Abstract
The tropism of breast cancer cells for bone and their tendency to induce an osteolytic phenotype are a result of interactions between breast cancer cells and stromal cells and are of paramount importance for bone metastasis. However, the underlying molecular mechanisms remain poorly understood. We hypothesize that tumor-stromal interaction alters gene expression in malignant tumor cells and stromal cells creating a unique expression signature that promotes osteolytic breast cancer bone metastasis and that inhibition of such interactions can be developed as targeted therapeutics. Microarray analysis was performed to investigate gene expression profiling at the tumor-bone (TB) interface versus the tumor alone area from syngenic mice injected with three different syngenic mammary tumor cell lines that differ in their metastatic potential. We identified matrix metalloproteinase 13 (MMP13), receptor activator of NF-κB ligand (RANKL), and integrins binding sialoprotein to be genes upregulated at the TB interface and validated. To determine the functional role of MMP13 in tumor-induced osteolysis, mice with Cl66 mammary tumors were treated with MMP13 antisense oligonucleotides (MMP13-ASO) or control scrambled oligonucleotides (control-ASO). Knockdown of MMP13 expression at the TB interface leads to significant reduction in bone destruction and in the number of activated osteoclasts at the TB interface. Further analysis to evaluate the mechanism of MMP13-dependent osteolytic bone metastasis revealed that MMP13-ASO treatment decreased active MMP9, RANKL levels, and transforming growth factor-β signaling at the TB interface. Together, our data indicate that upregulation of MMP13 at the TB interface is important in tumor-induced osteolysis and suggest that MMP13 is a potential therapeutic target for breast cancer bone metastasis. Cancer Res; 70(9); 3494–504. ©2010 AACR.
Introduction
Breast cancer is the most common cancer and the second leading cause of cancer-related death in women in the United States (1). Most complications of breast cancer are attributed to metastasis to distant organs, including lymph nodes, liver, lung, and bone (2, 3). In advanced stages of the disease, nearly all breast cancer patients suffer with bone metastasis. Bone metastases in breast cancer are predominantly osteolytic and also cause skeletal lesions including pathologic fracture, intractable bone pain, nerve compression, and hypercalcemia (4, 5). These complications not only increase the risk of mortality but also cause a significant decrease in the quality of life (3).
Breast cancer cells show a strong predilection for bone (5). Arrival of tumor cells in the bone microenvironment initiates a “vicious cycle” of bidirectional interactions between tumor cells and stromal cells (3, 6). Tumor cells produce various factors such as parathyroid hormone-related peptide (7, 8), interleukin-8 (IL-8), and IL-1 to stimulate osteoblasts to induce expression of receptor activator of NF-κB ligand (RANKL) to induce bone resorption (9). Increased bone resorption causes the release of sequestered factors that favor the growth of malignant tumor cells including bone-derived growth factor, fibroblast growth factor, and transforming growth factor β (TGFβ; ref. 3). The underlying molecular mechanisms of tumor-bone (TB) interaction are poorly understood. In this report, we hypothesize that tumor-stromal interaction in the bone microenvironment alters gene expression in malignant tumor cells and stromal cells creating a unique expression signature that promotes osteolytic bone metastasis and that inhibition of such interactions can be targeted for development of novel therapeutics.
Extracellular matrix (ECM) degradation, mediated by matrix metalloproteinases (MMP), is an essential step in the growth, invasion, and metastasis of malignant tumors. MMPs are a family of human zinc endopeptidases that can degrade virtually all ECM components (10). Apart from their ECM degradation functionality, latest research in MMPs reveals their specific roles in cleaving several extracellular and membrane-associated proteins and regulating cellular signaling pathways. MMP7 promotes osteolytic bone metastasis in prostate cancer through generation of soluble RANKL (sRANKL) from membrane bound RANKL (11). MMP2 and MMP9 have been associated with tumor angiogenesis (12). Expression of these proteases is also associated with poor clinical outcome in various malignancies, such as bladder, breast, lung cancer and head and neck squamous cell carcinomas (SCC; refs. 13, 14).
MMP13 was first identified from overexpressing breast carcinomas (15). IL-1α and IL-1β are potential candidates for inducing expression of MMP13 in breast carcinomas (16). In case of SCC, MMP13 is predominantly expressed by the tumor cells at the invading front and to some extent by stromal fibroblasts surrounding tumor cells (17, 18). Expression of MMP13 in the head and neck SCCs correlates with the invasion and metastatic capacity (17, 18). In laryngeal and vulvar carcinomas, the expression of MMP13 colocalizes with the expression of MT1-MMP and MMP2, suggesting that these three MMPs form a proteolytic cascade that leads to potent extracellular collagenolytic activity (19). In non–small cell lung carcinoma, tumor cells expressing MMP13 have a potential to shed from the primary tumor and aggregate in the bone marrow and associated with poorer survival rates (20). But the specific role of MMP13 in malignant breast cancer remains unclear. Our study is focused on elucidating the role of MMP13 in the tumor-stromal interaction, with particular attention to the TB microenvironment.
We identified that MMP13, RANKL, and integrins binding sialoprotein (IBSP) were the genes upregulated at the TB interface. Moreover, we showed that knockdown of MMP13 expression at the TB interface leads to a significant reduction in bone destruction and in the number of activated osteoclasts at the TB interface. MMP13-ASO treatment decreased the RANKL/OPG ratio, active MMP9, and TGFβ levels at the TB interface. Together, our data showed that upregulation of MMP13 at the TB interface is important for regulation of tumor-induced osteolysis and suggest that MMP13 might be a potential novel therapeutic target for breast cancer bone metastasis.
Materials and Methods
Cells and animals
Three murine mammary adenocarcinoma cell lines 4T1 (highly metastatic), Cl66 (moderately metastatic), and Cl66M2 (poorly metastatic), differing in their metastatic potential, were used in this study (21, 22). Cells were maintained in DMEM (Mediatech) with 5% serum supreme (Biowhitaker), 1% vitamins, 1% l-glutamine, and 0.08% gentamicin (Invitrogen).
Animal experiments were approved by Institutional Animal Care and Use Committee of University of Nebraska Medical Center. Eight-week-old female BALB/c mice (National Cancer Institute) were used in this study. For in vivo experiments, tumor cells (5 × 104/50 μL) mixed in growth factor–reduced Matrigel (BD Biosciences) were injected directly onto the calvaria to mimic the close association of tumor cells and bone. Four weeks after implantation of tumor cells, mice were sacrificed and tumor alone and TB interface samples were collected. For immunohistochemical analysis, the samples were fixed in 4% paraformaldehyde at 4°C for 48 hours. The tissues were then transferred into a decalcification solution [15% EDTA with glycerol (pH 7.4)] for 4 weeks and were subsequently paraffin embedded and processed for histology.
RNA and protein extraction were done by homogenization of tissue samples in liquid nitrogen. Total RNA was extracted using Trizol (Invitrogen) following the manufacturer's instructions. The RNA concentration was quantified using a NANO drop ND-1000 Spectrophotometer (Nano Drop Technologies).
Protein was extracted using T-PER tissue protein extractor solution (Pierce) following the manufacturer's provided protocol. Protein samples were quantified using a bicinchoninic acid protein assay kit (Pierce). Proteinase activity assays were performed with protein samples without protease inhibitors.
Microarray analysis and quantitative real-time PCR
Calcified frozen sections were serially sectioned in 10-μm-thick slices, and at least 10 slides per mouse were microdissected with careful separation of the TB interface and the tumor alone areas, as described earlier (22, 23). Total RNA was extracted from each microdissected population and pooled, and an equal amount of RNA was amplified using a probe amplification kit (Affymetrix). An Affymetrix Mouse Expression Array 430 was used for comparing gene expression profiles between the TB interface and the tumor alone areas. A complete detection and analysis of signals for each chip was performed using Affymetrix GeneChip Operating Software to generate raw expression data. A signal log ratio algorithm was used to estimate the magnitude of change of a transcript when two arrays were compared (experimental versus baseline). It was calculated by comparing each probe pair on the experimental array, the TB interface, with the corresponding probe pair on the baseline array, the tumor alone area, and considering the mean of the log ratios of probe pair intensities across the two arrays. The change is expressed as the log2 ratio. Thus, a signal log ratio of 1.0 indicates an increase of transcript level by 2-fold and −1.0 indicates a decrease by 2-fold. For each set of tissues from 4T1, Cl66, and Cl66M2, the signal log ratio of the TB interface versus the tumor alone area was calculated, and the genes were ordered from highest to lowest expression levels.
Gene expression analysis was confirmed using quantitative real-time PCR (qRT-PCR) for the TB interface and tumor alone area samples. RNA (5 μg) from each sample was used to synthesize first-strand cDNA. Diluted first-strand cDNA (1:100, 2 μL) was amplified in a 20-μL reaction with SYBR green master mix (Roche) and 10 mmol/L primer mix using a Bio-Rad iCycler (Bio-Rad). The following reaction conditions were used: initial denaturation at 95°C for 3 minutes, followed by amplification cycles with denaturation at 95°C for 60 seconds, annealing at 60°C for 60 seconds, extension at 72°C for 60 seconds, and finally a long extension at 72°C for 2 minutes. Primers used for validation of gene expression are included in Supplementary Table S1. The fluorescence intensity of double-strand specific SYBR green, reflecting the amount of formed PCR product, was monitored at the end of each elongation step. The Ct value for each gene was normalized with glyceraldehyde-3-phosphate dehydrogenase (GAPDH) expression for relative gene expression analysis.
Immunohistochemistry and tartrate-resistant acid phosphatase staining
MMP13 protein expression was evaluated by immunohistochemistry on tumor sections using a MMP13-specific antibody (Santa Cruz Biotechnology). The sections were deparaffinized using EZ dewax solution (Biogenex). For antigen retrieval, the sections were boiled in 10 mmol/L citrate buffer (pH 6.0) for 10 minutes, and endogenous peroxidase activity was blocked using 3% H2O2 for 5 minutes. The sections were then blocked in antibody diluent for 1 hour at room temperature. MMP13 antibody was diluted 1:100 in blocking solution, and sections were incubated overnight at 4°C. After washing, the slides were incubated with antigoat biotinylated antibody for 30 minutes at room temperature. After washing, immunoreactivity was detected using Vectastain avidin-biotin complex method and 3,3′-diaminobenzidine substrate kits (Vector Laboratories). Sections were counterstained with hematoxylin, dehydrated, and permanently mounted.
For in vivo evaluation of TGFβ signaling, we performed immunohistochemistry for phosophorylated Smad-2 (p-Smad-2; refs. 24, 25).Sections were blocked using goat serum diluted 1:500 for 1 hour at room temperature. Sections were then incubated overnight at 4°C with antibody directed against p-Smad-2 (Ser465/467, Cell Signaling Technology) diluted 1:50 in blocking solution. After washing, sections were incubated for 1 hour at room temperature with biotinylated antirabbit IgG diluted 1:500. TRAP staining was performed to detect activated osteoclasts in vivo according to the manufacturer's instructions (Sigma Chemicals). Briefly, deparrifinized slides were rinsed with deionized water before incubating with TRAP containing buffer at 37°C for 1 hour, rinsed with deionized water, and counterstained with Gill3 hematoxylin solution for 2 minutes followed by aqueous mounting. Immunostained sections were examined under a Nikon light microscope, and the number of TRAP-positive multinucleated cells at the TB interface was assessed at a magnification of 400× for each lesion. The total number of osteoclasts was then divided by the length of the TB interface to get the number of osteoclasts per millimeter of TB interface.
Antisense oligonucleotide treatment for inhibition of tumor-induced osteolysis
Antisense oligonucleotides (ASO) used in the therapeutic protocol were obtained from Isis Pharmaceuticals. ASOs designed specifically to target MMP13 were used throughout this study. 2'-Methoxyethyl modified chimeric ASOs with a phosphorothioate backbone were synthesized as previously described (26). Briefly, these oligonucleotides are modified at the 2' sugar position of the five bases at both the 3′ and 5′ ends with a methoxyethyl group. This modification greatly increases the stability of the oligonucleotides and the affinity for its target mRNA while reducing the amount of immune stimulatory and inflammatory effects that can be seen with oligonucleotides in mice (27). Active ASOs were identified by screening 46 ASOs designed to be specific for MMP13. The efficacy of these ASOs was confirmed in concentration-response experiments, and the most potent ASO was used for further experiments. Control-ASO did not match any known mRNA in the mouse genome.
Animals bearing Cl66 mammary tumors were randomly divided into two treatment groups (control-ASO and MMP13-ASO). The oligonucleotides were dissolved in physiologic saline (0.9% NaCl) and were given by i.p. injection at a dose of 50 mg/kg/d starting at day 7 following tumor implantation for 5 days with 2 days off followed by another 4 days. Tumor growth was monitored, and mice were sacrificed on day 28. Tumor alone and TB interface samples were collected and processed for further analysis.
Gelatin zymography
Total protein (50 μg) isolated from either the TB interface or tumor alone area from animals implanted with Cl66 tumors was subjected to electrophoresis on a 10% (w/v) polyacrylamide SDS gel containing 1 mg/mL porcine gelatin (Sigma-Aldrich). At the completion of electrophoresis, the gel was washed with 2.5% Triton X-100 buffer for 30 minutes. After rinsing, the gel was incubated for 12 hours at 37°C in incubation buffer containing 50 mmol/L Tris-HCl (pH 7.4), 150 mmol/L NaCl, 10 mmol/L CaCl2, and 0.05% (w/v) NaN3. After rinsing, the gel was then stained using 0.025% Coomassie brilliant blue (Bio-Rad) and photographed using a Multi Image Light Cabinet (Alpha Innotech Corporation). The volume of gelatinolytic activity was evaluated using ImageQuant 5.1 (Molecular Dynamics).
Pro-MMP9 ELISA
TB lysates from control-ASO–treated and MMP13-ASO–treated mice (400 ng/μL) were used for analysis. The concentration of pro-MMP9 in these samples was determined by Quantikine Mouse pro-MMP9 ELISA according to the manufacturer's instructions (R&D Systems). This assay is a quantitative “sandwich” enzyme immunoassay. Based on a curve of the absorbance of samples to the standard curve (plotted using recombinant pro-MMP9 protein), we determined the concentrations of pro-MMP9 in the tumor lysates.
Statistical analysis
For in vitro studies, the Student's t test was used for statistical comparison. For in vivo studies, the Mann-Whitney U test was used for statistical comparison. A P < 0.05 was considered significant.
Results
Gene expression profile at the TB interface
We analyzed gene expression patterns at the TB interface compared with the tumor alone area using cDNA microarray. Mammary tumor cells with different metastatic potential [4T1 (high), Cl66 (moderate), and Cl66M2 (low)] were transplanted onto the calvaria of BALB/c mice. Histologic analysis showed that all tumors exhibited tumor-induced osteolysis and osteoclast activation. Microarray analysis revealed the upregulation of 414 genes and the downregulation of 27 genes at the TB interface compared with the tumor alone area. The highly upregulated genes were IBSP, RANKL, MMP13, insulin growth factor binding protein 5, Lumican, Lysyl oxidase (Lox), Kinesin family 5B (Kif5b), and Wnt inhibitory factor 1 (Wif1; Fig. 1A). The common upregulation of these genes in all three cell lines further suggested that they may play an important role in mammary tumor-induced osteolysis.
We then used qRT-PCR with gene specific primers to confirm gene expression at the TB interface. Our data confirmed the upregulation of mRNA expression of MMP13 (Fig. 1B, i), IBSP (Fig. 1B, ii), Lumican (Fig. 1B, iii), Lox (Fig. 1B, iv), Kif5b (Fig. 1B, v), and Wif1 (Fig. 1B, vi) at the TB interface.
Upregulation of MMP13 at the TB interface
Recognizing the important role of proteases in tumor progression, we further evaluated the functional role of MMP13 in mammary tumor–induced osteolysis. We examined MMP13 protein expression at the TB interface by immunohistochemistry. MMP13-specific antibody staining revealed a significant upregulation of MMP13 in Cl66 tumors at the TB interface (Fig. 2A, b) but not at the tumor alone area (Fig. 2A, a). We observed low levels of immunoreactivity in normal calvaria (Fig. 2A, c). Antibody controls did not show any nonspecific staining (data not shown). This protein level analysis further confirmed the earlier observation of increased MMP13 transcript levels by cDNA microarray and qRT-PCR analyses. In addition, our immunohistochemical analysis reveals that MMP13 is expressed by tumor cells, osteoblasts, and stromal cells at the TB interface, which are interacting with cells of bone microenvironment (Fig. 2A, b).
Kinetics of MMP13 mRNA expression and tumor-induced osteolysis
In the next set of experiments, we examined the kinetics of MMP13 expression and its association with tumor-induced osteolysis and the number of activated osteoclasts. Mice bearing Cl66 tumors were killed 2, 3, or 4 weeks after tumor implantation, and tumors were examined for bone destruction, osteoclast number, and MMP13 expression at the TB interface and tumor alone areas. We quantified the severity of lesions by measuring bone destruction index (BDI), which is a ratio of the length of the bone that is destroyed by the tumor to the total length of the bone at the TB interface. Results shown in Fig. 2B show an increase in bone destruction and osteoclast number over time. We observed higher levels of MMP13 mRNA expression at the TB interface compared with tumor alone areas (Fig. 2C).
Inhibiting MMP13 expression abrogates mammary tumor–induced osteolysis and number of activated osteoclasts at the TB interface
Next, we examined whether knocking down MMP13 expression in Cl66 tumor-bearing mice using ASOs inhibited mammary tumor–induced osteolysis. We did not observe any weight loss or toxicity in any of the treatment groups (data not shown). We observed smaller tumors in MMP13-ASO–treated group compared with control-ASO–treated group; however, the decrease in tumor size was not significant (data not shown).
H&E staining of tumor sections showed severe bone destruction in control-ASO–treated tumors (Fig. 3B, i), whereas MMP13-ASO–treated tumors showed no osteolysis (Fig. 3B, ii). We observed a significant decrease in osteolysis in the MMP13-ASO–treated group compared with the control-ASO–treated group (Fig. 3B, iii). Similarly, we observed a significant decrease in the number of activated osteoclasts lining the TB interface in the MMP13-ASO–treated group (Supplementary Fig. S1; Fig. 3C).
We examined the expression of MMP13 in tumors from MMP13-ASO–treated and control-ASO–treated mice using qRT-PCR. The expression of MMP13 at the TB interface was significantly lower in the MMP13-ASO–treated group compared with that in the control-ASO–treated group (Fig. 3D). We observed very low levels of MMP13 expression in the tumor alone area, which was not altered by MMP13-ASO treatment (Fig. 3D).
Inhibition of MMP13 decreases RANKL expression and regulates the RANKL/OPG ratio at the TB interface
Previous observations from our laboratory have shown the upregulation of RANKL at the TB interface in mammary tumor–induced osteolysis (28). RANKL interacts with its receptor RANK and activates osteoclasts. We evaluated RANKL mRNA levels in MMP13-ASO–treated and control-ASO–treated mice and observed a significant decrease in RANKL expression at the TB interface in the MMP13-ASO treatment group (Fig. 4A).
The RANKL/osteoprotegrin (OPG) axis has been shown to play a pivotal role in osteolytic bone metastasis (29). We examined how the RANKL/OPG ratio at the TB interface was altered due to MMP13-ASO treatment. RANKL levels were significantly reduced due to MMP13-ASO treatment (Fig. 4B), whereas we did not observe any significant difference in OPG levels between the two treatment groups (Fig. 4C). With the decrease in the RANKL levels, we observed a significant decrease in the RANKL/OPG at the TB interface in the MMP13-ASO treatment group (Fig. 4D). These observations suggest a functional role for MMP13 in tumor-induced osteolysis mediated by the regulation of the RANKL/OPG axis.
Targeting MMP13 inhibits MMP9 activation and TGFβ signaling
Previously, we have shown that MMP9 is also upregulated at the TB interface in mammary tumor–implanted mice (22). We observed a decrease in MMP9 mRNA expression at the TB interface in the MMP13-ASO–treated group compared with the control-ASO–treated group (Fig. 5A). We evaluated the activity of MMP9 at the TB interface of Cl66 tumor-bearing mice treated with MMP13-ASO compared with control-ASO. We observed a significant decrease in gelatinolytic activity (at 92 kDa) in the MMP13-ASO–treated mice. We observed a significant decrease in active/pro-MMP9 enzyme levels in MMP13-ASO–treated mice (Fig. 5C). We did not observe significant difference in pro-MMP9 levels at the TB interface between control-ASO–treated and MMP13-ASO–treated group (Fig. 5B).
We have previously reported that upregulation of TGFβ signaling during TB interaction promotes tumor-induced osteolysis (23). We observed a significant decrease in tumor-induced osteolysis with MMP13-ASO treatment. To examine whether MMP13 acts via TGFβ signaling in mammary-induced osteolysis, we evaluated TGFβ expression and activity at the TB interface of MMP13-ASO–treated and control-ASO–treated mice. We observed downregulation of TGFβ mRNA expression in MMP13-ASO–treated mice (Fig. 6B). We evaluated TGFβ signaling using immunohistochemistry for p-Smad-2 (30). We observed a significant decrease in the p-Smad-2 staining index and TGFβ signaling at the TB interface of mice with MMP13-ASO treatment (Fig. 6A and C).
Discussion
In this study, we evaluated the gene expression signature at the TB interface and tumor alone areas in three different mammary adenocarcinoma cell lines differing in their metastatic potential. We identified MMP13, RANKL, IBSP, Lumican, Kif5B, LOX, and Wif1 as potentially important genes involved in tumor-induced osteolysis, which are commonly upregulated at the TB interface in all three tumor types. Furthermore, overexpression of MMP13 at the TB interface promotes tumor-induced osteolysis and knockdown of MMP13 with ASO results in a significant inhibition of bone destruction. These data show that interaction of breast cancer cells with bone stromal cells results in an altered gene expression pattern that is critical for tumor-induced osteolysis.
We identified genes that are potentially involved in the modulation of TB interaction during osteolytic bone metastasis using microarray analysis. We confirmed the microarray data by performing qRT-PCR analysis showing higher expression of MMP13, RANKL, IBSP, Lox, Lumican, Wif1, and Kif5b mRNA expression at the TB interface compared with the tumor alone area. Our previous studies have established the importance of RANKL upregulation during TB interaction in activating osteoclasts and promoting tumor-induced osteolysis (22, 23). IBSP is a secreted, noncollagenous glycoprotein of bone matrix and is expressed in breast, lung, thyroid, and prostate cancers that metastasize to bone (31). Expression of IBSP is associated with development of metastasis and poor prognosis (32, 33). Lumican is a small leucine-rich proteoglycon abundantly present in breast tissue, and it has been shown that lumican expression parallels tumor progression in breast carcinoma with higher lumican expression associated with higher tumor grade and lower estrogen receptor levels in the tumor (34). However, their role in bone metastasis remains unclear. LOX is an ECM remodeling enzyme, and studies delineating the role of LOX in metastasis showed that it is essential for hypoxia-induced metastasis and inhibition of Lox proved to be promising in eliminating metastasis in mice with orthotopically grown tumors (35, 36). Silencing of WIF1 is associated with increased susceptibility to osteosarcoma (37). The role of the Kif5B gene in cancer progression is not well established. However, a recent report suggested a possible role for Kif5B as an oncofusion kinase (38). These studies suggest possible roles for genes upregulated at the TB interface; however, little is known about their functional role in TB interaction during osteolytic metastasis.
MMPs have been implicated in tumor progression and metastasis in several tumor types (11, 39–41). Our present data showed the upregulation of MMP13 at the TB interface in three different mammary tumors. MMP13 expression was higher in both tumor cells and osteoblasts at the TB interface. Previous studies have shown expression of MMP13 in several cancers including breast (15), head and neck squamous carcinoma (18), melanoma, and chondrosarcoma (42). A previous report suggests involvement of MMP13 in the degradation of basement membrane during bone metastasis of breast cancer (41). We have observed an association between MMP13 expression, the number of osteoclasts, and tumor-induced osteolysis. Recent study has shown the upregulation of MMP13 in clinical bone metastatic samples from breast cancer patients (43). Our present data and the previous reports suggest an important role for the expression of MMP13 at the TB interface (11, 39–41, 44); hence, in this report, we focused on delineating the functional analysis of MMP13 in tumor-induced osteolysis.
To further strengthen our findings, we used MMP13-ASOs to target MMP13 expression in vivo. Treatment of mice with MMP13-ASOs significantly inhibited tumor-induced bone resorption as well as number of activated osteoclasts at the TB interface. The mechanism underlying MMP13-dependent osteoclast activation remains unclear. Previous reports have shown that RANKL/RANK/OPG signaling is important in osteoclast activation and subsequent bone destruction (45). It is possible that MMP13 inhibition might alter the expression of RANKL and OPG at the TB interface. We observed that targeting MMP13 decreased RANKL expression at TB interface without affecting OPG levels. As explained previously, the relative RANKL/OPG ratio is an important factor in driving osteolysis, and we observed a significant decrease in the RANKL/OPG ratio at the TB interface in MMP13-ASO–treated mice. These observations suggest the potential role of MMP13 in RANKL-dependent osteoclast activation and tumor-induced osteolysis. The actual mechanism through which MMP13 is involved in the regulation of the RANKL/OPG axis at the TB interface merits further investigation.
MMP13 has been shown to play a central position in the MMP activation cascade (46). MMP13 activates MMP9 by cleaving pro-MMP9 (47). In our previous studies, we have shown the upregulation of pro-MMP9 and active MMP9 during TB interaction (22). In this report, we observed a significant decrease in active MMP9 levels at the TB interface in MMP13-ASO–treated mice. MMP9 has been shown to recruit osteoclasts during development of long bones (48). These studies suggest that MMP13 might indirectly potentiate osteoclast recruitment and activation by regulating activation of MMP9 at the TB interface during TB interaction.
In a recent report, we showed the role of TGFβ signaling during TB interaction in promoting mammary tumor growth and osteoclast activation (23). In this report, we analyzed the effect of MMP13 knockdown on TGFβ signaling by p-Smad-2 staining and observed a significant decrease in the p-Smad2 activity in the MMP13-ASO–treated group compared with control-treated group. The decrease in TGFβ signaling could be due to lower levels of active MMP9 at the TB interface, which has been shown to be critical in TGFβ activation (49, 50). In our previous report, we did not observe an increase in TGFβ mRNA expression at the TB interface (23). However, TGFβR1 expression and signaling was increased at the TB interface compared with the tumor alone area (23). Our data suggest that increased MMP13 levels at the TB interface potentiate TGFβ signaling via activation of MMP9. In our previous study, we evaluated the functional role of cathepsin G in osteolytic bone metastasis of breast cancer and observed that cathepsin G was involved in activating MMP9 by cleaving pro-MMP9. Inhibition of cathepsin G resulted in increased latent TGFβ levels. Thus, cathepsin G–mediated activation of MMP9 led to activation of TGFβ and ultimately promotes tumor-induced osteolysis (50). Findings from our previous and present study allow us to speculate that MMP9 might be playing as a central molecule in tumor-induced osteolytic cascade, where it is activated by several factors including cathepsin G and MMP13 and leading to enhancement of TGFβ signaling at the TB interface, ultimately contributing to the osteolytic bone metastasis.
In conclusion, our present study showed that TB interaction during osteolytic bone metastasis alters the gene expression signature at the TB interface, which further promotes bone resorption and establishment of osteolytic bone metastasis. These findings delineate the potential role of MMP13 as one of the key regulators, commonly upregulated at the TB interface, during TB interaction in osteolytic bone metastasis. MMP13 expression contributes to the osteolytic process by regulating RANKL/OPG levels, activating MMP9, and increasing TGFβ signaling. Our study provides data for understanding the mechanistic role of MMP13 in osteolysis observed during bone metastasis. Additional studies are required to understand the regulation of MMP13 expression during osteolytic bone metastasis and the development of targeted therapeutics.
Disclosure of Potential Conflicts of Interest
No potential conflicts of interest were disclosed.
Acknowledgments
Grant Support: Susan G. Komen for the Cure grant KG090860, Cancer Glycobiology Program from Nebraska Research Initiative and grant CA72781 (R.K. Singh), Cancer Center Support grant P30CA036727 from National Cancer Institute, NIH, and Department of Defense Breast Cancer Research Program Predoctoral Traineeship Award BC083293 (K.C. Nannuru).
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