Advances in Understanding the Expression and Function of Dipeptidyl Peptidase 8 and 9

Published OnlineFirst September 13, 2013; DOI: 10.1158/1541-7786.MCR-13-0272

Molecular Cancer Review Research

Hui Zhang, Yiqian Chen, Fiona M. Keane, and Mark D. Gorrell

Abstract DPP8 and DPP9 are recently identiﬁed members of the dipeptidyl peptidase IV (DPPIV) enzyme family, which is characterized by the rare ability to cleave a post-proline bond two residues from the N-terminus of a substrate. DPP8 and DPP9 have unique cellular localization patterns, are ubiquitously expressed in tissues and cell lines, and evidence suggests important contributions to various biological processes including: cell behavior, cancer biology, disease pathogenesis, and immune responses. Importantly, functional differences between these two proteins have emerged, such as DPP8 may be more associated with gut inﬂammation whereas DPP9 is involved in antigen presentation and intracellular signaling. Similarly, the DPP9 connections with H-Ras and SUMO1, and its role in AKT1 pathway downregulation provide essential insights into the molecular mechanisms of DPP9 action. The recent discovery of novel natural substrates of DPP8 and DPP9 highlights the potential role of these proteases in energy metabolism and homeostasis. This review focuses on the recent progress made with these post-proline dipeptidyl peptidases and underscores their emerging importance. Mol Cancer Res; 11(12); 1487–96. 2013 AACR.

Introduction metabolic processes of obesity and diabetes, the immune – Proteases are efficient processing molecules involved in the system, and cancer biology (7,12 15). Structurally related to physiological maintenance of all cells. They can no longer DPPIV and with overlapping proteolytic activity, DPP8 and simply be thought of as essential for protein degradation and DPP9 remain less understood but are gaining increasing recycling; they are also vital regulators and signaling mole- research attention to decipher their unique proteolytic roles. cules (1). They control all aspects of cellular life by modifying To identify and characterize DPP8 and DPP9, both their targets to alter location and function. In doing so, they proteases were cloned, sequenced, and discovered to display have crucial roles in both normal and pathological processes considerable sequence homology with DPPIV and thus also and are thus important targets for drug treatment. likely structural similarity (2,3). DPP8 and DPP9, however have a different cellular localization to that of DPPIV, as well One such target is the ubiquitous serine protease, dipep- fi tidyl peptidase IV (DPPIV), with inhibitors of this enzyme as some differences in their expression pro le and are now in clinical use to treat type 2 diabetes. DPPIV and therefore likely to have distinct roles. In the past, treatment with a broad DPP family inhibitor produced functional related enzymes are members of the S9b protease subfamily, fl with a conserved catalytic triad of serine, aspartate, and changes in diabetes, the immune system, the in ammatory histidine (2,3) and a rare ability to cleave a post-proline response, and cancer biology (13,16,17). Although these bond 3 residues from the N-terminus of a substrate (2, 4–7). effects were attributed to DPPIV at the time, it is now DPPIV rapidly cleaves and inactivates insulinotropic hor- thought that inhibition of DPP8 and DPP9 contributed to the above outcomes. With more selective DPP8/9 inhibitors mones (incretins) and thus inhibitors of this enzyme are the – basis for the treatment of type 2 diabetes (8,9). Other being developed (18 20), there is now an opportunity to members of this family include DPP8, DPP9, and fibroblast delineate the roles of these enzymes and further explore the therapeutic potential of their chemical inhibitors. The search activation protein (FAP), which are of interest in cancer fi research. FAP is generally expressed at low levels in normal for DPP8- and DPP9-speci c substrates is ongoing, with the adult tissue but is greatly upregulated in diseased and challenge remaining to determine those that are most phys- iologically relevant. Although DPPIV has been reviewed at remodeling tissue as well as in tumors (10,11). Indeed, both fi FAP and DPPIV are multifunctional and have roles in length, few authors have focused speci cally on DPP8 and DPP9. Here we outline the molecular and biochemical characteristics of DPP8 and DPP9 so as to gain a compre- Authors' Affiliation: Molecular Hepatology, Centenary Institute and hensive understanding of these post-proline dipeptidyl pep- Sydney Medical School, University of Sydney, NSW, Australia tidases (Fig. 1). Corresponding Author: Mark D. Gorrell, Molecular Hepatology, Centenary Institute, Locked Bag No. 6, Newtown, NSW 2042, Australia. Phone: 61-2- 95656156; Fax: 61-2-95656101; E-mail: [email protected] Structural Properties of DPP8 and DPP9 doi: 10.1158/1541-7786.MCR-13-0272 Initial identification and cloning of DPP8 was established 2013 American Association for Cancer Research. by Abbott and colleagues (2). DPP9 was then identified by

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Proliferation Apoptosis ECM interaction adhesion EGF migration Akt Cell behavior Immune Lung organs Spleen Thymus Immunology Inflammation Figure 1. Overview of organs and Lymphocyte processes in which DPP8 and Colitis DPP9 have potential functions, along with molecules that interact with these proteases. DPP8 DPP9

H-Ras H-Ras

Tumor Tumor Alcoholic liver disease Liver biology cell lines Liver fibrosis disease Chronic Biliary cirrhosis Testicular lymphocytic tumors leukemia

BLAST search and sequence alignment with DPP8. With Gene Properties growing interest in these novel homologues of DPPIV, DPP8 and DPP9 have been localized by ﬂuorescence in several research groups have cloned DPP8 and DPP9 situ hybridization to human chromosome 15q22 and (3,21–23). Comparisons of the main physical attributes 19p13.3, respectively (2,3). A variety of diseases have of DPPIV, FAP, DPP8, and DPP9 are summarized been mapped to these loci, such as pulmonary ﬁbrosis in Table 1. (27), ovarian adenocarcinomas (28), and Bardet–Biedl

Table 1. Comparative overviews of the 4 enzyme members of the DPPIV family

Attribute DPPIV FAP DPP8 short DPP8-v3 long DPP9 short DPP9 longa Synonyms CD26 Seprase DPRP1 DPRP2 GenBank accession M74777 U09278 AF221634 AF354202 AY374518 AF542510 References (24) (4,25,26) (2) (21) (3,23) (23) Gene location Human 2q24.3 2q23 15q22.32 19q13.3 Mouse 2:62330073 2:62500945 9:65032458 17:56186682 Number of exons 26 26 20 22 19 22 Gene size (kb) 81.8 72.8 71 ? 47.3 48.6 mRNA (bp) 2924 2814 3127 3030 2592 3006 Number of amino 766 760 882 898 863 971 acids Monomer mobility 110 kDa 95 kDa 100 kDa 103 kDa 95–110 kDa ? Dimer HH HH Transmembrane HH domain Catalytic triad Ser630-Asp708-His740 Ser624-Asp702-His734 Ser739-Asp817-His849 Ser730-Asp808-His840 Enzyme activity Dipeptidyl peptidase Dipeptidyl peptidase Dipeptidyl peptidase Dipeptidyl peptidase and endopeptidase Cellular localization Cell surface and Cell surface and Intracellular Intracellular soluble soluble

H, yes; , no; ?, not known. aThe long form is a splice variant.

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syndrome type 4, which has the clinical manifestation of obesity (29). Thus, DPP8 and DPP9 are potentially associated with the pathogenesis of such diseases, but an underlying mechanism has not been elucidated. Adoles- cent idiopathic scoliosis maps to 19p13.3, but it is not associated with DPP9 (30). The human DPP4 gene spans 81.8 kb and contains 26 exons ranging in size from 45 to 1,386 bp (31). In contrast, the human DPP9 gene spans only 48.6 kb of genomic sequence and comprises 22 exons that are 53 to 1431 bp in length (23). The human DPP8 gene spans 71 kb and consists of 20 exons (2). The sequence around the active-site serine is encoded by a single exon in DPP8 and DPP9, but the homologous region in DPP4 and FAP is encoded by 2 exons (3,31), which suggests that DPP8 and DPP9 are the more ancient genes of the 4. Protein Model Protein structure is crucial for substrate binding and specificity, as well as for interacting with binding partners. The primary structure of human DPP8 contains 882 amino acids with 27% identity and 51% amino acid similarity to human DPPIV (2). Two forms of DPP9 have been cloned. A ubiquitously expressed transcript encoding 863 amino acids (short form) displays 26% identity and 47% similarity to Figure 2. A model of DPP9 protein structure. DPP9 residues 51 to 863 are fi depicted in ribbon representation of the monomer. The a/b-hydrolase human DPPIV (23). The most signi cant homology is domain is in red and the 8-blade b-propeller domain in green. The observed between human DPP8 and DPP9 (61% identity catalytic triad (Ser-Asp-His) is shown as blue spheres and the 2 and 79% similarity at the amino acid level; ref. 23). The glutamates essential for catalysis as yellow spheres. An extended arm amino acid sequences of both proteins are highly homolo- (285VEVIHVPSPALEERKTDSYR304) in the propeller surface of DPP9 that gous between human and mouse, with human DPP9 having is critical in SUMO1–DPP9 interaction is colored pink. 92% identity and 95% similarity to mouse DPP9 and human DPP8 having 95% identity and 98% similarity to single-point mutations in the C-terminal loop, such as mouse DPP8 (3,23). Mouse DPP8 and DPP9 enzymes have F822A, V833A, Y844A, and H859A in DPP8 (34), or not been isolated for comparisons with their human counter- F842A in DPP9 (40) inactivate these proteases without parts in enzyme specificity and kinetics studies. Given that disrupting dimerization. Similarly, a deletion mutation the crystal structures of DPP8 and DPP9 have not yet been (residues 317-334) or a single-point mutation (Y334A) in solved, protein homology modeling has shown that DPP8 a propeller loop in DPP9 can cause decreased activity and DPP9 likely share a similar tertiary structure with without affecting dimerization (35). Thus, the C-terminal DPPIV and FAP (32,33). DPP8 (34) and DPP9 (35) are loop and the propeller loop are essential for DPP8 and DPP9 dimeric, with each monomer likely consisting of an enzyme activity but not for dimerization. Third, DPP8 and a/b-hydrolase domain and an 8 blade b-propeller domain DPP9 lack a transmembrane domain and are translated as and the active site of the peptidase locates at the interface of intracellular proteins (2,23), whereas DPPIV and FAP are these 2 domains (Fig. 2). The DPPIV family of proteins cell surface expressed type II membrane glycoproteins. share a conserved catalytic triad, with that of DPP8 con- fi sisting of Ser739, Asp817, and His849 and the equivalent Posttranslational Modi cation residues in DPP9 being Ser730,Asp808,andHis840 (3,23). Posttranslational modifications (PTM) regulate protein A distinguishing feature of the DPPIV protein family is activity, localization, and interaction with other cellular that 2 important glutamates lie near the start of a loop that molecules. The DPP8 sequence contains no N-linked or protrudes from the b-propeller domain and borders the O-linked glycosylation sites (2) and, despite identification of substrate entry site (32) and these 2 glutamates are 2 potential N-glycosylation sites in DPP9, no evidence of essential for catalysis in DPP8 (36), DPPIV (37), and glycosylation has been found (3,23,40). Lysine 51 and FAP (38). Lysine 314 in DPP9 have been identified as potential Despite the close sequence and structure homology with acetylation targets in 2 separate proteomic and mass spec- DPPIV, DPP8, and DPP9 possess several distinct structural trometric acetylation studies (41,42), but this has not been properties. First, DPP8 has a larger substrate pocket (S2) verified in vivo. Further investigation is thus necessary to than DPPIV (39). This suggests that DPP8 and DPP9 might understand PTMs of DPP8 and DPP9, which may lead have either an additional element of tertiary structure at the to alteration of their localization, function, and enzyme N-terminus and/or a larger b-propeller domain. Second, activity.

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DPP8 and DPP9 Activity Regulation position. An in vivo substrate study confirmed that the DPP8 and DPP9 specifically interact with SUMO1 (43) majority of substrates of DPP8 and DPP9 contain a proline and a novel SUMO1-binding arm in the propeller domain of in the P1 position preceded by an alanine in the P2 position DPP9 (Fig. 2) has been shown to be important for its (47). DPP8 and DPP9 hydrolyze the synthetic chromogenic enzymatic regulation, whereby SUMO1 binding results in substrates Gly-Pro-pNA, Ala-Pro-pNA, and Arg-Pro-pNA elevated DPP9 activity in vitro and gene silencing of equally well (refs. 2,22,23, and 39; Table 2). Using sub- SUMO1 leads to reduced DPP8 and DPP9 activity in cell strates Ala-Pro-pNA and Gly-Pro-pNA, the pH optimum extracts (43). SUMO modification mediates numerous for DPP9 is between pH 7.5 and 8.0 (40). DPP8 has a intracellular processes including transcription, DNA repair, neutral pH 7.4 optimum with little activity below pH 6.3 chromatin remodeling, and nuclear translocation (44). (2), similar to the pH 7.8 optimum of DPPIV (48). Therefore, it would be interesting to explore the conse- quences of SUMO1 interactions with DPP8 and DPP9. Natural Substrate Proteins Furthermore, the activity of purified DPP9 from bovine Certain naturally occurring peptides and chemokines can testes (45) and recombinant DPP8 and DPP9 produced by be cleaved by DPP8 and/or DPP9 in vitro but significantly insect cells (33) is dependent on the redox state of their slower than DPPIV (refs. 22 and 49; Table 2). However, cysteines, whereby enzyme activity is reversibly decreased by the physiological relevance of these substrates is uncertain oxidation and increased by reduction (33). because they are predominantly extracellular whereas DPP8 and DPP9 are intracellular. However, in rat brain Synthetic Substrates for Activity Determination extract, Neuropeptide Y (NPY) can be cleaved in the Proteases regulate various physiological processes by cleav- presence of a DPPIV inhibitor, indicating that DPP8 and ing substrates to cause inactivation or modified function. DPP9 can cleave NPY in vivo (50). NPY-driven tumor cell Therefore, identifying the substrates for DPP8 and DPP9 is death has been achieved by inhibiting DPP8 and DPP9, essential for understanding their biological functions. DPP8 indirectly indicating that DPP8 and DPP9 can cleave and DPP9 have similar substrate specificities, with both releasable NPY (51). Other natural substrates have also been enzymes preferring substrates with an aromatic or branched identified. Silencing or inhibition of DPP9 in intact cells aliphatic amino acid (46) or basic residue (34,40) in the P2 results in increased presentation of the RU134-42 peptide

Table 2. Kinetic parameters of DPP8 and DPP9 substrates

Km (mmol/L)

Substrate DPP8 DPP9 References H-Ala-Pro-pNAa 0.991 0.171 N/A (2) H-Gly-Pro-pNA 0.467 0.064 N/A H-Ala-Pro-AFCb 0.18 0.02 0.18 0.07 (23) H-Gly-Pro-pNA 0.33 0.01 0.37 0.003 (22) H-Ala-Pro-pNA 0.34 0.10 0.31 0.13 H-Val-Ala-pNA 0.76 0.08 1.22 0.13 H-Ala-Pro-pNA 0.30 0.02 N/A (39) H-Gly-Pro-pNA 0.4 0.04 N/A H-Arg-Pro-pNA 0.11 0.005 N/A

t1/2 (h)

Substrate DPP8 DPP9 References

GLP-1 3.8 6.1 (22) GLP-2 4.0 6.7 NPY 0.2 0.2 PYY 50 8.0 CXCL 12/SDF-1a (1-67) 4 0.5 N/A (49) CXCL 12/SDF-1b (1-72) 2 0.6 N/A CXCL 10/IP10 (1-77) 13 2.4 N/A CXCL 11/ITAC (1-73) >24 N/A

Abbreviation: N/A, not available. apNA ¼ para-nitroaniline. bAFC ¼ 7-Amino-4-triﬂuoromethyl coumarin.

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(VPYGSFKHV), which was the first natural substrate of as well as molecular tools. Isoindoline derivatives have been fi DPP9 to be identi ed (46). It is possible that many more reported as potent DPP8 inhibitors with IC50 values below proline-containing antigens present in the cytoplasm are 20 nmol/L (56,57), but were later found to inhibit both DPP8 substrates for DPP9. Recently, a number of potential natural and DPP9 (18). Recently, 1G244 has been modified into substrates for DPP8 and DPP9 have been discovered using a several substructures to obtain some selectivity toward either sophisticated terminal amine isotopic labeling of substrates DPP8 or DPP9 (19). Limited selectivity for DPP8 over proteomic method (47). In that study, calreticulin and DPP9 (roughly 10-fold) has been achieved in 2 compounds adenylate kinase 2 (AK2) were identified as potential sub- (12m and 12n), using methylpiperazine analogues of 1G244 strates of both DPP8 and DPP9 and acetyl-CoA acetyl- (19). The boroProline-based dipeptidyl boronic acids are transferase, which is important in fatty acid metabolism, was potent DPPIV inhibitors (20). Various substitutions at the identified as a potential substrate of DPP8 (47). As AK2 4-position of the boroProline ring alter the inhibitory activity. plays a key role in maintaining cellular energy homeostasis, Arg-(4S)-boroHyp (4q) shows the most potent inhibition DPP8 and DPP9 may regulate this process by cleaving AK2. against DPPIV, DPP8, and DPP9, whereas (4S)-Hyp-(4R)- Although the impact of cleavage by DPP8 and DPP9 on the boroHyp (4o) exhibits the greatest selectivity for DPPIV over functions of these substrates remains to be studied, their DPP8 and DPP9 (20). Such studies point to the structure– identification highlights the potential roles of DPP8 and activity relationship that will facilitate the future design of DPP9 in energy metabolism and homeostasis. inhibitors toward either DPP8 or DPP9 alone and thus help to differentiate DPP8 and DPP9 activity from each other and Inhibitor Development for DPP8 and DPP9 also from that of DPPIV. These enzyme studies used human The properties of potent and selective inhibitors for DPP8 DPP8 and DPP9. It is likely that the homologues from other and DPP9 are listed in Table 3. The compounds allo- species would have very similar properties, but this has not Ile-isoindoline and 1G244 are the most commonly used been directly examined. DPP8/9 selective inhibitors. Allo-Ile-isoindoline had IC 50 fi values of 50 nmol/L against DPP8 and DPP9 when first Expression Pro les of DPP8 and DPP9 fi reported (52) but later IC50 values have been found to be Expression pro les of DPP8 and DPP9 have been an 200 nmol/L (18, 53–55). The most potent inhibitory important focus of interest in characterizing these new effect is observed with 1G244 (IC50 values of 14 and 53 proteases and their ubiquitous expression pattern has been nmol/L against DPP8 and DPP9, respectively), which does confirmed at mRNA and protein levels (Table 4). not inhibit DPPIV (18). 1G244 is a slow-tight binding DPP9 cDNA has 2 forms, with sizes of 2,589 and 3,006 competitive inhibitor of DPP8 and a reversible competitive bp (23). Using Northern blot analysis, a predominant DPP9 inhibitor of DPP9 (18). mRNA transcript of 4.4 kb (AY374518; short form encod- It would be of great value to distinguish DPP8 from DPP9 ing 863 amino acids) was detected ubiquitously with the activity in many biological processes using selective inhibitors highest levels in liver, heart, and skeletal muscle (3,23). A less

Table 3. Properties of potent and selective inhibitors for DPP8 and DPP9

IC50 (nmol/L) Ki (nmol/L)

Compound DPP8 DPP9 DPPIV DPP8 DPP9 References Allo-Ile-isoindoline 38 55 30,000 N/A N/A (52) (DPP8/9-selective) 120 290 90,000 N/A N/A (54,55) 145 242 >100,000 13.7 33.7 (18) 1G244 14 53 >100,000 0.9 4.2 (18) 12 84 >50,000 N/A N/A (19) 1G244 analogue (compound 12 m) 21 260 >100,000 N/A N/A (19) 1G244 analogue (compound 12 n) 55 540 >50,000 N/A N/A (19) Bis(4-acetamidophenyl) Pyrrolidin-2-yl 910 (irreversible) N/A 8,000 N/A N/A (57) Phosphonate with Lys in P2 position (compound 7e) Bis(4-acetamidophenyl) Isoindolin-2-yl 530 (irreversible) N/A >500, 000 N/A N/A (57) Phosphonate with Lys in P2 position (compound 2e) (4S)-Hyp-(4R)-boroHyp (compound 4o) 530 240 16 N/A N/A (20) Arg-(4S)-boroHyp (compound 4q) 1.2 0.31 0.3 N/A N/A (20)

Abbreviation: N/A, not available.

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Table 4. Expression features of DPP8 and DPP9

Characteristic DPP8 DPP9 References mRNA expression in normal adult tissues Ubiquitous Ubiquitous (3,23,58) Protein expression in normal adult tissues Ubiquitous Ubiquitous (59) Expression in tumor tissues HH(23,59) Expression by hepatocytes HH(59–61) Expression by activated hepatic stellate cells (59) Expression by lymphocytes HH(59,61,62)

H, yes; , no.

abundant 5 kb transcript (AF542510; long form encoding pancreas, muscle, and brain, express DPP8 and DPP9 971 amino acids) is abundant in skeletal muscle (23). (ref. 59; Fig. 3). Generally, concordant data on DPP8 and Similarly, 3 mRNA transcripts of DPP8 have been iden- DPP9 expression has been obtained from the cynomolgus tified with intense signals in testis, prostate, and muscle monkey and Sprague-Dawley rat (64), in which mRNA and (2,58). In particular, a transcript variant of human DPP8, protein levels correlated with specific enzymatic activity with 16 more amino acids at the N-terminus (898 amino (64). In addition, DPP8 and DPP9 activity is approximately acids in total), is abundant in the adult testis (2,21). DPP8 10-fold lower than DPPIV activity in nonrenal rat and and DPP9 enzymatic activities are predominant in bovine monkey tissues (64). DPPIV and DPP8 are more abundant and rat testis and immunohistochemical studies have than DPP9 protein in rat primary endothelial cells, however, localized these 2 proteases in spermatozoids (63). A about 60% of the total DPP enzyme activity is derived from natural form of DPP9 has been purified from bovine DPP8/9 rather than DPPIV in these cells (65). DPP9 is the testes and identified as the short form (45). Overall, the only DPPIV-like enzyme detectable by immunohistochem- abundance of DPP8 and DPP9 in testis suggests that these istry in human carotid artery endothelial cells, whereas 2 proteases might participate in the regulation of the male DPPIV is the predominant DPPIV-like enzyme in ventric- reproductive system. ular microvasculature by in situ hybridization (65). Thus, the The ubiquitous expression of DPP8 and DPP9 in mam- regulated expression of individual DPPs in these cells is mals has been shown by in situ hybridization and enzyme indicative of a role in endothelia. Altered DPP8 and DPP9 assays of mouse, human, and baboon organs (59). Notably, expressions, at both mRNA and protein levels in diseased lymphocytes and epithelial cells from many organs, includ- liver, point to important regulatory roles in the pathogenesis ing lymph node, thymus, spleen, liver, lung, intestine, of liver diseases (59–61).

DPP8/DPP9 enzyme activity in mouse organs 140 Highest 120 High 100 Medium Figure 3. Ranking mouse organs by approximate intensity of DPP8 and 80 Low DPP9 enzyme activity. DPP activity in DPPIV gene knockout mice mU/g 60 with/without N-ethylmaleimide (NEM) was measured by Yu and 40 colleagues (59). NEM is an inhibitor of DPP8/9 enzyme activity. Subtracting NEM-inhibited activity 20 Negligible from total DPP activity was used here to estimate the activity derived 0 from DPP8 and DPP9.

Skin Liver Lung Brain Colon Testis Heart Ovary Uterus Kidney Spleen PBMCs Plasma Thymus Adrenal AdiposeProstate Pancreas

Lymph node Bone marrow Small intestine Skeletal muscle

Submaxillary gland

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Biological Functions of DPP8 and DPP9 experiments in which DPP9 is manipulated by overexpres- Enzymes of the DPPIV family are likely to participate in sion or gene silencing or enzyme inhibition have provided normal homeostasis as well as in the pathology of disease nonconcordant data and thus the pro- or antiapoptotic conditions that include tumor growth, type 2 diabetes, liver activity of DPP9 may depend on the cell-type and cell- cirrhosis, and inflammatory and autoimmune diseases culture environment. (13,16,66,67). As knowledge of the precise expression and DPP9 mRNA is generally abundant in human tumor localization of DPP8 and DPP9 advances, distinct physio- cell lines, such as melanoma (G-361), colorectal adeno- logical functions of these proteases are emerging. carcinoma (SW480), chronic myelogenous leukemia (K- 562), and HeLa cells (3). DPP8 mRNA expression is fi DPP8 and DPP9 in Cell Behavior and Tumor signi cantly greater than all the other DPPs in breast Biology cancer and ovarian cancer cell lines, but DPP8 and DPP9 protein expression is ubiquitous across those cell lines Intracellularly distributed DPP8 and DPP9 have previ- (70). DPP9 mRNA is also greatly upregulated in human fl ously been shown to in uence cell behavior, including testicular tumors (59). Interestingly, the highest level of – cell extracellular matrix (ECM) interactions, proliferation, DPP9 protein is in 2 estrogen receptor negative breast and apoptosis. HEK293T epithelial cells overexpressing cancer cell lines that are negative for DPPIV mRNA (70). in vitro DPP8 and DPP9 exhibit impaired cell adhesion, In many tumor tissues, including skin melanotic mela- migration, and monolayer wound healing, independent of noma,neuroblastoma,andleukemia,thelongformof enzyme activity (68). In HEK293T cells, DPP8 and DPP9 human DPP9 mRNA is expressed (23). DPP8 and DPP9 can enhance induced apoptosis independent of enzyme are more abundantly expressed than DPPIV and FAP in activity and DPP9 overexpression causes spontaneous apo- human meningiomas (71) and constitutive expression of ptosis (68). In HepG2 cells, the levels of active forms of DPP8 and DPP9 is found in B-cell chronic lymphocytic fi caspase 9 and caspase 3 can be signi cantly elevated follow- leukemia (CLL; ref. 72). DPP8 mRNA and protein ing overexpression of enzyme active DPP9 compared with an expression are significantly upregulated in CLL compared enzyme inactive mutant DPP9, indicating that the intrinsic with normal tonsil B lymphocytes (72). The ubiquitous apoptosis induced by DPP9 is mediated by the caspase 9/ but differential expression of DPP8 and DPP9 in tumor caspase 3 pathway (60). DPP9 overexpression attenuates the cell lines and tissues indicates that they may have roles – epidermal growth factor (EGF) mediated PI3K/Akt path- in tumor pathogenesis, perhaps in particular stages or way, resulting in augmented apoptosis and suppressed cell circumstances. proliferation (ref. 60; Fig. 4). This inhibitory effect on Akt Recently published data more clearly point to the roles of phosphorylation is largely dependent upon DPP9 enzyme DPP8 and DPP9 in tumor growth. A high-throughput fi activity (60), which is the rst indication of DPP9 enzyme screen has found that the DPP inhibitor vildagliptin syner- activity regulating cell behavior through the EGF signaling gistically enhances parthenolide's antileukemic activity in pathway. Perhaps surprisingly, knockdown of DPP8 or leukemia and lymphoma cell lines as well as in primary DPP9 can induce NPY-driven tumor cell death, mediated human acute myeloid leukemia (73). Inhibition of DPP8 by poly (ADP-ribose) polymerase-1 and apoptosis-inducing and DPP9, but not DPPIV, is responsible for vildagliptin's factor (51) and enzyme inhibition of DPP8 and DPP9 by enhancement of parthenolide's cytotoxicity (73). In addi- 1G244 can enhance macrophage apoptosis (69). Therefore, tion, inhibition of DPP8 and DPP9 probably contributes to the tumor regression induced by the compound Val-boro- Pro (74). Moreover, a separate study suggests that the DPPs act as survival factors in the Ewing Sarcoma Family of EGF Tumors (51). In such sarcoma cell lines, cell death can be EGFR enhanced by blocking DPPIV, DPP8, and DPP9 with either selective enzyme inhibition or siRNA knockdown. These H-Ras effects were blocked by NPY receptor antagonists, indicating Plasma membrane that the cell survival might be dependent on the NPY DPP9 PI3K Raf pathway (51). Perhaps paradoxically, overexpression of DPP9 is antiproliferative and enhances intrinsic apoptosis in epithelial tumor cell lines (60,68), which might indicate MEK that high levels of DPP9 can be detrimental. More probably, Akt different responses of DPP8 and DPP9 under different pathological conditions imply that the roles of DPP8 and ERK DPP9 may vary with tumor stage or tumor type. DPP8 and DPP9 in the Immune System Figure 4. Diagram of potential involvement of DPP9 in the EGF signaling in vivo in vitro pathway. Cytoplasmic DPP9 is associated with H-Ras. DPP9 attenuates Good evidence, from both and studies, the EGF mediated PI3K/Akt pathway but does not influence the Raf/MEK/ indicates that DPP8 and DPP9 participate in immunoreg- ERK pathway. ulation. As described earlier, extensive in vivo expression of

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DPP8 and DPP9 occurs in normal immunological tissues Many studies have the limitation that DPP8 and DPP9 (59). DPP8 and DPP9 are expressed by all major lymphocyte activities have been examined in the presence of DPPIV. subpopulations and are upregulated upon B or T lympho- DPPIV-deficient mice and DPP8/9 selective inhibitors cyte activation (2,61). Intriguingly, there is one report can overcome this issue. In addition, most studies are suggesting that a minor fraction of typically intracellularly confined to in vitro models. More in vivo evidence and localized DPP8 and DPP9 might also be loosely bound on clinical data are needed. The development of DPP8 or the surface of immune cells under certain circumstances DPP9-deficient animals would be a breakthrough for such (75). functional studies. In addition, enzyme activity of DPP8 and DPP9 is present in cultured human and mouse primary leukocytes and B and Concluding Remarks T cell lines (2,52,75,62). Effects of DPP8 and DPP9 on the The recent insights into the emerging members of the activation and proliferation of immune cells seem to be DPPIV family reveal very interesting biochemical and mediated by enzyme activity. A selective DPP8 and DPP9 biological traits of DPP8 and DPP9. Both DPP8 and DPP9 inhibitor can attenuate both proliferation of peripheral interact with SUMO1. DPP9 has a novel SUMO1-specific blood mononuclear cells and the release of cytokine inter- interacting motif that is important for its allosteric enzy- leukin (IL)-2 (52). Moreover, DNA synthesis of mitogen- matic regulation. Both proteases are redox regulated and stimulated splenocytes of both wild-type and DPPIV knock- probably not glycosylated, whereas only DPP9 is predicted out mice is suppressed by the selective inhibition of DPP8 to be acetylated. Recent advances in this field have begun to and DPP9 (17). Collectively, these data point to DPP8/9 show functional differences between the 2 proteases. DPP9 enzyme activity having important roles in immunoregula- has a stronger association with H-Ras, attenuates the EGF- tion, but the mechanisms involved require additional mediated PI3K/Akt pathway, and regulates cell behavior. investigations. The enzyme activity of DPP9, but not DPP8, is rate limiting for degradation of antigenic proline-containing fl DPP8 and DPP9 in the In ammatory Response peptides and thereby may have an important role in antigen DPPIV activity is clearly related to the induction of the presentation, whereas DPP8 is more important in gut inflammatory response (76,77). Therefore, it would be inflammation. Some potential natural substrates of DPP8 interesting to know the extent of involvement of DPP8 and and DPP9 have been identified recently using proteomics, DPP9, which have DPPIV-like activity, in inflammation. one of which was specific for DPP8 only. These exciting Both mRNA and the enzyme activities of DPP8 and DPP9 new functional data reinforce the importance of expanding are upregulated after asthma induction. Thus, the increased our knowledge of DPP8 and DPP9 expression patterns in expression of DPP8 and DPP9 and their specific localization both rodent and primate species. The development of suggests that these enzymes have important roles during selective inhibitors and substrates for DPP8 versus DPP9 allergic reactions (78). is needed and would be instrumental in the future explo- An experimental colitis model in mice has provided ration and exploitation of the roles that DPP8 and DPP9 further evidence for the participation of DPP8 in the exhibit in pathogenesis. inflammatory responses. After dextran sulfate sodium (DSS) treatment for 6 days to induce colitis, DPP8, but not DPP9, Disclosure of Potential Conflicts of Interest mRNA, and enzyme activity is elevated in the colons of both Mark D. Gorrell has an ownership interest in patents on DPP8 and DPP9. No fl wild-type and DPPIV knockout mice (79), with inhibition potential con icts of interest were disclosed by the other authors. of DPP activity impairing neutrophil infiltration at the site of inflammation (79). These data implicate DPP8 in the Authors' Contributions fl Conception and design: H. Zhang, Y. Chen, M.D. Gorrell in ammatory response in colitis. DPPIV, aminopeptidase Writing, review, and/or revision of the manuscript: H. Zhang, Y. Chen, N (APN), and DPPIV/APN-like proteases are involved in F.M. Keane, M.D. Gorrell ischemia-trigged inflammation (80). DPP9 mRNA is tran- Study supervision: F.M. Keane, M.D. Gorrell siently and significantly downregulated whereas DPP8 mRNA is slightly decreased at day 3 postcerebral ischemia. Acknowledgments The authors thank Ms. M.G. Gall for kind suggestions and proofreading. The DPPIV, DPP8, and APN are in the activated microglia and authors are grateful for support from the Australian National Health and Medical macrophages at day 3 postischemia and in astroglial cells at Research Council, grant 512282, Australian Postgraduate Award for H. Zhang and day 7 postischemia (80). Therefore, DPPIV, DPP8, and University of Sydney International Scholarship to Y. Chen. DPP9 have potential roles in cerebral inflammation. Most recently, abundant DPP8 and DPP9 have been found in Grant Support This work is supported by Australian National Health and Medical Research macrophage-rich regions of human atherosclerotic plaques Council Grant 512282; Australian Postgraduate Award for H. Zhang; and University (69). Upregulated DPP9 and unchanged DPP8 expression of Sydney International Scholarship for Y. Chen. have been observed during macrophage differentiation and The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked advertisement in accordance with activation (69). Inhibition of DPP8 and DPP9 decreases 18 U.S.C. Section 1734 solely to indicate this fact. proinflammatory cytokines IL-6 and TNF-a secretion activated by lipopolysaccharide and IFN-g, suggesting an anti- Received May 28, 2013; revised September 4, 2013; accepted September 5, 2013; inflammatory effect of the DPP8/9 inhibitor (69). published OnlineFirst September 13, 2013.

1494 Mol Cancer Res; 11(12) December 2013 Molecular Cancer Research

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1496 Mol Cancer Res; 11(12) December 2013 Molecular Cancer Research

Advances in Understanding the Expression and Function of Dipeptidyl Peptidase 8 and 9

Hui Zhang, Yiqian Chen, Fiona M. Keane, et al.

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