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The Proceedings of the American Thoracic Society 5:316-322 (2008)
© 2008 The American Thoracic Society
doi: 10.1513/pats.200711-166DR
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Matrix Metalloproteinase–induced Fibrosis and Malignancy in Breast and Lung

Derek C. Radisky1 and Jennifer A. Przybylo1

1 Mayo Clinic Cancer Center, Jacksonville, Florida

Correspondence and requests for reprints should be addressed to Derek Radisky, Ph.D., Mayo Clinic Cancer Center, Griffin Cancer Research Building, 4500 San Pablo Road, Jacksonville, FL 32224. E-mail: radisky.derek{at}mayo.edu

ABSTRACT

Fibrosis is a pathological condition in which tissue structure is disrupted by production of excess extracellular matrix (ECM), and chronic tissue fibrosis is associated with tumor development. Myofibroblasts are the principal mediators of fibrosis, producing abundant ECM as well as inflammatory and angiogenic factors. Myofibroblasts are also abundant in tumor stroma, where they facilitate tumor growth and progression. Matrix metalloproteinases (MMPs), enzymes that degrade and remodel the ECM, are believed to play a critical role in the development of fibrotic tissue, though the mechanism by which this occurs is unclear. Expression of MMP-3 in mammary epithelial cells of transgenic mice stimulates development of fibrosis and subsequent tumor formation. We have recently determined that exposure of mammary epithelial cells to MMP-3 induces a specialized form of epithelial–mesenchymal transition in which the cells acquire myofibroblast-like characteristics and that this process is dependent upon the generation of cellular reactive oxygen species (ROS). New data from culture models in which MMPs are inducibly expressed in human lung cell lines, and transgenic mouse models in which MMPs are inducibly expressed in lung alveolar epithelial cells, suggest that similar processes likely exist in the lung.

Key Words: matrix metalloproteinases • epithelial–mesenchymal transition • breast • fibrosis

As their name suggests, matrix metalloproteinases (MMPs) are zinc-dependent proteases that cleave proteins of the extracellular matrix (ECM). They can also break down molecules that mediate cell–cell and cell–ECM interactions and can cleave and activate growth factors and growth factor receptors (1). While MMP expression is a hallmark of physiological processes involving tissue remodeling, such as developmental morphogenesis, wound healing, and neoangiogenesis, overexpression of MMPs is associated with cancer and a variety of other pathological processes. In nearly all tumors, these proteases facilitate tumor angiogenesis, tissue invasion, and metastasis (1, 2). Recent clinical trials of MMP inhibitors as potential cancer treatments have proved disappointing, due in large part to their nonspecific inhibition of both cancer-specific pathways and essential physiological processes (3). The failure of such a promising new line of cancer therapeutics clearly underscores the need to improve our understanding of the specific mechanisms by which MMPs support tumor development and progression. Advancing our knowledge of how MMPs support tumor formation and growth will help make possible a new generation of therapeutic strategies based on targeting tumor-specific, MMP-dependent effects.

Previous investigations using transgenic mouse models showed that expression of MMPs in the mammary gland was sufficient to stimulate tumor development (4–6), demonstrating that these proteases act not only as secondary tumor products that facilitate tumor progression, but also as carcinogens that can induce tumor development at the earliest stages. Studies using cell culture models revealed that exposure to stromelysin-1/matrix metalloproteinase-3 (MMP-3) caused mammary epithelial cells to undergo epithelial–mesenchymal transition (EMT) (4, 7, 8), a fundamental programmatic change in which epithelial cells become uncharacteristically migratory and invasive. EMT has been observed in normal development as well as in tumor progression (9–11). This review will examine how MMP-3 and other MMPs act to stimulate and facilitate cancer progression and how epithelial–myofibroblast transdifferentiation participates in this process.

MMPS, FIBROSIS, AND CANCER

Myofibroblasts are the key cells involved in repair of epithelial injuries, producing MMPs and other ECM-degrading enzymes that digest the damaged tissue, as well as synthesizing and modulating elements of the wound provisional ECM (12–15). Normally, once the damaged tissues are repaired, the provisional ECM is degraded and the myofibroblasts are removed by apoptosis; however, when repair mechanisms are inappropriately maintained, chronic action of myofibroblasts stimulates a pathological repair process, leading to fibrotic ECM that is not easily degraded by MMPs (12). This aberrant ECM interferes with normal cell function, leading to cell metaplasia and increased proliferation, which is an ideal context for tumor formation and progression (2). The presence of fibrosis significantly increases the risk of subsequent development of tumor foci in the lung (16, 17), liver (18, 19), and breast (20, 21).

The linkage between fibrosis and cancer is best established in the lung (12). Incidence of persistent or idiopathic pulmonary fibrosis (IPF) in the United States alone has been estimated to be as high as 50,000 individuals, with a less than 3-year median survival time from diagnosis; nearly half of those affected by IPF may develop premalignant lesions (17). Breast fibrosis is a much less understood phenomenon. Clinical appearance of breast fibrosis after radiation therapy is often observed (22), but characterization of fibrosis in nonmalignant breast tissue has not been as precisely defined as with lung fibrosis. One indication that breast fibrosis might also predispose to cancer, however, is the identification of increased risk of tumor incidence in women with mammographically dense breasts (20); several investigations of dense breast tissue have shown that these areas contain increased collagen deposition, presence of activated fibroblasts, and other early markers of fibrosis (23–29). Another indication is the common appearance of myofibroblasts in association with localized breast cancers, as well as in breast cancer stroma (30, 31).

While MMPs are generally considered to be causally associated with cancer development (32), defining specifically how MMPs may act to promote tumor development has been challenging (33), even though correlative evidence suggests that a limited number of players may be involved. Matrilysin (MMP-7) is expressed in bleomycin-induced lung fibrosis (34), and MMP-7 along with several other MMPs are found in asbestos-induced lung fibrosis (35). By comparison, mice genetically deficient in the expression of the tissue inhibitors of metalloproteinases (TIMPs), which act to block the activity of MMPs, show predisposition to fibrosis formation (36), while fibrosis is limited in mice treated with MMP inhibitors (35, 37, 38) or in mice that lack expression of specific MMPs (34, 38). Several studies have targeted MMP activity as a potential antifibrosis therapy, but the lessons of the antitumor MMP studies should be taken into account, as MMPs are also required for a wide variety of normal processes and nonspecific inhibition of MMPs is likely to be detrimental. Moreover, MMPs appear to be specifically necessary for removal of excess collagen deposition and morphogenic activities (39–41). Thus, defining the fibrosis-specific effects of MMPs will be critical for developing effective therapeutic inhibitors.

Mechanistic insight into how MMPs can stimulate fibrosis and how this can lead to malignancy is found in the WAP-MMP-3 transgenic mouse model, in which expression of MMP-3 is controlled by the whey acidic protein promoter so as to be expressed in mammary epithelial cells during pregnancy and lactation (4, 42). In the normal mammary gland, mammary stromal cells are the primary source of MMP-3, and the greatest levels of expression occur during postlactational involution, when ECM remodeling is maximal and there is extensive loss of epithelial cells (43–45). Initial expression of MMP-3 in the transgenic mice activated a stromal response that included greatly increased levels of endogenous MMP-3 production, as well as development of fibrotic characteristics, including collagen deposition, tissue disruption, and neoangiogenesis (4, 42, 43, 46–48); similar effects were also seen in the MMTV-MT1-MMP transgenic mouse (5). Early induction of fibrosis was followed by tumorigenesis; neoplasias developed in a substantial fraction of the WAP-MMP-3 mice, and these showed extensive genomic rearrangements by comparative genomic hybridization (4). Thus, inappropriate expression of MMP-3 that stimulates development of fibrosis is sufficient to cause development of tumors and progression to full malignancy. While this is not likely to be a unique effect of MMP-3, it is relevant that increased expression of MMP-3 has been associated with a number of human malignancies, including breast cancer (Table 1). It should be noted that in this transgenic model, MMP-3 expression is being targeted to mammary epithelial cells, although in breast cancer, the primary source of MMPs is the stromal cells.


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  		TABLE 1. HUMAN CANCERS ASSOCIATED WITH MATRIX METALLOPROTEINASE-3

 
EMT AS A SOURCE OF MYOFIBROBLASTS IN FIBROSIS AND CANCER

Defining effective therapies to prevent fibrosis requires a complete understanding of the mechanisms involved in the development of the disease, and one mechanism of particular interest to the field is fibroblast development. Early studies suggested that myofibroblasts are derived from tissue fibroblasts, and indeed, cultured fibroblasts develop myofibroblast characteristics when stimulated with cytokines, including transforming growth factor-β (TGF-β) (15, 49). While it is certainly true that unactivated tissue fibroblasts may follow similar developmental pathways in vivo, other cell types may also develop into myofibroblasts. Studies in which animals are given bone marrow transplants with genetically labeled myeloid cells have suggested that myofibroblasts can be derived from this cell type (50–56). Emerging evidence, however, suggests that epithelial cells could be an even more important source of myofibroblasts (57). This process, known as epithelial–myofibroblast transdifferentiation, can be induced in cultured epithelial cells (58–64), and biopsies of idiopathic pulmonary fibrosis show that many of the cells that manifest myofibroblast markers appear to retain the expression of epithelial markers as well (64). These myofibroblasts may be derived from normal epithelial cells, or from the tumor cells themselves. Transgenic mice in which lung epithelial cells were genetically tagged for expression of a distinctive marker were used to show that myofibroblasts that develop in TGF-β–induced pulmonary fibrosis are largely derived from epithelial cells (58). Analysis of myofibroblasts in human breast cancers has indicated that these can share genetic abnormalities with the tumor cells (63). The accumulating evidence of epithelial–myofibroblast transdifferentation has particular relevance for our understanding of the processes involved in fibrosis and cancer development because it provides a mechanism by which normal or premalignant epithelial cells as well as epithelial-derived carcinoma cells can give rise to stromal cells that stimulate fibrosis and tumor progression, and suggests that identification of the mechanisms governing epithelial–myofibroblast transdifferentiation could inspire novel therapeutic strategies complementary to existing antitumor therapies.

Epithelial–myofibroblast transdifferentiation from epithelial cells is a specialized version of epithelial–mesenchymal transition (EMT), a physiological process in which epithelial cells can acquire the invasive and motile properties of mesenchymal cells (10). EMT plays a central role in development and many of the mediators identified as controlling developmental EMT have now been found to act at key points of tumor development and progression (65). EMT also acts in tumor progression by providing increased resistance to apoptotic agents (66, 67), and by producing supporting tissues that enhance the malignancy of the central tumor (63). Induction of EMT appears to be highly tissue- and cell type–specific (68), as factors that induce EMT under some circumstances can have quite different effects in others (69).

The mechanisms that control pathological EMT are most commonly studied in cultured cells; we and others have used the mouse mammary epithelial cell line SCp2 to investigate breast EMT (7, 8, 70). SCp2 cells display the characteristics of nonmalignant, untransformed mammary cells: they undergo alveolar-like development and can be induced to synthesize milk proteins when given the appropriate hormonal stimuli and the appropriate extracellular matrix cues (7, 70). However, SCp2 cells can be induced by MMPs to adopt malignant characteristics, including anchorage independence in soft agarose (71) and tumor formation in mice (4). We found that SCp2 cells, when treated with MMP-3, showed altered gene expression characteristic of EMT as well as a breakdown of epithelial morphology (Figure 1A), and increased expression of myofibroblast markers (Figure 1B) (72). We have more recently found that MMP-induced EMT is not limited to mouse mammary cells, as treatment of a subline of A549 human lung epithelial cells that displayed epithelial cell morphology with MMP-3 leads to a breakdown of epithelial cell structure and a conversion to a more spindle-like morphology (Figure 1C).


Figure 1
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  		Figure 1. Properties of matrix metalloproteinase (MMP)-3–induced EMT. (A) MMP-3–treated SCp2 cells were stained for cytokeratins (red), vimentin (green), and DNA (blue). (B) Marker transcript levels in cells treated with MMP-3 for 4 days; P < 0.01 for altered expression levels. (C) A549 human lung epithelial cells and SCp2 mouse mammary epithelial cells were exposed to MMP-3 for 3 days and photographed. Adapted from Reference 72 and unpublished data; portions of this figure are reprinted with permission from Reference 86.

 
In mouse mammary SCp2 cells, we found that induction of the splice variant Rac1b and subsequent increases in the levels of cellular reactive oxygen species (ROS) were key intermediates in the MMP-3–induced EMT (72). Cells treated with MMP-3 showed increased expression of Rac1b as assessed by pulldown assay (Figure 2A) or by quantitative RT-PCR (72). Rac1b is a splice variant of Rac1 that has been shown to lack intrinsic GTPase activity and thus to act as a constitutively activated variant of Rac1; Rac1b has also been found in breast and colorectal tumors (73–77). MMP-3–induced Rac1b was shown to be directly responsible for the increased levels of reactive oxygen species (ROS) in MMP-3–treated cells (Figure 2B). These ROS were derived from mitrochondrial activation (Figures 2C and 2D), but elevation of ROS by treatment with H2O2 was also sufficient to induce cell scattering and invasion (Figures 2E and 2F), and MMP-3/Rac1b–induced ROS were necessary for induction of vimentin (Figure 2G) and other myofibroblast markers (72). It is important to note that there are many different species of ROS within cells, and that each has distinct biochemical and signaling properties; however, due to facile interconversion of ROS in the cytoplasm (78), sustained production of a single reactive oxygen species can cause elevation of the levels of the other species. Though ROS are often seen as destructive molecules, ROS can also act as signaling molecules (79, 80), and have been specifically associated with EMT (81). Thus, we identified a mechanism by which MMPs, long associated with fibrosis induction as well as tumor progression, stimulate transdifferentiation to myofibroblast-like cells, effectors of fibrosis as well as key players in the generation of the tumor microenvironment.


Figure 2
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  		Figure 2. MMP-3–induced reactive oxygen species (ROS) activate epithelial–mesenchymal transition (EMT). (A) SCp2 cells treated with MMP-3 stimulate increased production of Rac1b, as assessed by Rac1 activity assay and Western blot with Rac1b-specific antibody. (B) Exposure to MMP-3 activates redox-sensitive fluorescent dye DCFDA in Rac1/Rac1b-dependent fashion. (C) MMP-3 treatment induces mitochondrial production of superoxide as shown by precipitation of nitrobluetetrazolium in cells treated with MMP-3 (b) as compared to untreated cells (a). (D) MMP-3 treatment induces mitochondrial depolarization as shown by loss of punctuate red staining of the J-aggregate and increased diffuse green staining of the monomeric form in the MMP-3–treated cells (b) as compared to untreated (a). (E and F) Exposure to H2O2 induces cell scattering (E) and invasiveness through Matrigel (F), as compared with MMP-3–treated cells. Scale bars: C and D, 10 mm; E, 50 mm. (G) MMP-3–induced up-regulation of mesenchymal vimentin expression is inhibited by NAC and is reproduced by exposure to H2O2 or by expression of Rac1b. Adapted from Reference 72 and reprinted with permission from Reference 86.

 
We also explored the processes involved in MMP-induced genomic instability in mammary epithelial cells. That exposure to MMP-3 was responsible for the genomic instability found in the tumors was suggested by the strikingly nonrandom patterns of genomic deletions found on chromosome 4 in tumor tissue derived from the WAP-MMP-3 transgenic mice (Figure 3A) and in cultured mouse mammary epithelial cells exposed to MMP-3 (Figure 3B) (4, 72). To identify the mechanism by which MMP-3 induces genomic alterations in mouse mammary epithelial cells, we used an assay in which cells are treated with N-(phosphonacetyl)-L-aspartate (PALA), as resistance to PALA is acquired through amplification of the carbamoyl-phosphate syntetase 2, aspartate transcarbamylase, and dihydroorotase (CAD) gene (82–85). CAD is a multifunctional gene involved in the production of pyrimidine nucleotides that is under strict transcriptional control; as PALA suppresses the production of pyrimidine nucleotides, increased resistance to PALA is conferred only through amplification of the CAD locus. We found that cells treated with MMP-3 for increasing periods of time and then selected by incubation with PALA in the absence of MMP-3 showed a progressive increase in PALA resistance (Figure 3), and that this effect was due to increased CAD locus copy number (Figure 3D). We also showed that MMP-3–mediated production of ROS is specifically responsible for both oxidative DNA damage and the induction of genomic instability, since both MMP-3–mediated DNA damage and genomic instability were inhibited by coincubation with NAC (Figure 3E and Reference 72), and both MMP-3–mediated DNA damage and genomic instability are produced in the absence of MMP-3 by treatment with H2O2 (Figure 3D and Reference 72). These experiments showed that induction of ROS is also necessary and sufficient for MMP-3–induced DNA damage and genomic instability. Taken together, these studies revealed a specific signaling pathway connecting MMP-3 with downstream induction of EMT and genomic instability (Figure 4).


Figure 3
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  		Figure 3. MMP-3 induces genomic instability that can be inhibited by quenching ROS. (A) Analysis of normal and tumor tissue from WAP-MMP-3 transgenic mice by cytogenetic comparative genomic hybridization (CGH) reveals common patterns of deletions (red) in chromosome 4. (B) SCp2 mouse mammary epithelial cells exposed to MMP-3 show deletions in chromosome 4, as revealed by array-based CGH. (C) Exposure to MMP-3 induces amplification of the carbamoyl-phosphate syntetase 2, aspartate transcarbamylase, and dihydroorotase (CAD) gene locus as shown by increased resistance to N-(phosphonacetyl)-L-aspartate. (D) Assessment of CAD gene amplification (red, CAD locus; blue, nuclear stain). (E) MMP-3–induced genomic instability is dependent upon ROS. Panel A is adapted from Reference 4; panels B–E are adapted from Reference 72.

 

Figure 4
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  		Figure 4. Model of MMP-3 induction of EMT and genomic instability. Treatment with MMP-3 leads to increased production of Rac1b, causing elevated levels of ROS. The ROS are necessary and sufficient to stimulate epithelial–myofibroblast transdifferentiation and induction of genomic instability.

 
CONCLUSIONS AND FUTURE DIRECTIONS

The MMP-induced processes discussed here prompt questions about several specific steps in the MMP-induced malignancy pathways. The effects induced by MMP-3 required proteolytic activity, as shown by experiments in which treatment with MMP inhibitors blocked the effects of MMPs or where use of catalytically inactive MMP mutants had no effect (72). Apparently, MMPs are targeting specific extracellular molecule(s) that are initiating the EMT process, although the identities of these molecule(s) are still unknown. E-cadherin is one attractive possibility, particularly as it has been shown to be a target of several MMPs, although it is not clear how cleavage of E-cadherin would be sufficient to induce all of the effects induced by MMPs. Other important issues include defining how cleavage of an extracellular molecule leads to increased production of the Rac1b alternative splice form, how Rac1b stimulates production of mitochondrial superoxide, and how this elevation of cellular ROS induces EMT. All of these represent potential points of intervention in the MMP-induced pathological processes.

Another question is the relevance of these processes to other organ systems. To assess the role of these pathways in lung, an organ system in which development of fibrosis is a major health problem, we have generated culture models in which exposure of A549 cells to MMP-3 leads to morphological EMT (Figure 1C), and transgenic mouse models in which expression of MMP-3 from lung type II alveolar epithelial cells leads to development of fibrotic characteristics (86). These results point to the potential generality of the mechanisms described for mammary epithelial cells, and the association of MMPs with development of fibrosis and cancer suggests that there may be a causal relationship for these molecules in other organs as well. The challenge now is to define the role of MMP-induced epithelial–myofibroblast transition in vivo, and the specific role of the signaling pathways described here in this process.

FOOTNOTES

Production of this manuscript has been supported by funds from the James and Esther King Biomedical Research Program and the State of Florida Department of Health.

Conflict of Interest Statement: Neither author has a financial relationship with a commercial entity that has an interest in the subject of this manuscript.

(Received in original form November 14, 2007; accepted in final form December 24, 2007)

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