Here we have examined the function of two proteases, MMP7 and MMP9, in a mouse model of spontaneously metastasizing breast cancer. Genetic ablation of either of these enzymes did not impact the development or growth of multifocal tumors in mammary glands. Lung metastasis was significantly attenuated in MMP9-null, but not MMP7-deficient animals. MMP9 was expressed in inflammatory cells, primarily neutrophils, in tumor-bearing lungs. Experimental metastasis assays allowed determination of a time-frame most relevant for the MMP9-mediated effect, which was in the later outgrowth stages of lung foci. In agreement with this observation, lung tumors from MMP9-deficient mice showed decreased angiogenesis. Surprisingly, the role of MMP9 appeared to be dependent on strain. In our studies, only mice that had significant genetic background derived from C57BL/6 showed reduced metastasis, whereas mice fully of the FVB/N background did not. These strain-specific responses were also observed in a study using a pharmacological inhibitor with high selectivity for MMP9, and thus suggest that tumor responses to MMP inhibition are controlled by genetic differences.
The function of several proteases from multiple enzyme families has now been investigated in the MMTV-PyVT model [28
] This was a model identified by the Protease Consortium, a group of investigators with expertise in different protease families, as a valid setting for assessing differential contributions of proteases to multistage tumor development [32
]. As with MMP9, two other proteases – uPA and plasminogen - were found to contribute to development of lung metastases, but not to mammary tumors [28
]. In contrast, the cysteine protease cathepsin B, which is expressed by tumor cells and macrophages, contributed both to growth and progression of mammary tumors as well as to development of lung metastases [30
]. A different pattern was seen with MMP14 (also known as MT1-MMP). In MMP14-null mammary glands, there was an increase in primary tumor growth rate, but this was followed by a reduction in lung metastasis [31
]. The data suggested that MMP14 facilitated tumor dissemination by collagen cleavage in the peritumoral environment. There are multiple ways in which MMP9 could affect metastasis. MMP9 has been identified as a critical component for priming of the “pre-metastatic niche”, whereby soluble signals from a primary tumor result in selective preparation of certain organ sites to facilitate survival and growth of metastatic lesions in those organs [33
]. Interestingly, we saw comparable effects irrespective of whether analyzing spontaneous metastasis or experimental metastasis, a result different to that reported by Hiratsuka et al., who reported effects of MMP9 ablation on metastasis only when a primary tumor was present [33
]. Moreover, the imaging analysis suggested that the MMP9-mediated effect was more important in later stages of tumor outgrowth rather than initial survival of metastasizing cells. This interpretation is further suggested by the overall smaller size of the metastatic foci in MMP9-/-
mice and the reduced vascular density. It should be remembered however, that we could only assess the temporal effect of MMP9 deletion in the experimental metastasis setting, where development of a pre-metastatic niche is not an issue. In the spontaneously metastasizing tumors of the MMTV-PyVT mice, it is possible that MMP9 contributes both to angiogenesis as our data indicates, but also to priming of a metastatic niche.
As has been reported in other settings [19
], the primary source of MMP9 in these studies was cells of the myeloid lineage, particularly neutrophils. A previous analysis of MMTV-PyVT mice carrying a lacZ reporter driven by a region of the MMP9 promoter, suggested that mammary tumor cells express MMP9 at a timepoint corresponding to increased invasive activity [18
]. We never saw any MMP9 expression by tumor cells, which may explain the lack of impact of MMP9 deficiency in the mammary glands. One potential reason for the disparate expression patterns is that the MMP9 promoter used in the lacZ analysis did not recapitulate the full in vivo
promoter, including possible distant enhancer elements so that its expression pattern was not completely accurate.
The most surprising finding in our study was the influence of strain on the impact of MMP9 ablation. There are many reports of strain-related polymorphisms differentially affecting phenotypes [35
], including cancer [36
] in mouse models. Indeed, analysis of the phenotype of mice carrying the MMTV-PyVT transgene in different genetic backgrounds indicates a strong effect of strain on tumor aggressiveness in this model [37
]. In particular, introduction of C57BL/6 increases tumor latency and extends the timing of metastasis to 120 days. The Hunter lab has invested a major effort in discovering potential host determinants of metastasis and have identified Sipa1
, and Rrp1b
as two genes, polymorphisms of which can impact susceptibility both in mice and humans [38
]. Adding our results to their data further expands the pool of strain-specific metastasis modifiers, although it is not clear if Mmp9
is itself the gene that is polymorphic. Indeed, analysis of strain-dependent modifiers of MMTV-PyVT metastasis indicated that chromosomes 6, 9, 13, 17 and 19 contained loci related to metastasis susceptibility [41
], not chromosome 2 where Mmp9
is located in the mouse.
Why MMP9 shows this strain dependent effect is unclear. The possibilities are that the enzyme itself, its relevant substrate in the lung environment or a modifier of its function is differentially regulated according to strain. When we examined neutrophil presence in the lungs of tumor-bearing mice, we detected no difference between the FVB/N and C57BL/6 backgrounds indicating that the primary source of MMP9 is not dissimilar. While we have identified the process of angiogenesis as the mechanism for the difference seen between MMP9 wild-type and null mice of the C57BL/6 strain, we have not determined the substrate responsible. It has been suggested that release of VEGF from matrix sequestration is one mechanism by which MMP9 promotes angiogenesis [24
]. In a pilot study with the VEGF/VEGFR1 complex-detecting antibody [42
] used in the Bergers study, we saw reduced complex formation in MMTV-PyVT; MMP9-/-
lung tumors compared to controls (data not shown), suggesting that this is a possible explanation for the angiogenesis differences observed. Other roles for MMP9 in the angiogenic process include processing or liberation of other angiogenic growth factors [43
]; degradation of basement membrane proteins to facilitate endothelial cell migration and tube formation [43
]; and recruitment of pericytes to stabilize newly-formed vasculature [25
In all our experiments in which FVB/N mice were used, deficiency of MMP9 or treatment with the gelatinase inhibitor SB-3CT appeared to be associated with enhanced tumor development. In the case of the MMP9-/-
mice, this effect did not reach statistical significance, although a clear trend was evident (). Following treatment with the inhibitor SB-3CT, a significant enhancement of luciferase signal, which corresponds to tumor burden, was seen. There have been reports previously of MMP9 ablation associated with enhanced tumor development. Coussens et al., reported that MMP9-null mice developed fewer, but more aggressive tumors in the K14-HPV16 skin cancer model [44
]. Interestingly, these mice were also on the FVB/N background. In a model of lung metastasis of colonic tumor cells, Chen et al showed that use of the MMP-inhibiting agent doxycycline, which resulted in lower levels of MMP9, led to decreased numbers of metastatic foci, but those foci that did develop were larger and more vascularized [45
]. Intriguingly, while there are multiple reports of increased MMP9 levels being a marker of poor prognosis, there is one clinical study suggesting that higher levels of MMP9 are associated with good prognosis in node-negative breast cancer [46
]. This may reflect the heterogeneity with respect to MMP9 function that is related to genetic modifiers present in human populations, and has important implications for the treatment of metastatic breast cancer. Our data suggest that certain patients could respond to an MMP inhibitor by worsening disease progression while others show reduced metastatic growth. In seeming agreement with this idea, one MMPI inhibitor trial in small cell lung cancer was halted because patients treated with the drug were dying of their disease faster than were placebo-treated patients [4
In conclusion, we have identified a metastasis-specific role for MMP9 in a transgenic mouse model of multistage mammary tumorigenesis that has been regarded as a credible model of human disease. The function of MMP9 appears to be in promotion of angiogenesis at the metastatic site; however this function is dependent on genetic background. MMP9 was a primary target of all synthetic MMPIs tested clinically in cancer patients, yet no successful inhibitor was identified. Our data would suggest that, while MMP9 may be a reasonable target, its contribution to tumor progression is highly influenced by genetic factors. The results from the drug studies reported here suggest that opposing results of MMP inhibition can be achieved i.e. more or fewer metastases, depending on genetic background. This finding clearly complicates interpretation of clinical studies of MMPIs and suggests that future clinical use of MMP9 inhibitors may depend on the identification of a “responsive haplotype” to allow selection of patients likely to respond.