The dipeptidyl peptidase IV family in cancer and cell biology

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1 REVIEW ARTICLE The dipeptidyl peptidase IV family in cancer and cell biology Denise M. T. Yu 1, Tsun-Wen Yao 1, Sumaiya Chowdhury 1, Naveed A. Nadvi 1,2, Brenna Osborne 1, W. Bret Church 2, Geoffrey W. McCaughan 1 and Mark D. Gorrell 1 1 A.W. Morrow Gastroenterology and Liver Centre, Royal Prince Alfred Hospital, Centenary Institute and Sydney Medical School, University of Sydney, Australia 2 Pharmaceutical Chemistry, Faculty of Pharmacy, University of Sydney, Australia Keywords cancer; dipeptidyl peptidase; distribution; enzyme; extracellular matrix; immune function; liver fibrosis; structure Correspondence M. D. Gorrell, Molecular Hepatology, Centenary Institute, Locked Bag No. 6, Newtown, NSW 2042, Australia Fax: Tel: m.gorrell@centenary.usyd.edu.au (Received 23 October 2009, revised 25 November 2009, accepted 30 November 2009) Of the 600+ known proteases identified to date in mammals, a significant percentage is involved or implicated in pathogenic and cancer processes. The dipeptidyl peptidase IV (DPIV) gene family, comprising four enzyme members [DPIV (EC ), fibroblast activation protein, DP8 and DP9] and two nonenzyme members [DP6 (DPL1) and DP10 (DPL2)], are interesting in this regard because of their multiple diverse functions, varying patterns of distribution localization and subtle, but significant, differences in structure substrate recognition. In addition, their engagement in cell biological processes involves both enzymatic and nonenzymatic capabilities. This article examines, in detail, our current understanding of the biological involvement of this unique enzyme family and their overall potential as therapeutic targets. doi: /j x Introduction Proteases are heavily involved in specialized biological functions and thus often play important roles in pathogenesis. The dipeptidyl peptidase IV (DPIV CD26) gene family has attracted ongoing pharmaceutical interest in the areas of metabolic disorders and cancer. Four of its members DPIV (EC ), fibroblast activation protein (FAP), DP8 and DP9 are characterized by a rare enzyme activity, namely hydrolysis of a prolyl bond two residues from the N-terminus. DPIV is the best-studied member of the family and has a variety of roles in metabolism, immunity, endocrinology and cancer biology. DPIV is a new and successful type 2 diabetes therapeutic target, and FAP is under investigation as a cancer target. Although the exact functions of the newer members, DP8 and DP9, are yet to be elucidated, thus far they have been found to have interesting biological properties and, like DPIV and FAP, are likely to be multifunctional and employ both enzymatic and extra-enzymatic modes of action. Although DPIV, FAP, DP8 and DP9 possess similar enzyme activity and are structurally conserved, they have varying patterns of expression and localization Abbreviations bfgf, basic fibroblast growth factor; DP, dipeptidyl peptidase; ECM, extracellular matrix; FAP, fibroblast activation protein; gko, gene knockout; HSC, hepatic stellate cell; IL, interleukin; IP, interferon-c-inducible protein; IRAK-1, IL-1 receptor-associated serine threonine kinase I; ITAC, interferon-inducible T-cell chemo-attractant; MAPK, mitogen-activated protein kinase; MMP, matrix metalloproteinase; NK, natural killer; NPY, neuropeptide Y; SDF-1, stromal cell-derived factor-1; Th, T helper; upar, urokinase plasminogen activator receptor FEBS Journal 277 (2010) ª 2010 The Authors Journal compilation ª 2010 FEBS

2 Denise M. T. Yu et al. DPIV family in cancer and cell biology and are therefore likely to play diverse roles. Some of the functional significance placed on DPIV research over decades is now being credited to the whole family, particularly the newer members, DP8 and DP9, and so modern selective DPIV pharmaceutical inhibitor design has placed value on the structure and function of the other DPs. The development and application of DPIV inhibitors as successful type 2 diabetes therapeutics has occurred over a relatively short period of time, in the span of about a decade. Thus, careful consideration of the biological properties of each DP is required in the application of DP inhibitors to treat other disorders. The DPIV gene family The DPIV gene family is a subgroup of the prolyl oligopeptidase family of enzymes (Table 1), which are specialized in the cleavage of prolyl bonds. As most peptide hormones and neuropeptides comprise one or more proline residues, this family of enzymes is useful for processing and degrading such peptides [1,2]. DPIV and FAP (also known as seprase) are closely related cell-surface enzymes, with DPIV-like enzyme activity. FAP is also a narrow-specificity endopeptidase [3 5]. FAP endopeptidase activity includes a type I collagen-specific gelatinase [6,7] activity and seems restricted to Gly-Pro-containing substrates [4], which is interesting because the DP activity of FAP is greater on H-Ala-Pro than on H-Gly-Pro-derived artificial substrates [3]. DP8 and DP9 are dimers with DPIV-like enzyme activity [8 11]. Although DP8 and DP9 are very closely related to each other and share similar distribution patterns [12], there are some differences in their cell biological effects (discussed later), perhaps related to their cytoplasmic localization, and so may play different roles [13]. The nonenzymatic members of the family DP6 (DPL1 DPX) and DP10 (DPL2 DPY) are modulators of voltage-gated potassium channels in neurons and are primarily expressed in brain [14 18]. Although structurally similar to DPIV [19,20], they lack the catalytic serine and other residues necessary for enzyme activity [17,21]. Thus, they are likely to exert effects via protein protein interactions [20], similarly to the enzyme DP members that also have extra-enzymatic abilities (Fig. 1). Distribution of DPs in normal and pathogenic tissue DPIV distribution DPIV is expressed by epithelial cells of a large number of organs, including liver, gut and kidney; by endothelial capillaries; by acinar cells of mucous and salivary glands and pancreas; by the uterus; and by immune organs such as thymus, spleen and lymph node [22 25] (Fig. 2). Our recent study using the DPIV selective inhibitor, sitagliptin, on wild-type and DPIV gene knockout (gko) mouse tissue homogenates has confirmed the presence of DPIV enzyme activity in a large number of organs [12]. DPIV is a potential marker for a number of cancers, but with variability among different types of cancers. DPIV is upregulated in a number of aggressive types of T-cell malignancies, such as T-lymphoblastic lymphomas, T-acute lymphoblastic leukaemias and Table 1. Criteria and subclans of the prolyl oligopeptidase (POP; EC ) family of enzymes (MEROPS the Peptidase Database; merops.sanger.ac.uk; [166]). Criteria for POP family members DNA sequence homology to prolyl endopeptidase (PEP POP) Subclans of the POP family S9A Prolyl endopeptidase (PEP POP) S9B Dipeptidyl peptidase IV (DPIV) S9C Acylaminoacyl peptidase S9D Glutamyl endopeptidase (plant) S9B subclan - DPIV gene family DPIV Fibroblast activation protein (FAP) DP8 DP9 Nonenzyme DPIV-related POP family members DP6 (DPX, DPL1) DP10 (DPY, DPL2) Effect? Protumorigenic or Anti-tumorigenic? Regulatory processes? DP ligand Pro DPi Cell type? ECM environment? Mechanism? Enzymatic, extraenzymatic? Effect of inhibitor? Fig. 1. Dynamics involved in DP biology. The DPs have both enzymatic and extra-enzymatic properties, and the outcomes of their action may lead to anti-tumorigenic or tumorigenic effects, depending on factors such as cell type, regulation and microenvironment. FEBS Journal 277 (2010) ª 2010 The Authors Journal compilation ª 2010 FEBS 1127

3 DPIV family in cancer and cell biology Denise M. T. Yu et al. Fig. 2. Overview of the distribution of the DPs in normal and pathogenic tissue and cell types. T-anaplastic large cell lymphomas [26], and is a marker of poor prognosis for T-large granular lymphocyte lymphoproliferative disorder [27]. DPIV is also upregulated in lung adenocarcinoma [28], oesophageal adenocarcinoma [29], thyroid carcinoma [30 32], prostate cancer [33] and B-cell chronic lymphocytic leukaemia [34, 35], and dysregulated in liver cirrhosis [36]. However, DPIV expression is progressively downregulated in endometrial adenocarcinoma [37]. Thus, some care needs to be taken in the use of DPIV as a target for different cancers. Further understanding of the biological anti-invasive effect of DPIV in vitro could be of importance in the control of certain carcinomas. FAP distribution The unique tissue distribution of FAP has made it a potential marker and target for certain epithelial cancers. FAP is generally absent from normal adult tissues [38]. In silico electronic northern blot analysis shows that normal tissues generally lack FAP mrna expression, with the exception of endometrium [39]. Also, typing of cancers by electronic northern blotting reveals predominant FAP signals in tumour types marked by desmoplasia [39]. In vivo, FAP is generally absent in normal adult epithelial, mesenchymal, neural and lymphoid cells [40], or in nonmalignant tumours, such as fibroadenomas, and in nonproliferating fibroblasts [38]. Nevertheless, a soluble form of FAP has been isolated from bovine serum [41] and from human plasma [42,43]. FAP expression is highly induced during inflammation, for example, within fibroblast-like synovial cells in rheumatoid arthritis and osteoarthritis [44,45]. FAP is also significantly upregulated at sites of tissue remodelling, such as the resorbing tadpole tail [46], during scar formation in wound healing [38] and at sites of tissue remodelling during mouse embryogenesis, including somites and perichondrial mesenchyme from cartilage primordia [47]. In addition, FAP is preferentially expressed by activated, but not by resting, hepatic stellate cells (HSCs) of cirrhotic liver, but not in normal human liver [6]. FAP-immunopositive cells are present in the early stages of liver injury, and the expression level of FAP mrna correlates with the histological severity of fibrosis in chronic liver diseases [48]. FAP co-localizes with fibronectin and collagen in cirrhotic liver, with collagen fibrils present alongside activated HSCs [49,50]. FAP is expressed only by myofibroblasts and activated HSCs at sites of tissue remodelling, which is the portal parenchymal interface of cirrhotic liver [6]. FAP is upregulated in most human cancers [51]. FAP is highly expressed by fibroblasts at the remodelling interface in human idiopathic pulmonary fibrosis [52]. Interestingly, FAP is highly upregulated on reactive stromal fibroblasts of over 90% of human epithelial tumours, but not in benign tumours [38]. As stromal fibroblasts are a common feature of epithelial cancers, including breast, colorectal, ovarian and lung carcinomas, FAP is a potential therapeutic target for 1128 FEBS Journal 277 (2010) ª 2010 The Authors Journal compilation ª 2010 FEBS

4 Denise M. T. Yu et al. DPIV family in cancer and cell biology multiple human epithelial cancers [38]. Previous FAPspecific cancer therapeutic investigations have included antibody targeting [53 55], FAP DNA vaccination [56], immunotherapy [57] and inhibitor therapies [58]. It is not yet clear whether inhibiting FAP enzyme activity alone can lead to anti-tumorigenic effects (Fig. 1), although the use of FAP enzyme-inactive mutants in tumour growth studies have supported this concept [59]. Recent alternative approaches that utilize or localize FAP enzyme activity have shown potential, including a FAP-activated promelittin protoxin that reduces tumour growth in mice [60], and a FAP-triggered photodynamic molecular beacon for the detection and treatment of epithelial cancers [61]. DP8 and DP9 distribution The distribution of DP8 and DP9 has been studied by our group in some depth. Ubiquitous DP8 and DP9 mrna expression was previously shown by a Master RNA dot-blot [62] and a multiple-tissue northern blot [8,9]. More recently, we confirmed ubiquitous expression using enzyme assays in the presence of a DP8 9 inhibitor, in situ hybridization and immunohistochemistry, particularly in immune cells, epithelia, brain, testis and muscle [12]. DP8 and DP9 enzyme levels are predominant over DPIV in mouse testis and brain. In situ hybridization and immunohistochemistry analyses on baboon and human tissues detected DP8 and DP9 in lymphocytes and epithelial cells in the gastrointestinal tract, skin, lymph node, spleen, liver and lung, as well as in pancreatic acinar cells, adrenal gland, spermatogonia and spermatids of testis, and in Purkinje cells and in the granular layer of cerebellum. The results of other studies are in agreement with these findings [63 66]. The significance of the three DP8 splice variants is not known [8,67]; however, one of the splice variants is upregulated in human adult testis compared with fetal testis [67]. There are two known DP9 transcripts a ubiquitously expressed transcript of 863 amino acids [9,68,69] and a larger 971-amino acid transcript in muscle, spleen and peripheral blood leukocytes [9]. The larger form appears to be expressed in tumours [9]. There is some early evidence suggesting that DP8 and DP9 expression may be associated with disease pathogenesis. The DP9 mrna levels are elevated in testicular tumours [12] and DP9 has also been shown to be upregulated in DNA arrays comparing nontumour and normal liver tissue [70]. In diseased liver, DP8 and DP9 mrna has been detected in infiltrating lymphocytes [12]. DP8 9 expression is higher in inflamed lung, probably also associated with activated lymphocytes [63]. These distribution patterns, as well as DP inhibitor studies (see a later section), support possible roles for DP8 and DP9 in inflammation and in the immune system. Biological functions of DPIV, FAP and DP8/9 The DPs have interesting roles in cell biology and in pathogenic processes. DPIV and FAP have been identified both as potential cancer markers and as proteases with anti-tumorigenic properties [71]. Their mechanisms of action generally fall into two categories, namely enzymatic and extra-enzymatic (protein protein interactions) (Fig. 1). The enzymatic roles relate to the substrates of the DPs, whereas the extra-enzymatic roles relate to their ligand-binding properties. DPIV Enzymatic activity of DPIV and its role in type 2 diabetes The ubiquitously expressed enzymatic action of DPIV covers a large range of physiological substrates involved in varied functions. DPIV is best known for its enzymatic ability to inactivate the incretin hormones glucagon-like peptide-1 and glucose insulinotropic peptide. In the treatment of type 2 diabetes, DPIV inhibitors extend incretin action, resulting in improved glucose metabolism via prolonged insulin release and trophic beta cell effects [72,73]. We have discussed this therapeutic application of DPIV inhibitors elsewhere [74]. Other physiological substrates of DPIV include neuropeptide Y (NPY), substance P and the chemokine stromal cell-derived factor-1 (SDF-1 CXCL12). NPY is involved in the control of appetite, energy homeostasis and blood pressure [75]. DPIV-truncated NPY is unable to bind to its Y1 receptor, instead binding to its Y2 and Y5 receptors, which promote angiogenesis [75] and inflammation [76]. Substance P, involved in pain perception and nociception, is inactivated by DPIV enzyme activity [77]. DPIV enzyme activity is effective on a number of chemokines in vitro (Table 2). DPIV in cell biology A number of DPIV-binding proteins have been identified, including adenosine deaminase [78,79], CD45 (protein tyrosine phosphatase) [80], caveolin-1 [81], CARMA1 [82], fibronectin III [83,84], plasminogen 2 FEBS Journal 277 (2010) ª 2010 The Authors Journal compilation ª 2010 FEBS 1129

5 DPIV family in cancer and cell biology Denise M. T. Yu et al. Table 2. Potential downstream effects of DPIV on chemokines. Chemokine Cell type Activity of DPIV-cleaved form Reference Gro b a (CXCL2) Acts on neutrophils and basophils unknown [139] GCP2 b (CXCL6) Acts on neutrophils no difference in chemotaxis of [167] neutrophilic granulocytes MIG c (CXCL9) Expressed by stimulated monocytes, macrophages and endothelial cells. Acts on Th1 d lymphocytes ablates chemotaxis of activated Th1 lymphocytes [168] IP10 e (CXCL10) ITAC f (CXCL11) SDF-1 g (CXCL12) Expressed by neutrophils, hepatocytes, endothelial cells, keratinocytes. Acts on CD4 + T cells, haematopoetic progenitor cells, lymphocytes Expressed by leucocytes, fibroblasts, endothelial cells, pancreas, liver astrocytes Acts on activated T cells Acts on lymphocytes, dendritic cells, haematopoetic cells less chemoattraction CD4 + T cells inhibits haematopoetic progenitor proliferation ablates chemotaxis of activated Th1 lymphocytes loss of Ca 2+ flux via CXCR3 less chemotaxis of activated Th1 and NK cells less tumour growth less lymphocyte chemotaxis ablates antiviral activity more chemoattraction of monocytes regulation of haematopoietic stem cell recruitment [169,170] [168,171] [131,172,173] LD78b (CCL3 L1) Expressed by T cells, B cells and monocytes more chemoattraction of monocytes [174] Eotaxin (CCL11) Acts on eosinophils, basophils, Th2 lymphocytes less chemotaxis of eosinophils [170,175,176] less binding signalling via CCR3 MDC h (CCL22) Acts on NK cells, T-cell subsets, monocytes, dendritic cells ablates chemotactic activity for lymphocytes less Ca 2+ mobilization via CCR4 [168,177,178] a Gro b, growth regulated protein b; b GCP, granulocyte chemotactic protein; c MIG, monokine-induced interferon-c; d Th, T helper; e IP10, interferon-c-inducible protein 10; f ITAC, interferon-inducible T-cell chemo-attractant; g SDF-1, stromal cell-derived factor 1; h MDC, macrophage-derived chemokine; i NK, natural killer. [85], Na + -H + exchanger isoform 3 [86] and glypican-3 [87]. Adenosine deaminase, CD45, caveolin-1 and CARMA1 are involved in the costimulation of T cells by DPIV, whereas fibronectin III, plasminogen 2 and glypican-3 may have roles in cancer biology. Binding of DPIV to fibronectin III is important for metastasis and colonization of breast cancer cells, implicating the role of DPIV in tumour progression [88]. In the human prostate tumour 1-LN cell line, direct binding of plasminogen 2 with cell-surface DPIV induces a signal transduction cascade that produces a rapid increase in the calcium ion concentration, subsequently resulting in the expression of matrix metalloproteinase 9 (MMP-9), which enhances the invasiveness of cells [89]. Overexpression of DPIV in cell lines results in interesting cell-behavioural effects. Our studies have found that 293T epithelial cells transfected with DPIV exhibited less cell migration on extracellular matrix (ECM)-coated plastic, and exhibited increases in both spontaneous and induced apoptosis [50]. Wesley et al. [90 93] found that DPIV overexpression in a number of cell lines (melanocytes, nonsmall cell lung, prostate and neuroblastoma cancer lines) caused anti-tumorigenic effects, such as inhibition of in vitro cell migration and cell growth, increased apoptosis and inhibition of anchorage-independent growth. Other studies have confirmed similar findings in melanoma cells and in ovarian carcinoma cells [94,95]. In vivo, nude mice injected with DPIV-overexpressing cancer cells showed inhibition of tumour progression compared with control cancer cells [91,93]. A number of signalling pathways have been associated with DPIV ECM interactions, including the basic fibroblast growth factor (bfgf) pathway, which is involved in cell proliferation, migration, cell survival, wound healing, angiogenesis and tumour progression. Overexpression of DPIV in prostate cancer cells blocks the nuclear localization of bfgf, lowers bfgf levels and subsequently affects downstream components of the bfgf pathway [mitogen-activated protein kinase (MAPK)-extracellular signal-regulated kinase 1 2 and urokinase plasminogen activator]. These changes are accompanied by the induction of apoptosis, cell cycle arrest and the inhibition of in vitro cell migration [92]. Sato et al. [96] have shown that DPIV mediates cell 1130 FEBS Journal 277 (2010) ª 2010 The Authors Journal compilation ª 2010 FEBS

6 Denise M. T. Yu et al. DPIV family in cancer and cell biology adhesion to the ECM via p38 MAPK-dependent phosphorylation of integrin b 1. Inhibition of DPIV expression using small interfering RNA in the T-anaplastic large cell lymphoma cell line Karpas 299 causes reduction of adhesion to fibronectin and collagen I. Also, DPIV-depleted Karpas 299 cells have reduced tumorigenicity compared with control Karpas 299 cells when injected into severe combined immunodeficient mice [96]. This finding contrasts that observed by Wesley et al., possibly reflecting cell-type differences. In the Burkitt B-cell lymphoma line, Jiyoye, DPIV overexpression results in increased phosphorylation of p38 MAPK but no accompanying increase in cell adhesion [96,97]. DPIV overexpression in neuroblastoma lines leads to induction of apoptosis mediated by caspase activation, and downregulation of the chemokine SDF-1 and its receptor CXCR4. SDF-1 downregulation, in turn, leads to induced cell migration and to decreased levels of phospho-akt and active MMP-9 [93]. Other molecules that are upregulated by DPIV overexpression include p21, CD44 [50,91], topoisomerase IIa [97] and the known cell-adhesion molecules E-cadherin and tissue inhibitor of matrix metalloproteinases [50,98]. DPIV in immune function Also known as CD26 T-cell differentiation marker, DPIV plays vital roles in immunology and autoimmunity [99]. It is expressed at detectable levels by some resting T cells, but the cell-surface expression increases by 5 to 10-fold following stimulation with antigen, anti-cd3 plus interleukin (IL)-2 or mitogens such as phytohaemagglutinin [25, ]. The strongest lymphocytic CD26 expression is found on cells co-expressing high densities of other activation markers, such as CD25, CD71, CD45RO and CD29 [ ]. The CD26 bright CD4 + population of T cells is the CD45RO + CD29 + memory helper subset, which responds to recall antigens, induces B-cell IgG synthesis and activates cytotoxic T cells [102,107,109]. In addition, CD26 bright CD4 + memory T cells preferentially undergo transendothelial migration [108,110]. CD26 has a costimulatory role in T-cell activation and proliferation. CD26 is mainly expressed on T helper (Th) 1 cells and its expression is induced by stimuli favouring the development of Th1 responses [ ]. Through its expression on T cells, CD26 is able to provide a costimulatory signal in lipid rafts [80,114,115] to augment the T-cell response to foreign antigens [109,116,117] (Fig. 3). Crosslinking of CD26 with antibody increases the recruitment of CD26 and CD45 to these rafts [80] and induces T-cell activation Fig. 3. Features of the effects of DP upon T-cell activation function. Cell-surface CD26 DPIV interacts with adenosine deaminase, CARMA-1 and caveolin-1. Stimulation of CD26 causes IRAK-1 and Toll-interacting protein to disengage from caveolin-1, resulting in the phosphorylation of IRAK-1 and the upregulation of CD86 expression. Interaction with caveolin-1 also results in the recruitment of lipid rafts, leading to events downstream that activate the nuclear factor-jb pathway. The roles of intracellular DP8 and DP9 in lymphocyte proliferation are likely to be enzymatic, in contrast to the role of DPIV, and they may modulate signalling molecules. [113,118]. The signal transduced by CD26 overlaps with the T-cell receptor CD3 pathway, increasing tyrosine phosphorylation of p56 lck, p59 fyn, ZAP-70, phospholipase C-c, MAPK and c-cb1 in that pathway [119,120]. CD26 on activated memory T cells binds to caveolin-1 on antigen-presenting cells at the immunological synapse for T cell antigen-presenting cell interactions [81]. Stimulation of CD26 also causes IL-1 receptor-associated serine threonine kinase I (IRAK-1) and Toll-interacting protein to disengage from caveolin-1. IRAK is phosphorylated in this process, which leads to the upregulation of CD86 expression [121]. The interaction between CD26 and caveolin-1 also leads to the recruitment of lipid rafts, which are important for modulating signal transduction. Additional recruitment of a complex including CARMA1 in lipid rafts leads to events downstream of the T-cell receptor complex to activate the nuclear factor-jb pathway [82]. Thus, the role of CD26 in lymphocyte activation is probably attributable to its extra-enzymatic ligandbinding properties, as further evidenced by in vitro studies. Stimulation with a combination of anti-cd3 and anti-cd26 IgGs induces more IL-2 production by FEBS Journal 277 (2010) ª 2010 The Authors Journal compilation ª 2010 FEBS 1131

7 DPIV family in cancer and cell biology Denise M. T. Yu et al. CD26-transfected Jurkat cells than by CD26 enzymenegative mutant transfected cells [116], suggesting that the proteolytic activity of CD26 is not essential in the costimulatory function of CD26. Moreover, soluble CD26 enhances the proliferation of activated peripheral blood lymphocytes by decreasing strong responses and increasing weak responses of T cells [122,123]. Studies involving CD26 enzyme-deletion mutants have shown that the costimulatory activity of CD26 involves the ligand-binding domain [124,125]. A recent in vivo study using a DPIV-selective inhibitor in DPIV gko mice has further shown independence of immune functions of CD26 from its enzyme activity [126]. CD26 has a role in the development of effector functions by CD8 + T cells [109,127,128] and is also upregulated in activated B cells [34,129,130]. CD26 overexpression in a B-cell line enhances p38 phosphorylation, suggesting that as in T cells, CD26 in B cells could be involved in the MAPK p38 signalling pathway to activate signaling molecules such as extracellular signal-regulated kinase, p56 lck, p59 fyn, ZAP-70, c-cbl and phospholipase C-c [97,119]. CD26 has nine chemokine substrates in vitro: eotaxin (CCL11), macrophage-derived chemokine (CCL22), growth-regulated protein b (CXCL2), LD78b (CCL3 L1), granulocyte chemotactic protein 2 (CXCL6), monokine-induced interferon-c (CXCL9), interferon-c-inducible protein (IP-10 CXCL10), interferon-inducible T-cell chemo-attractant (ITAC CXCL 11) and SDF-1 (CXCL12) (Table 2). Of these, SDF-1 is the only verified chemokine substrate in vivo [131]. By enzyme cleavage, CD26 reduces the inflammatory properties of these chemokines by altering or abrogating the ability to trigger a signal via the cognate receptors, and in some cases the cleaved chemokine also blocks binding by the corresponding intact chemokine molecule. FAP The endopeptidase activity of soluble FAP cleaves a2-antiplasmin [42,43, 132], which is involved in blood clotting. The gelatinase activity of FAP is likely to be associated with its expression in ECM remodelling. One putative FAP ligand, urokinase plasminogen activator receptor (upar), has been reported in LOX malignant melanoma cells [133,134]. Because the upar ligand, urokinase plasminogen activator, is able to convert plasminogen to plasmin, which degrades fibrin and certain ECM proteins, formation of the heterogeneous proteolytic complex between FAP and upar might enhance the invasive and metastatic abilities of tumour cells [133,134]. FAP expression is associated with normal or excessive wound healing, and with malignant tumour growth and chronic inflammation [38], including human liver cirrhosis [6]. All of these processes involve ECM degradation. Proteolytic degradation of ECM components facilitates angiogenesis and or tumour cell migration. Many proteases, including secreted and cellsurface metalloproteinases, and some serine peptidases, have roles in these processes. The gelatinase activity of FAP, specifically collagenolytic activity towards type I collagen fragments, suggests that FAP could in this way contribute to ECM degradation [6,7,135]. Like DPIV, overexpression of FAP frequently leads to anti-tumorigenic effects. In overexpression studies using the HEK293T epithelial cell line, FAP had decreased adhesion on collagen I, fibronectin and Matrigel, and exhibited increases in both spontaneous and induced apoptosis [50]. Overexpression of FAP in melanoma cells leads to suppression of the malignant phenotype in cancer cells, specifically cell cycle arrest at the G0 G1 phase, increased susceptibility to stress-induced apoptosis and restoration of contact inhibition [136]. Overexpression of FAP abrogates tumorigenicity in nude mice; surprisingly, enzymatically inactive FAP further abrogates tumorigenicity [136]. In contrast to the above described anti-tumorigenic effects, FAP-overexpressing HEK293 cells, when xenografted into severe combined immunodeficient mice, result in a significantly greater incidence of tumour development and growth compared with controls, including an enzyme-inactive mutant [59,137]. FAP overexpression in the hepatic stellate cell line, LX-2, enhances adhesion and migration on collagen and fibronectin on ECM substrata in vitro [50]. These data suggest that FAP has a critical role in liver fibrosis, probably by influencing the functions of activated hepatic stellate cells and or by interacting with the ECM. FAP expression is stimulated by transforming growth factor-b and retinoic acid, which also stimulate HSC and myofibroblasts [134]. Moreover, transforming growth factor-b1 is a major stimulus for epithelial mesenchymal transition, a contributor of myofibroblasts in chronic liver injury [138]. DP8 and DP9 DP8 and DP9 have no confirmed physiological substrates, but do have the ability to cleave the DPIV substrates glucagon-like peptide-1, glucagon-like peptide-2, NPY and peptide YY in vitro [11,65]. In addition, DP8 can cleave four chemokines [139]. No ligands of DP8 and DP9 have been reported FEBS Journal 277 (2010) ª 2010 The Authors Journal compilation ª 2010 FEBS

8 Denise M. T. Yu et al. DPIV family in cancer and cell biology DP9 overexpression studies in the HEK293T epithelial cell line have revealed roles for DP9 in cell adhesion, in in vitro wound healing, in cell migration, and in proliferation and apoptosis, and roles for DP8 have been found in wound healing, in cell migration and in apoptosis enhancement [13]. DP9 overexpression impaired cell behaviour on a wider range of ECM components than for DP8. Despite their close sequence relatedness, DP8 and DP9 exert some differences in their cellular effects. Therefore, these two proteins are likely to have different functions and ligands. The mechanism of action of intracellular DP8 and DP9 remains unknown. Many cytoplasmic events are involved in cell ECM interactions, causing changes to cell behaviour, so it is difficult to predict which events are influenced by cytoplasmic DP8 and DP9. The observed decreases in DP9-overexpressing cells of the ECM-interacting molecules discoidin domain receptor 1, a kinase activated by collagen binding, and the MMP inhibitor, tissue inhibitor of matrix metalloproteinase-2, suggest possible DP9 target pathways [13]. The discovery of DP8 and DP9 as reactive oxygen species-responsive molecules may provide an indication of a cytoplasmic function [140]. DP8 and DP9 might be mammalian H 2 O 2 -sensing proteins that are important in intracellular processes where H 2 O 2 is regulated, such as phosphorylation, signaling pathways, apoptosis, cancer and immune function [ ]. While DP9, but not DP8, overexpression is associated with spontaneous apoptosis, both elevate induced apoptosis. Apoptosis is an important process in tissue remodelling, including recovery from liver injury [145]. At a biochemical level, apoptosis is a complex cellular event involving the coordinated action of proteins, several different peptidases, nucleases and membrane-associated ion channels and phospholipid translocases. As DP8 and DP9 activities are dependent on the redox state of their cysteines, the redox states of DP8 and DP9 may be a molecular switch in the regulation of apoptotic pathways [140]. In addition, as cytoplasmic DPIV can be phosphorylated [146], DP8 and DP9 may also be phosphorylated in signalling pathways, and, in fact, phosphorylation sites in DP8 and DP9 are identifiable using the NetPhos server [147]. Studies involving the use of nonselective CD26 inhibitors in CD26-deficient systems suggest that DP8 and DP9 are likely to play immune roles previously attributed to CD26, for example, in in vitro proliferation [148], arthritis [149] and haematopoiesis [150]. Recently, there has been more direct evidence of DP8 and DP9 immune function, and their potential as targets for inflammatory diseases. As previously outlined, DP8 and DP9 are present in leucocytes and leucocyte cell lines [8,12,151]; DP8 mrna is upregulated in asthma-induced lung [63]; and an inhibitor of DP8 and DP9 attenuates T-cell proliferation [152] and suppresses DNA synthesis in mouse splenocytes from both wild-type and DPIV gko mice [153]. The use of inhibitors in these studies has suggested that while the CD26 immune system function appears to be extra-enzymatic, DP8 and DP9 immune functions appear to be enzymatic, although the mechanisms are yet to be elucidated. We have reported four chemokine substrates of DP8 in vitro, namely SDF-1a, SDF-1b, IP10 and ITAC [139], although it is unclear whether intracellular DP8 makes physical contact with chemokines in vivo. DP8 could potentially be released to the extracellular space upon cell death in inflammatory lesions, whereby it could retain its activity and process chemokines involved in these pathological lesions [139]. In addition, IP10 and ITAC have crucial roles in hepatitis C virus infection, and DP8 is expressed in B-cell chronic lymphocyte leukaemia, various tumours and activated T cells [9]. This selective chemokine inactivation might have implications for cancer biology and immunobiology. The reactive oxygen species responsiveness of DP8 and DP9 enzyme activities may have an involvement in apoptosis induction of activated T cells [141]. Insights of DP biological functions from DP-deficient animals DPIV gko and FAP gko mice are healthy. Moreover, DPIV gko mice have increased glucose clearance after a glucose challenge, compared with wild-type mice [154]. The same effect is found with DPIV-inhibitor-treated wild-type mice, but not with DPIV-inhibitor-treated DPIV-deficient mice, showing that the mechanism is DPIV enzyme-activity dependent. DPIV gko mice resist diet-induced obesity and associated insulin resistance, probably through the activation of peroxisome proliferator-activated receptor-a, which is involved in fatty acid oxidation, downregulation of sterol regulatory element-binding protein-1c (which is involved in lipid synthesis) and reduced appetite [155]. DPIV-deficient animals also appear to have a mildly altered lymphocyte phenotype. DPIV gko mice have a decreased number of natural killer (NK) T lymphocytes in peripheral blood, suggesting that DPIV may be involved in the development, maturation and migration of NK T cells [130]. Moreover, NK cell cytotoxicity against breast adenocarcinoma cells has been found to be decreased in CD26-deficient rats, suggesting that DPIV activity is associated with NK cytotoxicity [156]. Studies on the DPIV-deficient Fischer rat have shown FEBS Journal 277 (2010) ª 2010 The Authors Journal compilation ª 2010 FEBS 1133

9 DPIV family in cancer and cell biology Denise M. T. Yu et al. age-dependent alteration of thymic emigration patterns and leucocyte subsets [157]. A recent report in DPIV gko mice treated with a DPIV-selective inhibitor demonstrated that DPIV selective inhibition does not impair T-dependent immune responses to antigenic challenge [126]. The FAP gko mouse has a normal phenotype in histological and haematological analysis [158], and the lack of FAP expression does not impair development or tissue remodelling in embryos [47]. Therefore, other compensatory pathways are likely to exist involving molecules with functions similar to FAP, which could include other DPIV family members or MMPs. Studies on tissue-remodelling models of FAP gko mice may help to elucidate its roles, in greater detail, in extracellular matrix interactions, liver fibrosis and cancer. Our studies have indicated that FAP gko mice have reduced fibrosis in a liver injury model [159]. Care should be taken when interpreting the results of studies on the DPIV and FAP gko mice because these mice have dual ablation of enzymatic and extraenzymatic activities, and therefore the results may not accurately reflect the effect of DP inhibitors, which only inhibit enzymatic functions. The gko mice could still be useful to prove that the effect of an inhibitor is DPIV or FAP specific and not caused by the nature of the compound. In any case, it is essential to carefully distinguish the enzymatic roles of a DP from its extraenzymatic roles in any given cell type. The apparently normal phenotype of the DPIV gko and FAP gko mice suggests that targeting either or both enzymatic and extra-enzymatic functions of DPIV and FAP is likely to produce few, if any, additional off-target effects. It appears that either all roles of DPIV and FAP in vivo are not critical, or perhaps that compensatory upregulation of another DP occurs in their absence, or both. Our enzyme distribution study suggests that in some DPIV gko mouse organs, a DP activity was detected that is probably not DP8 9 derived, but is present at low levels [12]. The adjacent position of the DPIV and FAP genes causes a DPIV FAP double knockout mouse to be very difficult to generate, and DP8 and DP9 knockout animals have not been reported. Implications for DPs in cell biology and cancer targeting Overall, there appears to be evidence for both extraenzymatic and enzymatic functions of DPs in cell biology. The two functions may work synergistically, in opposition or even independently, depending on the microenvironment and cell type. In many overexpression studies, similar effects have been found with both enzyme-inactive DP mutants and wild-type DP. However, a change in expression level of a DP in response to a stimulus is likely to have downstream enzymatic effects; for instance, in neuroblastoma cell lines, SDF-1-mediated migration is attenuated in the presence of overexpressed DPIV [93]. There has been some interest in the use of DP inhibitors for cancer therapy. The nonselective DP inhibitor, PT100 (Val-boro-Pro), slows growth of syngeneic tumours derived from fibrosarcoma, lymphoma, melanoma and mastocytoma cell lines to the same extent in both wild-type and DPIV gko mice [58], and reduces myeloma growth and bone disease [160]. In these cases it is not clear which DP(s), when inhibited, have antitumorigenic effects, or whether inhibiting multiple DPs has a synergistic effect. Further study using specific inhibitors is required to understand the mechanisms involved. It may be that in some cancers DP inhibition attenuates tumour growth, while extra-enzymatic DP functions have no effect, or extra-enzymatic and enzymatic activities are synergistic. There is some evidence in the literature that DPIVand FAP-exerted effects on cell behaviour are cell-type dependent. For example, while FAP overexpression in 293T cells was associated with decreased cell adhesion and cell migration, contrasting effects, namely increased cell adhesion and migration, were found with the LX-2 human stellate cell line [50]. In other instances, while anti-tumorigenic effects were associated with increased DPIV expression in melanocytes, nonsmall cell lung carcinoma, prostate cancer and neuroblastoma cell lines [90 92,95], anti-tumorigenic effects were conversely associated with decreased DPIV expression in the Karpas 299 T-anaplastic cell lymphoma line [96]. Another comparative study found that activation of DPIV in hepatic carcinoma cell lines induces cell apoptosis, but DPIV in Jurkat T cells conversely plays a role in cell survival [161]. DPIV expression is variable in cancers, being upregulated in certain cancer types and downregulated in others. As DPIV and FAP have multifunctional properties, their expression levels and mechanisms or sites of action in various cell types may depend on the particular requirement of the cell and on the surrounding environmental factors at any given time (Fig. 1). This seems to be the case for a number of proteases [71]. Structure of the DPIV gene family proteins and therapeutic considerations There are a number of favourable factors in considering the design and application of DP inhibitors in 1134 FEBS Journal 277 (2010) ª 2010 The Authors Journal compilation ª 2010 FEBS

10 Denise M. T. Yu et al. DPIV family in cancer and cell biology therapeutics. First, the relatively small size of the enzyme family can make it easier to specifically target the enzyme of interest and distinguish it from other members of the family. Second, as indicated by the phenotype of the gko mice, neither the enzymatic nor the extra-enzymatic roles of DPIV and FAP appear to play critical survival roles, which reduces the likelihood of side effects. Third, although similar in structure, the DPs have some differences at their active site [3,140,162], so it is likely that individual DPs can be specifically targeted through careful drug design. Fourth, although they have overlapping properties, the DPs appear to play different roles to each other in vivo. This is apparent by differences in their distribution [12] and in vitro biological effects [13,50], and in the absence of compensatory upregulation of DP8 and DP9 in the DPIV gko mouse [12]. Yet another potential advantage is that in certain cell microenvironments, extra-enzymatic functions could be anti-tumorigenic, while enzymatic functions may have pro-tumorigenic properties. Thus, targeting DP enzyme activity may be useful in certain therapies without disrupting the beneficial effect of extra-enzymatic functions. The crystal structures of DPIV and FAP [3,163], and the predicted structures of DP8 and DP9 [140,162], at first glance reveal almost identical fold and general topology amongst the family members. These proteins are composed of an N-terminal b-propeller domain and a C-terminal a b-hydrolase domain. The a b-hydrolase domain, containing the catalytic triad, is highly conserved throughout this protein family. The b-propeller domain, which is associated with extra-enzymatic functions, is variable. The active site, buried deep within the protein, is formed by amino acids from each domain and includes the catalytic triad and both conserved and variable residues. These proteins are hollow and accommodate the substrate, which is stabilized within the active site by these structures (Fig. 4A). For development of inhibitors selective for each DP, it is helpful to carefully consider structural differences, particularly around the active site. In order to develop selective inhibitors, current research has therefore focused on these variable regions and on the diversity of each DPIV gene family protein (Fig. 4B). Variable regions around the active site include two loop regions one forming the P2 pocket (P2-loop) and the other forming a substrate-binding region connected to the glutamate-rich region (EE-helix) and stabilized by salt bridges (R-loop) [162,164]. Analysis of the crystal structures of DPIV, FAP and DP6, and of the models of DP8 and DP9, indicate that the R-loop is ideal for A B Fig. 4. Ribbon representation of the sitagliptin-bound DPIV monomer (PDB ID 1X70). (A) Variable and conserved structural features of the DPIV gene family proteins. The C-terminal a b-hydrolase domain and the b-propeller domain are coloured blue and grey respectively. The DPIV inhibitor sitagliptin (shown as a green stick) denotes the location of the active site. Some residues in front of the figure, which would otherwise obscure the active site, have been omitted to indicate the hollow cavity found in this protein family. The regions of the active site are represented as follows: the catalytic triad Ser, Asp and His (conserved region) as magenta, grey and blue spheres, respectively; the P2-loop (variable region) in cyan; the R-loop (variable region) in dark green; and the glutamaterich EE-helix (conserved) in red [162]. The double-glutamate motif is shown as red spheres. The yellow sphere denotes the acidic region caused by the presence of Asp663 in DPIV (conserved in DP8 and DP9, equivalent to Ala657 in FAP). (B) Close-up view of the sitagliptin-bound active site of DPIV. Sitagliptin is shown in stick representation, with carbon in green, nitrogen in blue, oxygen in red and fluorine in grey. Polar interactions between sitagliptin and the surrounding structural motifs are denoted by black dotted lines. Rational drug design for the DPIV gene family proteins focuses on the variable regions presented by the P2-loop (cyan) and the R-loop (dark green), and on the acidic pocket presented by the Asp (yellow sphere) [162]. The image was generated using PYMOL (DeLano WL: http: selectivity and provides a structural basis for the design of enzyme-selective inhibitors [162]. At present a number of DP8 9 selective inhibitors have been FEBS Journal 277 (2010) ª 2010 The Authors Journal compilation ª 2010 FEBS 1135

11 DPIV family in cancer and cell biology Denise M. T. Yu et al. reported [152,153,165], but none can distinguish DP8 from DP9. Comparison of the crystal structures of FAP and DPIV (52% amino acid identity) reveal one major difference in the vicinity of the active site. Ala657 in FAP, instead of Asp663 in DPIV, lessens the acidity and increases the size of this pocket, making FAP capable of endopeptidase activity [3]. Inhibitors for FAP have been developed that utilize these different characteristics at the active site and are selective through its predominant endopeptidase activity versus its DP activity [4]. Concluding remarks The recent spotlight on the multifunctional DPIV family as therapeutic targets has highlighted their interesting biological properties as enzymes in metabolism, cell biology and immunology (Fig. 5) and the need for further insight into the therapeutic potential of DP inhibitors in pathogenic conditions, such as cancer. To gain a better understanding of the effectiveness and outcomes of therapeutic DP inhibitors, it will be Fig. 5. Multifunctionality of the DPIV gene family. The DPIV gene family is associated with a variety of processes in cell biology, immunology, metabolism and disease. valuable to assess in detail the individual distribution and localization of each DP, the cell type of interest, structure function relationships and the balance between extra-enzymatic and enzymatic properties, as well as their overall contribution to biological processes. Acknowledgements The authors thank Lingsi Lu for graphics services, Ana Julia Vieira de Ribeiro for critical reading and the National Health and Medical Research Council of Australia for a postgraduate scholarship to DMTY and grants to MDG and GWM. TWY and NAN hold Australian Postgraduate Awards. References 1 Cunningham DF & O Connor B (1997) Proline specific peptidases. Biochim Biophys Acta 1343, Mentlein R (1988) Proline residues in the maturation and degradation of peptide hormones and neuropeptides. FEBS Lett 234, Aertgeerts K, Levin I, Shi L, Snell GP, Jennings A, Prasad GS, Zhang Y, Kraus ML, Salakian S, Sridhar V et al. (2005) Structural and kinetic analysis of the substrate specificity of human fibroblast activation protein Alpha. J Biol Chem 280, Edosada CY, Quan C, Tran T, Pham V, Wiesmann C, Fairbrother W & Wolf BB (2006) Peptide substrate profiling defines fibroblast activation protein as an endopeptidase of strict Gly(2)-Pro(1)-cleaving specificity. FEBS Lett 580, Edosada CY, Quan C, Wiesmann C, Tran T, Sutherlin D, Reynolds M, Elliott JM, Raab H, Fairbrother W & Wolf BB (2006) Selective inhibition of fibroblast activation protein protease based on dipeptide substrate specificity. J Biol Chem 281, Levy MT, McCaughan GW, Abbott CA, Park JE, Cunningham AM, Muller E, Rettig WJ & Gorrell MD (1999) Fibroblast activation protein: a cell surface dipeptidyl peptidase and gelatinase expressed by stellate cells at the tissue remodelling interface in human cirrhosis. Hepatology 29, Park JE, Lenter MC, Zimmermann RN, Garin-Chesa P, Old LJ & Rettig WJ (1999) Fibroblast activation protein: a dual-specificity serine protease expressed in reactive human tumor stromal fibroblasts. J Biol Chem 274, Abbott CA, Yu DMT, Woollatt E, Sutherland GR, McCaughan GW & Gorrell MD (2000) Cloning, expression and chromosomal localization of a novel human dipeptidyl peptidase (DPP) IV homolog, DPP8. Eur J Biochem 267, FEBS Journal 277 (2010) ª 2010 The Authors Journal compilation ª 2010 FEBS

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