On How Mammary Gland Reprogramming Metalloproteinases Couple Form with Function

Khokha and Werb (2011) present a comprehensive overview of the roles of metalloproteinases in extracellular proteolytic pathways that are critical to mammary gland biology along with the deregulation of these enzymes that is associated with tumorigenesis. Indeed, many metalloproteinases have been confirmed to play causal roles in extracellular proteolysis. This has been shown for mammary gland development in knockout mice that lack these proteases and for mammary tumorigenesis by crossing metalloproteinase-null mice with transgenic mice predisposed to develop mammary tumors. The findings do not rule out causal roles for other proteases, however, including intracellular proteases. Such roles seem likely because there is functional redundancy among the >500 proteases in the human degradome and the >600 proteases in the murine degradome (Puente et al. 2003). Defining the role of any one protease is complex and there are examples of intracellular and membrane proteases that may, in addition to the metalloproteinases, contribute to mammary development and tumorigenesis. Khokha and Werb note that apoptosis, autophagy, and phagocytosis, all of which involve intracellular proteolysis, are critical to mammary gland biology. The primary mediators of the apoptotic cell death pathway are caspases, intracellular cysteine proteases that cleave after aspartic acid residues (Pop and Salvesen 2009). A variety of other intracellular proteases also have been implicated in apoptosis, including threonine proteases such as the proteasome (Dalton 2004); aspartic proteases such as cathepsin D (Conus and Simon 2008); serine proteases such as granzyme B (Kurschus et al. 2005); and cysteine cathepsins, such as cathepsins B and L (Vasiljeva and Turk 2008). In the case of the cysteine cathepsins, studies in a diverse array of cell lines indicate that they do not initiate apoptosis, but rather amplify caspase-dependent apoptosis (Droga-Mazovek et al. 2008; Oberle et al. 2010). Autophagy is an alternative cell death pathway that is central to both normal developmental processes and diseases, including cancer (for review, see Eskelinen and Saftig 2009). Essential in autophagy are autophagins, cysteine proteases involved in delipidation of other autophagic proteins and the development of autophagic vesicles (Marino et al. 2003), and lysosomal proteases that digest vesicle contents. An essential role for the lysosomal cysteine protease cathepsin L has been confirmed; in tissues of mice lacking this enzyme there is reduced autophagic degradation and an associated increase in large autophagolysosomes (Dennemarker et al. 2010). Phagocytosis is a form of endocytosis in which, like autophagy, lysosomal proteases degrade vesicle contents. For example, collagens can be degraded intracellularly by lysosomal proteases following uptake by the endocytic receptor urokinase plasminogen activator receptor-associated protein (uPARAP) or Endo180 (Kjoller et al. 2004). This intracellular degradation of collagens is linked to mammary tumorigenesis as the size of mouse mammary tumor virus (MMTV)-PyMT-induced mammary tumors is reduced in uPARAP-null mice (Curino et al. 2005). The studies on uPARAP show an indirect role for lysosomal proteases in mammary tumorigenesis, whereas other studies show a direct role for the lysosomal cysteine cathepsins. In cathepsin-B-null mice crossed with MMTV-PyMT mice, there is a reduction in high-grade invasive ductal carcinomas (Vasiljeva et al. 2008). The lysosomal cysteine protease cathepsin X (Z) becomes localized on the plasma membranes of the MMTV-PyMT mammary tumors in the cathepsin-B-null mice, compensating in part for an absence of cathepsin B on the tumor membrane (Sevenich et al. 2010). Cathepsin B is known to be present and active on the cell membrane in a wide variety of tumors, including mammary tumors (for review, see Mohamed and Sloane 2006). Further evidence that cathepsin X can compensate for loss of cathepsin B and vice versa comes from studies of MMTV-PyMT mammary tumors in mice lacking both enzymes. In these mice, synergistic delays in tumor development and reductions in the number and sizes of lung metastases are observed (Sevenich et al. 2010). The existence of such reciprocal compensatory mechanisms between two proteases indicates how difficult it is to define the function of any individual protease in a developmental or pathobiological process. In general, as indicated by Khokha and Werb, the metalloproteinases that play roles in mammary development also contribute to mammary tumorigenesis when they are deregulated. In contrast, although roles in tumorigenesis have been shown for other proteases, they have not been shown to play roles in mammary development. This absence of information may reflect the experimental design and focus of the investigator, the presence of a severe phenotype in tissues other than the mammary gland, and embryonic or perinatal lethality of mice lacking the protease. The membrane serine protease matriptase provides an illustrative example, as mice lacking it die shortly after birth owing to a defect in epidermal integrity (List et al. 2002). Matriptase is expressed at high levels in the terminal end buds of growing mammary ducts (Lee et al. 2010), which is consistent with a role for this enzyme in mammary development. As it is not possible to study mammary development in matriptase-null mice, Lee and colleagues used 3D cultures of mammary epithelial cells in basement membrane in which they decreased matriptase expression by RNA interference (RNAi) or matriptase activity by using a small molecule inhibitor. Activation of pro-HGF (hepatocyte growth factor) by matriptase resulted in the formation of acini with lumens, lobular, and ductal structures. Reduced expression or activity of matriptase abrogates these effects, consistent with matriptase-mediating mammary gland morphogenesis through HGF activation. In vitro models, even organotypic models, do not recapitulate all of the cell–cell interactions critical to development or tumorigenesis. Therefore, an alternative approach would be the use of tissue-specific and conditional knockouts to study the roles of proteases that cannot be studied in null mice owing to embryonic or perinatal lethality. Khokha and Werb describe protumorigenic roles for metalloproteinases; however, some metalloproteinases in addition to serine and cysteine proteases play antitumor or protective roles (for review, see Lopez-Otin and Matrisian 2007). MMP-8 was the first protease reported to play a tumor-suppressive role. MMP-8 reduces the incidence of skin tumors (Balbin et al. 2003) and breast cancer metastases (Decock et al. 2008; Gutierrez-Fernandez et al. 2008). Moreover, some proteases can be both anti- and protumorigenic. MMP-3 promotes side branching of mammary ducts during normal development and mammary tumors when an active form is expressed in the mammary glands of transgenic mice (Sternlicht et al. 1999), yet protects against chemically induced squamous cell carcinoma and mammary tumors (Lopez-Otin and Matrisian 2007). Although aspartic proteases had not previously been reported to be antitumorigenic, Yamamoto and colleagues (Kawakubo et al. 2008) found that the mammary glands of mice lacking cathepsin E, an intracellular endolysosomal aspartic protease, show a grossly abnormal morphology that is consistent with cathepsin-E-regulating mammary gland differentiation. The genes that are up-regulated (e.g., amphiregulin, IGFBP2, C-C, and C-X chemokine ligands) or down-regulated (e.g., antiapoptotic genes) in the mammary glands of the cathepsin-E-null mice are also associated with mammary gland tumorigenesis. Thus, cathepsin E is the first aspartic protease shown to play an antitumorigenic role in mammary tissue. The mechanisms by which the absence of cathepsin E exerts this effect are unknown. In this regard, Overall and colleagues are proponents of a systems biology approach (for review, see Rodriguez et al. 2010). They propose that we should not be analyzing protease-knockout mice in which a complex organism is responding over time to the absence of a single protease such as in the cathepsin E study. Instead, we need to use approaches such as high-content degradomics and bioinformatics to identify proteolytic networks and follow those with “specifically designed animal models for final validation.” This will be our challenge for the future.