Abstract

Introduction

Breast cancer progression is accompanied by increased expression of proteases that are capable of degrading the extracellular matrix (ECM) leading to cancer cell invasion and eventually to dissemination of cancer cells to other organs. The proteolytic capability of breast cancers is reminiscent of the inherent proteolytic capability of the healthy mammary gland. Extracellular proteases are necessary for the dynamic tissue remodeling that takes place during normal mammary gland development and pregnancy cycling (reviewed in [1]). During development, the invasive phenotype of terminal end buds is associated with a controlled proteolytic digestion of ECM in the fat pad, and side branching of the newly formed ducts requires a focal degradation of basement membrane. The proliferative response of the epithelium during pregnancy and lactation is accompanied by limited proteolytic activity. In contrast, the involution of the lactating gland to a resting gland after weaning restructures the entire gland and requires extensive degradation of ECM and basement membranes. Extracellular proteases are thus more abundant during involution than at any other stage during development and the pregnancy cycle. The similarity between the proteolytic repertoire of the normal gland and the proteolytic capability of mammary cancers is best illustrated by the fact that the cellular expression patterns of individual proteolytic components are very alike, especially when comparing involution and cancer [2]. In terms of extracellular proteolysis, breast cancer in many ways resembles a mammary gland in a state of continuous and uncontrolled tissue remodeling.

Matrix Metalloproteases and Plasminogen Activators

Two families of extracellular proteases have been extensively studied in relation to mammary gland morphogenesis and cancer. These are the matrix metalloproteases (MMPs) and the group of serine proteases that govern the activation of plasminogen (Plg). Figure 1 illustrates the connected extracellular proteolytic network of the MMPs and the Plg activation system (PA system).

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Figure 1

The PA and MMP systems in the mouse. Plasmin and MMPs are two major sources of extracellular proteolysis. Plg is converted to the active protease plasmin by one of three Plg activators, uPA, tPA, and pKal. The generation of plasmin is enhanced by the binding of uPA to the cell surface receptor uPAR, and by the binding of tPA to coagulated fibrin or to cell surface proteins (annexin II [86] and CKAP4 [87]). The conversion of pro-uPA to active uPA may be initiated by several proteases, depending on the tissue in question. Additional pro-uPA is then activated by plasmin in a positive feed-back loop. The PA system is tightly controlled by several serine protease inhibitors (serpins); the primary and most specific inhibitors are the Plg activator inhibitor PAI-1 that targets uPA and tPA, and the plasmin inhibitor α₂-antiplasmin (α₂AP). Major substrates of plasmin include fibrin, fibronectin, laminin, and other ECM proteins, latent TGF-β and other growth factors, and pro-MMPs. The 22 different mouse MMPs are all extracellular, and are either soluble or membrane-anchored. Like the serine proteases of the PA system, the MMPs are also synthesized as inactive proforms that require proteolytic activation. Ten of the 22 pro-MMPs, including the membrane-anchored MMPs, have a furin cleavage site and may be activated by furin-like proprotein convertases before they are secreted. The remaining pro-MMPs are activated extracellularly, typically by plasmin or by other MMPs. The MMP system is counterbalanced by a group of four inhibitors, TIMPs, that have varying specificities for individual MMPs. In addition, the membrane-anchored MMP inhibitor RECK regulates a subgroup of MMPs including MMPs-2, -9, and -14 [88]. An excess of plasmin or MMP activity may ultimately be cleared by α₂-macroglobulin (α₂M) which is a non-specific protease scavenger. The MMPs are collectively able to degrade any component of the ECM. Important substrates for the MMPs as a group include the native fibrillar collagens and all four major components of the basement membrane: collagen type IV, laminin, nidogen/entactin, and heparan sulfate proteoglycans.

The liver-derived proenzyme Plg is found in high concentration throughout the body. In contrast, expression of the specific urokinase- and tissue-type Plg activators (uPA and tPA) is induced locally and thus determines where and when Plg is converted to the active protease plasmin [3, 4]. Plasmin, activated by uPA, is important for a number of tissue remodeling processes that, in addition to pregnancy cycling of the mammary gland [5], include wound healing [6] and embryo implantation [7]. In contrast, tPA is acknowledged to be primarily involved in thrombolysis and neurobiology [3, 4] but is also active during wound healing [8]. A newly identified Plg activator, plasma kallikrein (pKal), is involved in adipocyte differentiation in the involuting mammary gland [9] and in wound healing [8].

The MMPs constitute a large family of 22 extracellular metalloproteinases in mice (24 in humans) [10, 11] (an updated list of human and mouse proteases is available at http://web.uniovi.es/degradome/). MMPs are essential for a number of physiological tissue remodeling processes during ontogenesis and adult life, including mammary gland development [12, 13], bone development [14], wound healing [15], and embryo implantation [7]. During mammary gland development MMP-2 is involved in ductal elongation, and MMPs-2 and -3 are involved in ductal branching [13]. MMP-3 is also rate limiting for adipocyte differentiation during post-lactational involution [16]. The ductal tree is eventually fully formed in mice that are deficient in MMP-2, -3 or several other MMPs, indicating that the remaining MMPs are able to replace their activity in a functional overlap.

Several members of the MMP and PA systems are involved in breast cancer progression. We will focus on the role played by these extracellular protease families during the various stages of breast cancer progression, from the initial hyperplastic lesion to disseminated disease, with particular emphasis on the insight gained from transgenic mouse models.

Mouse Models of Breast Cancer

The mouse mammary gland is ideally suited to study cancer progression for a number of reasons. First of all, there are many similarities between the human and mouse mammary glands (for detailed reviews see [17, 18]). Human and mouse mammary glands contain the same epithelial cell types: the ductal epithelial cells, luminal (milk secreting) epithelial cells, and the myoepithelial cells. They also contain adipose tissue and stroma, although the composition of the stroma differs between the human and mouse glands. One of the few differences between the species is that the epithelium in the mouse gland is surrounded by adipose tissue and a relatively sparse stroma, whereas the human mammary epithelium is encompassed by a specialized fibroblastoid stroma and is not in direct contact with adipocytes. The epithelial structure in the mature nulliparous mouse gland is also much less developed compared to the human gland and consists only of a branching but otherwise naked ductal tree. In contrast, the mature ductal tree in non-pregnant women contains groups of acini at the end of terminal ducts, named terminal ductal lobular units (TDLU). The mouse epithelium develops similar lobuloalveolar structures transiently during the estrus cycle in certain mouse strains [19] and always during early pregnancy, and in this way the resting human gland is structurally more similar to the pregnant mouse gland. In some transgenic breast cancer models, the earliest hyperplastic lesions may also resemble TDLU-like structures [20].

A second ideal feature of the mouse mammary gland is that it is readily accessible for monitoring tumor growth and for other experimental procedures. This has lead to the development of numerous transplanted models, chemically induced models and transgenic models. We will limit our discussion to the transgenic and chemically induced models since there are inherent problems of transplanted models that may introduce bias. Depending on the study design, these obstacles can include: (1) a species barrier between proteolytic components supplied by the transplant and the host (as documented for human and mouse uPA and its receptor uPAR [21]); and (2) a cellular inconsistency with respect to protease expression in the sense that the majority of proteolytic components are supplied by stromal cells in human breast cancers while epithelial-based cancer cell lines often express the very same proteolytic components [22]. This is presumably due to genetic alterations that have occurred during in vitro culture. Furthermore, (3) when gene-deficient mice are used as hosts for syngeneic transplantation, it is possible that a tumor-suppressive phenotype in the protease-deficient host is due to an immune response directed at the protease-producing transplant.

A third advantage of the mammary gland for cancer research is that there are more transgenic models of mammary cancer than for any other cancer form, primarily because relatively specific promoters are available for targeting oncogene expression to the mammary epithelium. The promoters most frequently used stem from the whey acidic protein (WAP) gene, and the mouse mammary tumor virus long terminal repeat (MMTV). The promoters are not equally active at all stages of mammary gland development and the pregnancy cycle [23]. The MMTV promoter is active during puberty but is greatly enhanced during pregnancy. The WAP promoter is primarily active from mid pregnancy and during lactation. Accordingly, cancer progression is accelerated by pregnancies in most transgenic models and some models only develop tumors after a number of pregnancies.

Finally, the tumor histopathology of several transgenic models is very similar to human breast cancers [17]. The histopathology seen in a single mouse model is, however, relatively uniform and often characteristic of the inducing oncogene, and in this way no single model replicates the variability of tumor phenotypes seen among different breast cancer patients [17, 24]. This drawback is to be expected, considering the fact that transgenic models rely on a well-defined initiating oncogene in mice with a homogenous genetic background (inbred strains). In contrast, human breast cancers arise from combinations of multiple mutations in women with heterogenous genetic backgrounds. A notable similarity between mouse and human tumors is the development of a reactive stroma and the appearance of extracellular proteolysis in the tumor microenvironment. There are, however, also significant drawbacks to the use of transgenic models, such as the fact that they express oncogenes in the entire mammary epithelium (or at least a large part of it) often giving rise to multiple tumors and in some models a general hyperplasia of the entire gland. By contrast, human tumors derive from a single site, therefore resulting in a single tumor in the breast.

A comprehensive list of transgenic breast cancer models is available at http://emice.nci.nih.gov/ and has recently been reviewed [23]. Here we will focus on models that have been studied in relation to extracellular proteolysis. Table 1 summarizes the studies that describe MMP or PA system intervention in transgenic and chemically induced breast cancer models. It is evident from Table 1 that the role of proteases has almost exclusively been studied in models, in which one of the four oncoproteins ErbB-2 (neu) [25], Ras [26], Wnt1 [27], or the polyomavirus middle T antigen (PymT) [28] are induced by the MMTV promoter. ErbB-2 is often overexpressed in human breast cancers. Ras and the downstream effector proteins of Wnt1 and PymT are also frequently activated in human breast cancer, although mutation/overexpression of Ras and Wnt1 are infrequent in breast cancer and there is no cellular counterpart of the PymT oncogene [23]. In all four transgenic models, tumor incidence is 100% or at least approaches 100% depending on the time allowed for observation [29–32]. Tumor incidence has only in a few cases [29, 32] been affected in protease intervention studies (Table 1). Tumor onset in the ErbB-2, Ras, and Wnt1 models can be accelerated by multiple pregnancies [25, 30, 32] and mice used for experiments are frequently mated continuously, at least in the Ras and Wnt1 models [30, 32]. The PymT model is exceptionally fast even in non-pregnant mice, where the average tumor latency is approximately 1.5 months [31] compared to 4–7 months for multiparous mice in the three other models [25, 30, 32]. In all four models the tumors metastasize primarily to the lungs although the models differ in aggressiveness. The PymT model is the most aggressive and the Wnt1 model the least aggressive [31, 32]. A notable exception to the use of transgenic models in protease studies is the chemically induced mammary tumors that are induced by orally administered 7,12-dimethylbenzanthracene (DMBA) with or without hormone supplementation [33, 34]. The DMBA model is primarily a carcinogenesis model, since the tumors generally do not progress to form metastases [33, 34].

A feature of any transgenic model that is particularly relevant to extracellular proteolysis is the transition from pre-invasive (carcinoma in situ, CIS) to invasive breast carcinoma. Most human pre-invasive lesions are ductal CIS (DCIS), and in these lesions the carcinoma cells accumulate in the ducts confined by the myoepithelial cell layer and the basement membrane. Proteolytic degradation of the basement membrane leads to depolarization of the myoepithelial cells and offers the carcinoma cells access to the surrounding periductal stroma [35, 36]. In some human breast carcinomas the tumor suppressive myoepithelium surrounding an emerging tumor can persist for a relatively long period of time. In the MMTV-PymT model the late stage tumors resemble human invasive ductal breast cancer, but the myoepithelium is lost very early in tumor progression and it is therefore a poor model of the CIS stage ([20] and unpublished data). The lack of an intact myoepithelium, as seen in the MMTV-PymT model, does not necessarily make the neoplastic cells invasive. These lesions fall in the broad category of mammary intraepithelial neoplasia (MIN), which includes CIS-like lesions but is used more generally in mouse tumors to describe pre-malignant epithelial cells that have significant nuclear atypia and are surrounded by an intact basement membrane [37]. A few transgenic breast cancer models have been described to develop CIS lesions with morphologic similarities to human DCIS and later progress into invasive breast cancer. These include the C3(1)-SV40-T prostate cancer model that also develops mammary lesions [38], the WAP-SV40-T model [39], and the MMTV-neu models [17]. However, the CIS stage has not been the focus of protease intervention studies so far.

Protease Expression in Transgenic Breast Cancer Models

Extracellular proteases are generally absent in resting mammary glands but are present as the gland undergoes the pregnancy cycle. The levels of a number of extracellular proteolytic components are particularly upregulated during post-lactational mammary gland involution. These include uPA, tPA, MMPs-2, -3, -9, -11, and their corresponding inhibitors PAI-1 and TIMP-1 [5, 40–42]. The upregulated proteases and protease inhibitors are presumably required for the orderly restructuring of the lactating gland to a virgin-like state (discussed later). During breast cancer development, a similar proteolytic program is activated in the diseased gland and extracellular proteases are abundant in invasive breast cancers of both humans and mice. Figure 2 gives a schematic comparison of the expression patterns of MMP and PA system components in human breast cancer and in the MMTV-PymT transgenic breast cancer model, which has been used to analyze the expression of several extracellular proteases. One study used laser microdissection to isolate cancer cells and stromal cells followed by quantitative RT-PCR to examine the expression level of MMPs and the PA system [43]. The study concluded that stromal tissue adjacent to cancer cells expresses higher levels of uPA, PAI-1 and MMPs-2, -3, -11, -13, and -14 than the cancer tissue. The predominantly stromal expression patterns of all these components in the MMTV-PymT model has been confirmed by in situ hybridization [31, 43–46]. Using immunohistochemistry, uPAR expression was found primarily in fibroblasts, endothelial cells, and in some macrophages in the MMTV-PymT model (unpublished data). In situ hybridization was used in another study on MMTV-PymT tumors to determine the expression pattern of four of the membrane-type MMPs: the transmembrane MMPs-14, -15, and -16 and the GPI-linked MMP-17 [46]. MMPs-14 and -16 were detected in the stroma whereas MMP-15 was the only protease found predominantly in the epithelium. MMP-17 expression was not observed in the MMTV-PymT tumors. The majority of these findings reflect the mRNA expression data for human breast cancer, since uPA [47], PAI-1 [48], and MMPs-2 [49], -3 [50], -11 [49], -13 [35], and -14 [51] all are found predominantly in the stroma (see suppl. data to review by Egeblad and Werb 2002 [10]). Immunoreactivity for uPAR is also primarily found in the stroma [52]. Figure 3 provides two examples of this close correlation between human and transgenic mouse tumors: MMP-13 is focally expressed in stromal cells in patient material [35] and in MMTV-PymT transgenic tumors [43] (Fig. 3c–d). Similarly, the primary uPA inhibitor PAI-1 is expressed in stromal cells in MMTV-PymT mice [45] and in breast cancer patients [48] (Fig. 3e–f). The PAI-1-producing stromal cells were identified as myofibroblasts in the human samples. It is of note that this cell type is uncommon in the normal mouse and human glands but is abundant in tumors from both transgenic mice (unpublished data) and breast cancers patients [53].

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Figure 2

Expression of MMPs and PA components in breast tumors in humans and in MMTV-PymT transgenic mice. MMPs and PA components are expressed by cancer cells or stromal cells in breast tumors or are present as ubiquitous plasma-derived proteins. The predominant source of each mRNA/protein is illustrated for human ductal breast cancer and for the MMTV-PymT model in mice. Only those proteases and related components that have been analyzed in the MMTV-PymT model are included in the comparison. Only mRNA analyses are included except for uPAR, which was analyzed by immunohistochemistry in both species. The “Not detected” category includes proteases that were sporadically expressed in the tumors. The data for the MMTV-PymT model: uPA [31]; PAI-1 [45]; uPAR, MMPs-7, -9, -10 (unpublished data); MMPs-2, -3, -11, -13, -14, uPA, PAI-1 [43]; MMPs-14, -15, -16, -17 [46]. The data for invasive ductal breast cancer: uPA [47]; PAI-1 [48]; uPAR [52]; MMP-2 [49]; MMP-3 [50], MMP-7 [89]; MMP-9 [72]; MMP-10 [89]; MMP-11 [49]; MMP-13 [35]; MMP-14 [51].

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Figure 3

Examples of protease expression in breast cancer from transgenic mice and humans. Expression of MMP-13 during early invasion in mouse C3(1)-SV40-T breast carcinoma (a) and in human ductal carcinoma in situ (DCIS) (b). MMP-13 is focally expressed in periductal fibroblast-like cells (arrows in a, a’, and b’) in areas with early cancer cell invasion (inv) and is not expressed in adjacent non-invasive areas with in situ carcinoma (CIS). Expression of MMP-13 in invasive MMTV-PymT carcinoma (c) and in human invasive ductal carcinoma (IDC) of the breast (d). Focal expression of MMP-13 is seen in fibroblast-like cells located in the stroma surrounding invasive cancer cells in both mouse and human breast cancers (arrows in c and d). Expression of PAI-1 in invasive MMTV-PymT carcinoma (e) and in human IDC of the breast (f). The expression of PAI-1 is seen in fibroblast-like cells located in the stroma surrounding invasive cancer cells in both the mouse and human breast cancers (arrows in e and f). All panels are in situ hybridization using 35S-labeled probes. Panels a, c–f are counterstained with haematoxylin and eosin, whereas b is combined with pan-cytokeratin immunohistochemistry and (b’) with CK14 immunohistochemistry for myoepithelial cells [35]. Bars: a,c, 200 μm; b, 100 μm; d–f, 150 μm.

Recently, the MMTV-Wnt1 model has also been used to study the expression levels of a number of MMPs during mammary tumor formation by RT-PCR and in situ hybridization [32]. MMPs-2, -3, -9, -13, and -14 displayed increased expression levels in hyperplastic glands and mammary tumors. MMPs-2, -3, and -9 were exclusively expressed in the stroma, while MMPs-13 and -14 were found predominantly in the cancer cells and to a lesser extent also in stromal cells. Again, most of these MMPs have been reported with similar expression patterns in human breast cancer, although MMPs-13 and -14 are predominantly stromal in ductal breast cancer [35, 51]. It is noteworthy that the MMTV-PymT and MMTV-Wnt1 models have broadly similar but not identical localization and expression of MMPs.

The initial expression of proteases in human breast cancer may be associated with the transition from DCIS to invasive ductal carcinoma. The transition to invasive cancer can be identified histopathologically in some DCIS lesions, particularly facilitated by immunohistochemical staining of myoepithelial cells and intraductal neoplastic cells. Expression studies of MMPs in human DCIS material have shown that MMP-13 is focally upregulated in microinvasive foci (Fig. 3b) in contrast to MMPs-2, -11, and -14, which are all more broadly expressed in the samples [35]. All four of these MMPs are expressed in periductal (myo)fibroblasts, indicating an intense stromal involvement during early invasion. Brummer et al. found that TIMP-1 is also upregulated in areas of early invasion [54]. Mimicking the human situation, the C3(1)-SV40-T model develops DCIS-like lesions, in which MMP-13 is upregulated specifically in those DCIS-like structures that show signs of invasion (Fig. 3a). In human samples, uPA and uPAR are also focally upregulated in areas of microinvasion together with MMP-13, suggesting that plasmin-directed proteolysis coincides with MMP activity in the transition to invasive cancer [36].

Thus, the extracellular proteases expressed in transgenic breast cancer models generally mirror those identified in human breast cancer both in terms of expression and localization. These findings suggest that transgenic models are useful tools for the study of protease involvement in breast cancer invasion and metastasis.

Functional Studies of Proteases in Breast Cancer Progression

Conclusion and Future Perspectives

Transgenic breast cancer models are invaluable research tools because they recapitulate the entire process from the initial genetic events in normal cells to metastatic disease. Furthermore, the very similar expression patterns of extracellular proteases in human and mouse breast cancer samples suggest that transgenic mouse models are ideally suited to study the role of proteases in carcinogenesis and the progression to invasion and metastasis.

We have only begun to examine gene deficiencies and transgenic overexpression of the various PA and MMP components in transgenic models. The studies that have been performed so far suggest that no single MMP or PA system component is likely to be all-important for any stage in tumor progression. Practically all of the protease intervention studies report partial phenotypes such as delayed carcinogenesis, slower tumor growth or reduced metastasis, if they report any phenotype at all. The absence of any absolute phenotypes, such as prevention of metastasis, suggests that many of the proteases have mutually overlapping functions. The challenge for developing novel therapeutics in the future will be to identify those proteases that can be targeted simultaneously to obtain a synergistic tumor-suppressive effect.

Another area that has so far only been thoroughly examined in one or two transgenic models is the expression patterns of proteases. Using protease array data, it may be possible to draw some more detailed parallels between individual mouse models and certain histopathological subtypes of human breast cancers. It is very possible that the collective of transgenic breast cancer models available, rather than any single transgenic, will be needed to adequately model the heterogenous human disease. Another open question is whether certain proteases or inhibitors are up- or downregulated in response to certain gene deficiencies. This may provide vital information on functional overlap between individual proteases.

Transgenic breast cancer mice may prove to be very reliable models not only for basic research but also for testing experimental anti-proteolytic therapeutics, due to the high degree of similarity with human breast cancer. However, the transgenic models also present some significant challenges. A key issue is that the experimental drug has to cross-react with the mouse version of the protease. Therefore, it may be necessary in some cases to develop two drugs in parallel, one for each species. Due to the extracellular nature of the PA and MMP systems, these represent excellent targets for therapeutic monoclonal antibodies. An elegant method for targeting extracellular proteases in transgenic mice is the development of monoclonal anti-mouse antibodies by immunizing protease-deficient mice with the target protease. In conclusion, transgenic breast cancer models will further the identification of extracellular proteases critical to cancer invasion and metastasis, and aid the development of new agents against the spread of cancer.

Acknowledgments

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References