Abstract

Ectodomain shedding of extracellular and membrane proteins is of fundamental importance for cell–cell communication in neoplasias. A Disintegrin And Metalloproteinase (ADAM) proteases constitute a family of multifunctional, membrane-bound proteins with traditional sheddase functions. Their protumorigenic potential has been attributed to both, essential (ADAM10 and ADAM17) and ‘dispensable’ ADAM proteases (ADAM8, 9, 12, 15, and 19). Of specific interest in this review is the ADAM proteinase ADAM8 that has been identified as a significant player in aggressive malignancies including breast, pancreatic, and brain cancer. High expression levels of ADAM8 are associated with invasiveness and predict a poor patient outcome, indicating a prognostic and diagnostic potential of ADAM8. Current knowledge of substrates and interaction partners gave rise to the hypothesis that ADAM8 dysregulation affects diverse processes in tumor biology, attributable to different functional cores of the multidomain enzyme. Proteolytic degradation of extracellular matrix (ECM) components, cleavage of cell surface proteins, and subsequent release of soluble ectodomains promote cancer progression via induction of angiogenesis and metastasis. Moreover, there is increasing evidence for significance of a non-proteolytic function of ADAM8. With the disintegrin (DIS) domain ADAM8 binds integrins such as β1 integrin, thereby activating integrin signaling pathways. The cytoplasmic domain is critical for that activation and involves focal adhesion kinase (FAK), extracellular regulated kinase (ERK1/2), and protein kinase B (AKT/PKB) signaling, further contributing to cancer progression and mediating chemoresistance against first-line therapies. This review highlights the remarkable effects of ADAM8 in tumor biology, concluding that pharmacological inhibition of ADAM8 represents a promising therapeutic approach not only for monotherapy, but also for combinatorial therapies.

Introduction

A disintegrin and metalloproteinase (ADAM) 8 (ADAM8) is a type I transmembrane (TM) glycoprotein consisting of an N-terminal prodomain, followed by a metalloproteinase-, disintegrin (DIS)-, cysteine-rich-, epidermal growth factor (EGF)-like- and TM domain and a cytoplasmic tail [1,2]. The human ADAM8 gene is located on chromosome 10q26.3, consists of 23 exons and encodes a protein of 824 amino acids [2]. ADAM8 was first identified in monocytic immune cells [1,2] and subsequently detected in B cells, dendritic cells [3], and granulocytes [4,5], exhibiting a selective expression profile, which is in contrast with the ubiquitous occurrence of the most other ADAM family members. ADAM8 was initially considered to be an immune-specific ADAM, based on its inducibility by inflammatory stimuli, such as bacterial lipopolysaccharide (LPS), interferon-γ (IFN-γ) [2], tumor necrosis factor α (TNF-α) [6], interleukin (IL)-1β, 4 and 13 [7,8], and peroxisome proliferator-activateds receptor-γ (PPAR-γ) [9]. Expression levels of ADAM8 in normal tissue is typically low and limited to a few distinct cell types in the lymphatic organs as components of the immune system [10], the central nervous system [6], in bone [11] and in lung [7]. Only under the aforementioned pathological stimuli, ADAM8 be induced to significant protein levels in disease so that ADAMs become potentially relevant for pathophysiology. A thorough phenotypical analysis of ADAM8-deficient mice revealed no evident spontaneous developmental or pathological defects [10,12], demonstrating that ADAM8 is non-essential for normal development and homeostasis.

Under inflammatory conditions, several diseases are associated with ADAM8 overexpression, including respiratory diseases such as asthma [7,13–15], bone destruction [8] and aseptic loosening of total hip replacements [16,17], liver injury [18,19], and neurodegeneration/neuroinflammation [6,12,20]. Functional data suggest an important role of ADAM8 in immunomodulation and inflammatory disease (reviewed in [21–23]). Once up-regulated, ADAM8 is proteolytically active and can potentially overlap with the substrate spectrum of two major shedding enzymes, ADAM10 (e.g. for the low affinity IgE receptor CD23) and ADAM17 (e.g. for L-selectin and TNF-R1), which results in enhanced shedding of cell adhesion molecules, cytokine receptors and extracellular matrix (ECM) components [24]. Many of these substrates are not only mediators of inflammation, but also of cancer. Whereas in inflammatory processes ADAM8 expression is controlled by cytokines and growth factors, increased ADAM8 expression in tumor cells is considered to be constitutive and can be further stimulated by hypoxia and inflammatory mediators [25,26]. Consistent with the findings for other catalytically active ADAM family members, such as ADAM 9, 12, 17, and 19 (reviewed in [27,28]), increased expression of ADAM8 in several tumors is correlated with severity and invasiveness, although the mechanisms underlying these processes remain less clear. Current knowledge of substrates and interaction partners gave rise to the hypothesis that ADAM8 is an important mediator of key events in tumorigenesis.

This review aims to summarize the current investigations on ADAM8 as a significant player in highly invasive tumor entities. As a ’dispensable’ ADAM proteinase [10,12], specific inhibition of ADAM8 could be a promising therapeutic strategy with minor expected side effects, not only for inflammatory diseases where it was initially implicated, but also for many tumor entities.

Processing of ADAM8

Based on the canonical domain structure consisting of an inhibitory prodomain (Pro), a metalloprotease (MP) domain (MP), a DIS-like (DIS), a cysteine-rich (Cys), and an EGF-like domain (EGF) followed by the TM region and the cytoplasmic tail (CD, Figure 1), ADAM8 is synthesized as an inactive 120 kDa proform and requires prodomain removal for activation [24,29]. Unlike other ADAM proteinase family members, pro-ADAM8 is activated by autocatalysis in the trans-Golgi network and not by furine-like convertases [24]. Autocatalytic prodomain removal is dependent on homophilic multimerization of the DIS and Cys domains of at least two ADAM8 monomers enabling intermolecular cleavage [24,29]. The 90-kDa active form of ADAM8 can be further processed leaving a remnant form of 60 kDa on the cell surface and releasing a soluble MP domain (∼30 kDa, Figure 1).

Domain structure of human ADAM8

Figure 1
Domain structure of human ADAM8

Domain borders, processing sites, and respective domain functions are summarized. Pre-processing at Glu158 fractures the prodomain (Pro, light blue) before terminal activation by removal of the putative Cysteine-switch (Cys167) at Val185 occurs. The MP (blue) domain contains the canonical trihistidine motif (His334, His338, and His344) that co-ordinates the active site zinc ion (Zn2+). The crystal structure of the MP domain complexed with batimastat (BB-94) (lavender) is shown below. Similar to other ADAM family members, ADAM8 has a characteristic central five-stranded b-sheet, which is flanked by four long and two short a-helices. The S1′ specificity loop (residues 355–375) produces local conformational differences that may contribute to substrate selectivity, and thus could allow structure-based rational drug design of selective inhibitors. One additional short a-helix lies near the C-terminus and constitutes part of the Ca2+-binding site. ADAM-8 is stabilized by three disulphide bridges formed by cysteine residues Cys310–Cys395, Cys351–Cys379, and Cys353–Cys362. The DIS domain (red) is supposed to interact with integrins and is critical for homophilic ADAM8–ADAM8 dimerization. A homology model of ADAM8 DIS-domain based on the integrin-binding region of ADAM15 is shown below. The amino-acid motif ‘KD’ is exposed toward the outer aspect of the DIS domain and is potentially accessible to peptidomimetics such as BK-1361 (magenta). The Cysteine-rich (CYS, violet) and EGF-like domain (EGF, yellow) were shown to co-ordinate autocatalytic activation of pro-ADAM8. Via the TM region (cyan) ADAM8 is attached to the cell membrane. The cytoplasmic tail (CD, gray) contains five potential SH3-binding motifs and two phosphorylation sites (Ser758 and Tyr766) that may transduce intracellular signaling involving MAPK pathway. Recently, direct interaction of TOCA-1, SNX33, and CIP4 with ADAM8 via SH3-binding motifs was demonstrated, however, the functional relevance of these interactions in cancerogenesis remains to be determined (modified from [22,29,32,37]). Abbreviations: CIP4, Cdc42-interacting protein-4; MAPK, mitogen-activated protein kinase; SH3, Src homology 3; SNX33, sorting nexin-33; TOCA-1, transducer of Cdc42-dependent actin assembly protein 1.

Figure 1
Domain structure of human ADAM8

Domain borders, processing sites, and respective domain functions are summarized. Pre-processing at Glu158 fractures the prodomain (Pro, light blue) before terminal activation by removal of the putative Cysteine-switch (Cys167) at Val185 occurs. The MP (blue) domain contains the canonical trihistidine motif (His334, His338, and His344) that co-ordinates the active site zinc ion (Zn2+). The crystal structure of the MP domain complexed with batimastat (BB-94) (lavender) is shown below. Similar to other ADAM family members, ADAM8 has a characteristic central five-stranded b-sheet, which is flanked by four long and two short a-helices. The S1′ specificity loop (residues 355–375) produces local conformational differences that may contribute to substrate selectivity, and thus could allow structure-based rational drug design of selective inhibitors. One additional short a-helix lies near the C-terminus and constitutes part of the Ca2+-binding site. ADAM-8 is stabilized by three disulphide bridges formed by cysteine residues Cys310–Cys395, Cys351–Cys379, and Cys353–Cys362. The DIS domain (red) is supposed to interact with integrins and is critical for homophilic ADAM8–ADAM8 dimerization. A homology model of ADAM8 DIS-domain based on the integrin-binding region of ADAM15 is shown below. The amino-acid motif ‘KD’ is exposed toward the outer aspect of the DIS domain and is potentially accessible to peptidomimetics such as BK-1361 (magenta). The Cysteine-rich (CYS, violet) and EGF-like domain (EGF, yellow) were shown to co-ordinate autocatalytic activation of pro-ADAM8. Via the TM region (cyan) ADAM8 is attached to the cell membrane. The cytoplasmic tail (CD, gray) contains five potential SH3-binding motifs and two phosphorylation sites (Ser758 and Tyr766) that may transduce intracellular signaling involving MAPK pathway. Recently, direct interaction of TOCA-1, SNX33, and CIP4 with ADAM8 via SH3-binding motifs was demonstrated, however, the functional relevance of these interactions in cancerogenesis remains to be determined (modified from [22,29,32,37]). Abbreviations: CIP4, Cdc42-interacting protein-4; MAPK, mitogen-activated protein kinase; SH3, Src homology 3; SNX33, sorting nexin-33; TOCA-1, transducer of Cdc42-dependent actin assembly protein 1.

Appropriate processing of ADAM8 in the secretory pathway and localization on the cell surface is critically dependent on N-glycosylation [30], but not on ADAM8 catalytic activity [24]. Human ADAM8 has four N-glycosylation sites in positions Asn76, Asn91, Asn436, and Asn612. Site-directed mutagenesis revealed that post-translational N-glycosylation of Asn91 and Asn612 is responsible for correct cellular localization on the cell surface [30]. Glycosylation of Asn436 affects ADAM8 stability, whereas Asn67 in the prodomain had only modest effects on processing or activity [30].

Shedding activity of ADAM8

As a metzincin, the MP domain of ADAM8 contains the typically conserved trihistidine consensus signature of MPs (HEXGHXXGXXHD) that co-ordinates the active-site zinc ion required for catalytic activity [31]. The canonical zinc-binding motif forms the base of a cleft, which is maintained by an α-helix (residues 327–335), a β-strand (residues 301–304) with the entrance loop (residues 298–301), and the S1′ specificity loop (residues 365–375) (Figure 1). These structural key features define a pocket into which the target-peptide sequence could bind for proteolytic cleavage [32]. All ADAMs cleave their substrates next to the cell surface, however, they exhibit no substrate specificity or functional relationship of substrates [33]. The S1′ specificity loop in ADAM8 produces local conformational differences that may contribute to substrate selectivity, but so far, no exclusive substrate of ADAM8 has been identified. Rather, the substrate spectrum of ADAM8 overlaps with the ubiquitous ADAM proteinases ADAM10 and ADAM17, thereby confirming a dispensable role of ADAM8 under physiological conditions. Tumor relevant ADAM8 substrates are summarized in Table 1 and will be discussed below.

Table 1
Summary of ADAM8 substrates linked to cancer progression
Type of substrate Substrate Prospective function in cancer biology Cleavage analysis* References 
Autocatalytic ADAM8 prodomain Proteolytic activity synPep, cell-based [24,29,34
ECM molecules Collagen I Invasion, migration, and metastasis protein [34
Fibronectin protein [39
Periostin protein [40
Receptors CD23 Immunosurveillance, cell motility, angiogenesis, metastasis cell-based [48
TNF-R1 synPep, cell-based [12
IL-1 RII synPep [88
Flt-1 cell-based [45
Flk-1 cell-based [45
Tie-2 cell-based [45
EphB4 cell-based [45
LRP6 protein [89
Ligands PSGL-1 Transmigration, metastasis synPep, protein [35,90
Adhesion molecules CHL-1 Cell detachment and motility, extravasation protein [47
VE-Cadherin cell-based [45
CD31 cell-based [45
E-Selectin cell-based [45
L-Selectin protein [4
Cytokines/chemokines TNF-a Immunomodulation synPep [88
CXCL1 syn Pep [47
Type of substrate Substrate Prospective function in cancer biology Cleavage analysis* References 
Autocatalytic ADAM8 prodomain Proteolytic activity synPep, cell-based [24,29,34
ECM molecules Collagen I Invasion, migration, and metastasis protein [34
Fibronectin protein [39
Periostin protein [40
Receptors CD23 Immunosurveillance, cell motility, angiogenesis, metastasis cell-based [48
TNF-R1 synPep, cell-based [12
IL-1 RII synPep [88
Flt-1 cell-based [45
Flk-1 cell-based [45
Tie-2 cell-based [45
EphB4 cell-based [45
LRP6 protein [89
Ligands PSGL-1 Transmigration, metastasis synPep, protein [35,90
Adhesion molecules CHL-1 Cell detachment and motility, extravasation protein [47
VE-Cadherin cell-based [45
CD31 cell-based [45
E-Selectin cell-based [45
L-Selectin protein [4
Cytokines/chemokines TNF-a Immunomodulation synPep [88
CXCL1 syn Pep [47
*

Characterization of substrates is based on either cleavage of synthetic peptide (synPep), recombinant protein (protein), or on cleavage in cell-based assays (cell-based).

Signaling functions of ADAM8

Given the distinct processing steps that ADAM8 undergoes, it seems likely that ADAM8 has functions beyond ectodomain shedding, i.e. as a remnant form containing the DIS/cysteine-rich/EGF-like domain. In addition, the cytoplasmic domain harbors cell signaling motifs close to the C-terminus (Figure 1). ADAM8 expression in cancer cells involves β1-integrin activation by virtue of a central motif (amino acids K, D, and M in the corresponding positions of R, G, and D in human ADAM15) in the so-called ‘integrin-binding loop’ and induces focal adhesion kinase (FAK), extracellular regulated kinase (ERK1/2), and AKT/PI3 kinase signaling [34–36]. Phosphorylation of these downstream effectors was associated with tumor cell invasiveness and critically depends on the cytoplasmic domain, but not on an active MP domain in ADAM8 [34,35]. ADAM8 contains two potential phosphorylation sites (Ser758 and Tyr766) and five Src homology 3 (SH3) motifs [37], which may bind adaptor proteins and affect intracellular signaling pathways. Recently, direct interactions of transducer of Cdc42-dependent actin assembly protein 1 (TOCA-1), sorting nexin-33 (SNX33), SNX9, Tec, Src and Cdc42-interacting protein-4 (CIP4) with ADAM8 via SH3-binding motifs was demonstrated, however, the functional relevance of these interactions in cancerogenesis remains to be determined [37].

ADAM8 in tumorigenesis

Malignant transformation of normal cells into tumor cells is a multistep process that requires complex alterations in cell physiology, summarized as the hallmarks of cancer [38]. High ADAM8 expression levels have been reported in various tumor entities and are correlated with invasiveness and poor prognosis (Table 2), providing evidence for an active role of ADAM8 in cancerogenesis. The MP, DI, and CD domains have been shown to constitute independent functional cores of ADAM8 activity (discussed above), thus, dysregulation of ADAM8 in cancer is postulated to promote tumorigenesis through different molecular mechanisms (Figure 2).

Prospective roles of ADAM8 in cancer biology

Figure 2
Prospective roles of ADAM8 in cancer biology

ADAM8 expression correlates with high tumor grade, chemoresistance, and poor survival of cancer patients. Given the different functional cores of ADAM8, proteolytic and non-proteolytic pathways may be involved in ADAM8-mediated cancer progression. (1) Active ADAM8 on the cell surface releases chemokines and cytokines by ectodomain shedding. These soluble ligands diffuse into the surrounding stroma and may stimulate tumor-associated cells, such as inflammatory cells, fibroblasts, and endothelial cells, thereby cultivating a protumorigenic microenvironment. (2) Dysregulated ADAM8 expression in cancer cells contributes to invasiveness by the cleavage of ECM components, like collagen I and fibronectin. Via the DIS domain, ADAM8 can cluster with β1-Integrin and is translocated into the invasive protrusions of tumor cells. By auto-processing of ADAM8, a soluble module of ADAM8 is released extending the range of influence on ECM degradation by diffusing away from the cell membrane. (3) Furthermore, the interaction of ADAM8 and β1-Integrin is essential for downstream signaling involving the MAPK pathway that may induce chemoresistance and can regulate the extracellular activities of MMP-2, -9, and -14, which have been linked to tumor cell invasiveness and metastasis previously. (4) Proteolysis of cell adhesion proteins and ligands may modulate cell motility and adhesion, thereby promoting tumor cell migration. (5) Cleavage of ADAM8 substrates related to angiogenesis could induce sprouting of new vessels. Transmigration of tumor cells is dependent on ADAM8 expression and requires proteolytic degradation of tight junction molecules. Abbreviation: MAPK, mitogen-activated protein kinase.

Figure 2
Prospective roles of ADAM8 in cancer biology

ADAM8 expression correlates with high tumor grade, chemoresistance, and poor survival of cancer patients. Given the different functional cores of ADAM8, proteolytic and non-proteolytic pathways may be involved in ADAM8-mediated cancer progression. (1) Active ADAM8 on the cell surface releases chemokines and cytokines by ectodomain shedding. These soluble ligands diffuse into the surrounding stroma and may stimulate tumor-associated cells, such as inflammatory cells, fibroblasts, and endothelial cells, thereby cultivating a protumorigenic microenvironment. (2) Dysregulated ADAM8 expression in cancer cells contributes to invasiveness by the cleavage of ECM components, like collagen I and fibronectin. Via the DIS domain, ADAM8 can cluster with β1-Integrin and is translocated into the invasive protrusions of tumor cells. By auto-processing of ADAM8, a soluble module of ADAM8 is released extending the range of influence on ECM degradation by diffusing away from the cell membrane. (3) Furthermore, the interaction of ADAM8 and β1-Integrin is essential for downstream signaling involving the MAPK pathway that may induce chemoresistance and can regulate the extracellular activities of MMP-2, -9, and -14, which have been linked to tumor cell invasiveness and metastasis previously. (4) Proteolysis of cell adhesion proteins and ligands may modulate cell motility and adhesion, thereby promoting tumor cell migration. (5) Cleavage of ADAM8 substrates related to angiogenesis could induce sprouting of new vessels. Transmigration of tumor cells is dependent on ADAM8 expression and requires proteolytic degradation of tight junction molecules. Abbreviation: MAPK, mitogen-activated protein kinase.

Table 2
Clinical relevance and biological role of ADAM8 in different cancer types
Cancer type ADAM8 in cancer biology Clinical relevance References 
Breast cancer Migration and invasion Transendothelial migration Angiogenesis MAPK signaling • ADAM8 expression is associated with aggressive phenotype and poor outcome in BC patients.
• Inhibition of ADAM8 by a monoclonal antibody reduces tumor dissemination and metastasis in mice. 
[25,35
Brain cancer Migration and invasion
Chemoresistance
MAPK signaling 
• ADAM8 expression correlates with high tumor grade, poor survival, and temozolomide resistance in glioma patients.
• Up-regulation of ADAM8 in medulloblastoma is correlated with the advanced tumor progression and poor prognosis. 
[36,63,64,66
Leukemia Chemoresistance • Marker of residual CML
• ADAM8 expression is associated with tyrosine kinase inhibitor resistance in cancer stem cells of CML. 
[74
Gastric cancer Migration and invasion
Proliferation
MAPK signaling 
• Serum biomarker
• Independent predictor of lymph node metastases
• ADAM8 expression correlates with higher tumor stages and a shorter patient overall survival. 
[55,58–60,84
Pancreatic cancer Migration and invasion
Integrin signaling
MAPK signaling 
• ADAM8 expression is associated with increased invasiveness and reduced patient survival.
• Inhibition of ADAM8 using a small peptide inhibitor reduced tumor burden and infiltration in mice. 
[26,34,53,54
Hepatocellular carcinoma Migration and invasion
Proliferation 
• Serum biomarker
• High ADAM8 expression is correlated with higher tumor stage, metastasis and shorter overall survival.
• anti-ADAM8 treatment using a monoclonal antibody improved survival rate and slowed down disease progression in mice. 
[56,62,85,86
Colorectal cancer Proliferation • Expression of ADAM8 is associated with cell growth and poor survival. [57
Lung cancer Invasion and migration
Chemoresistance 
• Serum biomarker
• High levels of ADAM8 expression are associated with advanced lung adenocarcinomas and cisplatin resistance.
• A truncated isoform of ADAM8 derived from alternative splicing in lung cancer cells contributes to their aggressive bone metastatic phenotype in mice. 
[72,73
Head and neck tumors N/A • ADAM8 expression is associated with poor patient outcome.
• High ADAM8 expression levels in head and neck squamous carcinoma, but not in serum, have prognostic value. 
[78,87
Osteosarcoma N/A • High ADAM8 expression is an indicator of poor prognosis for patients with osteosarcoma. [77
Prostate cancer N/A • ADAM8 expression in prostate cancer is associated with parameters of unfavorable prognosis and higher Gleason scores. [76
Renal cancer N/A • mRNA expression of ADAM8 is associated with higher tumor grade and shorter survival of patients.
ADAM8 mRNA expression is a predictor of distant metastases. 
[75
Cancer type ADAM8 in cancer biology Clinical relevance References 
Breast cancer Migration and invasion Transendothelial migration Angiogenesis MAPK signaling • ADAM8 expression is associated with aggressive phenotype and poor outcome in BC patients.
• Inhibition of ADAM8 by a monoclonal antibody reduces tumor dissemination and metastasis in mice. 
[25,35
Brain cancer Migration and invasion
Chemoresistance
MAPK signaling 
• ADAM8 expression correlates with high tumor grade, poor survival, and temozolomide resistance in glioma patients.
• Up-regulation of ADAM8 in medulloblastoma is correlated with the advanced tumor progression and poor prognosis. 
[36,63,64,66
Leukemia Chemoresistance • Marker of residual CML
• ADAM8 expression is associated with tyrosine kinase inhibitor resistance in cancer stem cells of CML. 
[74
Gastric cancer Migration and invasion
Proliferation
MAPK signaling 
• Serum biomarker
• Independent predictor of lymph node metastases
• ADAM8 expression correlates with higher tumor stages and a shorter patient overall survival. 
[55,58–60,84
Pancreatic cancer Migration and invasion
Integrin signaling
MAPK signaling 
• ADAM8 expression is associated with increased invasiveness and reduced patient survival.
• Inhibition of ADAM8 using a small peptide inhibitor reduced tumor burden and infiltration in mice. 
[26,34,53,54
Hepatocellular carcinoma Migration and invasion
Proliferation 
• Serum biomarker
• High ADAM8 expression is correlated with higher tumor stage, metastasis and shorter overall survival.
• anti-ADAM8 treatment using a monoclonal antibody improved survival rate and slowed down disease progression in mice. 
[56,62,85,86
Colorectal cancer Proliferation • Expression of ADAM8 is associated with cell growth and poor survival. [57
Lung cancer Invasion and migration
Chemoresistance 
• Serum biomarker
• High levels of ADAM8 expression are associated with advanced lung adenocarcinomas and cisplatin resistance.
• A truncated isoform of ADAM8 derived from alternative splicing in lung cancer cells contributes to their aggressive bone metastatic phenotype in mice. 
[72,73
Head and neck tumors N/A • ADAM8 expression is associated with poor patient outcome.
• High ADAM8 expression levels in head and neck squamous carcinoma, but not in serum, have prognostic value. 
[78,87
Osteosarcoma N/A • High ADAM8 expression is an indicator of poor prognosis for patients with osteosarcoma. [77
Prostate cancer N/A • ADAM8 expression in prostate cancer is associated with parameters of unfavorable prognosis and higher Gleason scores. [76
Renal cancer N/A • mRNA expression of ADAM8 is associated with higher tumor grade and shorter survival of patients.
ADAM8 mRNA expression is a predictor of distant metastases. 
[75

Abbreviations: CML, chronic myeloid leukemia; MAPK, mitogen-activated protein kinase.

Uncontrolled tumor growth, penetration of the basement membrane, and neo-angiogenesis are key events in cancerogenesis [38]. Invasion and migration depend on remodeling of the ECM. ADAM8 has been shown to cleave important ECM components of the tumor stroma such as collagen I [34], fibronectin [39], and periostin [40] and thus could directly contribute to the invasiveness of tumor cells. Membrane-anchored ADAM8 can co-cluster with β1 integrin in protrusions of cancer cells and could direct tumor cell invasion through localized proteolytic ECM degradation in these invasive structures [25,34,35].

Autoprocessing on the cell surface releases a soluble protease module of ADAM8 that is postulated to enhance the levels of ECM degradation by diffusing away from the cell membrane to reach more distant substrates [34,40]. The distribution of soluble ADAM8 via the bloodstream may also prime secondary side organs for metastatic colonization. ADAM8-mediated cellular invasive activity could further be amplified through activation of the mitogen-activated protein kinase (MAPK) signaling pathway, resulting in secretion of other extracellular proteinases, such as matrix-MP (MMP)-2, -9 and -14, which have previously been linked to cancer progression [34,35]. The cross-talk with other MMPs may have an important role in shaping the tumor microenviroment by additional ECM degradation and sequestration of cytokines and growth factors into the pericellular space. The communication of tumor and tumor-associated cells is essential for successful tumor cell survival and colonization [41]. It is a well-established concept that sustained inflammation and cancer progression are closely linked. ADAM8 is abundantly expressed in immune cells, including monocytes [1,2], granulocytes [4,5], dendritic cells, and B cells with the exception of T cells [3]. Immunohistochemical stainings of adenocarcinomas revealed that in addition to its significant expression in tumor cells, ADAM8 is up-regulated in tumor-associated inflammatory cells in the desmoplasmic stroma (Figure 4). Leukocyte recruitment and transmigration into inflamed tissues is critically dependent on ADAM8 [42,43], and shedding of cytokine receptors, such as CD23, TNF-receptor 1, and IL-1 receptor 2, suggest that ADAM8 may contribute to tumor immunosurveillance and survival to apoptotic clues. However, tumor-associated inflammatory cells can be polarized into different activation states by which they elicit various pro- or antitumor functions. Selective aspects of leukocyte activity may limit cancer spread by attacking tumor cells. Particularly in this regard, the specific contribution of ADAM8 in the tumor microenvironment remains elusive and is a matter of intense research.

The induction of angiogenesis to provide oxygen and nutrients for the growing tumor mass is another characteristic of neoplastic cells [44]. ADAM8 is constitutively overexpressed in cancer cells, but can be further up-regulated upon hypoxia [25,26] and may exhibit proangiogenic potential by increasing ectodomain shedding of several membrane proteins with roles in angiogenesis, such as CD31, Tie-2, Flk-1, Flt-1, EphrinB2, EphB4, VE-cadherin, KL-1, E-selectin, and neuregulin-1β2 [25,45]. Following sprouting of new blood vessels into the primary tumor, malignant cells can enter the circulation and travel to distant organs. Detachment from the primary side is proposed to be the result of shedding of membrane-associated adhesion molecules such as L-Selectin [4], CHL-1 [46], APP [47], VCAM-1 [13], PSGL-1 [14], and CD-30 Ligand [48] by ADAM8. In particular, cleavage of PSGL-1 may promote migration through an endothelial layer and tumor cell rolling [35].

No exclusive ADAM8 substrate has been identified yet, however, ADAM8 may process other undetermined membrane-anchored molecules such as chemokines, cytokines, and their receptors, which are related to cancer cell proliferation and progression.

Clinical relevance of ADAM8 in tumors

Breast cancer

Breast carcinoma is the most frequent malignancy and second most common cause of cancer-related deaths in women worldwide [49]. Hormone blockers, chemotherapy, and targetted EGF receptor 2 antibodies significantly improved the post-surgical survival of breast cancer (BC) patients; however, long-term survival remains elusive due to the high risk of distant metastases formation. ADAM8 is abundantly expressed in breast tumors compared with normal breast tissue and is associated with an aggressive phenotype and poor patient outcome [25]. In primary breast tumors, ADAM8-positive tumor cells predominantly accumulate in the invasion zone and ADAM8 expression is maintained during metastasis formation, giving rise to the hypothesis that ADAM8 is a crucial player in breast tumorigenesis [25,35]. Consequently, knockdown of ADAM8 in triple-negative BC cell lines MDA-MB-231 and Hs578t decreased their ability to migrate, to invade through Matrigel in Boyden chamber assay, and to form branched colonies in 3D-Matrigel outgrowth assay in vitro [25]. Inoculation of ADAM8-deficient MDA-MB-231 cells in a mouse orthotopic BC model resulted in profoundly smaller tumors, decreased numbers of circulating tumor cells and lower frequencies of brain metastases, suggesting that ADAM8 promotes tumor growth and dissemination in vivo. The vessel density in tumors derived from ADAM8-deficient MDA-MB-231 cells was significantly reduced compared with tumors derived from MDA-MB-231wild-type cells, indicating that in the absence of ADAM8 tumor angiogenesis is impaired [25]. Confirming these in vivo observations, conditioned supernatants of ADAM8 knockdown, cells failed to stimulate the formation of vessel-like structures by endothelial cells in vitro. Cell binding assays to a confluent endothelial monolayer, mimicking the process of adhesion to the blood vessel wall during intra- and extravasation, revealed that endothelial adhesion of BC cells is critically dependent on ADAM8 and subsequent interaction with β1 integrin [25]. ADAM8 can change the adhesive properties of MDA-MB-231 cells to endothelia by influencing β1 integrin cell-surface localization and activation, thereby changing the cell morphology into an invasive phenotype [25,35,50]. ADAM8-dependent β1 integrin signaling can induce the secretion of miR-720 to maintain the migratory and invasive phenotype of BC cells [51]. Following vessel attachment, the tumor cells penetrate the endothelium to enter the bloodstream or a secondary site organ. ADAM8 overexpression in BC cells significantly promoted migration through an endothelial monolayer dependent on the cleavage of PSGL-1 by ADAM8 in vitro, whereas knockdown or blocking of ADAM8 effectively reduced transmigration [35]. The process of transmigration requires proteolytic degradation of tight junction proteins and may further be amplified by modulating MMP-9 expression via transcriptional mechanisms involving ERK1/2 and CREB signaling. The striking positive correlation of ADAM8 and MMP-9 in 17 different BC cell lines provides evidence for a general regulatory effect of ADAM8 on MMP-9 transcriptional activation [35]. Thus, it is likely that the co-regulation of MMP-9 in conjunction with ADAM8 proteinase activity is an important mechanism for BC progression and metastasis. In accordance with an essential role of ADAM8 in breast cancerogenesis, therapeutic ADAM8 inhibition by a monoclonal antibody targetting the ADAM8 ectodomain revealed promising results in an orthotopic BC mouse model. Intra-peritoneal injection of 0.5 mg/kg anti-ADAM8 antibody twice per week following tumor cell implantation into the mammary fat pad substantially decreased primary tumor burden and size, as well as the number and size of brain metastases [25]. Interestingly, anti-ADAM8 treatment of pre-existing tumors and maintenance treatment for 5 weeks after tumor resection on day 15 profoundly reduced the metastatic potential, closely resembling the clinical situation of BC patients upon initial diagnosis, neoadjuvant therapy, and surgical resection [25]. Following this line of evidence, a therapeutic approach specifically targetting ADAM8 could be of clinical value.

Gastrointestinal cancer

Gastrointestinal cancers are neoplasias of the gastrointestinal tract and the accessory organs of digestion. Amongst the gastrointestinal tumors, pancreatic cancer is one of the most aggressive malignancies with a grim prognosis and a 5-year survival rate less than 5% [52]. Pancreatic Ductal Adenocarcinoma (PDAC) is the most common form of pancreatic cancer and 95% of PDACs are associated with oncogenic mutations in the KRAS gene. The majority of pancreatic tumors strongly express ADAM8 in the ductal adenocarcinoma cells, the tubular complexes and degenerating acinar cells [53], as well as in the tumor-associated inflammatory cells in the desmoplastic stroma [34]. In normal pancreas, ADAM8 expression is very low and restricted to the plasma membrane of ductal cells and, to a lesser extent, to islets and acinar cells. [53]. Elevated ADAM8 mRNA and protein levels in PDAC tumor samples are correlated with a poor patient prognosis and reduced survival [53]. Based on clinical data, Valkovskaya et al. [53] suggested a tumor-promoting role for ADAM8, speculating that enhanced proteolytic activity may result in increased cell migration, invasion, and metastasis in PDAC. To support this hypothesis, functional analyses investigating ADAM8 silencing in the pancreatic cancer cell line AsPC-1 with a high endogenous ADAM8 expression revealed significantly reduced invasive activity that was dose-dependent in a Boyden chamber assay without influencing anchorage-dependent cell growth and proliferation [53]. On mRNA and protein levels ADAM8 was inducible upon experimental hypoxia in seven pancreatic cancer cell lines, indicating the biological relevance of ADAM8 up-regulation in rapidly growing tumors such as PDAC, which is commonly deprived of oxygen [26]. Not only hypoxia, but also co-culture of pancreatic cancer cells with anti-inflammatory macrophages activated ADAM8 expression and subsequently promoted invasive activity of tumor cells [54]. To link the clinical data on ADAM8 expression in PDAC to mechanistic and in vivo data, Schlomann et al. [34] established pancreatic cancer cell lines with a stable knockdown or overexpression of ADAM8, respectively. As described previously, the knockdown of ADAM8 in AsPC-1 cells profoundly reduced migration and invasion in vitro [34]. Accordingly, the overexpression of ADAM8 in PANC-1 cells with low endogenous ADAM8 enhanced the migratory and invasive activity, in particular into different ECM components such as collagen I, collagen IV, fibronectin, and Matrigel [34]. This observed effect on cellular invasiveness was not only attributable to degradation of ECM substrates by ADAM8. In addition, membrane localization of ADAM8 in invasive protrusions of ADAM8 overexpressing PANC-1 cells suggests that ADAM8 may complex with cellular integrins. Consequently, ADAM8 interactions were analyzed by FRET-FLIM analysis using fluorescence-labeled fusion proteins and revealed ADAM8–β1 integrin interactions. These interactions cause β1 integrin activation and intracellular signaling which involves FAK and ERK1/2 activation [34]. Pharmacological inhibition of these respective downstream kinases indicated that enhanced cell migration and invasiveness is also a result of ADAM8-dependent kinase signaling. ADAM8 overexpression and subsequent ERK1/2 activation may further potentiate the invasive potential of PANC-1 cells by increasing the extracellular activities of MMP-14, and to a lesser extent MMP-2 as demonstrated by a global protease activity assay [34]. Confirming the proposed role of ADAM8 in PDAC progression, ADAM8 caused increased invasiveness and tumor growth in vivo in a xenograft model. Mice orthotopically injected into the pancreas with PANC-1 cells overexpressing ADAM8 developed bigger tumors with significant infiltration of the peritoneum, the diaphragm and the spleen and higher frequencies of liver metastases compared with control PANC-1 cells. In accordance, the genetic knockdown of ADAM8 in AsPC-1 cells resulted in a reduction in tumor load and less infiltration of adjacent organs [34]. To confirm ADAM8 inhibition as a therapeutic strategy in PDAC, Bartsch and Koller [34] developed a peptidomimetic ADAM8 inhibitor, BK-1361 (described below), which efficiently reduced tumor load, infiltration, and metastasis of ADAM8 overexpressing PANC-1 cells following orthotopic cancer cell inoculation. To mirror the human pathology more accurately, the therapeutic effect of ADAM8 inhibition in vivo was analyzed in a genetically engineered PDAC mouse model (KrasLSL-G12D, Trp53R172H/+, PdxCre/+ (KPC)). Treatment of KPC with BK-1361 extended overall survival and reduced frequencies of metastases and organ infiltration, suggesting that ADAM8 inhibition might be an effective therapy option in PDAC.

Current knowledge on the role of ADAM8 in the pathophysiology of other gastrointestinal malignancies is less detailed, however, higher ADAM8 expression levels in gastric, colorectal, and hepatocellular cancer significantly correlated with higher tumor stages and a reduced patient survival [55–58]. In gastric cancer (GC), ADAM8 is predominantly expressed in the cytoplasm and membranes of tumor cells. High ADAM8 levels were correlated with higher T stages, N stages, vessel invasion, and a shorter survival time compared with patients with ADAM8 negative primary tumors [58]. ADAM8, together with Factor XIII B, TFIIH p89, COX-2, and CUL-1, was identified as an independent predictor of lymph node metastasis, which is one of the most significant indicators of GC prognosis [59]. Recently, Huang et al. [58] suggested, that ADAM8 overexpression in GC cell lines BGC823 and AGS promoted invasion, migration, and cell growth via p38/ERK-MAPK signaling pathway, but did not affect apoptosis induction. Consistently, ADAM8 silencing in MKN45 GC cells abolished the observed effects [58]. Shen et al. [60] attributed further promigratory stimulation of gastric tumor cells to ADAM8 up-regulation induced by hypoxia and tumor-associated macrophages. To date, no in vivo data exist confirming recent clinical and in vitro findings for GC.

In hepatocellular carcinoma (HCC), high ADAM8 expression rates were reported in 54.3–78.1% of the HCC cases. Elevated ADAM8 expression levels in HCC were associated with serum AFP elevation, higher tumor stage, histologically less-differentiated tumor, and higher frequencies of tumor recurrence and metastasis resulting in a significantly reduced survival [56,61]. Knockdown of ADAM8 in HepG2 HCC cell line inhibited cell growth in both 2D and 3D conditions, and reduced cell migration and invasion. Consistent with the in vitro data, orthotopic implantation of ADAM8-deficient HepG2 cells resulted in a significantly smaller tumor in an HCC xenograft model [56]. Pharmacologic inhibition of ADAM8 was shown to increase survival rate and reduce morbidity in a mouse model inducing HCC by diethylnitrosamine [62]. The mechanism for this effect, however, remains unclear, since no data on proteolytic activity of ADAM8 were presented.

In colorectal cancer (CRC), ADAM8 is increased in the cancerous tissue compared with adjacent normal tissues. Reduced overall survival was correlated to tumors with high ADAM8 expression [57]. In the same study, ADAM8 was silenced in two CRC cell lines, HT29 and SW480, resulting in proliferation inhibition and apoptosis induction [57]. Thus, in all tumor entities described, ADAM8 serves functions in tumor progression and therapy resistance.

Brain cancer

Gliomas are primary brain tumors originating from astrocytes, oligodendrocytes, or ependymal cells. Glioblastoma multiforme (GBM) is the most frequent and most malignant neoplasm of the brain in adults with a dismal mean survival of 15 months despite multimodal treatments. In glioma tissues, ADAM8 expression was correlated with higher histopathologic tumor grades and shorter patient survival [63,64]. ADAM8 was mainly detected in the cytoplasm of glioblastoma cells, whereas the expression was absent or low in non-neoplastic brain sections [63,64]. By using peptides based on known ADAM8 substrates, Wildeboer et al. [64] were able to demonstrate that ADAM8 mRNA and protein levels in glioma correlated with the biological ADAM8 activity in respective tumor extracts, suggesting that ADAM8 may contribute to glioma progression and invasiveness. In C6 glioma cells, migratory and invasive activity was inducible upon transfection of either human or mouse ADAM8, which was dependent on the proteolytic activity, since exchanging critical glutamate residues in the catalytic side of ADAM8 abolished the effect [64]. Dong et al. [36] demonstrated that ADAM8, together with MMP-1, -9, and -14, is up-regulated in recurrent glioblastoma compared with the primary tumor manifestation in patients following chemotherapeutic treatment with temozolomide (TMZ), suggesting that the induction of the respective proteases could be mechanistically relevant for chemoresistance and invasive behavior of recurrent GBM cells [36]. In vitro, both in established glioblastoma cell line U87 as well as in primary glioblastoma cells derived from patients, ADAM8 mRNA and activity were significantly induced following systemic chemotherapy with TMZ. Treatment of the respective GBM cells with TMZ combined with the effective MP broad-spectrum inhibitor batimastat (BB-94) sensitized cells dose-dependently within 3–5 days, whereas cotreatment of TMZ and marimastat (BB-2516), an ADAM8-sparing broad range MMP inhibitor did not affect TMZ-induced cell death [36]. Similarly, pharmacological ADAM8 inhibition using BK-1361 and genetic ADAM8 silencing in U87 cells (highly endogenous ADAM8) caused sensitization to TMZ in a cell culture model. Analysis of intracellular signaling mechanisms revealed that ADAM8 caused chemoresistance via activation of pERK1/2 and pAKT [36]. However, the TMZ-mediated induction of metalloproteinases including ADAM8 not only affected chemoresistance, but also contributed to glioma progression by increasing the invasive capacity of TMZ-resistant GBM cells in vitro, which is a major reason for inoperability of these tumors [36]. In preliminary experiments, ADAM8 expressed in GBM cells causes high proliferation rates and an increase in tumor mass, which was less pronounced in GBM cells deficient in ADAM8 (Conrad, Schlomann, Bartsch et al., unpublished results). In summary, these data suggest that ADAM8 could be a major protease in glioblastoma progression and may limit efficacy of standard GBM therapy. Thus, specific inhibition of ADAM8 in future therapy regimen could optimize TMZ chemotherapy and prevent formation of recurrent GBM.

Medulloblastoma is the most common malignant brain tumor of childhood, comprising ∼20% of all pediatric brain tumors [65]. Although survival has improved during the past decades, many patients suffer from therapy-related side effects including persisting neurological deficits, and an increased risk of developing secondary tumors. In immunohistological staining, 73% of the pediatric medulloblastomas analyzed were positive for ADAM8, in particular undifferentiated tumors with an aggressive phenotype. In 15 pairs of fresh frozen medulloblastoma and adjacent normal cerebellum samples, ADAM8 expression was significantly up-regulated on mRNA and protein levels in the neoplastic tissue [66]. Follow-up of 66 medulloblastoma patients revealed an association of high ADAM8 expression with advanced metastatic stage and reduced survival [66]. These data provide evidence that the up-regulation of ADAM8 in medulloblastoma tissues might be correlated with the advanced tumor progression and poor prognosis, however, functional and in vivo data are missing to date in this tumor entity.

Lung cancer

Lung cancer is one of the most common cancers worldwide and is the leading cause of cancer mortality [67,68]. Non-small cell lung cancer (NSCLC) accounts for 85% of all lung cancer cases, and adenocarcinoma is the most frequent histological subtype [69]. In healthy lung tissue, ADAM8 expression is weak in lung epithelial cells, and absent from smooth muscle and endothelial cells, however, the interstitial inflammatory cells are positive for ADAM8 in immunohistological sections [70]. In lung cancer, ADAM8 is overexpressed in tumor cells and is located at the plasma membrane and in the cytoplasm. The expression levels of ADAM8 in adenocarcinomas are associated with advanced tumor stages (IIIB–IV) and poor prognosis [71]. Serum levels of ADAM8 are significantly higher in lung cancer patients than in healthy controls, suggesting that ADAM8 might serve as a biomarker and a target for novel anticancer therapies [71]. Given the clinical relevance of ADAM8 in lung cancer, Hernández et al. [72] investigated the functional role of ADAM8 in lung cancer cell lines and identified two spliced isoforms (Δ18a and Δ14′) encoding truncated proteins, which were present in several tumor cell lines, but not in normal lung epithelial cells. The ADAM8 variant Δ18a results from an additional exon 18 of 179 bp within the EGF-like domain, encoding an alternative carboxy-terminal domain of 95 amino acids with a new possible N-glycosylation site [72]. The other alternatively spliced ADAM8 isoform Δ14′ encompassed 14 exons and retained intron 14, inducing a novel frameshift in the Cysteine-rich domain with a premature stop codon and deleting a possible N-glycosylation site [72]. These truncated ADAM8 isoforms showed different functions compared with the native protein. Full-length ADAM8, and both Δ18a and Δ14′ isoforms increased the invasive activity of lung cancer cells in vitro, as demonstrated by invasion assays using A549 and H460 cells retrovirally overexpressing the respective ADAM8 isoform. Interestingly, Δ14′ expression levels were markedly up-regulated following co-culture with stromal cells and the soluble Δ14′ isoform induced osteoclast formation in bone marrow macrophages, suggesting that tumor–stroma interactions may exacerbate Δ14′ ADAM8-mediated invasiveness and tumor-induced osteolysis [72]. In accordance, lung cancer cells overexpressing Δ14′ increased prometastatic activity with higher tumor burden and increased osteolysis in a murine model of bone metastasis in vivo. Thus, the expression of truncated forms of ADAM8 by lung cancer cells may up-regulate metastatic bone colonization [72].

Besides a metastasis promoting function of ADAM8 in lung cancer, Zhang et al. [73] suggested that ADAM8 may contribute to mediate chemoresistance of NSCLC cells. ADAM8 overexpression in A549 and H460 lung cancer cell lines, both with low endogenous ADAM8 levels, resulted in attenuation of cisplatin-induced cytotoxicity increasing IC50 values by 1.85-fold in A549 and 3.91 in H460 cells, whereas ADAM8 silencing in H647 lung cancer cells, with highly endogenous ADAM8 expression, sensitized the cells to cisplatin and increased cisplatin-induced apoptosis by activation of the STAT3 signaling pathway [73]. These results indicate that pharmacological ADAM8 inhibition may represent an additive therapeutic modality to overcome treatment failures of conventional chemotherapies due to development of chemoresistance in NSCLC.

Leukemia

Leukemia is a form of cancer originating from hematopoetic stem cells in the bone marrow. Based on the lineage of the neoplastic cell population (myeloid or lymphocytic) and the rate of disease progression, acute and chronic leukemia are distinguished by having different treatment options. Chronic myeloid leukemia (CML) is a myeloproliferative neoplasm usually growing slowly (chronic phase), but will eventually progress rapidly to the lethal phase (accelerated phase or blast crisis). Unlike the three other leukemia subtypes, the phenotype of CML cells is initiated and maintained by chromosomal translocation t(9;22) (q34;q11), resulting in the BCR-ABL oncogenic tyrosine kinase. Despite therapeutic improvements by specifically targetting the fusion protein BCR-ABL with tyrosine kinase inhibitors (TKI) during the chronic phase, the mechanisms of the genetic switch that drives CML progression under TKI treatment remain less known. Recently, Miyauchi et al. [74] identified elevated ADAM8 levels in primary samples of CML patients during the chronic phases and postulated ADAM8 as a marker of TKI-resistant stem cells and residual CML cells. In this study, hematopoietic cells derived from induced pluripotent stem cells of two CML patients with chromosomal translocation t(9;22) (q34;q11) resembling a CML stem cell phenotype were cultures and sorted for ADAM8 expression. ADAM8-positive TKI-resistant CML stem cell subpopulations showed reduced BCR-ABL expression, whereas BCR-ABL levels remained unaffected when ADAM8 was absent [74]. Lentiviral ADAM8 knockdown using shRNA in primary ADAM8-positive CML samples reversed TKI resistance in viability assays, suggesting an active role of ADAM8 in chemoresistance.

Other cancer types

Recently, data on other malignancies remain merely descriptive with no links to clinical findings and ADAM8-mediated mechanisms in tumorigenesis. An association of ADAM8 overexpression and clinical outcome in cancerous diseases was initially described for renal cell carcinoma (RCC) [75]. The study of Roemer et al. [75] revealed that the expression of ADAM8 in RCC was related to a shorter survival of patients and was the best predictor of distant metastases. Fritzsche et al. [76] demonstrated that ADAM8 protein expression in 128 prostate cancer tissues was significantly associated with higher tumor grade, positive lymph nodal status, higher Gleason score, and unfavorable prognosis. However, the authors did not determine significant prognostic value for prostate-specific antigen relapse-free survival with ADAM8 expression due to inconclusive correlations between mRNA expression and measured protein levels [76]. Li et al. [77] reported ADAM8 overexpression in 88.4% of osteosarcoma in a cohort of 69 patients, which was associated with metastasis, tumor recurrence, and a poorer overall and disease-free patient survival. A retrospective study in squamous cell head and neck including 148 patients indicated that ADAM8 overexpression in this tumor entity is associated with reduced survival and ADAM8 serum levels in the ELISA correlated well with immunohistochemistry expression levels. However, similar to the findings in prostate cancer, soluble ADAM8 levels did not show any prognostic or diagnostic properties [78].

ADAM8 in chemoresistance of cancer cells

Overcoming intrinsic and acquired drug resistance is a major challenge in the treatment of cancer patients experiencing recurrency and tumor dissemination. ADAM8 has been shown to provide a link to drugs causing activation of the DNA damage response (DDR) and inhibition of tyrosine kinases. As drugs causing DDR, TMZ is used as a standard therapy in gliomas [36], whereas cisplatin is used for treatment of lung and BC. ADAM8 knockdown caused sensitization of A549 lung carcinoma cells treated with cisplatin [73] and BC cells overexpressing ADAM8 are more resistant to chemotherapy with cisplatin and carboplatin (Goette, Conrad, Bartsch, unpublished results). In addition to mediating resistance to DNA damaging drugs, a recent article described ADAM8 as an antigen of TKI-resistant CML cells [74]. Knockdown or inhibition of ADAM8 caused re-sensitization of CML cells to the TKI imatinib [72], demonstrating that the metalloproteinase activity is required to confer chemoresistance. Thus, there might be two reasons for the observed chemotherapy resistance either dependent on the activation of ADAM8-dependent intracellular signaling pathways involving phosphorylation of ERK1/2 and Akt/PI3K in the case of DNA damaging drugs, or on shedding of (unknown) substrates that lead to imatinib resistance.

Evaluation of ADAM8 as biomarker

Early cancer detection and monitoring of disease progression are essential in cancer patient survey. Ideally, less invasive screening for soluble biomarkers in blood or other body fluids is favored but remains a challenge in molecular cancer diagnostics. Soluble molecules shed from the cell surface of cancer cells reflect the composition of the tumor and provide a complex source of potential biomarkers. In this context, ADAM8 has been initially described as a potential biomarker in lung cancer. In serologic samples from lung cancer patients, levels of soluble ADAM8 were significantly higher (431 ± 249 pg/ml) compared with healthy individuals (267 ± 56 pg/ml). Serum ADAM8 was already detectable in patients with low-stage tumors, suggesting that ADAM8 may be an early onset biomarker [71]. However, in prostate cancer and squamous cell head and neck tumors soluble ADAM8 levels did not show any prognostic or diagnostic properties [76,78]. These contradictive data suggest that mere ADAM8 protein expression does not reflect the biological activity of the molecule. In addition, ADAM8 is up-regulated by various inflammatory stimuli, thus, activation of the immune system under any pathological condition other than cancer may cause false positive results. Consequently, there has to be a clear delimitation of the usefulness of ADAM8 as tumor marker compared with a general inflammation marker. When considering ADAM8 compartmentation, i.e. in extracellular vesicles (EVs), it is more likely that ADAM8 can be useful as a biomarker (Figure 3).

ADAM8 autocatalytic activation and processing

Figure 3
ADAM8 autocatalytic activation and processing

For cellular activity, ADAM8 requires homophilic multimerization of at least two ADAM8 monomers on the cell membrane, resulting in autocatalytic removal of the prodomain in the trans-Golgi network (upper zoom). After membrane transport, ADAM8 can cleave membrane-bound proteins or autoprocess itself, resulting in removal of a soluble metalloproteinase module and formation of remnant ADAM8 (lower zoom). Alternatively, ADAM8 may be secreted in EVs by cancer cells (modified from [24,34]).

Figure 3
ADAM8 autocatalytic activation and processing

For cellular activity, ADAM8 requires homophilic multimerization of at least two ADAM8 monomers on the cell membrane, resulting in autocatalytic removal of the prodomain in the trans-Golgi network (upper zoom). After membrane transport, ADAM8 can cleave membrane-bound proteins or autoprocess itself, resulting in removal of a soluble metalloproteinase module and formation of remnant ADAM8 (lower zoom). Alternatively, ADAM8 may be secreted in EVs by cancer cells (modified from [24,34]).

ADAM8 expression in adenocarcinomas

Figure 4
ADAM8 expression in adenocarcinomas

High expression levels of ADAM8 in several tumor entities are associated with invasiveness and predict poor patient outcome. In the tumor lesion, ADAM8 is predominantly expressed in neoplastic cancer cells. Representative histologic stainings are shown for breast, pancreatic, and lung carcinoma (upper panel). Note that ADAM8 expression is not limited to tumor cells, but is also prominent in tumor-associated cells of the desmoplastic stroma (arrowheads, lower panel, matched) (scale bar: 100 μm).

Figure 4
ADAM8 expression in adenocarcinomas

High expression levels of ADAM8 in several tumor entities are associated with invasiveness and predict poor patient outcome. In the tumor lesion, ADAM8 is predominantly expressed in neoplastic cancer cells. Representative histologic stainings are shown for breast, pancreatic, and lung carcinoma (upper panel). Note that ADAM8 expression is not limited to tumor cells, but is also prominent in tumor-associated cells of the desmoplastic stroma (arrowheads, lower panel, matched) (scale bar: 100 μm).

Newer methodologies using fluorescence-based approaches to sensitively track real-time protease activities indicate that specific activation profiles could more precisely reflect whether a condition is benign or malignant. Assuming that distinct patterns of protease activation are characteristic for each condition, multiplexing activities may also allow for discrimination of cancer and inflammation. Roy et al. [79] demonstrated in a proof-of-concept study that fluorescent substrate cleavage activity in the urine of BC patients was feasible to predict disease status. By using substrates based on the physiological cleavage sites of proteins that are shed semi-selectively by particular ADAMs, the observed activities were attributed to ADAM8 and ADAM12. Notably, cleavage of CD23-based ADAM8 substrates in the urine samples discriminated healthy controls from BC patients most promisingly [79]. However, none of the individual substrates in this study was useful in detecting the presence of other stages of the disease. Thus, Miller et al. [80] developed a combined experimental and mathematical method based on time-lapse fluorescence measurements of a panel of moderately specific FRET-based polypeptides and proteinase inhibitors (Proteolytic Matrix Activity Assay (PrAMA)). In a pilot study, our group investigated simultaneous detection of metalloproteinase activities in cerebrospinal fluid (CSF) samples of patients with neoplastic meningitis (NM). Interestingly, PrAMA computational inference implied increased activities including ADAM8 in CSF samples from NM patients [81]. Although all these data suggest that ADAM8 activity may be a useful biomarker in cancer, further validation in extended studies and integration into clinical applications is required.

Therapeutic strategies to target ADAM8

From the findings presented above, it can be anticipated that specific ADAM8 inhibition would be beneficial in preventing invasion and probably immune evasion in cancer. Given the non-essential role of ADAM8 under physiological conditions, targetting ADAM8 represents a promising novel therapeutic approach with minor expected side effects.

Early MP inhibitors exhibited poor selectivity and high toxicity, therefore, one of the biggest challenges is to attain specific inhibition of particular disease-relevant ADAMs [82]. As hydroxamate inhibitors, such as batimistat (BB-94), are unlikely to work specifically on ADAM proteinases due to the conserved MP domain of the metzincins, new avenues have to be pursued to inhibit ADAM8 based on structural modeling (Figure 1). Crystallizing ADAM8 in a complex with batimastat revealed that the S1′ specifity loop (residues 355–375) in the MP domain may be selectively accessible to hydroxamate-based small molecule inhibitors [32]. An alternative approach is to target the DIS domain of ADAM8 using a monoclonal antibody that has been shown to be efficient in an orthotopic BC model [25].

Experimentally, a peptide-based approach aimed to disrupt ADAM8 multimerization by masking the integrin binding loop within the DIS domain, thereby potentially inhibiting subsequent ADAM8 autoactivation and downstream signaling selectively [34]. It was postulated that a unique three amino acid motif ‘KDM’, residues 470–472 of human ADAM8 and KDK in mouse ADAM8, located in the integrin binding loop of ADAM8 is exposed on the outside of the DI-domain and forms a contact surface accessible to small peptides. To target the exposed three amino acid motifs, a short hexamer peptide was designed in a cyclic conformation, cyclo(RLsKDK) (BK-1361), mimicking the structure of the integrin binding loop. BK-1361 blocked ADAM8 activation with high potency (IC50 = 182 nM) and high specificity in vitro, functionally resulting in reduced cell adhesion, migration, invasion, and β1 integrin activation in cell-based assays. Using the inhibitory peptide in both an orthotopic and a genetic model of pancreatic cancer demonstrated a reduction in tumor burden and metastasis that is an in vivo proof-of-concept. Interestingly, unpublished data of our group investigating BC cell lines suggest synergistic efficacy of BK-1361 when combined with traditional chemotherapies such as cisplatin, carboplatin, and taxanes. The pharmacological inhibition of ADAM8 may result in a loss of chemoresistance, possibly mediated by preventing the interaction with β1 integrin, which has been implicated in mediating therapy resistance previously. The inhibition of ADAM8 via the integrin binding loop is a promising strategy for cancer therapy, however, the development of more potent and more stable peptidomimetics or therapeutic antibodies that block the catalytic activity of ADAM8 might also be required to identify a valid drug candidate for clinical trials [83].

Despite encouring data obtained so far, it needs to be taken into account that general systemic ADAM8 inhibition without cancer cell specifity may also have undesired effects with regard to natural tumor defense mechanisms. Inflammatory cells have the potential to exacerbate, but also to limit cancer spread depending on the balance of protumor and antitumor immunity. Analyses of leukocyte subpopulations in ADAM8−/− mice revealed that the CD4:CD8 T-cell ratio is lower (Bartsch et al., unpublished results), suggesting that inhibition of ADAM8 could attenuate the antitumoral response mediated by cytotoxic T cells. Whether these hypothesized side effects have clinical relevance remains to be investigated. To avoid proposed side effects of general ADAM8 inhibition, it could also be feasible to specifically target ADAM8 positive tumor cells, i.e. by ADAM8 antibody–coupled liposome formulations with encapsulated cytotoxic drugs released after uptake.

Acknowledgements

This review is dedicated to the 80th birthday of Harald Jockusch, a very inspiring mentor.

Competing interests

The authors declare that there are no competing interests associated with the manuscript.

Author contribution

C.C., U.S., L.C., designed and performed the experiments from which the data are derived. C.C. and J.W.B., J.B. and K.D. conceived the conceptual idea, C.C. and J.W.B. wrote the manuscript and illustrated the graphical artwork and the tables. E.P.S., A.Z., and C.N. provided input to the development of the conceptual idea and revised the manuscript. All authors read and approved the final manuscript.

Funding

This work on ADAM8 was supported by the Deutsche Forschungsgemeinschaft [grant numbers BA1606/3-1 (to J.W.B. and U.S.), BA1606/4-1 (Project B2 of the CRC Unit 325) (to J.W.B. and E.P.S.)].

Abbreviations

     
  • ADAM

    A Disintegrin And Metalloproteinase

  •  
  • AFP

    Alpha-Fetoprotein

  •  
  • APP

    Amyloid Precusor Protein

  •  
  • BB-94

    Batimastat

  •  
  • BC

    Breast Cancer

  •  
  • CD

    Cytoplasmic Domain

  •  
  • CHL-1

    Neural Cell Adhesion Molecule L1-like Protein

  •  
  • CIP4

    Cdc42-interacting Protein-4

  •  
  • CML

    Chronic Myeloic Leukemia

  •  
  • CRC

    Colorectal Cancer

  •  
  • CREB

    cAMP Response Element-Binding Protein

  •  
  • DDR

    DNA Damage Response

  •  
  • DIS

    Disintegrin

  •  
  • ECM

    Extracellular Matrix

  •  
  • EGF

    Epidermal Growth Factor

  •  
  • ERK

    Extracellular Regulated Kinase

  •  
  • FAK

    Focal Adhesion Kinase

  •  
  • GBM

    Glioblastoma Multiforme

  •  
  • GC

    Gastric cancer

  •  
  • HCC

    Hepatocellular Carcinoma

  •  
  • IL

    Interleukin

  •  
  • MAPK

    Mitogen-activated Protein Kinase

  •  
  • MMP

    Matrix-Metalloprotease

  •  
  • MP

    Metalloprotease

  •  
  • NSCLC

    Non-small Cell Lung Cancer

  •  
  • PDAC

    Pancreatic Ductal Adenocarcinoma

  •  
  • PSGL-1

    P-Selectin Glycoprotein Ligand-1

  •  
  • RCC

    Renal Cell Carcinoma

  •  
  • SH3

    Src-Homology 3

  •  
  • SNX33

    Sorting Nexin-33

  •  
  • Src

    Sarcoma

  •  
  • STAT3

    Signal Transducer and Activator of Transcription 3

  •  
  • Tec

    Tyrosine-proteine Kinase Tec

  •  
  • TKI

    Tyrosine Kinase Inhibitor

  •  
  • TM

    Transmembrane

  •  
  • TMZ

    Temozolomide

  •  
  • TOCA-1

    Transducer of Cdc42-dependent Actin Assembly Protein 1

  •  
  • VCAM-1

    Vascular Cell Adhesion Protein 1

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