The matrix metalloproteinases MMP-2, MMP-3, MMP-7, MMP-9, and MMP-13 are highly expressed in the tumor-bone microenvironment, and, of these, MMP-7 and MMP-9 were found to be localized to bone-resorbing osteoclasts in human breast-to-bone metastases. In a bid to define the roles of host-derived MMP-7 and MMP-9 in the tumor-bone microenvironment, the tibias of MMP-7 and MMP-9 null mice were injected with osteolytic luciferase–tagged mammary tumor cell lines. Our data show that osteoclast-derived MMP-7 significantly contributes to tumor growth and tumor-induced osteolysis whereas osteoclast-derived MMP-9 had no effect on these processes. MMP-7 is capable of processing a number of nonmatrix molecules to soluble active forms that have profound effects on cell-cell communication, such as RANKL, a crucial mediator of osteoclast precursor recruitment and maturation. Therefore, the ability of osteoclast-derived MMP-7 to promote RANKL solubilization in the tumor-bone microenvironment was explored. Results revealed that levels of soluble RANKL were significantly lower in the MMP-7 null mice compared with wild-type (WT) controls. In keeping with this observation, MMP-7 null mice had significantly fewer osteoclast numbers at the tumor-bone interface compared with the WT controls. In summary, we propose that the solubilization of RANKL by MMP-7 is a potential mechanism through which MMP-7 mediates mammary tumor–induced osteolysis. Our studies indicate that the selective inhibition of MMP-7 in the tumor-bone microenvironment may be of benefit for the treatment of lytic breast-to-bone metastases. [Cancer Res 2009;69(16):6747–55]
- breast-to-bone metastasis
- matrix metalloproteinase
- receptor activator of nuclear κB ligand
Bone metastasis is a common event during breast cancer progression with the resultant lesions often being osteolytic ( 1). In the bone microenvironment, metastatic breast cancer cells hijack the normal bone remodeling process to induce aberrant activation of bone-resorbing osteoclasts ( 2). Increased bone resorption results in the release of sequestered growth factors from the bone matrix, such as transforming growth factor-β (TGF-β) and insulin-like growth factors (IGF). These factors subsequently promote tumor survival and growth, thus completing what has aptly been described as the vicious cycle of tumor-induced osteolysis ( 3).
Osteoclasts are critical for the completion of the vicious cycle, because they are the principal cells involved in the direct resorption of the mineralized bone matrix. Therefore, understanding how osteoclast precursors are recruited to areas requiring bone remodeling and understanding the mechanisms involved in controlling their maturation and activation is the key for the development of new therapies that can effectively stop the vicious cycle. To resorb bone, the osteoclast forms a resorptive seal on the mineralized bone matrix after retraction of the osteoblast canopy ( 4). Acidification of the resorption zone, in combination with collagenolysis, leads to the demineralization and degradation of the bone matrix, respectively ( 5). Osteoclasts express a variety of proteases, including the cysteine protease, cathepsin-K, and matrix metalloproteinases (MMP; ref. 6). Whereas cathepsin-K activity is critical for bone resorption ( 7), the role of osteoclast-derived MMPs is less clear. The MMPs are a family of enzymatic proteins that are often overexpressed in the tumor microenvironment ( 8). Collectively, MMPs are capable of degrading the entire extracellular matrix, but more recently, MMPs have been implicated as important mediators of cell-cell communication by virtue of their ability to process multiple nonmatrix molecules such as cytokines and growth factors to soluble forms, resulting in either enhanced or attenuated activities ( 9).
In the context of the tumor-bone microenvironment, preclinical animal studies have shown the efficacy of broad spectrum MMP inhibitors (MMPI) in preventing tumor growth and tumor-induced osteolysis ( 10– 12). However, the failure of MMPIs in human clinical trials prevents their application for the treatment of bone metastases ( 13). A main conclusion derived from these trials was the necessity for defining the precise roles of individual MMPs in disease processes that would allow for the generation of highly selective MMPIs. To this end, we have assessed the expression of MMPs in human clinical samples of osteolytic breast-to-bone metastasis. Whereas the expression of many MMPs was noted throughout the tumor/stroma, MMP-7 and MMP-9 were highly localized to bone-resorbing osteoclasts. Given the importance of the osteoclasts in driving the vicious cycle, the current study focused on determining if and how these osteoclast-derived MMPs affected tumor-induced osteolysis.
Materials and Methods
Reagents. All experiments involving animals were conducted after review and approval by the Office of Animal Welfare at Vanderbilt University. Deidentified human samples of frank osteolytic breast-to-bone metastasis (n = 11) were collected by curettage with institutional review board approval from Vanderbilt University from 2005 to 2008. Double null immunocompromised recombinase activating gene-2 (RAG-2) and MMP-7 mice were generated as previously described ( 14). Wild-type (WT) and MMP-9 null mice in the FVB/N-Tg background were kindly provided by Dr. Lisa Coussens, Department of Pathology, University of California San Francisco. A luciferase expressing syngeneic FVB mammary tumor cell line derived from the polyoma virus middle T model of mammary tumorigenesis, designated PyMT-Luc, was isolated in our laboratory and maintained as previously described ( 15). A luciferase tagged 4T1 mammary tumor cell line ( 16) was kindly provided by Dr. Swati Biswas of Vanderbilt Center for Bone Biology. All reagents were obtained from Sigma-Aldrich, except where specified.
Intratibial injection and in vivo quantitation of tumor growth. PyMT-Luc and 4T1-Luc tumor cells (105) in a 10-μL volume of sterile PBS were injected into the tibias of anesthetized immunocompetent or immunocompromised 6-wk-old mice that were WT or null for MMP-7 or MMP-9. The contralateral limb was injected with 10 μL of PBS alone and acted as a sham-injected control. The IVIS system (Caliper Life Sciences) was used to detect luminescence from PyMT-Luc and 4T1-Luc cells after intratibial injection. Firefly luciferin (120 mg/kg in sterile PBS, Gold Biotechnology, Inc.) was delivered retro-orbitally 1 to 2 min prior imaging. Mice were imaged at 24 h and every 3 d after surgery until day 9, which was previously determined to be the time point before tumor breach of the cortical bone in WT control mice. Living Image software (Calipers Life Sciences) was used to quantify the luminescence intensity in the tumor-bearing limb over time. Mice were sacrificed at 9 d postsurgery, and both the tumor-injected and contralateral control tibias were harvested. All animal studies were independently repeated at least twice.
Histology. Fresh human breast-to-bone metastases and tumor and sham-injected mouse tibias were fixed overnight in 10% buffered formalin and decalcified for 3 wk in 14% EDTA at pH 7.4 with changes every 48 to 72 h. Tissues were embedded in paraffin, and 5-μm thick sections were cut. For MMP-7, MMP-9, and tartrate-resistant acid phosphatase (TRAcP) localization, the following technique was used. Sections were rehydrated through a series of ethanols and then rinsed in TBS (10 mmol/L Tris at pH 7.4, 150 mmol/L NaCl) with Tween 20 (0.05%). For antigen retrieval, slides were immersed in a 20 μg/mL solution of proteinase K according to the manufacturer's instructions for 10 min at room temperature. After washing in TBS, tissue sections were blocked using standard blocking criteria for 1 h at room temperature. MMP-7 ( 17) or MMP-9 (Oncogene) antibodies at a dilution of 1:100 were added in blocking solution overnight at 4°C. Slides were washed extensively in TBST before the addition of a species-specific fluorescently labeled secondary antibody (Alexafluor 568 nm, Invitrogen) diluted 1:1,000 in blocking solution for 1 h at room temperature. Slides were washed in TBS and then equilibrated in an acetate buffer as described ( 18). The ELF97 TRAcP stain (Invitrogen) was diluted 1:1,000 in acetate buffer, and slides were incubated for 15 min at room temperature. After washing, slides were aqueously mounted in media (Biomeda Corp.) containing 2 μmol/L 4′,6-diamidino-2-phenylindole (DAPI) for nuclear localization.
TRAcP was also detected using a traditional colorimetric kit according to the manufacturer's instructions (Sigma-Aldrich). Gross anatomy of the mouse tibias was assessed by H&E staining. Proliferation (anti–phosphorylated histone H3, Millipore) and apoptosis (anti–caspase-3, Cell Signaling) were assessed by immunohistochemistry as previously described ( 14).
Micro–computed tomography, X-ray, and histomorphometric analyses. For gross analysis of trabecular bone volume (BV), formalin-fixed tibias were scanned at an isotropic voxel size of 12 μm using a microCT40 (SCANCO Medical). The tissue volume (TV) was derived from generating a contour around the metaphyseal trabecular bone that excluded the cortices. The area of measurement began at least 0.2 mm below the growth plate and was extended by 0.12 mm. BV included all bone tissue that had a material density of >438.7 mgHA/cm3. These analyses allowed for the calculation of the BV/TV ratio. The same threshold setting for bone tissue was used for all samples. Radiographic images (Faxitron X-ray Corp.) were obtained using an energy of 35 kV and an exposure time of 8 s. The tumor volume (TuV) was calculated as a function of the total TV of the tibial medullary canal using Metamorph software (Molecular Devices). For histomorphometry, three nonserial sections of tumor-bearing limbs were H&E stained to assess the BV/TV ratio or with TRAcP to assess osteoclast number per millimeter of bone at the tumor-bone interface using Metamorph.
Immunoprecipitation, immunoblotting, and ELISA. Tumor and sham-injected tibias from WT or MMP null animals were harvested 9 d postinjection and flash frozen in liquid nitrogen. Tissue homogenates were generated by mortar and pestle, and total protein was subsequently extracted using a standard protein lysis buffer containing a complete protease inhibitor cocktail (Roche). Protein concentration in isolated samples was quantitated using a bicinchoninic acid assay as per manufacturer's instructions (Pierce). For immunoprecipitation and quantitation of soluble RANKL in the tumor-bone microenvironments, equal concentrations of total protein (1 mg) in 1 mL of PBS were precleared with 10 μL of protein G–sepharose beads (Amersham Biosciences) for 1 h at 4°C. Precleared lysates were then incubated with 2 μg of antibody directed to the NH2 terminus of RANKL (Santa Cruz Biotechnology) for 1 h at 4°C. Subsequently, 10 μL of protein G–sepharose beads were added to the samples, and the bead-antibody-protein complexes were allowed to form overnight at 4°C. A nutator was used during all steps for agitation. The complexes were washed extensively [100 mmol/L NaCl, 50 mmol/L Tris-HCl (pH 7.5), 0.5% NP40] and then boiled in sample buffer [10% SDS, 0.5 mol/L Tris-HCl (pH 6.8), 30% glycerol, 1% β-mercaptoethanol, and 0.02% bromophenol blue] for 10 min before loading on to a 15% SDS-PAGE gel. Recombinant RANKL (462-TR-010/CF, R&D Systems) or MMP-7 solubilized RANKL [10 μg recombinant RANKL incubated with 100 ng active MMP-7 (Calbiochem) for 1 h at 37°C as previously described; ref. 14] were added as positive controls for the molecular weight of full-length and MMP-solubilized RANKL. Proteins were transferred to nitrocellulose membranes and blocked for 1 h at room temperature (5% milk powder in 1×TBS). The blots were then panned with an antibody directed to the NH2 terminus of RANKL (1:1,000 dilution; Axxora LLC in 5% milk in 1 × TBST) overnight with rocking at 4°C. The following day, blots were washed extensively with 1 × TBST before the addition of a secondary IR-labeled antimouse antibody (1:5,000 dilution in 1×TBST, Rockland, Inc.) for 1 h at room temperature. After washing in 1×TBST, blots were developed and bands of interest were quantitated using the Odyssey system (LI-COR Biosciences). ELISA was also used for the quantitation of soluble RANKL in samples according to the manufacturer's instructions (Quantikine, R&D Systems).
Statistical analyses. For in vivo data, statistical analysis was performed using ANOVA and Bonferroni multiple comparison tests. In vitro, statistical significance was analyzed using a Student's t test. A value of P < 0.05 was considered significant. Data are presented as mean ± SD.
MMP-7 and MMP-9 are expressed by osteoclasts in human breast-to-bone metastases. Previous observations using an animal model of tumor-bone interaction identified several MMPs as being highly expressed at the tumor-bone interface compared with the tumor area alone, namely MMP-2, MMP-3, MMP-7, MMP-9, and MMP-13 ( 14). 4 The expression of these MMPs was examined in human cases of frank breast-to-bone metastasis (n = 11). Interestingly, MMP-7 and MMP-9 were largely localized to the majority of mature TRAcP-positive multinucleated osteoclasts at the tumor-bone interface in human samples containing areas of osteolysis (10 of 11 samples; Fig. 1A-C and Supplementary Figs. S1 and S2). Other cells in the stromal compartment stained positively for MMP-7 and MMP-9, but remarkably, the tumor cells were negative for these metalloproteinases. MMP-2, MMP-3, and MMP-13 were also detected, but their expression was diffuse throughout the tumor/stroma compartment (data not shown). Because osteoclasts are the principal cells involved in bone resorption, we examined whether the ablation of host-derived MMP-7 or MMP-9 would affect the vicious cycle in terms of mammary tumor growth and/or mammary tumor–induced osteolysis.
Host-derived MMP-9 does not contribute to tumor growth or tumor-induced osteolysis. MMP-9 has previously been reported to be localized to osteoclasts, and MMP-9 null animals have been identified as having a delay in osteoclast recruitment during the development of long bones ( 19). Therefore, we initially tested the role of host-derived MMP-9 in tumor growth or tumor-induced osteolysis. Consistent with our observations in human samples, bone-resorbing osteoclasts in WT mice were positive for MMP-9 expression by immunofluorescent staining whereas, as expected, MMP-9 was not detected in MMP-9 null osteoclasts ( Fig. 2A ). Because MMP-9 null animals have a transient developmental bone phenotype, we determined the baseline trabecular BV as a function of TV (BV/TV) in WT and MMP-9 null animals at 6 weeks of age, which was the proposed time point for introduction of the PyMT-Luc tumor cells. No difference in the BV/TV between the WT and MMP-9 null animals was observed (Supplementary Fig. S3A).
To assess the contribution of host MMP-9 in mammary tumor growth in the bone microenvironment, the PyMT-Luc tumor cells, in which MMP-9 expression is undetectable in vivo ( 20), were injected into the tibia of syngeneic FVB WT or MMP-9 null mice. Surprisingly, quantitation of the bioluminescent signal from the tumor cells showed no difference in the tumor growth rate between the MMP-9 null and WT control mice ( Fig. 2B). With respect to tumor-induced osteolysis, analysis of the BV/TV ratio by high-resolution micro–computed tomography (μCT) showed that the tumor-injected tibias of WT and MMP-9 null were significantly lower (P < 0.05) than their respective sham-injected control counterparts ( Fig. 2C). However, a direct comparison of the BV/TV ratios between the WT and MMP-9 null tumor-injected limbs revealed no difference in BV/TV ratios ( Fig. 2C). Furthermore, no differences in tumor growth as assessed by phosphorylated histone H3 for proliferation and cleaved caspase-3 immunohistochemistry for apoptosis, trabecular BV, and osteoclasts/millimeter bone by histomorphometry were observed between the WT and MMP-9 null groups (data not shown). These experiments with similar-sized groups were repeated on several occasions with similar results. These results using the intratibial model suggest that host MMP-9 does not contribute to mammary tumor growth in the bone or tumor-induced osteolysis and are consistent with studies examining the role of host MMP-9 in the prostate cancer-bone microenvironment ( 21).
Host MMP-7 contributes to mammary tumor growth in the bone microenvironment. This is the first report to document the expression of MMP-7 in human breast-to-bone metastases and in human osteoclasts ( Fig. 1), although MMP-7 has previously been identified in rodent osteoclasts by our group ( 14). Recapitulating observations in human clinical samples, MMP-7 expression was identified in WT murine osteoclasts and not in MMP-7 null osteoclasts ( Fig. 3A ). Given that MMP-7 expression by osteoclasts is a relatively recent observation, studies into defining roles for MMP-7 in skeletal development have not been explored thus far. Therefore, before testing the effect of host-derived MMP-7 on the vicious cycle, the trabecular BV in noninjected, 6-week-old immunocompromised WT and MMP-7 null animals was examined using high-resolution μCT. Our results revealed no significant difference in the BV/TV ratio between WT and MMP-7 null animals, suggesting that, at this time point, MMP-7 null animals do not display an obvious bone phenotype compared with the WT controls (Supplementary Fig. S3B).
To determine the contribution of host MMP-7 to mammary tumor growth in the bone microenvironment, PyMT-Luc cells were injected into 6-week-old WT or MMP-7 null mice. Quantitation of the bioluminescent signal from the PyMT-Luc cells showed a significant decrease in the tumor growth rate in MMP-7 null mice compared with the WT controls ( Fig. 3B). These experiments with similar-sized groups in terms of animal numbers were independently repeated on four occasions, and similar observations were noted. To further investigate the potential role of MMP-7 in tumor growth, tumor proliferation and apoptosis were assessed by immunohistochemistry for phosphorylated histone H3 and cleaved caspase-3, respectively, in multiple sections from at least five animals per group ( Fig. 3C and D). Surprisingly, no difference in tumor proliferation was observed between the WT and MMP-7 null groups; however, tumor apoptosis was significantly higher in MMP-7 null mice compared with the WT controls (P < 0.05). Similar findings with respect to the effect of host MMP-7 on tumor growth using the 4T1-Luc cell line were also observed (Supplementary Fig. S4A–C). These results suggest that host-derived MMP-7 significantly contributes to mammary tumor growth in the bone by enhancing tumor cell survival.
Host-derived MMP-7 contributes to mammary tumor–induced osteolysis. The vicious cycle of tumor-bone interaction suggests that tumor growth/survival is dependent on osteoclast-mediated bone resorption. Because MMP-7 is primarily localized to bone-resorbing osteoclasts in the tumor-bone microenvironment, we assessed whether a lack of MMP-7 in osteoclasts affected tumor-induced osteolysis. Analysis of the BV/TV ratios from WT and MMP-7 null tumor-injected tibias using μCT ( Fig. 4A ) and histomorphometry ( Fig. 4B) revealed that the MMP-7 null group had a significantly higher amount of trabecular bone, which is in keeping with our tumor growth data, i.e., less tumor growth in the MMP-7 null animal would lead to less osteolysis. X-ray analysis also revealed a significantly lower TuV in the MMP-7 null animals compared with WT controls ( Fig. 4C). Studies using the 4T1-Luc cell line also showed that host-derived MMP-7 significantly affected tumor-induced osteolysis (Supplementary Fig. S5A and B). These results show that host-derived MMP-7 significantly affects mammary tumor–induced osteolysis.
MMP-7 mediates RANKL solubilization in the tumor-bone microenvironment. Next, we explored the potential molecular mechanism through which osteoclast-derived MMP-7 was affecting tumor-induced osteolysis. Given the acidity of the resorption lacunae (pH < 4) and the neutral activity profile of MMP-7, we suggest that MMP-7 does not function in direct bone matrix degradation but in the processing of factors that affect cell-cell communication within the tumor-bone microenvironment. MMP-7 has previously been shown to process a number of growth factors and cytokines to soluble active forms, including members of the tumor necrosis factor family TNF-α, Fas ligand (FasL), and RANKL ( 14, 22, 23). RANKL is essential for osteoclastogenesis and is a potent chemotactic molecule for monocytes and osteoclast precursor cells ( 24, 25). Therefore, we investigated if MMP-7 solubilization of RANKL was relevant in our model.
ELISA analysis revealed lower levels of total RANKL (membrane bound and soluble) in the tumor-injected tibias of MMP-7 null mice compared with WT control mice ( Fig. 5A ), whereas no difference was observed in the sham-injected control counterparts of each group (data not shown). Similar levels of osteoprotegerin, a soluble decoy receptor of RANKL, were found in the WT and MMP-7 null animals and were not present at a high enough concentration to interfere with the detection of RANKL by ELISA (data not shown). Immunoprecipitation and immunoblotting for soluble RANKL revealed significantly lower levels of soluble RANKL in PyMT-Luc or 4T1-Luc tumor-injected MMP-7 null animals compared with WT controls as assessed by densitometry ( Fig. 5B, P < 0.05 and Supplementary Fig. S5C, P < 0.05).
Interestingly, soluble RANKL could still be detected in the tumor-bearing limbs of MMP-7 null animals. This suggests that RANKL solubilization is still occurring in the absence of MMP-7. We and others have previously identified that other metalloproteinases, such as MMP-1, MMP-3, MMP-14, a disintegrin and metalloproteinase-17 (ADAM-17), and the serine protease cathepsin G are capable of processing RANKL to a soluble active form, and therefore, these proteases may also be playing a role in the solubilization of RANKL in our model ( 14, 26– 28). However, because the levels of RANKL are significantly lower in the MMP-7 null mice, we suggest that MMP-7 is the dominant protease involved in RANKL solubilization.
Next, because a decrease in the amount of soluble RANKL was detected in the tumor-bearing limbs of the MMP-7 null animals, we asked if there was concomitant decrease in the number of osteoclasts in the MMP-7 null tumor-bone microenvironment. We observed significantly lower numbers of TRAcP-positive multinucleated osteoclasts per unit length of tumor-bone interface in the MMP-7 null animals compared with the WT controls ( Fig. 5C). Significantly lower numbers of osteoclasts were also recorded in MMP-7–deficient animals injected with 4T1-Luc cells compared with WT controls (Supplementary Fig. S5D). Given the importance of RANKL in mediating osteoclastogenesis, these data suggest that MMP-7 mediates mammary tumor–induced osteolysis by affecting the availability of a key factor for osteoclastogenesis, RANKL.
Understanding the molecular mechanisms that control the vicious cycle is the key for the development of new therapeutics that will be effective not only in treating bone metastases but also in curing them. In the current study, we found that, in human cases of breast-to-bone metastasis, osteoclasts were a rich source of MMP-7 and MMP-9. Interestingly, our studies using two unrelated osteolytic-inducing tumor cell lines (PyMT-Luc and 4T1-Luc) revealed that only MMP-7 seemed to contribute to mammary tumor growth and tumor-induced osteolysis in the bone microenvironment. Furthermore, our data suggest that MMP-7 solubilization of the osteoclastogenic factor RANKL is the principal molecular mechanism underlying these observations. Previously, we have identified that MMP-7 processing of RANKL results in the generation of an active soluble form that can promote osteoclast maturation and activation ( 14). Therefore, in the context of breast-to-bone metastasis, we hypothesize that, in the absence of MMP-7–solubilized RANKL, there is a resultant decrease in osteoclast maturation and bone resorption at the tumor-bone interface, which in turn results in a decrease in bone-derived growth factors, such as TGF-β and IGF, which affect tumor survival and growth ( Fig. 6 ).
Our results show that an osteoclast-derived protease, MMP-7, can promote osteoclast activation in the tumor-bone microenvironment by generating an active soluble form of the osteoclastogenic factor RANKL and suggest that selective inhibition of MMP-7 may be of benefit for the treatment of lytic metastases. Several studies support the rationale for the development of selective MMPIs for the treatment of bone metastases. Broad spectrum MMPIs, such as batimastat, have been identified as being effective in preventing tumor growth and tumor-induced osteolysis in the bone environment using animal models ( 10– 12). However, conclusions from human clinical trials with the same inhibitors identified the necessity for highly selective MMPIs, which lack the deleterious side effects of broad spectrum inhibitors before their application in clinical settings ( 13). This requires an understanding of the precise roles of MMPs in the context of particular diseases, and in this regard, our studies suggest MMP-7 as an attractive target for the treatment of lytic metastases.
Whereas MMP-7 solubilization of RANKL is predicted here to be a mechanism underlying our observations, MMP-7 may contribute via other mechanisms. For example, MMP-7 processing of apoptotic factors, such as Fas ligand in the tumor-bone microenvironment, may directly affect tumor survival ( 22). In addition, the direct processing of the bone matrix by MMP-7 may also be a possibility. Acidification and cathepsin-K secretion into osteoclast resorption lacunae allows for the demineralization and collagenolysis of the bone matrix, respectively ( 6). By a process known as transcytosis, the osteoclast mediates the removal of bone products from the area of the bone undergoing resorption ( 29). Given the punctate localization of MMP-7 by immunofluorescent staining ( Figs. 1A and 3A) it is tempting to speculate that MMP-7 contributes to the further processing of bone matrix components, such as osteopontin ( 30), or the release of growth factors from bone matrix components, such as TGF-β ( 31) and IGFs ( 32), within these transcytotic vesicles. The expression of MMP-7 from other cellular sources may also be a possibility. In the tumor-bone microenvironment, we observed that MMP-7 expression was largely confined to osteoclasts. However, MMP-7 has also been shown to be expressed by macrophages, and given the role of macrophages in tumor-induced osteolysis, the contribution of macrophage-derived MMP-7 in our model or in humans cannot be discounted ( 33, 34).
Given the apparent role of MMP-7 in osteoclast function in the pathologic setting of tumor-induced osteolysis, it is surprising that MMP-7 null animals seem to have a normal skeletal phenotype. Data presented here using μCT scan analysis show a similar BV/TV ratio between MMP-7 null and WT control mice at 6 weeks of age. Whereas a role for MMP-7 in bone development has not been explored, a number of reports have revealed that the phenotype of the MMP-7 null animals often becomes apparent in response to injury/challenges or disease. For example, in nonpathologic conditions such as herniated disc resorption, macrophage-derived MMP-7 is critical for the resorption of the herniated disc and in mammary and prostate involution; MMP-7 processing of FasL is important for initiating apoptosis ( 22, 23, 35). More often, phenotypes in the MMP-7 null animals have been observed in pathologic conditions such as pancreatitis, colon tumorigenesis, mammary gland tumorigenesis, and in innate defense wherein MMP-7 null animals show significant delays in disease progression or in response to infection ( 36– 39). Therefore, although MMP-7 null mice lack an apparent skeletal phenotype, in the context of tumor-bone microenvironment, it is clear based on the results in the current study that host MMP-7 plays an important role in osteoclast biology. In addition, our observations defining a role for MMP-7 in bone diseases are consistent with previous reports that implicate roles for host MMP-7 in prostate cancer–induced osteolysis, osteoarthritis, and cartilage/periarticular bone destruction ( 14, 40, 41).
Although MMP-9 was localized to human and murine osteoclasts, the ablation of host MMP-9 did not seem to affect PyMT-Luc tumor growth and bone resorption compared with the WT controls. Analogous results were obtained by Nabha and colleagues, using the same intratibial model but in the context of prostate cancer progression in the bone ( 21). Given the importance of MMP-9 in osteoclast migration and recruitment in developing long bones ( 19), these results were surprising. It seems that, in the tumor-bone microenvironment, MMP-9 is not critical for osteoclast function. The possibility that tumor-derived MMP-9 could overcome the absence of host MMP-9 exists in our model; however, in vivo studies by our group have shown that MMP-9 expression by the PyMT-Luc tumor cells is not detectable ( 20). Therefore, the ability of tumor-derived MMP-9 to circumvent the loss of host-derived MMP-9 and affect tumor progression in the bone is unlikely. However, due to functional overlap among members of the MMP family, other MMPs produced by osteoclasts and other stromal cells in the tumor microenvironment may compensate for the absence of host MMP-9.
Whereas our data point toward MMP-9 as not being critical for mammary tumor growth or induced osteolysis, it is important to note that MMP-9 could contribute to other steps of metastasis that are not taken into account with the intratibial model. These include extravasation from the sinusoidal vasculature in the bone and initial survival, the latter of which has been shown to be an important role for host-derived MMP-9 in early lung metastasis ( 42). Furthermore, MMP-9 has been implicated in tumor angiogenesis by mediating the release of matrix-sequestered vascular endothelial growth factor ( 43). In the context of the prostate tumor-bone microenvironment, Nabha and colleagues showed a decrease in angiogenesis in MMP-9 null animals compared with WT controls ( 21). Therefore, the selective inhibition of MMP-9 may still prove useful in preventing the establishment and angiogenesis of bone metastases.
In conclusion, this study shows that osteoclast-derived MMP-7, but not MMP-9, significantly contributes to tumor-induced osteolysis by affecting osteoclast activation. We suggest that MMP-7–mediated solubilization of RANKL is a potential mechanism underlying this observation. Our data support the rationale for the generation of selective MMPIs for the treatment of osteolytic bone metastases and implies that the development of such reagents would expand the therapeutic options available to patients suffering with this incurable disease.
Disclosure of Potential Conflicts of Interest
No potential conflicts of interest were disclosed.
Grant support: NIH grant 1 R01 CA84360 (L.M. Matrisian) and Susan G. Komen Foundation grant PDF 02 1394 (C.C. Lynch). C.C. Lynch and S. Thiolloy are supported by the Department of Defense awards W81XWH-07-1-0208 and BC051038, respectively.
Views and opinions of, and endorsements by, the authors (S. Thiolloy and C.C. Lynch) do not reflect those of the U.S. Army or Department of Defense.
The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked advertisement in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.
We thank James Edwards and Steve Munoz of Vanderbilt Bone Center for their expertise.
Note: Supplementary data for this article are available at Cancer Research Online (http://cancerres.aacrjournals.org/).
↵4 S. Thiolloy and C.C. Lynch, unpublished observation.
- Received October 14, 2008.
- Revision received April 21, 2009.
- Accepted June 9, 2009.
- ©2009 American Association for Cancer Research.