Pericellular proteolysis provides a significant advantage to developing tumors through the ability to remodel the extracellular matrix, promote cell invasion and migration, and facilitate angiogenesis. Recent advances demonstrate that pericellular proteases can also communicate directly to cells by activation of a unique group of transmembrane G-protein–coupled receptors (GPCR) known as protease-activated receptors (PAR). In this review, we discuss the specific roles of one of four mammalian PARs, namely PAR-2, which is overexpressed in advanced stage tumors and is activated by trypsin-like serine proteases that are highly expressed or otherwise dysregulated in many cancers. We highlight recent insights into the ability of different protease agonists to bias PAR-2 signaling and the newly emerging evidence for an interplay between PAR-2 and membrane-anchored serine proteases, which may co-conspire to promote tumor progression and metastasis. Interfering with these pathways might provide unique opportunities for the development of new mechanism-based strategies for the treatment of advanced and metastatic cancers.

G-protein–coupled receptors (GPCR) are a large family of cell surface receptors that react to extracellular molecules to activate internal signaling pathways, facilitating a wide range of physiologic responses (1). Dysregulation of GPCR functions and their ligands are linked to tumorigenesis, angiogenesis, and metastasis (2). A unique class of GPCRs, known as the protease-activated receptors or PARs, sense and respond to active proteases in the cell microenvironment (3, 4). Uniquely, the proteolytic nature of PAR activation is irreversible, distinct from many other GPCRs. The four PARs found in mammals are activated by various different protease agonists. PAR-1, PAR-3, and PAR-4 are main targets for the coagulation protease thrombin, orchestrating physiologic responses to vascular injury, thrombosis, and inflammation (5–9). PAR-2, on the other hand, is activated by trypsin, several trypsin-like serine proteases (3, 10, 11), and synthetic soluble PAR-2–activating peptides (12), signaling to various downstream pathways that modulate cell proliferation, migration and invasion, cytokine production, stimulation of angiogenesis, and other functions promoting tumor development (2).

This review concerns the roles of PAR-2 and a network of membrane-anchored serine proteases in cancer. There are several excellent comprehensive reviews of PARs in cancer and other diseases (13–16), as well as reviews on membrane-anchored serine proteases in development, tissue homeostasis, and tumor progression (17–21). Here, we focus on recent evidence in support of an interplay between PAR-2 and membrane-anchored serine proteases in proximity on the tumor cell surface that could significantly modulate the magnitude, duration, and nature of PAR-2 signaling, as well as restrict PAR-2 signaling to local membrane microdomains. Their overexpression and dysregulation in tumors have the potential to cooperate to promote aggressive disease through cell–surface interactions, integration of extracellular signals, and induction of intracellular signaling pathways.

Unlike trypsin and other secreted, soluble serine proteases, members of the family of membrane-anchored serine proteases, are synthesized as catalytically inactive or near-inactive proenzymes (zymogens) that are converted into active serine proteases by proteolytic cleavage after an arginine or lysine amino acid residue that is positioned in a conserved activation motif within the catalytic domain (22). These proteases possess domains that tether the extracellular catalytic serine protease domain directly to the cell surface, allowing cleavage of cell surface and pericellular substrates (Fig. 1; refs. 19, 20, 22–24). The manner in which they are linked to the cell surface may be through type I or type II single-pass transmembrane domains or linked via glycophosphatidylinositol (GPI)-anchors. The serine protease domains of these enzymes are structurally highly conserved and contain a triad of amino acids (serine, histidine, and aspartate) required for catalytic activity (25). Overexpression of many of the 20 human members of this family has been documented in many cancers, and several membrane-anchored serine proteases have been shown to promote experimental malignant transformation when aberrantly expressed in tumor cells or in in vivo tumor models (21, 26). In this review, we will focus on those membrane-anchored serine proteases that have been identified to date to be associated with tumor biology and linked to the PAR-2 signaling axis, namely matriptase, hepsin, prostasin, TMPRSS2, testisin, and the membrane-associated pathway triggered by tissue factor (TF), factor VIIa, and factor Xa (TF:FVIIa/FXa).

In the majority of studies to date, PAR-2 has been reported to have oncogenic activities, functioning as a positive regulator of tumor growth and/or progression. Initial evidence that PAR-2 may drive tumorigenesis came from experimental studies showing that PAR-2 indirectly enhances thrombin-dependent tumor cell migration and metastasis (27). Increased PAR-2 expression has been reported in a diverse set of human cancers such as breast, ovarian, prostate, and gastric cancer, when compared with normal patient tissue specimens (28–31). In addition, a recent survey of PAR family member expression in human tumor samples of various cancer types from The Cancer Genome Atlas (TCGA) and the Genotype-Tissue Expression projects (GTEx) reveals upregulated PAR-2 in 15 different cancer types compared with normal tissues (16). A global transcriptome array analysis of PAR expression in over 1,000 ovarian cancer and normal tissue samples showed that human epithelial ovarian cancers predominantly overexpress PAR-2, followed closely by PAR-1, with minimal detection of PAR-3 and PAR-4 (32). Consistent with this, increased PAR-2 is associated with poor prognosis and decreased progression-free and overall survival in patients with ovarian, cervical, and hepatocellular carcinoma (30, 33–35). Increased PAR-2 expression and activation is also correlated with the degree of invasiveness exhibited by both primary and metastatic tumors (29, 30, 36). Protumorigenic activities attributed to PAR-2 signaling include chemokinesis, cell proliferation, invasion and migration, inflammatory signaling, and increased angiogenesis (Fig. 1) in several tumor types including breast, oral, renal, pancreatic, gastric, lung, and esophageal cancers (36–39). PAR-2 may also modulate transactivation of other cell surface receptors (i.e., EGF, TGFβ, and Met tyrosine kinase receptors) that are frequently drivers of tumor progression (34, 40–42). In contrast, a few studies have demonstrated tumor-suppressive functions of PAR-2 (43, 44). In a DMBA-induced mouse model of skin carcinogenesis, PAR-2–deficient mice displayed increased tumor number and increased blood vessel infiltration, which was attributed to modulation of tumor-suppressing TGFβ-1 secretion (44). The specific protease agonists associated with the tumor-suppressive and oncogenic roles of PAR-2 in various tumor types are poorly characterized.

PARs are activated by a tethered ligand that is revealed by proteolytic cleavage of an N-terminal sequence and that can bind to an extracellular docking domain to cause a receptor conformational change that triggers intracellular signaling. There are several modes of regulation for PAR-2 activation and signaling. Different ligands can stabilize unique conformations of the cleaved PAR-2 that activate distinct signaling pathways, a phenomenon referred to as biased agonism or functional selectivity (45–47). A study of mutations within the tethered ligand sequence of PAR-2 (48) revealed that the nature of the tethered ligand sequence and the mode of its presentation to the receptor determine biased signaling by PAR-2. In addition, different proteases that cleave PAR-2 at distinct sites activate divergent patterns of receptor signaling and trafficking (reviewed in refs. 42, 47). Signaling outcomes are diverse and can activate pathways leading to release of proinflammatory cytokines and angiogenic factors, increased cell motility and migration, and increased inflammatory responses (Fig. 1; refs. 47, 49).

Activation of PAR-2 by trypsin and other soluble proteases has been most widely studied. Trypsin cleavage of PAR-2 involves hydrolysis at the canonical cleavage site R36↓S37, which reveals the tethered ligand SLIGKV (human; ref. 50) or SLIGRL (mouse; ref. 51). The exposed tethered ligand interacts with the second extracellular domain of the cleaved receptor and can trigger MAP kinase/ERK1/2 activation, calcium mobilization via Gαq activation, cAMP formation via Gαs activation, and Rho-kinase activity via Gα12/13 activation (Fig. 1; refs. 47, 49). The binding of β-arrestin to phosphorylated residues on the PAR-2 C-terminal tail uncouples and terminates G-protein signaling (52, 53), results in endocytosis of the complex (52), and mediates early endosomal signaling via scaffolding complexes containing Raf1 and activated cytosolic ERK1/2 (54).

β-Arrestins are not only active participants in signaling by internalized PAR-2, but can also direct receptor trafficking to regulate the duration and magnitude of PAR-2 signaling. PAR-2 signal termination occurs by direct ubiquitination and trafficking of PAR-2 to lysosomes for degradation by distinct components of the ESCRT machinery, a process that is unique to PAR-2 within the PAR family (42, 55, 56). The pathways that regulate β-arrestin–mediated signaling versus signal termination are incompletely understood.

The specific signaling pathways activated by PAR-2 in the context of cancer in vivo have received limited attention and will likely depend on the protease activator(s) and the (patho)biological context. For example, in murine asthma models, disease-promoting PAR-2 proinflammatory signaling is dependent on β-arrestin-2, whereas G-protein–dependent signaling is beneficial (57, 58). Several in vitro, in vivo, and human patient data suggest that dysregulation of β-arrestin expression, localization, and/or phosphorylation is associated with increased migration and invasion and ultimately poorer outcomes in various types of cancer (59, 60). This may be attributed not only to direct tumorigenic signaling through β-arrestin but also other selective pathways of PAR-2 signaling. The contributions of various G-proteins and β-arrestin signaling downstream of membrane-anchored serine protease activation of PAR-2 in vivo are not yet well characterized.

Numerous in vitro and in vivo studies identify matriptase (encoded by ST14) to be a potent activator of PAR-2. Matriptase (Fig. 1) is a type II single-pass transmembrane serine protease with a unique extracellular stem region containing various conserved protein-binding domains (SEA, CUB, and LDLR-a repeats), which are involved in matriptase activation as well as interaction with its cognate inhibitor hepatocyte growth factor activator (HAI)-1 (61–66) and other proteins. Matriptase is expressed as a precursor or zymogen form that may be proteolytically processed first within the SEA domain, and then activated by further cleavage at a highly conserved R↓VVGG motif by pericellular serine proteases or by autoactivation by matriptase itself (67). Interestingly, the zymogen form of matriptase, unusual among trypsin-like serine proteases, possesses measurable enzymatic activity and was recently shown to be capable of executing the in vivo developmental and homeostatic functions of the proteolytically processed protease (68).

Matriptase is widely expressed in normal epithelial tissues, where it plays a critical role in maintaining epithelial barrier homeostasis (69–72). Matriptase was first discovered in breast cancer cell lines (73), and its expression is associated with breast cancer progression (74, 75). Matriptase is also upregulated in many other tumors of epithelial origin, namely prostate, cervical, gastric, esophageal, renal cell, skin, oral squamous cell, ovarian, and cervical carcinomas (reviewed in refs. 76, 77). Upregulation of matriptase expression in many of these cancers is associated with poor outcomes (reviewed in ref. 21).

Substantial molecular and cellular data identify matriptase to be a direct proteolytic activator of PAR-2. Early studies using the human HaCaT transformed keratinocyte cell line, which endogenously expresses PAR-2, showed that treatment with soluble recombinant matriptase (protease domain only) stimulates canonical PAR-2 cleavage (R36↓S37) and potent PI hydrolysis (78). In KOLF cells (which do not express endogenous PARs, matriptase, or hepsin), PI hydrolysis in response to recombinant matriptase was observed only upon PAR-2 transfection, indicating direct and specific PAR-2 activation (78). These findings have since been confirmed in several other in vitro studies using PAR-2–expressing KOLF or HEK293 cells and soluble or coexpressed matriptase (68, 79–82).

In vivo studies using murine transgenic models of matriptase and PAR-2 deficiencies have provided compelling evidence for a matriptase–PAR-2 signaling axis, specifically during normal embryonic development and placental barrier function (78, 83). Is it possible that a matriptase–PAR-2 signaling pathway regulates global epithelial integrity during homeostasis, and that this pathway becomes dysregulated in cancer? In support of this, PAR-2 has been shown to be critical for matriptase-mediated tumor progression using several in vivo tumor models in which matriptase is overexpressed. In a transgenic mouse model of squamous cell carcinoma (SCC) where matriptase is overexpressed in the epidermis via a keratin-5 promoter (K5-matriptase), animals developed spontaneous multistage SCCs (84) and displayed protumorigenic inflammatory cytokine release that was PAR-2 dependent (81). The downstream effects of matriptase activation of PAR-2 were attributed to selective signaling through Gαi and NFκB-directed cytokine release (81). In this model, matriptase also induced the activation of a HGF/c-Met–dependent pathway, and both c-Met and PAR-2 signaling were independently required for tumor initiation (81).

Matriptase activity seems to be critical for the regulation of inflammatory signaling via the matriptase–PAR-2 axis. Matriptase expression and trafficking, activity, and shedding are controlled by Kunitz-type serine protease inhibitors, specifically HAI-1/SPINT1 and HAI-2/SPINT2, and downregulation of these endogenous matriptase inhibitors increases aberrant matriptase activity (62, 85–88). Interestingly, the specific HAI required for proper matriptase trafficking is cell type dependent (reviewed in ref. 89). The ratio of matriptase to its inhibitors, or the protease–inhibitor balance, is important: loss of or decreased endogenous HAI-1 or HAI-2 enables increased matriptase activity and this has been shown to promote in vitro tumorigenesis in several studies (90–96). In human SCCs, increased matriptase expression is correlated with diminished expression of matriptase–HAI-1 complexes and with reduced PAR-2 expression (97), possibly due to PAR-2 overactivation induced by deregulated matriptase activity. The importance of protease–inhibitor balance has also been demonstrated in vivo in murine transgenic and xenograft models, where loss or decreased levels of endogenous HAI-1 or HAI-2 and increased matriptase activity promotes carcinogenesis, which can be effectively reversed by increased expression of the inhibitor (84, 98, 99). Recent studies also suggest that matriptase can function in a paracrine manner to activate PAR-2 (100). Pericellular matriptase activity on the surface of oral SCC, caused by insufficient HAI-1, was shown to activate PAR-2 on the surface of cancer-associated fibroblasts (CAF), leading to enhanced CAF migration and infiltration (100).

In human colon cancer, matriptase was originally designated as a tumor suppressor gene due to its loss of heterozygosity (101). Consistent with this, mice with tissue-specific deletion of matriptase in the gastrointestinal tract show increased intestinal permeability, spontaneously develop chronic colitis and ultimately inflammation-induced colon cancer (102). These data suggest that complete loss of matriptase can lead to colon carcinogenesis in the context of inflammation. Although matriptase expression may be downregulated in human colon cancer, additional studies using a specific antibody (A11) that targets active matriptase show that there is increased active matriptase in human colon cancer tissues and in a patient-derived colon cancer xenograft model (92, 103). The presence of increased active matriptase in human colon cancer is also supported by studies on the ratio of matriptase:HAI-1 levels, which suggest that although both matriptase and HAI-1 are downregulated during carcinogenesis, the matriptase:HAI-1 ratio increases during cancer progression, resulting in a population of active matriptase on the cell surface (90, 92). In support of this, the presence of HAI-1 in intestinal epithelium was shown to be protective in two murine models of intestinal carcinogenesis (104). Whether these sequelae are related to matriptase mediated–PAR-2 signaling is an unexplored area.

Several other members of the membrane-anchored serine protease family were originally reported to functionally activate PAR-2 signaling, namely prostasin (encoded by PRSS8), hepsin (encoded by HPN), and TMPRSS2 (encoded by TMPRSS2). Recent studies indicate that these proteases indirectly trigger PAR-2 activation through the matriptase–PAR-2 axis (Fig. 1). Hepsin and TMPRSS2 are type II transmembrane serine proteases (18), while prostasin is anchored to the plasma membrane via a GPI anchor (105).

Hepsin was shown to indirectly activate PAR-2 and trigger PI hydrolysis only in the presence of matriptase in HaCaT cells. This was attenuated by a specific matriptase-blocking antibody, suggesting that hepsin is capable of activating the matriptase zymogen, which can then activate PAR-2 (78). In the same study, PAR-2 activation induced by recombinant prostasin was only observed in cells that also expressed catalytically active matriptase, indicating that prostasin is capable of functioning as an indirect activator of PAR-2 signaling via activation of the matriptase zymogen (78). TMPRSS2 was originally thought to activate PAR-2 directly, resulting in calcium mobilization in prostate cancer cell lines (106). In a later study, stable overexpression of TMPRSS2 in a variety of prostate cancer cell lines was shown to induce matriptase activation and to increase the metastasis of orthotopic xenografts (107). These results identify matriptase as a possible substrate of TMPRSS2 and indicate that like hepsin, TMPRSS2 activates the matriptase–PAR-2 axis.

The functional interactions between prostasin, matriptase, and PAR-2 activation have been most extensively studied; however, these interactions have proved to be complex and are still incompletely understood. In normal tissues, matriptase and prostasin are ubiquitously coexpressed in epithelial cells, whereas during the progression of multi-stage epithelial carcinogenesis, they are found to be expressed in separate tumor cell compartments, possibly indicating altered regulation or activation requirements during tumor progression (108). Results from several studies highlight the importance of tissue distribution and subcellular localization for the function and regulation of these two membrane anchored proteases in other disease contexts (108–111). It is possible that interactions between their extracellular domains as well as tightly regulated membrane distribution and subcellular localization all contribute to regulating the activation of the matriptase–PAR-2 axis as well. When ectopically expressed in the skin of transgenic mice, prostasin was shown to induce epidermal hyperplasia, ichthyosis, and inflammation, phenotypes that are completely negated when superimposed on a PAR-2–null background, establishing PAR-2 as a pivotal downstream mediator of prostasin inflammatory activity (112). Matriptase may be involved in this activity, because matriptase and prostasin are found to be capable of forming a reciprocal zymogen activation complex stimulating the activation of the zymogen forms of each other (80). The matriptase zymogen, which has a low rate of catalytic activity, has been shown to be capable of activating prostasin (80). Utilizing detailed cell-based analyses and genetically modified animals, Friis and colleagues (68) recently demonstrated that the matriptase zymogen can induce PAR-2 activation in the presence of prostasin, and that this activity requires catalytically active and membrane-anchored prostasin. This finding may indicate that matriptase zymogen–activated prostasin can execute the activation site cleavage of PAR-2 directly or that the intrinsic catalytic activity of the matriptase zymogen is stimulated effectively by catalytically active prostasin (68).

Hepsin, prostasin, and TMPRSS2 upregulation in epithelial breast, prostate, and ovarian cancer cell lines, mouse models, and patient samples are believed to contribute to increased proliferation, tumor growth, metastasis, ascites formation, and various other invasive processes (107, 113–125). Overexpression of hepsin in prostate epithelium in a prostate tumor model (LPB-Tag mice) resulted in increased basement membrane disorganization and tumor metastasis to distant organs, which did not occur in control mice, indicating that hepsin is capable of promoting prostate cancer metastatic processes (119). Expression of TMPRSS2 is also associated with prostate cancer progression, as knockdown of TMPRSS2 in prostate cancer cell lines results in decreased invasion, tumor size, and incidence in xenograft models (107). In the transgenic adenocarcinoma mouse prostate (TRAMP) model of prostate cancer, mice with TMPRSS2 deficiency exhibited larger tumors but a lower incidence of distant metastasis (125). In contrast, prostasin, like matriptase, is reported to function as a tumor suppressor in colon cancer, with reduced expression correlating with more aggressive clinical stages and shorter patient survival time (114, 116). In many of these cancer contexts, it is not yet known whether the tumorigenic processes attributed to protease expression or activity occur via PAR-2 activation.

In addition to these membrane-anchored serine proteases, the membrane-localized coagulation complex containing TF:FVIIa/FXa was also originally reported to activate PAR-2 directly (126), but was later shown to trigger the activation of matriptase zymogen, which mediates the cleavage and activation of PAR-2 (82) (Fig. 1). In experimental studies using the spontaneous mammary tumor virus (MMTV) promoter-driven model of breast cancer in mice, the PAR-2–deficient phenotype was similar to that of mice with a truncated cytoplasmic domain of TF, suggesting an interplay between TF cytoplasmic domain signaling and PAR-2 in promoting breast cancer progression (127). Interestingly, in this study, PAR-2 deficiency led to a significant delay in the transition from adenomas to invasive carcinoma and was associated with less tumor vascularization and reduced immune cell infiltration. Reconstitution of the PAR-2–deficient tumor cells with PAR-2 mutated at the β-arrestin–binding site restored proangiogenic chemokine induction, tumor growth and increased vessel density (127), demonstrating that the tumor promoting activities of PAR-2 activation in this model were due to G-protein, rather than β-arrestin signaling. The involvement of matriptase in this tumor-associated TF–PAR-2 signaling activity is not known.

Testisin (encoded by PRSS21) is a GPI-anchored membrane serine protease (128–130) that has been found to induce PAR-2 activation (Fig. 1). Exposure of PAR-2 overexpressing HeLa (human cervical carcinoma) cells to soluble recombinant testisin results in potent intracellular calcium mobilization, ERK1/2 phosphorylation, and NFκB activation (131). Furthermore, coexpression of testisin and PAR-2 results in inflammatory cytokine (IL8, IL6) induction and a decrease in PAR-2 surface expression via receptor internalization (131). The data indicate that testisin is capable of PAR-2 cleavage and activation affecting signaling responses important for tumor cell motility, proliferation, and inflammation. The potential involvement of matriptase or the matriptase zymogen in testisin-mediated PAR-2 activation is not known. Interestingly, testisin shows very limited normal tissue distribution, but is overexpressed in human epithelial ovarian, cervical, and lung carcinomas (132–134). Aberrant overexpression of testisin in epithelial ovarian tumor cells was shown to promote malignant transformation, increase tumor growth, and tumor formation in subcutaneous xenograft models (129). The involvement of PAR-2 activation in the in vivo tumor phenotypes is not known.

Noncanonical PAR-2 cleavage

Cleavage of the N-terminal PAR-2 sequence by membrane-anchored serine proteases at a noncanonical cleavage site has not yet been reported. However, several soluble secreted proteases alter PAR-2 signaling responses via cleavage at noncanonical sites. Cleavage of PAR-2 at residues C-terminal to the tethered ligand domain sequence has the effect of removing the tethered ligand sequence and effectively “disarming” PAR-2 to prevent its activation by trypsin-like proteases and membrane-anchored serine proteases (Fig. 1). The neutrophil proteases cathepsin G and proteinase 3 cleave PAR-2 at F65↓S66 and V62↓D63, respectively, silencing trypsin-induced calcium mobilization and MAPK signaling, presumably by removing the tethered ligand sequence (135). Neutrophil elastase cleaves PAR-2 at S68↓V69, which, unlike trypsin, does not induce calcium mobilization via Gαq activation, recruitment of β-arrestin or receptor internalization (135). On the other hand, neutrophil elastase does activate MAPK signaling independent of the tethered ligand binding, suggesting that it instead stabilizes a unique receptor conformation that favors a distinct signaling profile (135). Thus, in a protease-rich environment, noncanonical PAR-2 cleavage adds another level of complexity in PAR-2 signaling that would be expected to impact the activities of membrane anchored serine proteases in the context of cancer. Whether noncanonical protease activators significantly impact biased signaling that influences tumor cell behavior is an interesting area for future study.

Activation of PAR-2 by PAR-1

O'Brien and colleagues (136) were the first to show that the PAR-1–tethered ligand domain is capable of transactivating PAR-2, indicating a possible role for transactivation of PAR-2 induced by protease-mediated cleavage of PAR-1. Thrombin- and PAR-1–dependent migration of melanoma and prostate cancer cells was found to be dependent on indirect activation of PAR-2. This effect of thrombin on migration and chemokinesis required PAR-1 activation and transactivation of PAR-2 by the PAR-1–tethered ligand, independent of PAR-2 cleavage (27). In vivo, PAR-1 transactivation of PAR-2 was shown to contribute to altered responses in late stages of sepsis (137). This mechanism was further characterized and attributed to the formation of PAR-1–PAR-2 heterodimers. Thrombin was shown to cleave PAR-1 at its canonical site to reveal its tethered ligand, which can also bind to and activate PAR-2 (136). A PAR-1 cleavable but nonsignaling variant, with a mutation in the second extracellular ligand-binding domain, was shown to donate its cleaved tethered ligand to wild-type PAR-2, which triggered thrombin-mediated signaling in COS-7 cells. This suggests that transactivation of PAR-2 by the PAR-1–tethered ligand facilitates PAR-1–associated signaling when present as a heterodimer (136). PAR-1–PAR-2 transactivation can furthermore recruit β-arrestin, cointernalize, and activate nuclear ERK1/2 signaling, all of which are different from the trafficking and signaling in response to PAR-1 activation alone (138). The impact and mechanisms by which PAR-1–PAR-2 heterodimers influence protumorigenic signaling is an understudied area but provides yet another pathway of biased PAR-2 signaling in response to indirect activation by thrombin, and perhaps other PAR-1–activating proteases, such as the cancer-associated protease plasmin and the coagulation protease FXa. To date, no membrane-anchored serine proteases are known to activate PAR-1 directly, but how the coexpression of PAR-2–activating membrane-anchored serine proteases may influence PAR-1–PAR-2 transactivation in these contexts has not been investigated.

Although considerable progress has been made in our understanding of mechanisms by which proteases and synthetic agonists activate PARs, there is much to learn about the interactions between membrane-anchored and soluble proteases, and specific protease–receptor interactions that influence PAR-2 signaling. This is important because PAR-2 is a signaling receptor with established links to cancer progression and metastasis, and whose protease-dependent activation can signal substantial changes in cell behavior. The emerging data implicate PAR-2 and membrane-anchored serine proteases in proximity on the tumor cell surface as co-conspirators in regulating the nature of PAR-2 signaling responses and as determinants of biased signaling in cancer.

Membrane anchored serine proteases may impact PAR-2 signaling bias in multiple and divergent ways. Biased PAR-2 signaling has mostly been studied with regard to cleavage at different sites by various protease agonists or antagonists. However, although membrane-anchored serine protease activators of PAR-2 identified to date cleave PAR-2 at the trypsin canonical site, other mechanisms that modulate differential PAR-2 activities are likely. The extracellular domains of membrane-anchored serine proteases (Fig. 1) offer unique opportunities for allosteric modulation of PAR-2, potentially by inducing conformational changes that can impart functional selectivity. It is also possible that cell surface and membrane localization may limit signaling responses to specific areas or local microdomains, whereas soluble proteases that cleave and activate PAR-2, such as trypsin, mast cell tryptase, kallikreins, and gingipains, would induce transient activation independent of surface location. In nontransformed cells, matriptase is localized to cell–cell junctions and basolateral surfaces of polarized epithelium (139), prostasin is found on apical membranes (111) and hepsin is associated with desmosomes (121), while testisin is found in lipid rafts (130). The influence of these protease specific localizations on PAR-2 activation and signaling responses is currently unknown. Furthermore, these distributions may be expected to significantly impact and be impacted by epithelial-to-mesenchymal and mesenchymal-to-epithelial transitions associated with tumor progression and metastasis.

PAR-2 present on tumor cells could be considered to act as a “protease sensor” of the tumor cell microenvironment, responding to circulating protease agonists that unmask the tethered ligand as well as circulating proteases that act indirectly as antagonists by “disarming” the receptor. Because membrane-anchored serine proteases are overexpressed and their inhibitors are downregulated in many cancers, these overactive proteases in direct contact with PAR-2 molecules on the cell membrane are poised to induce sustained signaling responses via continual surveillance of the cell membrane, activating PAR-2 present on the surface as well as newly synthesized receptors that repopulate the cell membrane in their vicinity. Such activities would not only affect the type, magnitude, and duration of signaling responses, but would also be expected to modulate canonical activation by soluble proteases in the cell environment. A lack of protease-specific inhibitors, activity assays, and other tools confounds studies aimed at teasing out these mechanistic details. These analyses are further complicated by proteolytic cleavage or “shedding” of protease domains from several of the membrane-anchored serine proteases, potentially releasing protease activity in a soluble form. The emerging evidence for membrane serine protease zymogens with functional activities adds another level of complexity. Undoubtedly, it will be a challenge to understand the factors that determine which proteases cleave PAR-2 in a protease-rich environment, which may be disease or cell-type dependent and will also depend on the local milieu of protease inhibitors present. Membrane-anchored serine protease–mediated allosteric modulation of PAR-2 signaling has not yet been investigated and could potentially facilitate tumor-suppressive or oncogenic activities depending on the tumor cell context.

One approach to interfering with the protumorigenic activities of membrane-anchored serine proteases is the development of specific inhibitors. This strategy has had limited success, likely because there is redundancy and/or overlap in the catalytic specificities and substrates in physiologic and pathologic contexts. An early approach to the development of inhibitors that showed promise was the targeting of selective conformational changes required for protease activity. The human recombinant antibody termed A11, developed against a protease surface loop of matriptase that is less conserved among trypsin-fold proteases, was shown to selectively recognize the active form of matriptase over the zymogen form and is a specific inhibitor of its activity (103, 140). Other strategies to inhibit matriptase activity include treatment with soluble recombinant HAI-1, synthetic small molecules, peptides, and mAbs, all of which have been shown to inhibit matriptase to varying degrees in vitro but do not address therapeutic limitations of specificity and optimal serum half-lives in vivo (141–144). A recent approach for inhibition of matriptase consists of an engineered variant of natural HAI-1, utilizing a Kunitz-domain 1/Kunitz-domain 2 chimera to replace the less-specific Kunitz-domain 2 of HAI-1, fused to an antibody Fc domain to increase putative binding sites to matriptase (144). This fusion protein was shown to inhibit matriptase activation of pro-HGF and matriptase activity on the surface of cancer cells (144). The activities of membrane-anchored serine proteases have also been exploited as functional biomarkers for imaging tumorigenesis. A11 has been used to visualize aberrant matriptase activity in epithelial tumors in vivo (92, 140), demonstrating a potential application in noninvasive tumor imaging and monitoring of disease progression. Fluorescent nanoparticles targeted against hepsin allosteric binding peptides have also been shown to bind specifically to hepsin-expressing LNCaP xenografts (145). Such reagents hold promise for the diagnostic detection and experimental manipulation of protease activities that contribute to disease progression, with potential clinical applications.

An alternative strategy is to potentially manipulate the functional selectivity of PAR-2 to develop novel cancer therapies. In the broader context of GPCR signaling, therapeutic strategies are now being aimed at shifting the signal bias in the appropriate direction to mitigate disease progression (146, 147). It is likely that membrane-anchored serine proteases in proximity to PAR-2 will be a critical consideration in the development of such therapies. Many attempts to develop PAR-2–biased agonists (148) and antagonists (15, 37) have been modeled on analogues of the PAR-2–activating peptide, some of which are capable of preferentially modulating Ca2+ or ERK1/2 signaling (148). Other small molecules and antibodies are being developed to target binding pockets and transmembrane regions, as well as allosteric sites on PAR-2, in an effort to prevent conformational changes required for receptor activation upon proteolytic cleavage or to prevent tethered ligand binding to the peptide-binding site (149–151).

Several hallmarks of aggressive cancer are a direct result of proteolytic activity, including tumor cell invasion into the stroma, angiogenesis, and metastasis (152), with the roles of proteases traditionally focused on protein degradation and extracellular matrix remodeling. Advances in our understanding of membrane-anchored serine proteases as modulators of PAR-2 activation and signaling are anticipated to uncover novel avenues for pharmaceutical intervention that could be used to selectively tune receptor activity for the treatment of cancer progression.

No potential conflicts of interest were disclosed.

This work was supported by NIH Grants R01 CA196988, R01 HL118390, and T32CA154274.

1.
O'Hayre
M
,
Degese
MS
,
Gutkind
JS
. 
Novel insights into G protein and G protein-coupled receptor signaling in cancer
.
Curr Opin Cell Biol
2014
;
27
:
126
35
.
2.
Dorsam
RT
,
Gutkind
JS
. 
G-protein-coupled receptors and cancer
.
Nat Rev Cancer
2007
;
7
:
79
94
.
3.
Gieseler
F
,
Ungefroren
H
,
Settmacher
U
,
Hollenberg
MD
,
Kaufmann
R
. 
Proteinase-activated receptors (PARs) - focus on receptor-receptor-interactions and their physiological and pathophysiological impact
.
Cell Commun Signal
2013
;
11
:
86
.
4.
Adams
MN
,
Ramachandran
R
,
Yau
MK
,
Suen
JY
,
Fairlie
DP
,
Hollenberg
MD
, et al
Structure, function and pathophysiology of protease activated receptors
.
Pharmacol Ther
2011
;
130
:
248
82
.
5.
Coughlin
SR
. 
Thrombin signalling and protease-activated receptors
.
Nature
2000
;
407
:
258
64
.
6.
O'Brien
PJ
,
Molino
M
,
Kahn
M
,
Brass
LF
. 
Protease activated receptors: theme and variations
.
Oncogene
2001
;
20
:
1570
81
.
7.
Ossovskaya
VS
,
Bunnett
NW
. 
Protease-activated receptors: contribution to physiology and disease
.
Physiol Rev
2004
;
84
:
579
621
.
8.
Coughlin
SR
. 
Protease-activated receptors in hemostasis, thrombosis and vascular biology
.
J Thromb Haemost
2005
;
3
:
1800
14
.
9.
Ramachandran
R
,
Hollenberg
MD
. 
Proteinases and signalling: pathophysiological and therapeutic implications via PARs and more
.
Br J Pharmacol
2008
;
153
:
S263
82
.
10.
Coughlin
SR
,
Camerer
E
. 
PARticipation in inflammation
.
J Clin Invest
2003
;
111
:
25
7
.
11.
Cottrell
GS
,
Amadesi
S
,
Schmidlin
F
,
Bunnett
N
. 
Protease-activated receptor 2: activation, signalling and function
.
Biochem Soc Trans
2003
;
31
:
1191
7
.
12.
Maryanoff
BE
,
Santulli
RJ
,
McComsey
DF
,
Hoekstra
WJ
,
Hoey
K
,
Smith
CE
, et al
Protease-activated receptor-2 (PAR-2): structure-function study of receptor activation by diverse peptides related to tethered-ligand epitopes
.
Arch Biochem Biophys
2001
;
386
:
195
204
.
13.
Arora
P
,
Ricks
TK
,
Trejo
J
. 
Protease-activated receptor signalling, endocytic sorting and dysregulation in cancer
.
J Cell Sci
2007
;
120
:
921
8
.
14.
Schaffner
F
,
Ruf
W
. 
Tissue factor and PAR2 signaling in the tumor microenvironment
.
Arterioscler Thromb Vasc Biol
2009
;
29
:
1999
2004
.
15.
Ramachandran
R
,
Noorbakhsh
F
,
Defea
K
,
Hollenberg
MD
. 
Targeting proteinase-activated receptors: therapeutic potential and challenges
.
Nat Rev Drug Discov
2012
;
11
:
69
86
.
16.
Arakaki
AKS
,
Pan
WA
,
Trejo
J
. 
GPCRs in cancer: protease-activated receptors, endocytic adaptors and signaling
.
Int J Mol Sci
2018
;
19
.
17.
Netzel-Arnett
S
,
Hooper
JD
,
Szabo
R
,
Madison
EL
,
Quigley
JP
,
Bugge
TH
, et al
Membrane anchored serine proteases: a rapidly expanding group of cell surface proteolytic enzymes with potential roles in cancer
.
Cancer Metastasis Rev
2003
;
22
:
237
58
.
18.
Bugge
TH
,
Antalis
TM
,
Wu
Q
. 
Type II transmembrane serine proteases
.
J Biol Chem
2009
;
284
:
23177
81
.
19.
Antalis
TM
,
Bugge
TH
,
Wu
Q
. 
Membrane-anchored serine proteases in health and disease
.
Prog Mol Biol Transl Sci
2011
;
99
:
1
50
.
20.
Szabo
R
,
Bugge
TH
. 
Membrane-anchored serine proteases in vertebrate cell and developmental biology
.
Annu Rev Cell Dev Biol
2011
;
27
:
213
35
.
21.
Tanabe
LM
,
List
K
. 
The role of type II transmembrane serine protease-mediated signaling in cancer
.
FEBS J
2017
;
284
:
1421
36
.
22.
Antalis
TM
,
Buzza
MS
,
Hodge
KM
,
Hooper
JD
,
Netzel-Arnett
S
. 
The cutting edge: membrane-anchored serine protease activities in the pericellular microenvironment
.
Biochem J
2010
;
428
:
325
46
.
23.
Antalis
TM
,
Conway
GD
,
Peroutka
RJ
,
Buzza
MS
. 
Membrane-anchored proteases in endothelial cell biology
.
Curr Opin Hematol
2016
;
23
:
243
52
.
24.
Hooper
JD
,
Clements
JA
,
Quigley
JP
,
Antalis
TM
. 
Type II transmembrane serine proteases. Insights into an emerging class of cell surface proteolytic enzymes
.
J Biol Chem
2001
;
276
:
857
60
.
25.
Hedstrom
L
. 
Serine protease mechanism and specificity
.
Chem Rev
2002
;
102
:
4501
24
.
26.
Murray
AS
,
Varela
FA
,
List
K
. 
Type II transmembrane serine proteases as potential targets for cancer therapy
.
Biol Chem
2016
;
397
:
815
26
.
27.
Shi
X
,
Gangadharan
B
,
Brass
LF
,
Ruf
W
,
Mueller
BM
. 
Protease-activated receptors (PAR1 and PAR2) contribute to tumor cell motility and metastasis
.
Mol Cancer Res
2004
;
2
:
395
402
.
28.
Caruso
R
,
Pallone
F
,
Fina
D
,
Gioia
V
,
Peluso
I
,
Caprioli
F
, et al
Protease-activated receptor-2 activation in gastric cancer cells promotes epidermal growth factor receptor trans-activation and proliferation
.
Am J Pathol
2006
;
169
:
268
78
.
29.
Black
PC
,
Mize
GJ
,
Karlin
P
,
Greenberg
DL
,
Hawley
SJ
,
True
LD
, et al
Overexpression of protease-activated receptors-1,-2, and-4 (PAR-1, -2, and -4) in prostate cancer
.
Prostate
2007
;
67
:
743
56
.
30.
Jahan
I
,
Fujimoto
J
,
Alam
SM
,
Sato
E
,
Sakaguchi
H
,
Tamaya
T
. 
Role of protease activated receptor-2 in tumor advancement of ovarian cancers
.
Ann Oncol
2007
;
18
:
1506
12
.
31.
Su
S
,
Li
Y
,
Luo
Y
,
Sheng
Y
,
Su
Y
,
Padia
RN
, et al
Proteinase-activated receptor 2 expression in breast cancer and its role in breast cancer cell migration
.
Oncogene
2009
;
28
:
3047
57
.
32.
Chanakira
A
,
Westmark
PR
,
Ong
IM
,
Sheehan
JP
. 
Tissue factor-factor VIIa complex triggers protease activated receptor 2-dependent growth factor release and migration in ovarian cancer
.
Gynecol Oncol
2017
;
145
:
167
75
.
33.
Aman
M
,
Ohishi
Y
,
Imamura
H
,
Shinozaki
T
,
Yasutake
N
,
Kato
K
, et al
Expression of protease-activated receptor-2 (PAR-2) is related to advanced clinical stage and adverse prognosis in ovarian clear cell carcinoma
.
Hum Pathol
2017
;
64
:
156
63
.
34.
Hugo de Almeida
V
,
Guimaraes
IDS
,
Almendra
LR
,
Rondon
AMR
,
Tilli
TM
,
de Melo
AC
, et al
Positive crosstalk between EGFR and the TF-PAR2 pathway mediates resistance to cisplatin and poor survival in cervical cancer
.
Oncotarget
2018
;
9
:
30594
609
.
35.
Sun
L
,
Li
PB
,
Yao
YF
,
Xiu
AY
,
Peng
Z
,
Bai
YH
, et al
Proteinase-activated receptor 2 promotes tumor cell proliferation and metastasis by inducing epithelial-mesenchymal transition and predicts poor prognosis in hepatocellular carcinoma
.
World J Gastroenterol
2018
;
24
:
1120
33
.
36.
Wojtukiewicz
MZ
,
Hempel
D
,
Sierko
E
,
Tucker
SC
,
Honn
KV
. 
Protease-activated receptors (PARs)–biology and role in cancer invasion and metastasis
.
Cancer Metastasis Rev
2015
;
34
:
775
96
.
37.
Ungefroren
H
,
Witte
D
,
Fiedler
C
,
Gadeken
T
,
Kaufmann
R
,
Lehnert
H
, et al
The role of PAR2 in TGF-beta1-Induced ERK activation and cell motility
.
Int J Mol Sci
2017
;
18
:
pii:E2776
.
38.
Kularathna
PK
,
Pagel
CN
,
Mackie
EJ
. 
Tumour progression and cancer-induced pain: a role for protease-activated receptor-2?
Int J Biochem Cell Biol
2014
;
57
:
149
56
.
39.
Sedda
S
,
Marafini
I
,
Caruso
R
,
Pallone
F
,
Monteleone
G
. 
Proteinase activated-receptors-associated signaling in the control of gastric cancer
.
World J Gastroenterol
2014
;
20
:
11977
84
.
40.
Kaufmann
R
,
Oettel
C
,
Horn
A
,
Halbhuber
KJ
,
Eitner
A
,
Krieg
R
, et al
Met receptor tyrosine kinase transactivation is involved in proteinase-activated receptor-2-mediated hepatocellular carcinoma cell invasion
.
Carcinogenesis
2009
;
30
:
1487
96
.
41.
Chung
H
,
Ramachandran
R
,
Hollenberg
MD
,
Muruve
DA
. 
Proteinase-activated receptor-2 transactivation of epidermal growth factor receptor and transforming growth factor-beta receptor signaling pathways contributes to renal fibrosis
.
J Biol Chem
2013
;
288
:
37319
31
.
42.
Soh
UJ
,
Dores
MR
,
Chen
B
,
Trejo
J
. 
Signal transduction by protease-activated receptors
.
Br J Pharmacol
2010
;
160
:
191
203
.
43.
Kaufmann
R
,
Schafberg
H
,
Nowak
G
. 
Proteinase-activated receptor-2-mediated signaling and inhibition of DNA synthesis in human pancreatic cancer cells
.
Int J Pancreatol
1998
;
24
:
97
102
.
44.
Rattenholl
A
,
Seeliger
S
,
Buddenkotte
J
,
Schon
M
,
Schon
MP
,
Stander
S
, et al
Proteinase-activated receptor-2 (PAR2): a tumor suppressor in skin carcinogenesis
.
J Invest Dermatol
2007
;
127
:
2245
52
.
45.
Galandrin
S
,
Oligny-Longpre
G
,
Bouvier
M
. 
The evasive nature of drug efficacy: implications for drug discovery
.
Trends Pharmacol Sci
2007
;
28
:
423
30
.
46.
Hollenberg
MD
,
Mihara
K
,
Polley
D
,
Suen
JY
,
Han
A
,
Fairlie
DP
, et al
Biased signalling and proteinase-activated receptors (PARs): targeting inflammatory disease
.
Br J Pharmacol
2014
;
171
:
1180
94
.
47.
Zhao
P
,
Metcalf
M
,
Bunnett
NW
. 
Biased signaling of protease-activated receptors
.
Front Endocrinol
2014
;
5
:
67
.
48.
Ramachandran
R
,
Mihara
K
,
Mathur
M
,
Rochdi
MD
,
Bouvier
M
,
Defea
K
, et al
Agonist-biased signaling via proteinase activated receptor-2: differential activation of calcium and mitogen-activated protein kinase pathways
.
Mol Pharmacol
2009
;
76
:
791
801
.
49.
Rothmeier
AS
,
Ruf
W
. 
Protease-activated receptor 2 signaling in inflammation
.
Semin Immunopathol
2012
;
34
:
133
49
.
50.
Nystedt
S
,
Emilsson
K
,
Larsson
AK
,
Strombeck
B
,
Sundelin
J
. 
Molecular cloning and functional expression of the gene encoding the human proteinase-activated receptor 2
.
Eur J Biochem
1995
;
232
:
84
9
.
51.
Nystedt
S
,
Emilsson
K
,
Wahlestedt
C
,
Sundelin
J
. 
Molecular cloning of a potential proteinase activated receptor
.
Proc Natl Acad Sci U S A
1994
;
91
:
9208
12
.
52.
Ricks
TK
,
Trejo
J
. 
Phosphorylation of protease-activated receptor-2 differentially regulates desensitization and internalization
.
J Biol Chem
2009
;
284
:
34444
57
.
53.
Jung
SR
,
Seo
JB
,
Deng
Y
,
Asbury
CL
,
Hille
B
,
Koh
DS
. 
Contributions of protein kinases and beta-arrestin to termination of protease-activated receptor 2 signaling
.
J Gen Physiol
2016
;
147
:
255
71
.
54.
DeFea
KA
,
Zalevsky
J
,
Thoma
MS
,
Dery
O
,
Mullins
RD
,
Bunnett
NW
. 
beta-arrestin-dependent endocytosis of proteinase-activated receptor 2 is required for intracellular targeting of activated ERK1/2
.
J Cell Biol
2000
;
148
:
1267
81
.
55.
Jacob
C
,
Cottrell
GS
,
Gehringer
D
,
Schmidlin
F
,
Grady
EF
,
Bunnett
NW
. 
c-Cbl mediates ubiquitination, degradation, and down-regulation of human protease-activated receptor 2
.
J Biol Chem
2005
;
280
:
16076
87
.
56.
Hasdemir
B
,
Murphy
JE
,
Cottrell
GS
,
Bunnett
NW
. 
Endosomal deubiquitinating enzymes control ubiquitination and down-regulation of protease-activated receptor 2
.
J Biol Chem
2009
;
284
:
28453
66
.
57.
Nichols
HL
,
Saffeddine
M
,
Theriot
BS
,
Hegde
A
,
Polley
D
,
El-Mays
T
, et al
beta-Arrestin-2 mediates the proinflammatory effects of proteinase-activated receptor-2 in the airway
.
Proc Natl Acad Sci U S A
2012
;
109
:
16660
5
.
58.
Walker
JK
,
DeFea
KA
. 
Role for beta-arrestin in mediating paradoxical beta2AR and PAR2 signaling in asthma
.
Curr Opin Pharmacol
2014
;
16
:
142
7
.
59.
Sobolesky
PM
,
Moussa
O
. 
The role of beta-arrestins in cancer
.
Prog Mol Biol Transl Sci
2013
;
118
:
395
411
.
60.
Song
Q
,
Ji
Q
,
Li
Q
. 
The role and mechanism of betaarrestins in cancer invasion and metastasis (Review)
.
Int J Mol Med
2018
;
41
:
631
9
.
61.
Takeuchi
T
,
Shuman
MA
,
Craik
CS
. 
Reverse biochemistry: use of macromolecular protease inhibitors to dissect complex biological processes and identify a membrane-type serine protease in epithelial cancer and normal tissue
.
Proc Natl Acad Sci U S A
1999
;
96
:
11054
61
.
62.
Oberst
MD
,
Williams
CA
,
Dickson
RB
,
Johnson
MD
,
Lin
CY
. 
The activation of matriptase requires its noncatalytic domains, serine protease domain, and its cognate inhibitor
.
J Biol Chem
2003
;
278
:
26773
9
.
63.
Lee
MS
,
Tseng
IC
,
Wang
Y
,
Kiyomiya
K
,
Johnson
MD
,
Dickson
RB
, et al
Autoactivation of matriptase in vitro: requirement for biomembrane and LDL receptor domain
.
Am J Physiol Cell Physiol
2007
;
293
:
C95
105
.
64.
Kojima
K
,
Tsuzuki
S
,
Fushiki
T
,
Inouye
K
. 
Roles of functional and structural domains of hepatocyte growth factor activator inhibitor type 1 in the inhibition of matriptase
.
J Biol Chem
2008
;
283
:
2478
87
.
65.
Kojima
K
,
Tsuzuki
S
,
Fushiki
T
,
Inouye
K
. 
Role of the stem domain of matriptase in the interaction with its physiological inhibitor, hepatocyte growth factor activator inhibitor type I
.
J Biochem
2009
;
145
:
783
90
.
66.
Inouye
K
,
Tomoishi
M
,
Yasumoto
M
,
Miyake
Y
,
Kojima
K
,
Tsuzuki
S
, et al
Roles of CUB and LDL receptor class A domain repeats of a transmembrane serine protease matriptase in its zymogen activation
.
J Biochem
2013
;
153
:
51
61
.
67.
Lin
CY
,
Tseng
IC
,
Chou
FP
,
Su
SF
,
Chen
YW
,
Johnson
MD
, et al
Zymogen activation, inhibition, and ectodomain shedding of matriptase
.
Front Biosci
2008
;
13
:
621
35
.
68.
Friis
S
,
Tadeo
D
,
Le-Gall
SM
,
Jurgensen
HJ
,
Sales
KU
,
Camerer
E
, et al
Matriptase zymogen supports epithelial development, homeostasis and regeneration
.
BMC Biol
2017
;
15
:
46
.
69.
List
K
,
Haudenschild
CC
,
Szabo
R
,
Chen
W
,
Wahl
SM
,
Swaim
W
, et al
Matriptase/MT-SP1 is required for postnatal survival, epidermal barrier function, hair follicle development, and thymic homeostasis
.
Oncogene
2002
;
21
:
3765
79
.
70.
List
K
,
Kosa
P
,
Szabo
R
,
Bey
AL
,
Wang
CB
,
Molinolo
A
, et al
Epithelial integrity is maintained by a matriptase-dependent proteolytic pathway
.
Am J Pathol
2009
;
175
:
1453
63
.
71.
Buzza
MS
,
Netzel-Arnett
S
,
Shea-Donohue
T
,
Zhao
A
,
Lin
CY
,
List
K
, et al
Membrane-anchored serine protease matriptase regulates epithelial barrier formation and permeability in the intestine
.
Proc Natl Acad Sci U S A
2010
;
107
:
4200
5
.
72.
Netzel-Arnett
S
,
Buzza
MS
,
Shea-Donohue
T
,
Desilets
A
,
Leduc
R
,
Fasano
A
, et al
Matriptase protects against experimental colitis and promotes intestinal barrier recovery
.
Inflamm Bowel Dis
2012
;
18
:
1303
14
.
73.
Lin
CY
,
Wang
JK
,
Torri
J
,
Dou
L
,
Sang
QA
,
Dickson
RB
. 
Characterization of a novel, membrane-bound, 80-kDa matrix-degrading protease from human breast cancer cells. Monoclonal antibody production, isolation, and localization
.
J Biol Chem
1997
;
272
:
9147
52
.
74.
Zoratti
GL
,
Tanabe
LM
,
Varela
FA
,
Murray
AS
,
Bergum
C
,
Colombo
E
, et al
Targeting matriptase in breast cancer abrogates tumour progression via impairment of stromal-epithelial growth factor signalling
.
Nat Commun
2015
;
6
:
6776
.
75.
Zoratti
GL
,
Tanabe
LM
,
Hyland
TE
,
Duhaime
MJ
,
Colombo
E
,
Leduc
R
, et al
Matriptase regulates c-Met mediated proliferation and invasion in inflammatory breast cancer
.
Oncotarget
2016
;
7
:
58162
73
.
76.
Uhland
K
. 
Matriptase and its putative role in cancer
.
Cell Mol Life Sci
2006
;
63
:
2968
78
.
77.
List
K
. 
Matriptase: a culprit in cancer?
Future Oncol
2009
;
5
:
97
104
.
78.
Camerer
E
,
Barker
A
,
Duong
DN
,
Ganesan
R
,
Kataoka
H
,
Cornelissen
I
, et al
Local protease signaling contributes to neural tube closure in the mouse embryo
.
Dev Cell
2010
;
18
:
25
38
.
79.
Szabo
R
,
Uzzun Sales
K
,
Kosa
P
,
Shylo
NA
,
Godiksen
S
,
Hansen
KK
, et al
Reduced prostasin (CAP1/PRSS8) activity eliminates HAI-1 and HAI-2 deficiency-associated developmental defects by preventing matriptase activation
.
PLoS Genet
2012
;
8
:
e1002937
.
80.
Friis
S
,
Uzzun Sales
K
,
Godiksen
S
,
Peters
DE
,
Lin
CY
,
Vogel
LK
, et al
A matriptase-prostasin reciprocal zymogen activation complex with unique features: prostasin as a non-enzymatic co-factor for matriptase activation
.
J Biol Chem
2013
;
288
:
19028
39
.
81.
Sales
KU
,
Friis
S
,
Konkel
JE
,
Godiksen
S
,
Hatakeyama
M
,
Hansen
KK
, et al
Non-hematopoietic PAR-2 is essential for matriptase-driven pre-malignant progression and potentiation of ras-mediated squamous cell carcinogenesis
.
Oncogene
2015
;
34
:
346
56
.
82.
Le Gall
SM
,
Szabo
R
,
Lee
M
,
Kirchhofer
D
,
Craik
CS
,
Bugge
TH
, et al
Matriptase activation connects tissue factor-dependent coagulation initiation to epithelial proteolysis and signaling
.
Blood
2016
;
127
:
3260
9
.
83.
Szabo
R
,
Peters
DE
,
Kosa
P
,
Camerer
E
,
Bugge
TH
. 
Regulation of feto-maternal barrier by matriptase- and PAR-2-mediated signaling is required for placental morphogenesis and mouse embryonic survival
.
PLoS Genet
2014
;
10
:
e1004470
.
84.
List
K
,
Szabo
R
,
Molinolo
A
,
Sriuranpong
V
,
Redeye
V
,
Murdock
T
, et al
Deregulated matriptase causes ras-independent multistage carcinogenesis and promotes ras-mediated malignant transformation
.
Genes Dev
2005
;
19
:
1934
50
.
85.
Lin
CY
,
Anders
J
,
Johnson
M
,
Dickson
RB
. 
Purification and characterization of a complex containing matriptase and a Kunitz-type serine protease inhibitor from human milk
.
J Biol Chem
1999
;
274
:
18237
42
.
86.
Szabo
R
,
Hobson
JP
,
List
K
,
Molinolo
A
,
Lin
CY
,
Bugge
TH
. 
Potent inhibition and global co-localization implicate the transmembrane Kunitz-type serine protease inhibitor hepatocyte growth factor activator inhibitor-2 in the regulation of epithelial matriptase activity
.
J Biol Chem
2008
;
283
:
29495
504
.
87.
Friis
S
,
Sales
KU
,
Schafer
JM
,
Vogel
LK
,
Kataoka
H
,
Bugge
TH
. 
The protease inhibitor HAI-2, but not HAI-1, regulates matriptase activation and shedding through prostasin
.
J Biol Chem
2014
;
289
:
22319
32
.
88.
Oberst
MD
,
Chen
LY
,
Kiyomiya
K
,
Williams
CA
,
Lee
MS
,
Johnson
MD
, et al
HAI-1 regulates activation and expression of matriptase, a membrane-bound serine protease
.
Am J Physiol Cell Physiol
2005
;
289
:
C462
70
.
89.
Kataoka
H
,
Kawaguchi
M
,
Fukushima
T
,
Shimomura
T
. 
Hepatocyte growth factor activator inhibitors (HAI-1 and HAI-2): Emerging key players in epithelial integrity and cancer
.
Pathol Int
2018
;
68
:
145
58
.
90.
Vogel
LK
,
Saebo
M
,
Skjelbred
CF
,
Abell
K
,
Pedersen
ED
,
Vogel
U
, et al
The ratio of Matriptase/HAI-1 mRNA is higher in colorectal cancer adenomas and carcinomas than corresponding tissue from control individuals
.
BMC Cancer
2006
;
6
:
176
.
91.
Oberst
MD
,
Johnson
MD
,
Dickson
RB
,
Lin
CY
,
Singh
B
,
Stewart
M
, et al
Expression of the serine protease matriptase and its inhibitor HAI-1 in epithelial ovarian cancer: correlation with clinical outcome and tumor clinicopathological parameters
.
Clin Cancer Res
2002
;
8
:
1101
7
.
92.
LeBeau
AM
,
Lee
M
,
Murphy
ST
,
Hann
BC
,
Warren
RS
,
Delos Santos
R
, et al
Imaging a functional tumorigenic biomarker in the transformed epithelium
.
Proc Natl Acad Sci U S A
2013
;
110
:
93
8
.
93.
Tsai
CH
,
Teng
CH
,
Tu
YT
,
Cheng
TS
,
Wu
SR
,
Ko
CJ
, et al
HAI-2 suppresses the invasive growth and metastasis of prostate cancer through regulation of matriptase
.
Oncogene
2014
;
33
:
4643
52
.
94.
Wu
SR
,
Teng
CH
,
Tu
YT
,
Ko
CJ
,
Cheng
TS
,
Lan
SW
, et al
The Kunitz Domain I of hepatocyte growth factor activator inhibitor-2 inhibits matriptase activity and invasive ability of human prostate cancer cells
.
Sci Rep
2017
;
7
:
15101
.
95.
Nonboe
AW
,
Krigslund
O
,
Soendergaard
C
,
Skovbjerg
S
,
Friis
S
,
Andersen
MN
, et al
HAI-2 stabilizes, inhibits and regulates SEA-cleavage-dependent secretory transport of matriptase
.
Traffic
2017
;
18
:
378
91
.
96.
Sun
P
,
Jiang
Z
,
Chen
X
,
Xue
L
,
Mao
X
,
Ruan
G
, et al
Decreasing the ratio of matriptase/HAI1 by downregulation of matriptase as a potential adjuvant therapy in ovarian cancer
.
Mol Med Rep
2016
;
14
:
1465
74
.
97.
Bocheva
G
,
Rattenholl
A
,
Kempkes
C
,
Goerge
T
,
Lin
CY
,
D'Andrea
MR
, et al
Role of matriptase and proteinase-activated receptor-2 in nonmelanoma skin cancer
.
J Invest Dermatol
2009
;
129
:
1816
23
.
98.
Ye
J
,
Kawaguchi
M
,
Haruyama
Y
,
Kanemaru
A
,
Fukushima
T
,
Yamamoto
K
, et al
Loss of hepatocyte growth factor activator inhibitor type 1 participates in metastatic spreading of human pancreatic cancer cells in a mouse orthotopic transplantation model
.
Cancer Sci
2014
;
105
:
44
51
.
99.
Sales
KU
,
Friis
S
,
Abusleme
L
,
Moutsopoulos
NM
,
Bugge
TH
. 
Matriptase promotes inflammatory cell accumulation and progression of established epidermal tumors
.
Oncogene
2015
;
34
:
4664
72
.
100.
Kanemaru
A
,
Yamamoto
K
,
Kawaguchi
M
,
Fukushima
T
,
Lin
CY
,
Johnson
MD
, et al
Deregulated matriptase activity in oral squamous cell carcinoma promotes the infiltration of cancer-associated fibroblasts by paracrine activation of protease-activated receptor 2
.
Int J Cancer
2017
;
140
:
130
41
.
101.
Zhang
Y
,
Cai
X
,
Schlegelberger
B
,
Zheng
S
. 
Assignment1 of human putative tumor suppressor genes ST13 (alias SNC6) and ST14 (alias SNC19) to human chromosome bands 22q13 and 11q24→q25 by in situ hybridization
.
Cytogenet Cell Genet
1998
;
83
:
56
7
.
102.
Kosa
P
,
Szabo
R
,
Molinolo
AA
,
Bugge
TH
. 
Suppression of Tumorigenicity-14, encoding matriptase, is a critical suppressor of colitis and colitis-associated colon carcinogenesis
.
Oncogene
2012
;
31
:
3679
95
.
103.
Schneider
EL
,
Lee
MS
,
Baharuddin
A
,
Goetz
DH
,
Farady
CJ
,
Ward
M
, et al
A reverse binding motif that contributes to specific protease inhibition by antibodies
.
J Mol Biol
2012
;
415
:
699
715
.
104.
Hoshiko
S
,
Kawaguchi
M
,
Fukushima
T
,
Haruyama
Y
,
Yorita
K
,
Tanaka
H
, et al
Hepatocyte growth factor activator inhibitor type 1 is a suppressor of intestinal tumorigenesis
.
Cancer Res
2013
;
73
:
2659
70
.
105.
Verghese
GM
,
Gutknecht
MF
,
Caughey
GH
. 
Prostasin regulates epithelial monolayer function: cell-specific Gpld1-mediated secretion and functional role for GPI anchor
.
Am J Physiol Cell Physiol
2006
;
291
:
C1258
70
.
106.
Wilson
S
,
Greer
B
,
Hooper
J
,
Zijlstra
A
,
Walker
B
,
Quigley
J
, et al
The membrane-anchored serine protease, TMPRSS2, activates PAR-2 in prostate cancer cells
.
Biochem J
2005
;
388
:
967
72
.
107.
Ko
CJ
,
Huang
CC
,
Lin
HY
,
Juan
CP
,
Lan
SW
,
Shyu
HY
, et al
Androgen-Induced TMPRSS2 activates matriptase and promotes extracellular matrix degradation, prostate cancer cell invasion, tumor growth, and metastasis
.
Cancer Res
2015
;
75
:
2949
60
.
108.
List
K
,
Hobson
JP
,
Molinolo
A
,
Bugge
TH
. 
Co-localization of the channel activating protease prostasin/(CAP1/PRSS8) with its candidate activator, matriptase
.
J Cell Physiol
2007
;
213
:
237
45
.
109.
Lee
SP
,
Kao
CY
,
Chang
SC
,
Chiu
YL
,
Chen
YJ
,
Chen
MG
, et al
Tissue distribution and subcellular localizations determine in vivo functional relationship among prostasin, matriptase, HAI-1, and HAI-2 in human skin
.
PLoS One
2018
;
13
:
e0192632
.
110.
Lai
CH
,
Chang
SC
,
Chen
YJ
,
Wang
YJ
,
Lai
YJ
,
Chang
HD
, et al
Matriptase and prostasin are expressed in human skin in an inverse trend over the course of differentiation and are targeted to different regions of the plasma membrane
.
Biol Open
2016
;
5
:
1380
7
.
111.
Friis
S
,
Godiksen
S
,
Bornholdt
J
,
Selzer-Plon
J
,
Rasmussen
HB
,
Bugge
TH
, et al
Transport via the transcytotic pathway makes prostasin available as a substrate for matriptase
.
J Biol Chem
2011
;
286
:
5793
802
.
112.
Frateschi
S
,
Camerer
E
,
Crisante
G
,
Rieser
S
,
Membrez
M
,
Charles
RP
, et al
PAR2 absence completely rescues inflammation and ichthyosis caused by altered CAP1/Prss8 expression in mouse skin
.
Nat Commun
2011
;
2
:
161
.
113.
Mok
SC
,
Chao
J
,
Skates
S
,
Wong
K
,
Yiu
GK
,
Muto
MG
, et al
Prostasin, a potential serum marker for ovarian cancer: identification through microarray technology
.
J Natl Cancer Inst
2001
;
93
:
1458
64
.
114.
Selzer-Plon
J
,
Bornholdt
J
,
Friis
S
,
Bisgaard
HC
,
Lothe
IM
,
Tveit
KM
, et al
Expression of prostasin and its inhibitors during colorectal cancer carcinogenesis
.
BMC Cancer
2009
;
9
:
201
.
115.
Yan
BX
,
Ma
JX
,
Zhang
J
,
Guo
Y
,
Mueller
MD
,
Remick
SC
, et al
Prostasin may contribute to chemoresistance, repress cancer cells in ovarian cancer, and is involved in the signaling pathways of CASP/PAK2-p34/actin
.
Cell Death Dis
2014
;
5
:
e995
.
116.
Bao
Y
,
Li
K
,
Guo
Y
,
Wang
Q
,
Li
Z
,
Yang
Y
, et al
Tumor suppressor PRSS8 targets Sphk1/S1P/Stat3/Akt signaling in colorectal cancer
.
Oncotarget
2016
;
7
:
26780
92
.
117.
Tamir
A
,
Gangadharan
A
,
Balwani
S
,
Tanaka
T
,
Patel
U
,
Hassan
A
, et al
The serine protease prostasin (PRSS8) is a potential biomarker for early detection of ovarian cancer
.
J Ovarian Res
2016
;
9
:
20
.
118.
Tanimoto
H
,
Yan
Y
,
Clarke
J
,
Korourian
S
,
Shigemasa
K
,
Parmley
TH
, et al
Hepsin, a cell surface serine protease identified in hepatoma cells, is overexpressed in ovarian cancer
.
Cancer Res
1997
;
57
:
2884
7
.
119.
Klezovitch
O
,
Chevillet
J
,
Mirosevich
J
,
Roberts
RL
,
Matusik
RJ
,
Vasioukhin
V
. 
Hepsin promotes prostate cancer progression and metastasis
.
Cancer Cell
2004
;
6
:
185
95
.
120.
Xuan
JA
,
Schneider
D
,
Toy
P
,
Lin
R
,
Newton
A
,
Zhu
Y
, et al
Antibodies neutralizing hepsin protease activity do not impact cell growth but inhibit invasion of prostate and ovarian tumor cells in culture
.
Cancer Res
2006
;
66
:
3611
9
.
121.
Miao
J
,
Mu
D
,
Ergel
B
,
Singavarapu
R
,
Duan
Z
,
Powers
S
, et al
Hepsin colocalizes with desmosomes and induces progression of ovarian cancer in a mouse model
.
Int J Cancer
2008
;
123
:
2041
7
.
122.
Xing
P
,
Li
JG
,
Jin
F
,
Zhao
TT
,
Liu
Q
,
Dong
HT
, et al
Clinical and biological significance of hepsin overexpression in breast cancer
.
J Investig Med
2011
;
59
:
803
10
.
123.
Tervonen
TA
,
Belitskin
D
,
Pant
SM
,
Englund
JI
,
Marques
E
,
Ala-Hongisto
H
, et al
Deregulated hepsin protease activity confers oncogenicity by concomitantly augmenting HGF/MET signalling and disrupting epithelial cohesion
.
Oncogene
2016
;
35
:
1832
46
.
124.
Vaarala
MH
,
Porvari
K
,
Kyllonen
A
,
Lukkarinen
O
,
Vihko
P
. 
The TMPRSS2 gene encoding transmembrane serine protease is overexpressed in a majority of prostate cancer patients: detection of mutated TMPRSS2 form in a case of aggressive disease
.
Int J Cancer
2001
;
94
:
705
10
.
125.
Lucas
JM
,
Heinlein
C
,
Kim
T
,
Hernandez
SA
,
Malik
MS
,
True
LD
, et al
The androgen-regulated protease TMPRSS2 activates a proteolytic cascade involving components of the tumor microenvironment and promotes prostate cancer metastasis
.
Cancer Discov
2014
;
4
:
1310
25
.
126.
Camerer
E
,
Huang
W
,
Coughlin
SR
. 
Tissue factor- and factor X-dependent activation of protease-activated receptor 2 by factor VIIa
.
Proc Natl Acad Sci U S A
2000
;
97
:
5255
60
.
127.
Schaffner
F
,
Versteeg
HH
,
Schillert
A
,
Yokota
N
,
Petersen
LC
,
Mueller
BM
, et al
Cooperation of tissue factor cytoplasmic domain and PAR2 signaling in breast cancer development
.
Blood
2010
;
116
:
6106
13
.
128.
Hooper
JD
,
Nicol
DL
,
Dickinson
JL
,
Eyre
HJ
,
Scarman
AL
,
Normyle
JF
, et al
Testisin, a new human serine proteinase expressed by premeiotic testicular germ cells and lost in testicular germ cell tumors
.
Cancer Res
1999
;
59
:
3199
205
.
129.
Tang
T
,
Kmet
M
,
Corral
L
,
Vartanian
S
,
Tobler
A
,
Papkoff
J
. 
Testisin, a glycosyl-phosphatidylinositol-linked serine protease, promotes malignant transformation in vitro and in vivo
.
Cancer Res
2005
;
65
:
868
78
.
130.
Honda
A
,
Yamagata
K
,
Sugiura
S
,
Watanabe
K
,
Baba
T
. 
A mouse serine protease TESP5 is selectively included into lipid rafts of sperm membrane presumably as a glycosylphosphatidylinositol-anchored protein
.
J Biol Chem
2002
;
277
:
16976
84
.
131.
Driesbaugh
KH
,
Buzza
MS
,
Martin
EW
,
Conway
GD
,
Kao
JP
,
Antalis
TM
. 
Proteolytic activation of the protease-activated receptor (PAR)-2 by the glycosylphosphatidylinositol-anchored serine protease testisin
.
J Biol Chem
2015
;
290
:
3529
41
.
132.
Shigemasa
K
,
Underwood
LJ
,
Beard
J
,
Tanimoto
H
,
Ohama
K
,
Parmley
TH
, et al
Overexpression of testisin, a serine protease expressed by testicular germ cells, in epithelial ovarian tumor cells
.
J Soc Gynecol Investig
2000
;
7
:
358
62
.
133.
Bignotti
E
,
Tassi
RA
,
Calza
S
,
Ravaggi
A
,
Bandiera
E
,
Rossi
E
, et al
Gene expression profile of ovarian serous papillary carcinomas: identification of metastasis-associated genes
.
Am J Obstet Gynecol
2007
;
196
:
245
.
134.
Yeom
SY
,
Jang
HL
,
Lee
SJ
,
Kim
E
,
Son
HJ
,
Kim
BG
, et al
Interaction of testisin with maspin and its impact on invasion and cell death resistance of cervical cancer cells
.
FEBS Lett
2010
;
584
:
1469
75
.
135.
Ramachandran
R
,
Mihara
K
,
Chung
H
,
Renaux
B
,
Lau
CS
,
Muruve
DA
, et al
Neutrophil elastase acts as a biased agonist for proteinase-activated receptor-2 (PAR2)
.
J Biol Chem
2011
;
286
:
24638
48
.
136.
O'Brien
PJ
,
Prevost
N
,
Molino
M
,
Hollinger
MK
,
Woolkalis
MJ
,
Woulfe
DS
, et al
Thrombin responses in human endothelial cells. Contributions from receptors other than PAR1 include the transactivation of PAR2 by thrombin-cleaved PAR1
.
J Biol Chem
2000
;
275
:
13502
9
.
137.
Kaneider
NC
,
Leger
AJ
,
Agarwal
A
,
Nguyen
N
,
Perides
G
,
Derian
C
, et al
‘Role reversal’ for the receptor PAR1 in sepsis-induced vascular damage
.
Nat Immunol
2007
;
8
:
1303
12
.
138.
Lin
H
,
Trejo
J
. 
Transactivation of the PAR1-PAR2 heterodimer by thrombin elicits beta-arrestin-mediated endosomal signaling
.
J Biol Chem
2013
;
288
:
11203
15
.
139.
Hung
RJ
,
Hsu Ia
W
,
Dreiling
JL
,
Lee
MJ
,
Williams
CA
,
Oberst
MD
, et al
Assembly of adherens junctions is required for sphingosine 1-phosphate-induced matriptase accumulation and activation at mammary epithelial cell-cell contacts
.
Am J Physiol Cell Physiol
2004
;
286
:
C1159
69
.
140.
Darragh
MR
,
Schneider
EL
,
Lou
J
,
Phojanakong
PJ
,
Farady
CJ
,
Marks
JD
, et al
Tumor detection by imaging proteolytic activity
.
Cancer Res
2010
;
70
:
1505
12
.
141.
Quimbar
P
,
Malik
U
,
Sommerhoff
CP
,
Kaas
Q
,
Chan
LY
,
Huang
YH
, et al
High-affinity cyclic peptide matriptase inhibitors
.
J Biol Chem
2013
;
288
:
13885
96
.
142.
Owusu
BY
,
Bansal
N
,
Venukadasula
PK
,
Ross
LJ
,
Messick
TE
,
Goel
S
, et al
Inhibition of pro-HGF activation by SRI31215, a novel approach to block oncogenic HGF/MET signaling
.
Oncotarget
2016
;
7
:
29492
506
.
143.
Colombo
E
,
Desilets
A
,
Duchene
D
,
Chagnon
F
,
Najmanovich
R
,
Leduc
R
, et al
Design and synthesis of potent, selective inhibitors of matriptase
.
ACS Med Chem Lett
2012
;
3
:
530
4
.
144.
Mitchell
AC
,
Kannan
D
,
Hunter
SA
,
Parra Sperberg
RA
,
Chang
CH
,
Cochran
JR
. 
Engineering a potent inhibitor of matriptase from the natural hepatocyte growth factor activator inhibitor type-1 (HAI-1) protein
.
J Biol Chem
2018
;
293
:
4969
80
.
145.
Kelly
KA
,
Setlur
SR
,
Ross
R
,
Anbazhagan
R
,
Waterman
P
,
Rubin
MA
, et al
Detection of early prostate cancer using a hepsin-targeted imaging agent
.
Cancer Res
2008
;
68
:
2286
91
.
146.
Rankovic
Z
,
Brust
TF
,
Bohn
LM
. 
Biased agonism: An emerging paradigm in GPCR drug discovery
.
Bioorg Med Chem Lett
2016
;
26
:
241
50
.
147.
Bologna
Z
,
Teoh
JP
,
Bayoumi
AS
,
Tang
Y
,
Kim
IM
. 
Biased G protein-coupled receptor signaling: new player in modulating physiology and pathology
.
Biomol Ther
2017
;
25
:
12
25
.
148.
Jiang
Y
,
Yau
MK
,
Kok
WM
,
Lim
J
,
Wu
KC
,
Liu
L
, et al
Biased signaling by agonists of protease activated receptor 2
.
ACS Chem Biol
2017
;
12
:
1217
26
.
149.
Cheng
RKY
,
Fiez-Vandal
C
,
Schlenker
O
,
Edman
K
,
Aggeler
B
,
Brown
DG
, et al
Structural insight into allosteric modulation of protease-activated receptor 2
.
Nature
2017
;
545
:
112
5
.
150.
Yau
MK
,
Liu
L
,
Fairlie
DP
. 
Toward drugs for protease-activated receptor 2 (PAR2)
.
J Med Chem
2013
;
56
:
7477
97
.
151.
Yau
MK
,
Liu
L
,
Suen
JY
,
Lim
J
,
Lohman
RJ
,
Jiang
Y
, et al
PAR2 modulators derived from GB88
.
ACS Med Chem Lett
2016
;
7
:
1179
84
.
152.
Mason
SD
,
Joyce
JA
. 
Proteolytic networks in cancer
.
Trends Cell Biol
2011
;
21
:
228
37
.