Trypsins and their role in carcinoma growth

E XP E RI ME N TA L CE L L RE S E A RCH 3 1 2 ( 2 00 6 ) 1 2 1 9 –12 2 8

a v a i l a b l e a t w w w. s c i e n c e d i r e c t . c o m

w w w. e l s e v i e r. c o m / l o c a t e / y e x c r

Review Article

Trypsins and their role in carcinoma growth Pia Nyberg a , Merja Ylipalosaari a , Timo Sorsa c , Tuula Salo a,b,⁎ a

Department of Diagnostics and Oral Medicine, Institute of Dentistry, University of Oulu, Oulu, Finland Oulu University Hospital, Oulu, Finland c Institute of Dentistry, University of Helsinki, Department of Oral and Maxillofacial Diseases, Helsinki University Hospital, Helsinki, Finland b

ARTICLE INFORMATION

ABS T R AC T

Article Chronology:

There are more than 100 distinct types of cancer, and subtypes can be found within specific

Received 23 September 2005

organs. Cancer progression is a complex multi-step process. These steps reflect alterations

Revised version received

that drive the progressive transformation of normal cells into highly malignant ones. One

5 December 2005

critical step in tumor growth and invasion is the proteolytic processing of the extracellular

Accepted 24 December 2005

matrix environment. The degradation of the extracellular matrix not only enables cell

Available online 2 February 2006

migration, invasion, and metastasis formation, but also affects cell behavior in multiple ways; on one hand by cleaving extracellular matrix bound growth factors and on the other

Keywords:

hand by inhibiting angiogenesis into the tumor by liberating cryptic endogenous inhibitors

Serine proteases

of angiogenesis. Serine proteases and matrix metalloproteases are families of proteolytic

Carcinoma

enzymes involved in physiological and pathological extracellular matrix and basement

Proteolytic activation

membrane processing. In this review, we will focus on the role and activation of

Trypsinogens

trypsinogens, a family of serine proteases, in cancer progression.

Matrix metalloproteases

© 2005 Elsevier Inc. All rights reserved.

⁎ Corresponding author. Department of Diagnostics and Oral Medicine, Institute of Dentistry, PO Box 5281, University of Oulu, FIN-90014 Oulu, Finland. Fax: +358 8 537 5560. E-mail address: [email protected] (T. Salo). 0014-4827/$ – see front matter © 2005 Elsevier Inc. All rights reserved. doi:10.1016/j.yexcr.2005.12.024

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Abbreviations: ADAM, a disintegrin and metalloprotease protein API, α-1-proteinase inhibitor BM, basement membrane ECM, extracellular matrix EGF, epidermal growth factor FAK, focal adhesion kinase hKs, human tissue kallikreins MMP, matrix metalloprotease MT, membrane type PAI, plasminogen activator inhibitor PAR, protease activated receptor PSA, prostate-specific antigen SCC, squamous cell carcinoma TAT, tumor-associated trypsinogen TATI, tumor-associated trypsinogen inhibitor TGF, transforming growth factor TIMP, tissue inhibitor of matrix metalloprotease uPA, urokinase-type plasminogen activator VEGF, vascular endothelial growth factor

Contents Introduction . . . . . . . . . . . . . . . . . . . . Protease families involved in cancer progression Trypsinogens . . . . . . . . . . . . . . . . . . . . Tumor-associated trypsinogens. . . . . . . . . . The complex roles of proteases in cancer . . . . Acknowledgments . . . . . . . . . . . . . . . . . References . . . . . . . . . . . . . . . . . . . . .

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Introduction A critical step in tumor growth and invasion is the proteolytic processing of the extracellular matrix (ECM). In addition to providing structural support to cells and tissues, this ECM supports cell adhesion, transmits signals, binds, stores, and presents growth factors (reviewed by [1]). Basement membranes are specialized thin sheet-like matrix structures. The backbone constituent of basement membranes is type IV collagen that forms a network together with other basement membrane molecules, such as laminins, fibronectin, type XV and XVIII collagens, and heparan sulfate proteoglycans [2]. Basement membranes function as barriers, they can polarize epithelial cells, shape tissue structures, guide and support migrating cells, and act as selective filters in the kidney [1]. The constituents of the basement membranes can vary depending on the location. Different cells and even different types of tumor cells secrete a characteristic pattern of matrix proteins [3]. The degradation of the extracellular matrix promotes cell migration in many cases. As early as in 1949, investigators

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noted that there was an association between protease activities and the invasive behavior of tumor cells [4]. On the other hand, ECM degradation can reveal cryptic molecules that have an inhibitory effect on migration [5–7]. There are five major groups of proteases: the aspartate and cysteine enzymes, which are active at low pH and are mainly involved in the intracellular proteolysis within lysozymes, and the serine and metal-dependent enzymes that function at neutral pH and are responsible for extracellular proteolysis and the threonine proteases [8]. Although representatives of all classes of proteolytic enzymes have been implicated in tumor invasion and metastasis (Table 1), matrix metalloproteases and serine proteases have been studied the most and affect tumor progression in many stages (Fig. 1).

Protease families involved in cancer progression Matrix metalloproteases (MMPs) are a family of endopeptidases, whose involvement in tumor progression is probably

E XP E RI ME N TA L CE L L RE S E A RCH 3 1 2 ( 2 00 6 ) 1 2 1 9 –12 2 8

Table 1 – Protease families involved in tumor invasion and metastasis Serine proteases

Metalloproteases

Other proteases

Cathepsins G and E Chymase

Matrix metalloproteases Collagenases (MMP-1, -8, and -13)

Chymotrypsin

Gelatinases (MMP-2, -9) Matrilysins (MMP-7, -26) MT-MMPs (MMP-14, -15, -16, -17, -24, and -26) Stromelysins (MMP-3, -10, and -11) Other MMPs (MMP-12, -19, -20, -23, -27, and -28) ADAMs

Aspartate proteases (cathepsins D and E) Cysteine proteases (cathepsins B, H, K, L, M, N, O, and S) Threonine proteases

Membrane-bound serine proteases Elastase

Plasmin Plasminogen activators (Tumor-associated) trypsins Tryptase Human tissue kallikreins

most widely studied. At present, 24 different vertebrate MMPs have been identified, 23 of which have been found in humans (reviewed by [9]). In addition to the conserved zinc-binding catalytic site, all MMPs have a propeptide domain that maintains the enzyme in latent inactive stage until it is removed or disrupted [9]. Individual MMPs are referred to with their common names or according to a sequential numeric nomenclature based on the order of discovery. On the basis of specificity, sequence similarity and domain organization vertebrate MMPs are divided into six groups: collagenases (MMP-1, -8, and -13), gelatinases (MMP-2 and -9), stromelysins

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(MMP-3, -10, and -11), matrilysins (MMP-7 and -26), membrane-type MMPs (MMP-14, -15, -16, -17, -24, and -26), and other MMPs (MMP-12, -19, -20, -23, -27, and -28) (Table 1) [10]. MMPs are involved in various physiological and pathological conditions. They play a role in many events of reproduction, embryonic development, and organ morphogenesis, as well as in wound healing, angiogenesis, bone remodeling, nerve growth, inflammation, and apoptosis. In addition to normal physiological events, MMPs are associated with many tissue destructive diseases, such as cancer, arthritis, skin diseases, chronic wound healing, liver fibrosis, cardiovascular diseases, kidney diseases, periodontitis, and gastric ulceration. MMPs are inhibited by tissue inhibitors of metalloproteases (TIMPs) [7]. Numerous excellent reviews have been written about MMPs, of which only a few can be mentioned here [7,9–12]. A family of membrane anchored proteases called ‘a disintegrin and metalloprotease’ (ADAM) proteins have emerged as the major proteinase family that mediates ectodomain shedding, the proteolytic release of extracellular domains from their membrane-bound precursors. At present, 29 ADAM orthologues are found in mammals, of which ADAM17 and -10 seem to be the principal sheddases and are so far studied the most. ADAMs share a characteristic domain structure including metalloprotease, disintegrin, EGF-like, transmembrane, and cytoplasmic domains and a cysteinerich region, which contribute to the proteolytic, adhesion, and signalling activities of ADAMs. However, only half of ADAMs contain the conserved catalytic site typical for metalloproteases and thus have proteolytic activity. ADAMs bind to integrins, inhibit TIMP activity, and affect intracellular signalling through ectodomain shedding (reviewed by [13,14]). The serine proteases are another large and conserved proteolytic family implicated in tumor growth, invasion, and metastasis. They are characterized by a catalytic mechanism by which the hydroxyl group of the active site serine residue

Fig. 1 – Schematic presentation of the many steps of carcinoma growth and invasion affected by serine proteases and matrix metalloproteases (adapted from Acta Universitatis Ouluensis, Nyberg, P: Matrix degrading proteases and collagen-derived angiogenesis inhibitors in the regulation of carcinoma cell growth, http://herkules.oulu.fi/isbn9514276612/).

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acts as the nucleophile that attacks the peptide bond (reviewed by [6,15,16]). Plasmin and plasminogen activators are so far the most thoroughly studied tumor-associated serine proteases. They form a potent mechanism for generating localized pericellular proteolytic activity that is needed for tumor growth, metastasis, and angiogenesis. The plasminogen activator/plasmin system consists of the serine proteases plasminogen and the corresponding active form plasmin, two plasminogen activators, tissue-type plasminogen activator (tPA) and urokinase-type plasminogen activator (uPA), plasminogen activator inhibitor (PAI), and cell surface receptors (PAR). Plasmin is involved in the proteolysis of the fibrin clot, but it is also able to degrade various extracellular matrix components (reviewed by [16–18]). Plasmin can also activate some MMPs, such as progelatinase A (MMP-2) [19,20], prostromelysin-1 (MMP-3) [21], and prometalloelastase (MMP12) [22]. Plasminogen has important functions in metastasis, as the spread of tumor from the primary origin is significantly delayed in plasminogen-deficient mice [23]. High levels of uPA, uPAR, and PAI-1 in breast cancer tissue are associated with poor prognosis [24]. Consequently, uPA/uPAR/plasmin antagonists are currently being developed as therapeutic strategies to inhibit tumor angiogenesis and progression [25]. Human tissue kallikreins (hKs) are secreted serine proteases that have only recently arrived on the cancer proteolysis scene. They are primarily known for their clinical applicability as cancer biomarkers, the hK3, also known as prostate-specific antigen (PSA), probably being the best known. Accumulating evidence indicates the importance of kallikreins in many cancer-related processes, including a role in protease activation cascades, cell-growth regulation, angiogenesis, invasion, and metastasis (reviewed by [6]). Not all serine proteases are secreted to the extracellular milieu as soluble proteins. A rapidly expanding subgroup of serine proteases are membrane anchored serine proteases. They are anchored to the plasma membrane either via a carboxyl-terminal transmembrane domain, carboxyl-terminal glycosyl-phosphatidylinositol linkage, or amino-terminal transmembrane domain. The first membrane anchored serine protease discovered was enteropeptidase (enterokinase), and matriptase-3 is one of the most novel ones. The expression of many of these membrane anchored serine proteases is widely dysregulated during tumor growth and progression (reviewed by [15]).

Trypsinogens Trypsinogens are serine proteases that play a significant role in tumor progression. Trypsinogen and its active form trypsin were one of the first enzymes to be isolated and characterized; it was crystallized from human pancreas as early as in 1936 [26]. Eight trypsinogen genes have been found, but only three of them, T4, T8, and T9, have been demonstrated to encode a protein [27–31]. Figarella et al. purified two trypsinogen isoforms from pancreatic juice and named them trypsinogen-1 and -2 [32]. These isoforms were later called cationic and anionic trypsinogen, respectively, and a third isoform, mesotrypsinogen, was found [33]. Scheele et al. called these same pancreatic isoforms trypsinogen-3, -1, and -2, respectively [34]. A fourth trypsinogen isoform was found in the brain in 1993

Table 2 – Tumor-associated and pancreas-derived trypsin(ogen)s Protein

Trypsinogen-1 TAT-1 Trypsinogen-2 TAT-2 Trypsinogen-3

Identified first from

Relative molecular mass (kDa)

Reference

Pancreas Ovarian tumors Pancreas Ovarian tumors Pancreas

28 25 26 28 26.7

[32,34] [43] [32,34] [43] [34]

[29]. To clarify the nomenclature of trypsinogen isoforms, from now on the isoform (trypsinogen-1/cationic/trypsinogen-3) encoded by T4 gene is called trypsinogen-1, the second isoform (trypsinogen-2/anionic/trypsinogen-1) encoded by T8 gene is called trypsinogen-2, and the isoforms encoded by the T9 gene (mesotrypsinogen/trypsinogen-2 and trypsinogen-4) are called trypsinogen-3 and -4 (Table 2). The latent trypsinogens need to be activated into catalytically competent trypsins. In the digestive tract, secreted trypsinogens are activated by the serine protease enterokinase/enteropeptidase [35]. Most normal tissues produce small amounts of enterokinase-like protease [36]. Enterokinase has previously been found to be capable of activating proMMP-9 in vitro to some extent [37]. Trypsin-1 and -2 are also able to autoactivate trypsinogen-1 and -2 [38]. Trypsinogens can be activated at least in vitro by cathepsin B, and in vivo cathepsin B participates in the premature activation of trypsinogen during pancreatitis [39,40]. Pancreatic cancer cells produce trypsinogen activity stimulating factor (TASF) that is active uPA [41]. Trypsin hydrolyzes the peptide bond on the carboxylterminal side of arginine or lysine residues [42]. In addition to digesting dietary proteins, trypsinogens and the corresponding active forms trypsins from various sources are involved in proteolytic cascades by activating other proteases, such as pro-uPA [43], proMMP-9 [44,45] and -2 [44], proMMP-14 [46], proMMP-1, -3, -8, and -13 [47], and a serine proteinase PSA (prostate-specific antigen) [48]. However, it should be noted that many of these activation studies are done only in vitro and thus these activation events might not have major biological significance in vivo. The physiological role of trypsinogen-3 has been controversial, but recently, it has been suggested that digestive degradation of trypsin inhibitors is its predominant function [49]. Trypsinogens are not only expressed in the pancreas, as pancreatectomized patients have detectable levels of trypsinogens in the serum [50]. Trypsinogen expression has been detected in vascular endothelial cells [51], epithelial cells of the skin, esophagus, stomach, small intestine, adult and preterm lung, kidney, liver, bile ducts, splenic, and neuronal cells [52–54], and male genital track and seminal fluid [48] as well as in many tumors and tumor cell lines that are described in the next chapter.

Tumor-associated trypsinogens Trypsinogens were first thought to be pancreatic enzymes only involved in the digestive process, but in the early 1980s,

E XP E RI ME N TA L CE L L RE S E A RCH 3 1 2 ( 2 00 6 ) 1 2 1 9 –12 2 8

LaBombardi et al. identified a trypsin-like protease in the cell membrane of carcinoma cells [55]. It is currently known as tumor-associated trypsinogen (TAT) [43,56]. There are two isoforms, TAT-1 and TAT-2, the latter being the more predominant one [57]. TAT-2 and trypsin-2 derived from pancreas have identical aminoterminal sequence, molecular weight, and immunoreactivity, but they differ in their activity against synthetic substrates, enzyme stability, and isoelectric point. These differences are probably due to post-translational modification that is different in pancreas and carcinomas [43,44,50,58]. The nucleotide sequence of TAT-2 and pancreatic trypsin-2 was identical except for one base substitution (G to A) [44]. TAT-2 was identified for the first time in ovarian neoplasms, when investigators were looking for the target molecule for the tumor-associated trypsin inhibitor (TATI) that is known to be a marker molecule in ovarian carcinomas [43,59]. Later, tumor-associated trypsinogens have also been shown to be expressed in other carcinomas, such as pancreatic cancer [60], hepatocellular and cholangiocarcinomas [61], lung neoplasms [62] and colorectal cancers [63], and by various cancer cell lines, such as colon carcinoma, fibrosarcoma, erythroleukemia [58], and gastric cancer [36,64,65]. A highly invasive tongue carcinoma cell line produces TAT-2, whereas a less invasive tongue carcinoma line does not [66]. In oral squamous cell carcinoma samples, TAT-2 was localized especially in the peripheral tumor cells, but also throughout the cancer island. Moreover, positive immunohistochemical staining is found sporadically in fibroblasts of the surrounding stroma (Vilen et al. unpublished data). The overexpression of trypsinogens stimulates cell growth and adhesion and correlates with the malignant phenotype. Particularly, the production of TAT-2 has been shown to correlate with the malignant phenotype of cancers. In fact, TAT-2 is the predominant isotype in malignant cancers. The level of TAT-2 correlates with the malignancy and the metastatic potential of tumors [57]. Other studies have shown that the amount of TAT-1 is also up-regulated in colorectal tumors with a more malignant phenotype, although TAT-2 is the dominant trypsinogen isoform in colon tissue [67]. TAT-2 complexed with α-1-proteinase inhibitor (API) is a strong prognostic marker in advanced epithelial ovarian cancer [68]. On the other hand, although the trypsinogen family members are highly homologous to each other, trypsinogen-4 has been reported to possess a tumor-suppressive role in cancer progression [69]. There is evidence of several possible mechanisms on how tumor-associated trypsins increase tumor aggressiveness. Addition of trypsin increases the growth of various cells lines by activating their thrombin receptors and plasminogen receptor, and possibly also other cell surface receptors, such as growth factor receptors, integrins [36,70], and proteinase activated receptor PAR-2 [71]. Integrins in particular may be significant, as esophagus carcinoma cells transfected with TAT-1 attached more to vitronectin and fibronectin, and treatment of human melanoma cells with trypsin, chymotrypsin, or plasmin stimulates the integrin-mediated cell attachment to vitronectin and fibronectin [36,72]. More specifically, trypsin treatment exposes more integrin αvβ3 on the cell surface, but it does not up-regulate the amount of integrin αvβ3 on melanoma cells [72]. Furthermore, trypsin promotes

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integrin α5β1-mediated adhesion to fibronectin, and less efficiently integrin αvβ3-mediated adhesion to vitronectin, and regulates the adhesion and proliferation of human gastric carcinoma cells by inducing PAR-2/G protein signalling [73]. On the other hand, long-term exposure of cells to trypsin liberates large amounts of integrin β1 and makes the cells more susceptible to H2O2-induced damage. Presumably, other adhesion molecules are also removed by long-term trypsinization of the adherent cells [74]. One important reason for the ability of trypsin to make cells more invasive is its effect on the degradation of the extracellular matrix either on its own or by activating other proteases [36,64]. TAT-2 can initiate protease cascades by activating proMMPs [44,47], and thus it may promote tumor spreading. It can activate proMMP-9 in vitro at an extremely low molar ratio. Furthermore, TAT-2 activated proMMP-9 complexed with the tissue inhibitor of matrix metalloproteases, which is thought to be the major MMP form in vivo [44,75]. TAT-2 was further shown to degrade TIMP1. TAT-2 also activates proMMP-2 in vitro, but much less efficiently [44]. This phenomenon has also significance in vivo; our results strongly suggest that overexpression of TAT-2 activates MMP-9 in malignant oral carcinoma cell line, and that the intravasation, the first step of invasion, of these cells increased in an in vivo model [66]. Furthermore, the higher the amount of trypsin-2 is in ovarian tumor cyst fluids, the higher the level of MMP-9 activation becomes. On the other hand, trypsin-2 had no effect on the activation of MMP-2 in these experiments [66,76]. Recently, we have shown that trypsin-2 can also activate other proMMPs, MMP-1, -3, -8, and -13, and it can directly degrade native type I collagen on its own [47]. Chemically modified tetracyclines down-regulate TAT-2 expression and reduce human colon adenocarcinoma cell migration suggesting that there might be an association between these two events [37]. Briefly, TAT-2 can contribute to matrix degradation and remodeling both directly and indirectly via activation of other proteases, and can play a role in controlling cell behavior via processing cell surface receptors. Tumor-associated trypsin is inhibited by tumor-associated trypsin inhibitor, TATI, a 6-kDa peptide originally detected in the urine of ovarian cancer patients [77]. It is expressed by several tumors and cancer cell lines [78] as well as in normal renal tissue [79]. TATI is also a useful serum marker for squamous cell carcinomas of the head and neck [80], ovarian cancers [59,81], and renal cell carcinomas [82]. In tumors, high concentrations of TATI may protect the ECM from trypsinmediated proteolysis, thus inhibiting invasion [78]. However, lower concentrations might only partially protect the ECM to an extent that is needed for cell adhesion. This might explain the findings that ovarian cancer patients with elevated amounts of TATI in serum sometimes have a worse prognosis than patients with normal TATI levels [81,83].

The complex roles of proteases in cancer Although in general it seems that the higher the amount of proteases in carcinomas, the more aggressive the phenotype of the carcinoma, it should be noted that the role of proteases in cancer is not as simple as that. For quite a while, the scientific community was convinced that the activity of proteases was

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mostly a bad thing in cancer, and as soon as specific and efficient enough inhibitors were discovered, it would be an efficient cancer treatment. Unfortunately, the story proved out to be more complicated. At first, proteases were considered to be important almost exclusively in invasion and metastasis. Multiple studies have implicated elevated levels of proteases in various cancers. In mouse transplantation assays, relatively benign cancers acquire malignant properties when protease expression is up-regulated. Highly malignant cells become less aggressive when protease expression or activity is reduced [10]. Furthermore, the expression and activity of proteases are increased in almost every type of human cancer, and this correlates with advanced tumor stage, increased invasion, and metastasis, as well as shortened survival. However, recent studies have revealed that they are involved in several steps of cancer progression. Not only do they degrade basement membranes and other physical barriers, enabling metastatic cells to migrate and spread, but they also affect cellular and immune processes [6,7]. Many angiogenesis inhibitors are stored as cryptic fragments within larger precursor matrix molecules that are not themselves antiangiogenic, and the regulation of proteolytic processing plays an important role in the vascularization of tumors [5]. Noteworthy, the activity of proteases on non-matrix substrates, such as chemokines, growth factors, growth factor receptors, adhesion molecules, and apoptosis mediators, is essential for the rapid and critical cellular responses required for tumor growth and progression [6,84]. Trypsins are widely expressed in various tissues and cancer cells [52,65]. The roles of trypsins seem to follow the similar dual paths as other proteinase families implicated in cancer. Trypsins not only contribute to the degradation of the ECM directly and indirectly by activating other proteases but also modulate cell behavior via cell surface receptors. Although there are four trypsin isoforms, TAT-2 seems to be the most predominant one in tumors. We have found that the overexpression of TAT-2 increases oral squamous cell carcinoma intravasation at least partially by activating MMP-9 [44,66]. Later, we have discovered that in addition to MMP-9 TAT-2 activates proMMP-1, -3, -8, -13, and to a lesser extent proMMP-2 at least in vitro [44,47], and others have confirmed the activation of proMMP-9 [45] and found in vitro activation of pro-uPA [43], proMMP-14 [46], and serine proteinase PSA [48]. This list of proteases that can be activated by trypsins at least in vitro suggests that trypsins play an important role in the beginning of protease activation cascades in cancer. In ovarian tumors, elevated levels of TAT-2 correlate with elevated levels of MMP-8, but surprisingly not with the levels of MMP-1 [85]. In addition to activating other proteases, trypsins also degrade many extracellular matrix components, such as fibronectin, laminin, gelatins, and type I, III, IV, and V collagens [64]. Therefore, it is not surprising that the concentration of trypsinogen-2 correlates with the malignancy of tumors [57,82]. Thus, TAT-2 might be a good candidate for a clinical prognostic marker that could identify patients with an aggressive disease. Especially, the level of TAT-2 complexed with API might be an important prognostic factor [68]. On the other hand, levels of TATI, the TAT-2 inhibitor, also seem to correlate with patient survival at least in renal cell carcinoma. Paradoxically, patients with elevated TATI levels had signif-

icantly shorter survival time that those with normal TATI levels [76]. However, a higher molar ratio of trypsinogen to TATI suggests poorer prognosis [82,86]. But it cannot always be concluded that trypsin activity promotes cancer progression. Interestingly, trypsinogen-4 seems to do the opposite as it plays a tumor suppressive role in human esophageal squamous cell carcinomas and gastric adenocarcinomas [69]. It should always be noted that, in addition to the contribution to ECM degradation, trypsins modulate the functions of cell surface receptors, such as integrins and PARs (proteinase activated receptors). The binding of integrins to extracellular matrix proteins, such as fibronectin and vitronectin, activates focal adhesion kinases (FAK), and the activated FAKs interact with a variety of cytoskeletal or intracellular signalling molecules. Low concentrations of exogenous trypsin stimulate cell growth and adhesion. This is at least partially due to increased integrin α5β1-mediated adhesion to fibronectin and integrin αvβ3-mediated adhesion to vitronectin promoted by trypsin. Trypsin can regulate gastric carcinoma cell adhesion and proliferation by inducing PAR-2/G-protein signalling [73]. Emerging evidence is nowadays supporting the idea that trypsin can act as a potent growth factor for cancer cells by acting at protease-activated receptor PAR-2. Trypsin stimulates cell proliferation via ERK phosphorylation pathway involving EFG-receptor tyrosine kinase activity in this ERK activation [87]. A novel mechanism of receptor activation was revealed with the discovery of PAR receptors. PARs are cleaved by serine proteases including trypsins at a specific site on N-terminal extracellular extension. The newly exposed N-terminus itself acts as a ligand and activates the receptor leading to numerous intracellular events [88,89]. Mechanistically, trypsin acts as a PAR-2 agonist transactivating EGF-receptor through a pathway that includes MMP-dependent cleavage and release of TGF-α, which in turn activates the EGF-receptor and downstream MAPK cascade leading to cell proliferation [87]. This cascade is also an example of how trypsin and MMPs are both needed for a certain biological response. In addition, ADAM proteins are also involved in EGF-receptor signalling [13]. Four PARs identified so far have distinct N-terminal cleavage sites and tethered ligand pharmacology. PAR-2 seems to be activated physiologically mainly by trypsin, although trypsin activates less efficiently also PAR-1 and PAR-4 [88,90]. Only recently, it has been explored how the various trypsin isoforms differ in their ability to activate PARs. Indeed, there are differences: the activity of trypsin-1 and -2 on PARs was comparable to that of bovine trypsin, but trypsin-3 and -4 were not able to activate epithelial PAR-1 and -2, but it weakly activates brain PAR-1 [90]. On the other hand, Cottrell et al. found trypsin-4 to be an agonist of both PAR-2 and -4 [91]. Nevertheless, TAT-2 is an activator of PAR-2 [71]. Thus, distinct trypsin isoforms can have specific activity depending on the cell type. Although it has not been demonstrated so far, in addition to activating cleavage of receptors, there is also the possibility of proteolytic cleavage by trypsins elsewhere in the receptor resulting in prevention of receptor activation by amputating or destroying the tethered ligand. The similar complex effect on cancer and cell growth is observed with other proteases as well, not just trypsins. Studies with the plasminogen system show that, in general,

E XP E RI ME N TA L CE L L RE S E A RCH 3 1 2 ( 2 00 6 ) 1 2 1 9 –12 2 8

uPA initiates a proteinase cascade at the cell surface, which in turn leads to breakdown of the extracellular matrix and thereby promotes cellular migration. Unexpectedly, high levels of PAI-1 are predictive of poor survival prognosis in patients suffering from a variety of different cancers [92]. Even though uPA usually promotes, and PAI-1 suppresses tumor growth in most available experimental tumor models, PAI-1 was recently found to promote tumor growth and angiogenesis [93]. It has been suggested that plasmin is involved in the formation of new vessels, but that PAI-1-mediated control of proteolytic breakdown is required, probably to allow vessel stabilization and maturation [25]. Plasminogen contributes to tumor angiogenesis also by being the parent molecule for angiogenesis inhibitor angiostatin [94,95]. Another good example of the complex network is MMP-9; it can be an initial modulator of the tumor angiogenic switch by promoting the release of VEGF [96] and an inhibitor of the angiogenic switch by releasing cryptic angiogenesis inhibitors from their parent matrix molecules [5]. Increased MMP-9 expression in fact reduced tumor growth and vasculature [97], and MMP-9deficient mice have accelerated growth of tumors [98]. On the other hand, metastasis has been shown to decrease in MMP-9 knockout mice and MMP-2-deficient mice show reduced tumor angiogenesis and growth [10,99]. MMP-8 is another example of the dual effect of MMPs; on one hand, it is involved in tissue remodeling and destruction in cancer progression and inflammation, but on the other hand, it has cancer protective and anti-inflammatory properties due to its ability to process anti-inflammatory chemokines [12,100]. In summary, proteases are of crucial importance in many biological and pathological processes. Trypsins are no exception to this phenomenon. Numerous studies show abnormal, often enhanced, protease expression and activity in carcinomas. Proteases contribute more to tumor progression and regulation than just by degrading physical ECM barriers and enabling cell migration and tumor invasion. They can release matrix-bound growth factors and endogenous angiogenesis inhibitors, process anti-inflammatory chemokines, expose cryptic integrin-binding sites, and affect cell–cell interactions and apoptosis. The complex roles of proteases in different stages of tumor growth should be kept in mind when designing protease inhibitors as cancer treatments.

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Acknowledgments This work has been supported by grants from the Academy of Finland, the Helsinki University Central Hospital EVO funds (TYH 5306 and TI 020Y0002), Maud Kuistila Memorial Foundation, Oulu University Hospital EVO-funds, Oulu University Pharmacy Foundation, Cancer Society of Finland, Cancer Society of Northern Finland, and Finnish Dental Society Apollonia.

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