Altered metalloproteinase activity has a direct impact on mammary gland physiology as controlled remodeling of mammary gland ECM through pericellular proteolysis is important for mammary morphogenesis, cyclical changes during the estrous cycle, and the differentiation necessary for lactation. Although ECM breakdown is required by epithelial cells, stromal cells, including fibroblasts, as well as inflammatory and immune cells are major producers of metalloproteinases 2. In addition, ADAM proteases operate as sheddases for cell surface substrates and participate in stromal-epithelial cross-talk through paracrine delivery of signals 10. Finally, TIMPs, as inhibitors of metalloproteinases, are critical regulators of matrix turnover in the mammary gland. The spatial localization of proteins of the metalloproteinase axis may be particularly important for the orchestration of these events.

During mammary morphogenesis in the mouse, MMP3 localizes to elongating ducts 11 and its overexpression results in supernumerary ductal branching 12. MMP2 and MMP14 deficient mice display diminished ductal elongation, whereas MMP9 deficiency has no effect 13. ADAM17 plays a role in paracrine communication involving epithelial-specific amphiregulin and stromally restricted epidermal growth factor receptor (EGFR) 14. Specifically, Amphiregulin^-/- mammary glands have impaired ductal outgrowth 15, while Adam17^-/- mammary glands have severe growth and branching retardation that phenocopies EGFR-deficient mammary glands 14. Manipulation of TIMP levels also leads to alterations in mammary morphogenesis. Reduction of TIMP1 expression through antisense RNA production leads to more extensive branching, increased ductal elongation, and increased proliferative index. Conversely, TIMP1 upregulation leads to inhibition of ductal elongation without affecting bifurcation or lateral budding 16. TIMP3 deficient mice also show accelerated ductal elongation but normal branching patterns 17. Orthotopically implanted TIMP-containing pellets result in inhibition by TIMP4, but promotion by TIMP2 of ductal outgrowth 17. Thus, individual members of the metalloproteinase axis are not necessary for gland development per se, but are required for refining the ductal and branching patterns within the mammary gland. This is emphasized by the fact that most phenotypes in genetic models of MMPs and TIMPs are transient; the glands become lactationally competent when brought to parturition.

Reversion of a lactating gland to a virgin-like state during involution requires an extensive remodeling of the ECM along with epithelial cell death. The first and second phases of involution have been designated protease-independent and protease-dependent stages, respectively, based on the expression of MMPs and TIMPs 18. Mammary gland involution is accelerated upon mammary-specific over-expression of an autoactivating form of MMP3, due to unscheduled apoptosis early in pregnancy 19. In contrast, excess TIMP1 delivered through an implanted pellet delays gland regression 20. TIMP3 is produced by epithelia and stroma and its loss leads to accelerated involution that cannot be rescued by reinitiation of suckling 21. The substrates of MMPs that have been identified during involution include components of the ECM, as well as proteins involved in cell-cell, and cell-ECM adhesion. MMP3 cleaves the ECM protein entactin, which interacts with other ECM proteins and integrins 19. TIMP3 deficiency leads to fibronectin fragmentation 21 and liberates the DIII fragment of laminin-5 during involution, which activates EGFR 22. Metalloproteinases fragment E-cadherin, releasing a degradation product that further destabilizes E-cadherin function, compromising epithelial integrity during involution 2324. MMPs and TIMPs are also implicated in regulating adipogenesis during the third phase of mammary gland involution. While genetic deletion of MMP3 in the gland does not affect epithelial apoptosis, immature adipocytes have increased differentiation, displaying accelerated adipogenesis; an effect phenocopied by TIMP1 overexpression 25. These studies highlight the importance of tissue interactions during involution, with the mammary stroma actively contributing towards epithelial cell death.

A review by Vorbach and colleagues 26 presented a concept that the mammary gland may have evolved from the innate immune system. This hypothesis suggests that the gland's initial function was the provision of innate immunity, and its nutritional role evolved later. Indeed, failure of passive transfer of immunity from mammary gland secretions to mammalian newborns can contribute to neonatal mortality. Various studies show that immune cells are present in the mammary stroma and are implicated in gland development. In humans, extramedullary hematopoietic cells have been found in the stroma of the infant breast anlage 27. Leukocytic infiltrates have been documented throughout pubertal and adult breast development 27. Colony stimulating factor (CSF)-1 mutant mice (Csf1^op/op) that lack macrophages, or mice deficient in eotaxin, a chemokine that recruits eosinophils, have impaired formation of terminal end buds, ductal invasion, and ductal branching 28. Whole body irradiation with a sublethal dose that depletes the bone marrow leads to impaired epithelial ductal elongation, suggesting a general role for immune cell involvement in murine gland development 28. Expression of inflammatory mediators and acute phase proteins, along with the presence of neutrophils, plasma cells, macrophages and eosinophils in involuting glands all point to a role for inflammation 2930. The local tissue deconstruction during this process may be facilitated by the activation of innate immune components, with macrophages likely performing a corpseclearing function 31. At present, very little is known about the presence or role of components of the adaptive immune system in mammary gland physiology. Further, the role of the metalloproteinase axis in mediating inflammation and immunity during morphogenesis and involution currently remains unexplored.

Genetic mouse models are powerful tools for understanding the role of specific genes in breast cancer development. MMP3 overexpression driven by the whey acidic protein promoter or MMP7 overexpression under the mouse mammary tumor virus (MMTV) promoter both lead to mammary tumor formation at low frequency 3233, while MMP7 deficiency results in 60% reduction of early mammary lesions in a chemical carcinogenesis model 34. MMTV-ras mice lacking MMP11 have significantly increased survival and a smaller tumor burden compared to wild type but develop significantly more metastatic lesions 35. Overexpression of the membrane-anchored MMP14 in the mammary epithelium leads to increased lymphocytic infiltrates, periductal fibrosis, ductal hyperplasia with dilated ducts, dysplasia, and adenocarcinoma in multiparous transgenic mice 36. The effect of TIMP1 on mammary tumorigeneis has been assessed in transgenic mice that either secreted TIMP1 systemically using an albumin promoter, or expressed it in a mammary-specific manner using the MMTV promoter 37. When subjected to the DMBA model of mammary carcinogenesis or crossed with MMTV-PyMT mice, systemic TIMP1 elevation reduced tumor burden by 70% and 44%, respectively. Metastasis was also inhibited. Interestingly, mammary-specific TIMP1 overexpression was ineffective against mammary tumorigenesis in both models. On the other hand, a recent report showed inhibition of MCF10A (non-transformed, immortalized mammary epithelial cells) apoptosis by recombinant TIMP1 in a metalloproteinase inhibitor-independent capacity 38. A recent study has revealed that TIMP2 overexpression in the mammary gland increases MMTV-Wnt1-induced mammary tumor latency, with tumors showing lower bromodeoxyuridine and CD31 positivity, and higher TUNEL (terminal deoxynucleotidyl transferasemediated dUTP nick end labeling) positivity compared to wild-type Wnt1 tumors 39. Thus far, genetic studies that address the role of TIMP3 or TIMP4 in mammary tumorigenesis are lacking, although several in vitro and clinical reports suggest that these remaining TIMPs may also be important. For instance, overexpression of engineered mutant TIMP3 protein that mimics the Sorsby's Fundus Dystrophy mutation promotes apoptosis in MCF-7 cells 40, and metastasis of TIMP3 overexpressing MDA-MB-435 breast cancer cells is significantly reduced 41. Notably, Timp3 is found silenced by promoter methylation in a panel of human cancer cell lines derived from primary breast cancers and metastases to brain 42434445. TIMP4 was originally identified in human breast cancer 46 and its overexpression in human breast cancer cells diminishes growth and metastasis in athymic mice 47. Individual members of the metalloproteinase axis investigated to date are able to function as tumor modifiers in a variety of breast cancer models, with increased MMP or decreased TIMP activity generally associated with tumor promotion. Future investigations exploring non-proteolytic functions of members of this axis, as well as characterizing newer members such as ADAMs and ADAMTSs, will better define their specific contribution to mammary tumorigenesis.

Classically, inflammation is associated with immune surveillance against neoplasms 48, and tumors are known to develop strategies to circumvent immune recognition and clearance. Although mouse models provide an opportunity to directly test the specific role of individual inflammatory and immune cell types and effector molecules such as cytokines in mammary tumourigenesis, there has been very little work addressing this important issue. A few studies using mice point to a protective role of immune cells in tumorigenesis: concurrent lack of the immune mediators granulocyte macrophage CSF (GM-CSF)1 and interferon-γ lead to spontaneous tumor formation in mice, including mammary adenocarcinoma 49; and the loss of neutrophil collagenase, MMP8, leads to increased susceptibility to skin cancer due to ineffectual neutrophil infiltration, indicating the importance of a timely inflammatory response in protecting against skin carcinogenesis 50. In contrast, other studies have pointed to a pro-tumorigenic role for inflammatory cells, specifically tumor-associated macrophages 51 and B cells 52. These cells are postulated to foster tumor growth and metastasis through release of cytokines and matrix remodeling enzymes. Genetic crosses of macrophage-deficient, osteopetrotic mice that are mutant for the macrophage growth factor CSF1 (Csf1^op/op) with MMTV-PyMT mice show reduced progression to malignancy and metastatic disease 53. Mice that are deficient in cyclooxygenase-2 have decreased levels of prostaglandin E2 and decreased tumor multiplicity 54 when crossed into the breast cancer model expressing the activated form of Neu/human epidermal growth factor receptor (HER)2 (MMTV-NeuNDL – neu deletion mutant).

A system to study the importance of a wide variety of immune and inflammatory cells as well as cytokines on mammary tumorigenesis exists in the MMTV-PyMT model. Beyond macrophages, we have observed other inflammatory and immune cell types, namely CD3+ T lymphocytes, B cells, mast cells, and neutrophils in and around the mammary tumors arising in MMTV-PyMT mice (Figure 1). The presence of these cells provides an opportunity to study the effect of specific cell types and effector molecules in mammary tumor progression by crossing this model to mice with the desired gene deficiencies. Although the MMTV-PyMT model has relatively high tumor multiplicity and short latency, histological analyses reveal that this model shares molecular and morphological characteristics with human breast cancer 55 as well as the immune and inflammatory cell populations shown in Figure 1. Additionally, the role of metalloproteinase axis members that are linked to regulation of inflammation can be functionally assessed using this model in combination with mice deficient in the protease or inhibitor of interest.

Many studies have attempted to correlate MMP, ADAM and TIMP expression profiles with breast cancer progression and common trends have emerged (reviewed in 5657). The levels of MMP expression usually correlate with aggressive breast tumors, those of individual TIMPs suggest a more complex association with breast cancer, while data on ADAM and ADAMTS expression in breast cancer are relatively recent 75758. High levels of MMP9, which degrades type IV collagen in basement membrane, is associated with poor prognosis in breast cancer independent of the cell type expressing this protease 5960. Patients with atypical ductal hyperplasia are at increased risk of developing invasive breast cancer. MMP1 protein was detectable in a subgroup of patients with atypical ductal hyperplasia that had a history of cancer 61 and this protease was found in ductal lavage, leading to the suggestion that MMP1 may identify atypical ductal hyperplasia patients that are at risk for developing breast cancer. Studies on TIMP1 expression in breast cancer show both a role for and against it as a positive prognostic factor 58, owing to its diverse effects on cellular proliferation, angiogenesis, and apoptosis, as detailed in a recent review by Cruz-Munoz and Khokha 7. Lipton and colleagues 62 measured plasma TIMP1 levels via ELISA (enzyme-linked immunosorbent assay) and correlated its elevation with higher serum HER2 levels, increased metastasis, and reduced survival in breast cancer patients. Real time-PCR analysis of breast cancer tissue correlated TIMP3 overexpression with success of adjuvant endocrine therapy 6364. Similarly, TIMP3 mRNA levels in breast tumors significantly associated with good prognosis and longer disease-free survival 65. In contrast, TIMP3 levels were found to be higher in mammographically dense breasts, which are considered to be at a higher risk for developing breast cancer 66. In another study, higher mRNA expression of the membraneanchored MMP inhibitor RECK (reversion-inducing cysteine-rich protein with Kazal motifs) in breast tumors was found to be an independent prognostic indicator associated with a longer relapse-free survival time 67.

In a tissue microarray study of primary invasive ductal carcinoma, high individual expression of MMP9, MMP11, TIMP1, and TIMP2 was significantly associated with increased incidence of metastasis at five years post-surgical resection 68. When the authors accounted for cell type-specific expression (tumor cells, fibroblasts, inflammatory mononuclear cells), additional specific members (MMP1, MMP7, MMP13, MMP14, TIMP3) had significant associations with developing metastastic disease 68. In a follow-up study 69, the most powerful indicator of distant relapse-free survival in breast cancer patients was a set of MMPs and TIMPs whose expression was specific to tumor-associated mononuclear inflammatory cells. Similarly, separating breast tumor tissue into different cellular components revealed that TIMP3 was not present in ductal carcinoma in situ or normal epithelium, but was significantly overexpressed in myofibro-blasts and myoepithelial cells surrounding ductal carcinoma in situ 70. Thus, such profiles were less informative when bulk tumor, fibroblasts, or tumor cells were analyzed, suggesting that monitoring inflammatory cell-specific expression may provide clinically important insights. Future studies must consider the cell- and stage-specific patterns of these proteins to resolve present evidence that is limited and at times contradictory with respect to the association of TIMP expression with breast cancer.

ADAM and ADAMTS metalloproteinases are becoming recognized as important factors in breast cancer. ADAM9, ADAM12, ADAM15, ADAM17, ADAM23, ADAM28, and ADAMTS1 have all been found in breast cancer 56. Levels of ADAM9 correlate positively with HER2 levels 6 and with positive response to Tamoxifen 5. A possible diagnostic role for the soluble form of ADAM12 has been proposed since urine levels of this metalloproteinase positively correlate with breast cancer progression 5. ADAM17 is overexpressed in breast tumors and its inhibition leads to decreased cell proliferation in vitro or tumor growth in xenograft models 5.

The critical role of ADAM17 in mediating tumor necrosis factor (TNF)-initiated inflammation 71 and/or its role in transactivating EGFR through the cleavage of EGF ligands such as transforming growth factor-α may underlie these effects 10. Although the biology of ADAMs is less understood than that of MMPs, their ability to shed cell surface molecules qualifies them and their substrates as candidates for breast cancer progression biomarkers.

The importance of cytokine signaling as a link between inflammation and cancer has been highlighted 72, and the bioavailability of many of these critical molecules is regulated by the metalloproteinase axis. Figure 2 illustrates the metalloproteinases and the potential substrates linked to specific aspects of an inflammatory or immune response, such as the generation of chemokine gradients, immune cell influx, lymphocyte activation and effector functions. Each of these aspects is described in greater detail by Murphy and colleagues 9. For instance, ADAM17 processes a number of cell surface proteins, including TNF, fractalkine and GM-CSF, all important recruiters and activators of macrophages. Ductal lavage of the breast shows the presence of macrophages 73 and tumor-associated macrophage density has been correlated with poor prognosis 74. CSF1, an important growth factor for macrophages, is over-expressed in human breast cancers and its expression correlates with high grade tumors and poor prognosis 51. Given these clinical observations, an intriguing avenue for investigation is the contribution of metalloproteinases to macrophage function in breast cancer.

Several experimental models have linked TIMP activity to inflammation, although such a function in breast cancer is unexplored. TIMP1 deficiency promotes neutrophil accumulation in an inflammatory model of bleomycin-induced lung injury 75, whereas TIMP2 deficiency has no effect. TIMP3 regulates the bioactivity of the inflammatory cytokine TNF through its physiological inhibition of the TNF sheddase, ADAM17/TNF alpha converting enzyme, which is critical for several physiological systems that depend on TNF 76777879. Increased numbers of neutrophils are observed in Timp3^-/- remodeling hearts in an otherwise non-inflammatory model of cardiac pressure overload 77. Timp3^-/- mice are also hyper-responsive to endotoxin, which causes systemic release of TNF in a model of innate immunity 78. Overall, these data point to select candidates of the metalloproteinase axis that can potentially participate in inflammation during breast cancer progression. Specifically, the coordinated action of TIMP3, ADAM17, and TNF in initiating signal transduction pathways essential to innate immune responses that can impact mammary tumourigenesis is currently under investigation in our lab.

In addition to the metalloproteinase-mediated generation of critical inflammation triggers, metalloproteinases are, in turn, utilized by immune cells to further propagate the inflammatory reaction. Of the MMPs, MMP9 is often implicated as an inflammation-related MMP, with reported roles in carcinogenesis models 8081. In breast cancer samples, MMP9 in the stroma is found in neutrophils, macrophages, and T lymphocytes 56. In a mammary tumorigenesis xeno-graft model, CD4+ T-cells in the periphery as well as within the breast tumor expressed high levels of MMP9 82. MMP3 is often present in infiltrating T-lymphocytes when found overexpressed in breast carcinoma 56. During inflammation, increased TNF has been shown to induce expression of collagenases 83. Notably, the roles of MMPs, such as the neutrophil collagenase MMP8 primarily produced by inflammatory cells 50, and macrophage elastase MMP12 84, have yet to be elucidated in mammary tumorigenesis.

To address the possible role of MMPs, TIMPs, and ADAMs in inflammation in breast cancer, we performed expression profiling of members of these families in the Oncomine database 85, which contains microarray expression data from a variety of human cancers. Of the 31 studies on breast cancer, only the study by van't Veer and colleagues 86 recorded lymphocytic infiltration as one of the many clinical parameters. This study profiled breast tumor mRNA from 117 patients, of which 89 were lymphocytic infiltrate-negative and 28 were lymphocytic infiltrate-positive. Lymphocytic infiltrate positivity correlated with BRCA mutant and estrogen receptor-negative status in an unsupervised two-dimensional clustering analysis 85. We found differential expression of specific MMPs, ADAMs, and TIMPs, when the sample set was stratified based on lymphocytic infiltration (Table 1). Of the 22 MMPs examined in their study, several showed differential expression. Specifically, mRNAs of the inflammation associated MMPs, MMP9 and MMP12, were upregulated in lymphocyte infiltrate-positive breast cancers. ADAM8, a reported sheddase for L-selectin, and ADAM17, the sheddase for TNF, were also upregulated, consistent with their suggested pro-inflammatory function. Interestingly, the mRNA expression of the membrane-type MMPs did not correlate with lymphocytic infiltrate status in this study, and ADAMTS expression was also variable. Low expression of TIMP1, TIMP3, TIMP4, and RECK mRNA significantly correlated with lymphocytic infiltrate positivity, while TIMP2 was comparable between groups. While this one study shows intriguing trends, further clinical studies that document lymphocyte involvement are needed to reveal the association between global gene expression patterns, inflammation, and breast cancer.