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Matrix metalloproteinases (MMPs) are a family of 23 extracellular proteases that are best known for their collective ability to degrade all components of the extracellular matrix. We previously demonstrated that genetic ablation of MMP-7 reduced tumour multiplicity in multiple intestinal neoplasia mice possessing a genetic alteration in the adenomatous polyposis coli gene, APC. These mice, commonly referred to as APC-Min mice, are a frequently used model of early intestinal tumourigenesis. To further examine the role of MMPs in intestinal tumour development; we generated APC-Min mice genetically deficient in MMP-2, -9, -12 or -19. Genetic ablation of MMP-2, -12 or -19 did not affect multiplicity or size of intestinal tumours when crossed into the APC-Min system. However, MMP-9 deficient animals developed 40% fewer tumours than littermate controls, though tumour size distribution was unaffected. Intestinal adenomas from MMP-9 deficient mice demonstrated a 50% decrease in proliferating cells compared to control tissues, with no difference in apoptosis. To determine the cellular origin of MMP-9 in these tumours immunofluorescent co-staining with markers for different leukocyte lineages was used to demonstrate that intratumoural MMP-9 is largely a product of neutrophils. These studies extend the potential targets for chemoprevention of intestinal adenomas to MMP-9 in addition to MMP-7, and exclude MMP-2,-12,-19 as attractive targets for intervention.
MMPs are a family of 23 enzymes best known for their ability to cleave virtually all components of the extracellular matrix (Egeblad and Werb, 2002). Due to the ability of MMP-2 and MMP-9 to degrade the basement membrane, they are frequently associated with tumour invasion and metastasis (Egeblad and Werb, 2002). However, roles for MMPs have been identified at virtually every stage of tumour development (Chambers and Matrisian, 1997), and recent evidence suggests that certain MMPs may function in a protective role at certain stages of tumour development (Jacobs, et al., 2007). Furthermore, in recent years a number of non-matrix substrates have been identified for MMPs including growth factors, chemokines, cytokines, angiogenic factors and pro-apoptotic regulators (Noel, et al., 2008). In light of this, rather than simply functioning in pro-metastatic capacity during later stages of tumour progression, MMPs are now known to be involved in both pro- and anti-neoplastic roles throughout all stages of tumour development.
Several MMP family members have been shown to be differentially expressed in both human colorectal tumours and tumours from the APC-Min mouse (multiple intestinal neoplasia), a commonly used model organism for studying intestinal neoplasia (Martinez, et al., 2005, Wagenaar-Miller, et al., 2004) (Moser, et al., 1990). Due to a missense mutation in the tumour suppressor gene APC (Su, et al., 1992), 100% of mice heterozygous for the APCMin allele develop numerous spontaneous tumours throughout the small and large intestine (Moser, et al., 1990). Etiologically, genetic alterations in the human orthologue of APC have been shown to be a causative agent of both sporadic (Nishisho, et al., 1991) and familial (Familial Adenomatous Polyposis [FAP]) (Kinzler, et al., 1991) human colorectal tumours.
A number of genetic modifiers of tumourigenesis in the APC-Min system have been identified which including both coding (Dietrich, et al., 1993) and non-coding (Kwong, et al., 2007) regions. Previously, our lab has shown that genetic ablation of MMP-7, a secreted endopeptidase produced by adenomatous epithelial cells, reduces tumour multiplicity in the APC-Min system by >50% suggesting a pro-tumourigenic role for MMP-7 (Wilson, et al., 1997). Human colonic tumours commonly express several matrix metalloproteinases (MMPs) not detected in normal colonic tissue that are predictors of both positive and negative clinical response and survival (Wagenaar-Miller, et al., 2004).
We chose the APC-Min model of early tumour development to investigate the potential functions of 4 different MMPs expressed by both stromal and tumour cells in the APC-Min system. MMP-2, MMP-9, and MMP-12 were previously reported to be overexpressed by intestinal polyps of the APC-Min mouse (Martinez, et al., 2005). In contrast, MMP-19 is produced by normal intestinal epithelium, but expression is lost upon transformation (Bister, et al., 2004).
MMP-2 (common name “gelatinase A”) is expressed by various stromal cell populations in both human (Poulsom, et al., 1992) and murine (Wilson, et al., 1997) intestinal polyps. Normally, in the APC-Min mouse model only a subset of tumours express MMP-2. However, upon genetic ablation of MMP-7, all tumours that do form express MMP-2 suggesting that in the absence of MMP-7, MMP-2 may function in a compensatory role in promoting tumourigenesis (Wilson, et al., 1997). MMP-2 has been shown to contribute to tumour growth in several experimental systems (Itoh, et al., 1997, Bergers, et al., 2000), making it an attractive target for further study.
Macrophage metalloelastase, MMP-12, is largely expressed by macrophages, though it can be expressed by tumour epithelial cells (Lavigne and Eppihimer, 2005). An elevated level of macrophage derived MMP-12 is commonly associated with an improved prognosis in intestinal neoplasia (Asano, et al., 2007, Zucker and Vacirca, 2004), though the mechanism behind this protective effect is obscure.
MMP-19, also known as RASI-1, was first identified as an autoantigen associated with rheumatoid arthritis (Sedlacek, et al., 1998) and is expressed by a wide spectrum of tissues (Pendas, et al., 1997). In the context of intestine it has been detected on the surface of activated peripheral blood mononuclear cells, TH1 lymphocytes and is normally expressed throughout the intestine in enterocytes, stromal fibroblasts, and macrophages (Mueller, et al., 2000). Unlike most MMPs, expression of MMP-19 is decreased upon transformation of intestinal epithelial cells (Bister, et al., 2004).
Finally, MMP-9 (commonly known as gelatinase B), is produced by several stromal cell populations including neutrophils, mast cells, macrophages, fibroblasts, as well as by the tumour epithelial cells directly (Noel, et al., 2008). Previous studies using MMP-9-null mice in a model of squamous cell carcinoma have shown that MMP-9 deficient animals develop fewer tumours than do littermate controls (Coussens, et al., 2000). Additionally, in a correlative study examining human colorectal specimens, an increasing abundance of MMP-9 was found to correlate with the progression of normal intestinal mucosa to dysplastic adenomas and eventual invasive carcinomas (Herszenyi, et al., 2008). Taken together, these observations suggest that MMP-9 contributes to adenoma progression in a pro-tumourigenic fashion.
We pursued a genetic approach to determining the contribution of MMP-2, -9, -12, and -19 to the formation and growth of intestinal neoplasias in the APC-Min model system.
APC-Min mice (C57Bl/6/J-APCMin/+) and wild type littermates (C57Bl/6J) were bred in our laboratory from breeders obtained from The Jackson Laboratory (Bar Harbor, ME), and genotyped for the APC-Min mutation by PCR as recommended by the supplier. Mice were housed with littermates in microisolator cages lined with CareFresh bedding (Absorption Corp., Ferndale, WA).
Mice lacking MMP-2 (Itoh, et al., 1997) were the generous gift of Dr. T. Itoh. MMP-12 deficient mice (Shipley, et al., 1996) were purchased from The Jackson Laboratory. MMP-9 null mice (Vu, et al., 1998) were the generous gift of Dr. Z. Werb. MMP-19 knockout mice (Pendas, et al., 2004) were obtained from Dr. J. Caterina. All experimental mice were bred at Vanderbilt University and genotyped by PCR using previously described protocols.
Male APC-Min mice were bred to female MMP-2−/− mice, and ensuing heterozygotes were crossed to generate APC-Min-MMP2−/− mice (C57Bl/6/J-APCMin/+;MMP2−/−) and heterozygous and homozygous control littermates, with the Min allele carried along the paternal lineage. A similar breeding strategy was used to produce Min-MMP9−/− (C57Bl/6/J-APCMin/+;MMP9−/−), Min-MMP12−/− (C57Bl/6/J-APCMin/+;MMP12−/−), and Min-MMP19−/− (C57Bl/6/J-APCMin/+;MMP19−/−) mice. All mouse experiments were conducted according to protocols approved by the Institutional Animal Care and Use Committee.
Mice were maintained on a Purina 5015 chow, an 11% fat diet that has been shown to enhance tumour formation in the APC-Min model system (Wasan, et al., 1997). At 17 weeks of age mice were asphyxiated with CO2 and their intestines harvested, fixed overnight in 4% w/w formaldehyde diluted in PBS (Fisher Scientific, Pittsburgh, PA), and stored in 70% ethanol. Tumour number and size was measured using a binocular dissecting microscope by two independent investigators blinded to the genotype of the sample. Tumour multiplicity was compared using the Mann-Whitney U-test. Tumour size was determined by measuring along the longest diameter using digital calipers to the nearest 100μm. Tumour diameter measurements were log-transformed and analysed by a mixed models analysis of variance with an autoregressive correlation structure. For these studies only tumours macroscopically visible (at minimum 0.2mm in diameter) were included in these analyses.
Paraffin embedded, formalin-fixed sections were dewaxed and rehydrated through a series of graded alcohols. Sections were treated for 30 minutes with 0.6% hydrogen peroxide in methanol to destroy endogenous peroxidase prior to antigen retrieval. Antigen was retrieved by microwaving sections for 10 minutes in 10mM sodium citrate buffer. Non-specific binding was inhibited by incubation in a blocking solution (10mM Tris-HCl pH7.4, 0.1M MgCl2, 0.5% Tween20, 1% BSA, 5% Serum) for 1hr at room temperature. Rabbit polyclonal anti-mouse MMP-9 (Abcam, Cambridge, MA) was diluted 1:100 in blocking solution and applied at 4°C overnight. Appropriate IgG controls were used on adjacent sections to evaluate background staining. Sections were washed with TBS (150mM NaCl, 10mM Tris) and incubated with appropriate biotinylated secondary antibody for 1hr at room temperature. Positive cells were visualised with an avidin-biotin peroxidase complex (Vectastain Avidin-Biotin Complex kit, Vector Laboratories, Burlingame, CA) and 3,3′-diaminobenzidine tetrahydrochloride substrate (Sigma). Nuclei were counterstained with Mayer’s hematoxylin (Sigma), washed in TBS, dehydrated through alcohols, cleared in xylenes and mounted.
Proliferation and apoptosis were assessed by immunohistochemistry for phospho-Histone-H3(Ser10) (1:250 dilution; Millipore, Billerica, MA), and cleaved caspase-3 (1:500 dilution; Cell Signaling Technology, Danvers, MA), respectively. Phospho-Histone H3 positive cells were counted from two adjacent sections from multiple tumours of 4 each Min-MMP-9−/− and Min-MMP-9+/+ littermates. A proliferative index was calculated by determining the ratio of positive cells per constant arbitrary unit of area as determined by Metamorph software (Molecular Devices, Sunnyvale, CA). Apoptotic index was similarly calculated by quantifying cells positive for cleaved caspase-3 immunohistochemistry. Results are reported as mean ± standard deviation.
Five micron, paraffin embedded, formalin fixed sections were dewaxed and rehydrated through alcohols. Sections were microwaved in a 10mM sodium citrate solution to retrieve antigen for 10 minutes and allowed to cool. Non-specific staining was prevented by treating sections with a blocking solution for 1 hour at room temperature. Sections were simultaneously treated with a rabbit polyclonal antibody to detect MMP-9 and an antibody to detect leukocytes for 6 hours at room temperature. Neutrophils were stained using a monoclonal rat anti-neutrophil antibody (AbD Serotec, Oxford, UK) diluted at 1:100 in blocking solution. B cells were visualised using a 1:100 dilution of a rat monoclonal antibody recognizing CD45R/B220 (AbD Serotec). Macrophages were demonstrated by staining for F4/80 antigen using a rat monoclonal antibody (AbD Serotec) diluted at 1:100 in blocking solution. Sections were washed in PBS, and incubated with AlexaFluor 594 goat anti-rat (A-11007) or AlexaFluor 488 goat anti-rabbit (A-11008) fluorescently labeled secondary antibodies (Molecular Probes, Carlsbad, CA) and DAPI to visualise nuclei then mounted in aqueous mounting media (Biømeda, Foster City, CA).
To examine the role of various stromal MMPs in intestinal tumour development, we generated Min-MMP-2−/−, Min-MMP-9−/−, Min-MMP-12−/− and Min-MMP-19−/− mice and corresponding littermate controls. Mice were raised on an 11% fat diet to enhance tumourigenesis (Wasan, et al., 1997). Genetic ablation of MMP-2 is known to retard the growth rate of young mice (Mosig, et al., 2007), and mice lacking MMP-19 have been shown to be susceptible to diet induced obesity (Pendas, et al., 2004). However, at the 17-week time point chosen for this study, animals of both lineages were of normal size. Additionally all lineages of knockout animals examined herein had no obvious morphological, behavioral, or pathological differences when compared to wild-type Min littermates. Mice lacking MMP-9, but not those lacking MMP-2, -12 or -19 developed significantly fewer intestinal adenomas than littermate controls (Figure 1 A-D; Table 1). On average, Min-MMP-9−/− developed 24.5±16.1 tumours while wild type littermates developed 41.9±27.6 tumours, indicative of a 40% decrease in tumour multiplicity when MMP-9 is absent (p<0.05). Min-MMP-9+/− and Min-MMP-9+/+ mice developed a similar number of tumours, and comparison of Min-MMP-9−/− (n=21) and Min-MMP-9+/+ and+/− mice (n=26) resulted in a similar reduction in tumour multiplicity (p<0.005).
To determine if ablation of selected MMPs affected tumour size, tumours were measured throughout the intestinal track in five each of MMP single deficient animals and littermate controls and the size distribution was compared using a mixed model analysis (Figure 2 A-D). Mean tumour diameter and 95% confidence intervals presented in a tabular format are listed in Table 1. The mean diameter for all groups examined was 1.2mm, and diameter ranged from 0.2mm to 4.7mm. Based on these results, ablation of any of the tested MMPs did not significantly affect tumour size. Because no affect on tumour multiplicity or size was observed upon genetic ablation of MMP-2, -12 or -19, only Min-MMP-9 mice were further examined.
To examine if MMP-9 ablation affected the rate of tumour cell proliferation or apoptosis immunohistochemical staining was performed, and an index of positive cells per unit area was calculated using Metamorph image analysis software. Using a monoclonal antibody to phospho-HistoneH3(Ser10), a marker specific for late anaphase and mid-metaphase mitosis (Hendzel, et al., 1997), tumours from Min-MMP-9−/− mice had fewer than half as many actively proliferating cells as did Min-MMP-9+/+ littermates (50.3±27.1 vs. 103.2±55.4 cells/unit area) (Figure 3A). However, tumours from both Min-MMP-9−/− and Min-MMP-9+/+ mice had a similar rate of apoptotic cells as measured by immunohistochemical detection of cleaved caspase-3 (35.2±16.9 vs. 29.6±17.0 cells/unit area, Figure 3B), suggesting that MMP-9 functions as a pro-tumourigenic capacity by stimulating tumour cell mitosis.
Several different cellular lineages produce MMP-9 including tumour cells, endothelium, macrophages, neutrophils, mast cells, and fibroblasts (Noel, et al., 2008). To determine the cellular source of MMP-9 in APC-Min tumours, immunohistochemical staining was performed. No MMP-9 positive epithelial or endothelial cells were observed. Positive staining was restricted to stromal populations that morphologically appeared to be leukocytes within the vasculature or lymphatic vessels (Figure 4A). Immunofluorescent co-staining for MMP-9 and leukocyte markers revealed that neutrophils, but not macrophages or B cells, express MMP-9 in the context of our tumours (Figure 4B-D). However, neutrophil abundance was similar in both Min-MMP-9−/− and littermate controls (data not shown). Taken together, these data suggest that neutrophil derived MMP-9 promotes the development of APC-Min adenomas by enhancing epithelial cell proliferation.
In the current study we chose to examine the potential contribution of four members of the MMP family that have been shown to be differentially expressed in intestinal tumours to early tumour development. Importantly, the model system we chose to employ in this study is best suited for studying effects on early tumourigenesis, and would not assess roles for these enzymes during later stage tumour development such as malignant conversion, angiogenesis, and metastatic spread. Genetic ablation of three of these MMPs--MMP-2, -12, and -19--had no discernable effect on adenoma multiplicity or size. We did, however, observe a 40% reduction in tumour multiplicity upon genetic ablation of MMP-9, and a >50% reduction in tumour cell proliferation.
The effect observed here suggests a role for MMP-9 early in neoplastic development, i.e. the stages of tumour initiation and/or promotion. One potential way that MMP-9 could impact tumour initiation, in this case by the loss of the normal APC allele (Luongo, et al., 1994), is through the generation of reactive oxygen species (ROS), which are commonly associated with damage to both DNA and proteins (Kundu and Surh, 2008). An ROS-based mechanism of tumour initiation has been previously identified for MMP-3, which incidentally can also act as an activator of pro-MMP-9 (Inuzuka, et al., 2000). MMP-3 expression induces an alternatively spliced form of the small GTPase rac1 known as rac1b. This variant induces an increase in intracellular ROS, elevated levels of the transcription factor Snail, and ultimately genomic instability, though the initial cleavage product leading to this change has not yet been identified (Radisky, et al., 2005). Importantly, MMP-9 has been shown to be capable of substituting for MMP-3 in this same pathway (Radisky, et al., 2005).
In an animal model of pancreatic tumourigenesis, genetic ablation of MMP-9 has been shown to inhibit angiogenic switching, as well as reduce tumour multiplicity and growth (Bergers, et al., 2000). However, in the APC-Min model adenomas are in general less than 2mm in size and therefore unlikely to be dependent on angiogenesis for growth (Folkman, 1992). As expected, no difference in tumour angiogenesis or total vasculature was detected between MMP-9+/+ and MMP-9−/− adenomas (data not shown), suggesting that MMP-9 influences tumour cell proliferation prior to the angiogenic switch. Despite a reduction in proliferative index, the lack of a difference in tumour size between MMP-9+/+ and MMP-9−/− mice is consistent with the tumours being growth-limited by the lack of an angiogenic event in this tumour model.
MMP-9 co-localised with neutrophils, a finding consistent with previous findings that MMP-9 is stored in the tertiary granules of neutrophils (Opdenakker, et al., 2001). Although angiogenesis does not appear to contribute to the biological effect observed following MMP-9 ablation, neutrophils have been implicated as a key regulator of the initial angiogenic switch (Nozawa, et al., 2006) by virtue of their granule contents that uniquely contain TIMP-1 free MMP-9 (Ardi, et al., 2007). Defects in neutrophil migration associated with MMP-9 deficiency have been reported in some (Khandoga, et al., 2006), but not all (Felkel, et al., 2001) model systems. However, in our samples, there was no difference between the number or distribution of neutrophils in APC-Min tumours of wild-type and MMP-9 null mice, suggesting that MMP-9 is not essential for neutrophil recruitment or migration to tumours in the gastrointestinal tract.
The observed effect may be the result of MMP-9 activity influencing neutrophil degranulation, even though neutrophil numbers are not affected. Neutrophils generate reactive oxygen and nitrogen species capable of damaging adjacent cells (Klebanoff, 2005, Kuby, 1997), and, as discussed above, can result in tumour initiation (Josephy and Coomber, 1998). MMP-9 activity could be processing cytokines, a mechanism that has been shown to be a major regulator of neutrophil activity. For example, MMP-9 mediated cleavage of full length IL-8 (1-77) to a truncated form (7-77) enhances IL-8 activity by more than tenfold (Van den Steen, et al., 2000). This enhanced activity stimulates neutrophils via a positive feedback loop resulting in increased IL-8 binding, migration, production of MMP-9 (Opdenakker, et al., 2001) and ROS (Guichard, et al., 2005), and ultimately degranulation (Van den Steen, et al., 2000). Thus, it is possible that MMP-9 mediated differences in cytokine signaling are involved in modulating neutrophil-mediated genetic instability.
MMP-9 alters the proliferation of APC-Min adenoma cells, suggesting a likely effect on tumour promotion through the expansion of initiated cells that are lacking APC function. Although we observed difference in tumour multiplicity rather than size in Min-MMP-9−/− mice, this may be reflective of the fact that we only counted macroscopically visible tumours (>0.2mm). In addition to cleaving matrix components, MMPs have been demonstrated to act upon a number of non-matrix substrates including cytokines, cell surface makers, growth factors and their inhibitory binding proteins (Noel, et al., 2008, Sternlicht and Werb, 2001). Specifically, MMP-9 activity has been shown to liberate a number of growth factors from the matrix including vascular endothelial growth factor (VEGF) (Belotti, et al., 2003, Bergers, et al., 2000), transforming growth factor-β (TGF-β) (Mott and Werb, 2004), and basic fibroblast growth factor (bFGF) (Brauer, 2006). Further, MMP-9 has the ability to convert pro-TNFα to an active form (Brauer, 2006), and has been shown to degrade IGF-BPs thus allowing for higher levels of circulating insulin-like growth factors (IGFs). In particular, IGF-II (Hassan and Howell, 2000), VEGF (Alferez, et al., 2008, Goodlad, et al., 2006), and EGF (Alferez, et al., 2008) have all been shown to affect growth and multiplicity of APC-Min tumours.
The APC-Min model has been particularly useful for identifying targets for chemoprevention of colonic tumours (Corpet and Pierre, 2003, Hawk, et al., 2005). Here we have demonstrated that MMP-9 but not MMPs-2, -12, or -19 contributes to the development of intestinal adenomas. This would suggest that a selective inhibitor of MMP-9 in combination with inhibition of MMP-7, as identified in our previous work (Wilson, et al., 1997), may be a useful strategy for the prevention of adenoma formation. Conversely, broad-spectrum inhibition of multiple MMPs would not have additional benefit and may instead be associated with adverse effects (Fingleton, 2008).
In conclusion, we have identified MMP-9, but not the related MMP, MMP-2, or MMP-12 and -19 as a contributor to early intestinal tumourigenesis. This expands both the number of molecules important for early tumour formation and the potential roles of MMP-9.