Proprotein Convertases And Cancerrelated Substrates

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Numerous cancer-related associated proteins are PC substrates. These substrates play significant roles in biological processes that are paramount to tumor promotion and progression, i.e. cell locomotion, adhesion, invasiveness and growth. The most salient substrate groups included in this chapter are those associated with cell to cell and cell to ECM interactions (collagens, integrins, cadherins) as well as those associated with ECM degradation (metalloproteinases). Other PC substrates that also have a significant role during tumor development, namely growth factors and growth factor receptors, will only be reviewed when their activation was shown to impinge on the processes of ECM degradation and tumor cell invasiveness.

Cell locomotion, together with cell adhesiveness and secretion of proteases are crucial elements in the process of tumor cell invasiveness. PCs are involved in all these processes, including cell motility. For instance, furin is capable of processing of some of the collagens associated to membranes [29] as collagen XIII, XVII and XXII. These collagens are important components of hemidesmosomes, structures that participate in cell-cell and cells-stromal adhesion milieu [30]. After furin cleavage, these proteins ectodomains are shed, influencing negatively on cell adhesion, favoring cell motility and spreading.

PCs were shown to be capable of processing alpha6 [31] and alphaV integrins [32] that play a significant role in cell attachment to stromal components. The relationship between cell attachment and PC expression needs to be further explored to define whether PCs play a significant role in increased attachment to stromal structures and which are the mechanisms whereby increased integrin processing (or other substrates) result in increased attachment.

PCs involvement in the second step, basement membrane proteolysis has been explored in more detail and now it is a field that reached maturity. Overexpression and activation of matrix proteases, i.e., enzymes capable of disrupting the proteins composing the extracellular matrix is a common feature found in tumors. The main targets of these proteases include components of the extracellular matrix itself, such as collagens and laminins as well as other proteolytic enzymes that, after being activated, gain the ability to degrade these and other components of the extracellular matrix. Degradation of these components of the ECM initiates several mechanisms that contribute to an invasive phenotype. First, degradation of the ECM permits epithelial cancer cell access to the underlying connective tissue and eventually to reach blood and lymphatic vessels. On the other hand, many growth factors that are usually "trapped" in the ECM may be released, stimulating transformation and aiding in the cells' invasive behavior. More recently, novel approaches consider that the normal architecture of the basement membrane prevents invasion and, conversely, alteration of this structure is more permissive towards invasion.

The activity of metalloproteinases is an essentially destructive activity that must be highly regulated either at the transcriptional as at the post-translational level. Normal cells do not express high levels of these enzymes. However their expression is higher in wound-healing, tumor cell invasion, and metastasis. In addition, their activity is highly regulated since all of them are synthesized as inactive zymogens that require a further proteolytic step in order to become fully active. PCs activate several matrix metalloproteinases that play a crucial role in ECM degradation, invasion and metastasis. Furthermore, inhibition of PCs activity may constitute a novel therapeutic approach since targeting a single protein or group of protein leads to inhibition of several invasion-related pathways. On the other hand, some of the PCs are capable of degrading some components of the ECM directly, highlighting their role in invasion.

In summary PCs are capable of cleaving a series of substrates resulting in increased stromal degradation and enhanced motility.

4.1 PCs and Metalloproteinases

MT-MMPs, a family of six members, contain a trans-membrane domain that anchors them to the plasma membrane where it is expressed in an active form [24, 33]. All of them contain the cleavage sequence for PCs, namely RRK/RR between their pro and the catalytic domain. The active enzymes catalyze MMP-2 (one of the major collagen IV degrading enzymes, [34] activation [24, 35-37], collagen I processing [38, 39], direct degradation of ECM components such as the small rich-leucine proteoglycans lumican and decorin [40, 41], adhesion molecules such as integrins [42] and laminin y2 [41] and molecules involved in cell migration as syndecan [43]. Most of the MT-MMPs studies focused on MT1-MMP, the best known member of the family. Additional studies done with the other members of the MT-MMP family demonstrated that they share with MT1-MMP many biochemical and functional properties.

MT-1 MMP is synthesized as a ~63kDa zymogen that is subsequently processed and activated at least in some systems by furin or PC5 to a ~57-60kDa mature protein [44]. Since Sato et al demonstrated that recombinant MT1-MMP was efficiently cleaved in vitro by purified soluble furin [25], this concept has been challenged [42, 45, 46]. Interestingly, furin inhibition either with a synthetic inhibitor (CMK) or antisense technology inhibited Pro-MT1-MMP activation and MMP-2 activity in cardiac and uterine cervical fibroblasts [47, 48] but not in rabbit dermal fibroblasts [48]. These results suggest that furin involvement in MT1-MMP maturation and consequently in the regulation of collagenolytic modulation may depend on the species, tissues and/or cell types.

Inhibition of PCs resulted in inhibition of MT-MMPs processing. Furin trans-fection to low-grade SCC cell lines from head and neck, resulted in increased levels of MT1-MMP processing. After treatment of these furin overexpressing cells with CMK, furin-mediated MT1-MMP processing was greatly diminished [49]. Furthermore, several SCC head and neck cell lines become 50 to 70% less proficient in MT1-MMP processing after transfection with PDX- a high affinity inhibitor of furin and PC5 [50]. These results were supported with transfection with a second furin specific inhibitor, furin pro-segment (ppfur). Transfection with ppfur resulted in reduction of Pro MT1-MMP cleavage [51]. On the other hand, the extracellular PACE4 was found to cleave MT2-MMP in murine SCC cell lines. When PACE4 expressing SCC and papilloma cell lines were treated with a specific antibody, their ability to process MT2-MMP was diminished [52]. This behavior does not seem to be restricted to SCC since astrocytoma cell lines ability to process MT1-MMP and to invade in vitro and in vivo, was hampered by transfection with PDX [53]. Moreover, only the unprocessed MT1-MMP form was detected when A375 melanoma cell lines were transfected with this inhibitor [54]. Reduced MT-MMPs processing resulted in reduced invasion in vitro and in vivo [49, 50, 52, 53]. Cells, treated either with CMK, antibodies or transfected with the inhibitors showed a reduction in the ability to degrade and pass through Matrigel-composed by a reconstituted murine extracellular matrix. This behavior was also observed in vivo using the tracheal xenotransplantation assay. This assay permits the evaluation of invasion trough the connective tissue that lies between the ends of the C-shaped tracheal cartilages. In addition, cell proliferation and viability can be assessed. When PDX transfected cells were xenotransplanted into tracheal grafts in immunosuppressed mice, the tumor cells remained viable but their growth and invasive ability were markedly impaired when compared with the vector alone-transfected counterparts.

Other proteases have been suggested to activate MT1-MMP. First, MT1-MMP itself can function as a auto-convertase [46]. However, BB-94 a MT1-MMP inhibitor, was unable to prevent Pro-MT1-MMP cleavage in A375 melanoma cell lines, arguing against the autocatalytic cleavage, at least in this cell line [54]. These results confirm the pivotal role of PCs in metalloproteinase activation and highlight the importance of these proteases as possible targets for novel cancer therapy strategies. Other candidate, plasmin, whose activity is mainly extracellular, was able to cleave Pro-MT1-MMP in vitro [55]. However, plasmin, tested at various concentrations had no effect in the generation of active MT1-MMP on control or MT-1MMP overexpressing HT-1080 cell lines [56]. Moreover, incubation of MT1-MMP expressing cells in the presence of plasmin-depleted serum had no effect in the pro-collagenolytic activity of MT1-MMP, indicating that plasmin is not required for MT1-MMP activation [57].

In summary, furin or other PC convertases are likely to be the physiological activators of this group of metalloproteinases. This action seems to be constitutive although a model of the regulation of furin expression through TGF-P has been proposed suggesting that the activation of MT-MMPs might be dependent, in some systems, on the levels of serum TGF-P [58].

Recently, it has been shown in co-transfection systems, that furin cleaves MMP-2 directly in the trans-Golgi network, and the product of this proteolysis is an inactive metalloprotease [59]. This finding provides a novel view at the role of furin (and PCs) in invasion, since convertases are proposed as preventing rather that supporting invasion. Although further studies need to be performed, it can be speculated that furin contributes to directing the process of invasion by activating substrates in the compartment where they are needed (i.e., the extracellular) while preventing their putative devastating action in the intracellular compartments. In addition, it is probable that MMP-2 and furin are expressed in different cell types, i.e stromal and epithelial, and that the interaction between them, one secreting the pro-MMP, and the other cell type providing the machinery to activate this Pro-MMP, results in enhancement of cell invasion.

4.2 PC and Other Sustrates Related to Tumor Cell Invasiveness

IGF-1R is synthesized as a 200 kb precursor that, after furin cleavage, originates the a and P chains of the mature receptor [60] [61]. Cleavage of IGF-1R precursor is absolutely necessary for intracellular signaling (tyrosine kinase activity) and ligand (IGF-1) binding [62]. In addition, inhibition of furin activity results in decreased levels of receptor maturation, leading to resistance to IGF-1 mediated proliferation [49, 61] suggesting that furin-mediated cleavage is essential for IGF-1R functionality. Other PCs are capable of cleaving Pro-IGF-1R, albeit with much less efficiency as shown in LoVo cells, which do not express functional furin. In these cells, IGF-1R processing was greatly reduced but not totally abolished, pointing to the action of other (s) PC [62].

Binding of IGF-1R ligand, IGF-1, leads to increased proliferation as well as pro-survival signals. In addition, lung carcinoma cells over expressing this receptor showed enhanced invasion through Matrigel indicating that IGF-1R favored degradation of some components of the basement membrane, probably through induction of MMPs. In this context, Long et al demonstrated that overexpression of IGF-1R in M-27 lung cancer cell lines, which express low levels of the receptor, resulted in increased MMP-2, levels. Conversely, ablation of IGF-1R expression using antisense technology, lowered MMP-2 levels resulting in impaired invasive ability [63]. Recently, a dual regulation of MMP-2 levels through the IGF-1/IGF-1R signal transduction system has been elucidated [64]. Two major pathways contribute to MMP-2 regulation: the PI 3 kinase/Akt/mTOR and Ras/Raf/MEK pathaways. When H59 lung carcinoma cells were stimulated with IGF-1concentrations (10ng/ml) that favor cell proliferation and motility, activation of PI 3-kinase/Akt/mTOR resulted in increased MMP-2 mRNA and protein synthesis. The other pathway, the Ras/Raf/MEK axis, is also activated but in a transient and weak manner, partly due to Akt-mediated inactivation of Raf-1. Interestingly, at higher IGF-1 doses (100 ng/ml), the latter pathway is preponderant, leading mainly to MMP-2 suppression in these cells. These two contrasting activities exerted by different IGF-1 concentrations emphasizes IGF-1's role in fine-tuning the invasive ability of cancer cells.

Different cell lines may respond differently to IGF-1. In MCF-7 breast cancer cells, high doses of IGF-1 (100 ng/ml) activated the PI 3Kinase pathway, resulting in Raf phosphorylation at Ser 259, inhibiting its activity [65]. Although free (not bound to IGF binding proteins), IGF-1 concentrations in tumors may be difficult to assess. These results point to a critical role of this grow factor in the acquisition of the invasive phenotype. In a similar way, the number of active receptor in the plasma membrane should play a decisive role in MMP-2 induction. Furin, and to a lesser extent PC5, cleaves and activates IGF-1R [61]. Inhibition of furin activity led to reduced levels of active receptor, less proliferation, and resistance to IGF-1. Ideally, total ablation of IGF-1R functionality implies a complete irresponsiveness to its ligand, IGF-1, regardless of IGF-1 concentrations in tumor tissue. This will result in decreased PI3 kinase activation and subsequently decreased MMP-2 levels, thus inhibiting tumor cell invasiveness.

TGF-P remains one of the most controversial molecules in the field of cancer invasion and metastases. Numerous papers have been published supporting its role in increasing motility, invasion and metastasis. Equally number of works challenged this concept arguing that the opposite actually happens; decreased tumorigenesis and suppression of metastasis. To make matters worse, it is possible to find these contradicting results in similar tumor cell lines.

To cite two of the more recent and prominent works in the field, Muraoka-Cook et al. using a transgenic model where TGF-P is expressed conditionally in the mammary gland, demonstrated that expression of this growth factor resulted in >10 fold metastasis to lung whereas primary cells proliferation or tumor size was unaffected by the expression of the transgene [66]. Conversely, abolition of the TGF-P pathway, by disrupting TGF-P receptor II in the mouse mammary gland, resulted in increased lung metastasis in a conditional knockout model [67].

It is probable that these conflicting ideas may be reconciled by taking into account a considerable number of evidences pointing to a dual role of TGF-p suppressor of tumorigenesis in early stages of tumor progression, (pre-malignant or well-differentiated cells). On the other hand, it is an enhancer of tumor development, growth and metastasis in more aggressive or later stages [68-70].

Since 1995 when Dubois et al. provided convincing evidence that furin was responsible of the first step in TGF-P maturation in vitro and in vivo [71, 72], the field has been steadily growing. TGF-p induces the expression of metalloproteinases and integrins playing an important role in the acquisition of the malignant phenotype. It has been shown that TGF-P induces alpha 3 integrins in hepatocellular carcinoma leading to a more invasive behavior [73]. The fact that active TGF-P is only secreted in invasive hepatocellular carcinoma cell lines but not in non-invasive or normal liver cells [73] and that furin and TGF-P co-localized in regenerating rat liver after partial hepatectomy where TGF-P is required in an active form [74] suggest that the TGF-^-driven increase in cell growth and invasion may be furin-dependent.

Furin mediates increased MMP-2 activation, and hence invasion, through a MT-MMP independent, TGF-P dependent pathway [75]. This findings lead to the proposition of a new axis that considers TGF-P as a new mediator between MMPs and furin.

4.3 Miscellaneous Substrates

Many other proteins containing the furin-cleavage recognition sequence and at the same time putatively associated with invasion and metastasis have been identified. The ADAM (a disintegrin and metalloproteinase) and ADAMTS (a disintegrin and metalloproteinase domain, with thrombospondin type-1 modules) families are proteases associated with cell-surface proteolysis and adhesion. The inflammatory cytokine TACE or tumor necrosis factor-alpha converting enzyme was the first member of this family to be discovered. Interestingly it is processed and activated by furin and other PCs such as PACE4, PC1/PC2 and PC5 [76, 77]. After this first discovery, many other ADAMs were identified. Some of them are furin substrates and are overexpressed in cancer [78-80]. For instance, ADAM9, which may be putatively activated by furin, is expressed in non-small cell lung cancer and its expression correlates with brain metastasis. The mechanism of action involves modulation of adhesion molecules such as a3p: integrins [81].

Processing by furin is a pre-requisite for ADAM 10 [82] and 12 activity [83] and, probably for ADAM 15 [84]. These new findings linking furin to novel mechanisms of adhesion and proteolytic degradation open a new field that is currently actively explored. Recently, it has been demonstrated that furin and possibly other PCs are responsible for shedding membrane-anchored proteins, such as membrane-associated collagens, modifying the adhesion properties of tumor cells. Because of its significance for cell invasion and its relationship to furin more studies will be required in the future. Whether furin cleavage is necessary in order to alter the functionality of these collagens and whether or not this cleavage aids in tumor cell invasiveness awaits further clarification.. In this context, it has been shown that furin-dependent cleavage of collagen XXIII results in its shedding and multi-merization, a feature already observed in prostate cancer [85]. Others have argued that the furin "sheddase" activity may be ADAM-dependent, at least in the case of collagen XVII [86].

4.4 Crosstalk between PC-Activated Pathways

MT1-MMP can be induced in response to insulin like growth factor I (IGF-1) stimulation in a pathway that involves PI 3 kinase/Akt signaling [87]. In addition, furin cleaves pro-IGF-lR into a and P chains [61], a prerequisite for the receptor to be

Collagen) degradation Collagen IV degradation

Prill (Mi lytic deavagp

+ : Induction of protein expression

Figure 2. Crosstalk between PC-Activated Pathways functional. In this context, furin also contributes to MT1-MMP activity through increased MT1-MMP synthesis, providing additional reinforcement of the basic axis PC/MT-MMPs/MMP-2 activation cascade. Recently, it has been shown that IGF-1/PI 3 kinase pathway was involved in the induction of MT1-MMP in vascular smooth muscle cells [88]. Interestingly, IGF-1 mediated induction of MT1-MMP was inhibited by treatment with CMK, a general PC inhibitor in this system, suggesting that inhibition of PC may derive in diminishing MT1-MMP activity through lesser levels of functional IGF-1R and, thus reduced degradation of ECM components as collagen I and IV. Moreover, TGF-P induces the MMP-2 expression linking this growth factor to the furin/MT-MMPs/MMP-2 axis. Interestingly, TGF-P seems to regulate furin expression, positioning it upstream all these pathways related to invasion and metastasis. These relationships are summarized in Figure 2.

Stratum corneum

Stratum corneum

Figure 3. Collagen IV staining. Frozen sections from skins form wild type or PACE4 transgenic mice were stained with a monoclonal antibody against collagen IV. Collagen IV was visualized by immunofluorescense (shown in green). Nuclear stain was performed with Hoechst H 33342 (in blue). The collagen IV component of the basement membrane is markedly thinner and more disrupted (arrows) than the wild type that exhibits a continuous collagen IV layer. The fluorescence intensity was normalized to the staining of the stratum corneum, considered the internal control

Figure 3. Collagen IV staining. Frozen sections from skins form wild type or PACE4 transgenic mice were stained with a monoclonal antibody against collagen IV. Collagen IV was visualized by immunofluorescense (shown in green). Nuclear stain was performed with Hoechst H 33342 (in blue). The collagen IV component of the basement membrane is markedly thinner and more disrupted (arrows) than the wild type that exhibits a continuous collagen IV layer. The fluorescence intensity was normalized to the staining of the stratum corneum, considered the internal control

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