only a negligible effect on the intraosseous growth rate and tumor burden of C4-2B cells.
Effects on Bone in Vivo of C4-2B/luc+/Nog+ Clones
To evaluate the influence of forced noggin expression in C4-2B/luc+on the bone response induced by this osteoinductive CaP cell line in vivo, we analyzed by pQCT the bone structure of tibiae inoculated with either parental or Nog- or mock-transfected cells. In a first experiment, pQCT analysis was performed 56 days after intraosseous implantation (Figure 6A)
. Tibiae inoculated with the parental C4-2B/luc+, when compared with those sham-operated, showed an enlargement of the bone shaft associated with a thinning of the cortex and an increase in trabecular bone, as indicated by the increase in total bone area (TBA), trabecular bone area (TrBA), and content (TrBC) and by the decrease in the cortical bone area (CBA) and content (CBC). These modifications of the bone architecture, which are consistent with those already shown in Figure 4C
, suggest that the osteoinductive effect induced by the C4-2B/luc+ tumor is prevalently acting on the trabecular bone. The enlargement of the bone shaft associated with the thinning of the bone cortex may be a direct consequence of the altered bone remodeling and/or the effect of an attempt by the bone to compensate for the reduced mechanical competence of the tumor-induced trabecular bone.
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. This effect was also evident for Nog19 and Nog20 clones (not shown). This result clearly indicates that forced noggin expression in C4-2B/luc+ cells alone is sufficient to abolish their osteoinductive activity. The observation that Nog20 expressed in vivo only twice as much noggin mRNA compared with the Nog17 clone (Figure 5B)
may explain why both these Nog-cell clones exerted equivalent bone effects. Noggin does not increase tumor growth rate and burden (see above, Figure 5E
), in agreement with a previous report by others.56
This excludes that an increase in tumor burden may be responsible for the normalization of TrBA and TrBC.
In the experiment above none of the tibia implanted with the three mock-transfected clones (mock1, 2, and 4) showed an extent of modifications of the pQCT parameters equivalent to those seen in the tibiae implanted with the parental cells. This is most probably attributable to the fact that none of the mock clones developed within 8 weeks an intraosseous tumor burden and, consequently, an osteoinductive effect equivalent to that of the parental or Nog-transfected C4-2B/luc+ cells. Therefore, in a next experiment the period of observation was prolonged to 12 weeks after the intraosseous implantation of parental C4-2B/luc+ cells, Nog20, and mock4 clones. At this time point pQCT analysis showed equivalent bone architectural modifications for both mock cell- and parental cell-implanted tibiae. Consistent with the previous experiment, implantation of the Nog20 clone reverted the indices of excess of bone formation, such as high TrBA and TrBC, to the normal values of the sham-operated animals, as shown for the Nog20 clone (Figure 6B)
, corroborating that forced expression of noggin blunts completely the osteoinductive effect of the parental C4-2B/luc+ cells. In contrast to the previous experiment, CBA and CBC of bones inoculated with Nog20 cells did not attain the values of the sham-operated animals, although significantly different from those inoculated with parental cells. This was also reflected by the smaller TBA of bones implanted with Nog20 cells compared with all other bones. It is conceivable that the longer exposure to the noggin inhibitory activity on bone formation may have exerted a detrimental effect on the periosteal bone apposition responsible for the age-related cortical drift.57
Effects of C4-2B/luc+/Nog+ Clones on Osteoclasts
In the experiment in which intraosseous xenografts of parental C4-2B/luc+ cells, Nog (Nog17, 19, and 20)-, and mock (mock1, 2, and 4)-transfected clones were allowed to grow for 56 days, histomorphometry shows a significantly lower number of osteoclasts in bones implanted with Nog20 cells (P < 0.01) than in sham-operated bones and in bones xenografted with parental C4-2B/luc+ cells or mock-transfected (mock1 and 4) clones. A tendency to a lower osteoclast number was also seen in tibiae inoculated with Nog17 cells, secreting lower amounts of noggin (Figure 5A
and Table 3
) but not attaining statistical significance (Figure 6C)
. This result confirms previous reports suggesting that noggin possesses an anti-bone-resorbing activity in vitro and in vivo, which is mediated by an inhibitory effect on osteoclast generation.42,58
Histomorphometry also showed that the osteoclast number in bones xenografted with parental C4-2B/luc+ cells and mock-transfected (mock1 and 4) clones does not significantly differ from that of sham-operated bones. This strongly suggests that intraosseous growth of C4-2B/luc+ cells does not alter basal bone resorption.
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The mechanism(s) determining the exaggerated osteoblast response in osteosclerotic bone metastases is poorly understood.4,43,59 An increased secretion and/or activation of factors inducing osteoblast recruitment by the bone metastatic cancer cells is commonly believed to cause the excess of bone matrix deposition.4 Our findings suggest a novel mechanism by which the lack of antagonist molecules to osteoinductive factors, rather than the excess of osteoinductive factors, contributes essentially to the osteoblast response in bone metastasis. A similar mechanism is at the origin of sclerosteosis, the sole example for a human congenital disease characterized by a generalized excess of bone formation.23
Several BMPs have been shown to be produced by CaP cell lines.60,61 Here we show that at least one BMP species is expressed by all CaP and CaM cell lines independently of their osteoinductive or osteolytic potential. Noggin has been used as a tool to block BMP function because it is a specific inhibitor of BMP activity and does not seem to have actions independent of BMP binding. However, noggin possesses a rather promiscuous binding activity among various BMPs,62 which precludes a precise definition of the BMP(s) prevalently involved in determining the osteoblast response. Nevertheless, a recent report has provided evidence for a primary role of BMP-6 in the osteoinductive activity of the CaP cell line LuCaP 23.1.60 This study, showing that forced noggin expression abolished the osteoinductive activity in vivo of the CaP cell line C4-2B, expressing mainly BMP-6, further supports this view.
Absent or greatly reduced expression of bone resorbing cytokines in osteoinductive cancer cell lines has also been postulated to unbalance further the bone response in favor of an osteoblast reaction.43,59 The lack of expression of bone-resorbing cytokines in osteoinductive CaM and CaP cell lines shown in the present study supports this view. To provide experimental evidence in vivo for this hypothesis, we are currently investigating the possibility of inducing osteolytic potential in vivo by forced CSF-1 expression in an osteoinductive CaP cell line.
It has been suggested that bone metastatic CaP cells induce an initial phase of bone resorption, which in turn promotes their growth and osteoinductive activity59
and, conversely, that repression of their osteoinductive activity unmasks their original osteolytic potential.63
In the present study repression of the osteoblast response by forced noggin expression in vivo reverses the bone structural parameters to normal control values, suggesting that there is a return to physiological bone remodeling and that C4-2B cells do not produce additional osteolytic activity. This is also substantiated by the facts that, at least in vitro, none of the osteoinductive cancer cell lines express the osteolytic cytokines PTHrP, CSF-1, and IL-8, and that in bones xenografted with C4-2B cells there is no increase in osteoclast number when compared with sham-operated bones. The normalization of bone parameters cannot be referred to an enhanced tumor volume because forced noggin expression does not increase tumor burden. In addition, noggin exerts an inhibitory effect on osteoclast generation, as shown here and by others,16,42,58
which may counterbalance osteolytic effects, if any, induced by C4-2B cells. Therefore, we postulate a model in which the osteoblast response in bone metastasis is, at least partially, the result of the lack of noggin secretion by cancer cells and, consequently, of the unopposed effect of the excess of BMPs released locally by cancer cells themselves. This leads to an increased recruitment of osteoblasts laying down an excess of bone matrix at sites of physiological bone resorption, with a net increase in the bone mass adjacent to cancer cell deposits. Concomitant lack of production by cancer cells of factors stimulating osteoclast generation and activity may further favor this imbalance. In this model cancer cells do not need to induce an initial phase of bone resorption to promote their growth and osteoinductive activity, because the excess of bone formation is coupled to physiological bone resorption (Figure 7)
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Whether lack of noggin expression is also relevant for determination of the osteosclerotic feature of bone metastasis in CaP and CaM patients has not yet been established. Noggin is expressed at very low levels in adult tissues in general and in the adult skeleton.15 This has hampered our attempts to test by traditional methods of mRNA and protein detection in situ whether noggin is differentially expressed by cancer cells also in clinical samples of osteolytic and osteoblastic bone metastasis. On the other hand, noggin expression by the bone stromal component may interfere with its mRNA analysis by quantitative PCR when performed on bulk tissue samples of human bone metastasis. A preliminary analysis of noggin mRNA expression in a very limited sample number of pure cancer cells isolated by laser-capture microdissection of bone metastasis biopsies seems to confirm that noggin is expressed at lower levels in osteoblastic than osteolytic bone metastases. We are currently extending this analysis to a larger number of clinical specimens of bone metastasis.
A causal role for Wnt in the pathogenesis of osteosclerotic bone metastasis has been demonstrated. The canonical Wnt signaling is involved in determining bone mass.20-22 The extracellular Wnt-antagonist DKK-1 secreted by myeloma cells is responsible for the suppression of the osteoblast activity in the osteolytic lesions that characterize this neoplastic disease.27 Conversely, it has been demonstrated that lack of DKK-1 expression, and thus an unopposed osteoblast response to Wnt signaling, is responsible for the osteoinductive activity of bone metastatic CaP cells.63 In addition, here we show that lack of DKK-1 expression correlates also with the osteoinductive potential of cell lines of CaM origin. Here we propose that, by a similar mechanism, lack of noggin expression by cancer cells may lead to an unopposed osteoblast response to BMPs and, therefore, concur to the pathogenesis of osteosclerotic bone metastasis of both CaP and CaM origin. Taken together, these considerations suggest that both BMP and Wnt signaling pathways play an essential role as osteoinductive factors in osteosclerotic metastases. However, the exact hierarchy of these signaling pathways in determining the osteoblast response is not yet defined. BMP and Wnt signaling clearly cooperate and co-regulate each other in promoting osteoblast differentiation. However, it is still controversial whether they operate in parallel or whether BMP activity is required downstream of a Wnt stimulus.66 Our results and previous reports56,63 show that both noggin- and DKK-1-mediated inhibition of the BMP and Wnt signaling, respectively, can repress the cancer-induced osteoblastic response in vivo. This supports the concept of a parallel cooperation between BMP and Wnt signaling in bone formation.
Bones affected by either osteolytic or osteosclerotic metastases are prone to pathological fractures. The dual inhibitory effect of noggin on the osteoblast and osteoclast reactions in bone metastatic lesions may result in a decreased incidence of pathological bone fractures. In addition, noggin may also exert a direct inhibitory effect on tumor growth.67 Therefore, noggin could prove to be useful as an adjuvant drug in the therapy of bone metastasis.
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Supported by the European Commission (grant PRIMA-504587), the Swiss National Foundation (3200-068409.02), the Genera Foundation, and the Department of Clinical Research, University of Bern.
R.S. and C.A.R. have equally contributed to the study presented here.
Supplemental material for this article can be found on http://ajp.amjpathol.org.
Accepted for publication September 13, 2006.
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3). Equal amounts of CM were immunoblotted with noggin antibody. Cells were stimulated with 100 ng/ml BMP-2 or BMP-6 for 24 hours.
