A disintegrin and metalloproteinases (ADAMs) are a new gene family of proteins with sequence similarity to the reprolysin family of snake venomases that share the metalloproteinase domain with matrix metalloproteinases (MMPs). They are structurally classified into two groups: the membrane-anchored ADAM and ADAM with thrombospondin motifs (ADAMTS). These molecules are involved in various biological events such as cell adhesion, cell fusion, cell migration, membrane protein shedding and proteolysis. Studies on the biochemical characteristics and biological functions of ADAMs are in progress, and accumulated lines of evidence have shown that some ADAMs are expressed in malignant tumors and participate in the pathology of cancers. The activities of ADAMs are regulated by gene expression, intracytoplasmic and pericellular regulation, activation of the zymogens and inhibition of activities by inhibitors. Many ADAM species, including ADAM8, ADAM9, ADAM10, ADAM12, ADAM15, ADAM17, ADAM19, ADAM28, ADAMTS1, ADAMTS4 and ADAMTS5, are expressed in human malignant tumors. Many of them are involved in the regulation of growth factor activities and integrin functions, leading to promotion of cell growth and invasion, although the precise mechanisms of these are not clear at the present time. In this article, we review recent information about ADAM family members and their implications for cancer cell proliferation and progression. (Cancer Sci 2007; 98: 621–628)
Modulation of tissue microenvironment through degradation of the ECM, processing of growth factors and activation of cell adhesion molecules is essential to cancer cell proliferation and progression. MMPs may play a central role in these processes, and elevated expression and activation of MMPs have been reported in many human cancer tissues.(1) Biochemical and clinicopathological analyses have suggested that the expression and activation of proMMP-2 by membrane-type 1 MMP and proMMP-7 anchored to cell membranes by CD151, a member of the tetraspanin family, are important in cancer cell invasion and metastasis in human malignant tumors.(2)
In recent years, however, members of the ADAM family of proteins, an MMP-related metalloproteinase family, have attracted attention, and functional analyses of ADAMs are ongoing. ADAMs are multifunctional proteins involved in the proteolytic processing of other transmembrane proteins, cell adhesion and cell signaling events. Many transmembrane proteins are processed by one or several proteolytic steps to the biologically active configuration. Examples include growth factors such as EGF, HB-EGF and TGF-α, and cytokines such as TNF-α, all of which are synthesized as precursors. In addition, there are a number of cell surface receptors that undergo cleavage near the transmembrane domain, a process called ectodomain shedding. These include TNF-α receptor-I, TNF-α receptor-II, CD44, L-selectin and Erb4/HER4. The soluble, released ectodomains of the receptors may be part of the downmodulation in response to ligand activation, or they may have a function of their own. It has become clear over the past few years that ADAMs play a major role in these processes.
Another function of ADAMs lies in their ability to act as ligands for integrins by competing with matrix proteins.(3) In addition, ADAMs have cell adhesion properties mediated by the interaction of their cysteine-rich domains with other proteins such as syndecans(4) and fibronectin.(5) The cytoplasmic tails of some ADAMs contain SH3-binding sites, which can potentially activate SH3 domain-containing signaling molecules such as src and grb.(3) Furthermore, recent studies have demonstrated that several ADAMs are highly expressed in cancer cells and cancer tissues. Although information about the functions of ADAMs in cancers is still limited, it is worth reviewing the biochemical and biological characteristics of ADAMs and their involvement in human cancer cell proliferation and progression according to the data of our group and other studies.
Members of the ADAM gene family
There are two groups in the ADAM family: membrane-anchored ADAM (Table 1) and secreted-type ADAMTS (Table 2). ADAM members are composed of common domains including propeptide, metalloproteinase, disintegrin, cysteine-rich, EGF-like, transmembrane and cytoplasmic domains, whereas ADAMTS members contain thrombospondin motifs, cysteine-rich and spacer domains in addition to propeptide, metalloproteinase and disintegrin domains (Fig. 1). Several ADAM genes give rise to more than one protein due to differential splicing of mRNA. This causes the synthesis of secreted ADAM proteins, in addition to membrane-anchored forms, or variation in the length of the cytoplasmic tail of ADAM proteins.
Table 1. The a disintegrin and metalloproteinase (ADAM) gene family
Shedding of TNF-α, TGF-β, TNF-p75 receptor, ErbB4, TRANCE and HB-EGF, presence of RRKR sequence, cleavage of APP, Notch, L-selectin and CD44
Macrophage, various tissues
Formation of neuron, digestion of neuregulin
α4β1 and α5β1
Formation of sperm
Sperm–egg binding and fusion
e-MDC II, MDC-Lm, MDC-Ls
Digestion of myelin basic protein and IGFBP-3
α4β1, α4β7 and α9β1
Testis, lung, lymphocyte, pancreas, uterus
Mutation in bronchial asthma patients, cleavage of APP, KL-1 and insulin B chain
α4β1, α5β1 and α9β1
Lung (flbroblast, smooth muscle)
Table 2. The a disintegrin and metalloproteinase with thrombospondin motifs (ADAMTS) gene family
Functions and biochemical features
C3-C5, METH1, KIAA1346
Binding to heparin, presence of RRKR sequence, digestion of aggrecan and versican
Kidney, heart, cartilage
Procollagen N-proteinase, hPCPNI, PCINP
Processing of collagen I and II N-propeptides
Processing of collagen N-propeptides
KIAA0688, aggrecanase-1, ADMP-1
Digestion of aggrecan, brevican and versican
Brain, heart, cartilage
ADAMTS11, aggrecanase-2, ADMP-2
Digestion of aggrecan
Uterus, placenta, cartilage
Digestion of aggrecan, inhibition of angiogenesis
Digestion of aggrecan
Cleavage of von Willebrand factor
Liver, prostate, brain
Processing of collagen N-propeptides
Digestion of aggrecan
Liver (fetus), kidney (fetus)
Prostate, brain, uterus
Prostate, brain, liver
According to differences in the active site sequence of the metalloproteinase domain, 60% of the members are non-proteolytic ADAM molecules, which are not discussed in this review. In contrast, active sites in the metalloproteinase domain of the proteinase-type ADAM molecules (ADAM8, -9, -10, -12, -15, -17, -19, -20, -21, -28, -30 and -33) contain a common HEXGHXXGXXHD sequence with a ‘Met-turn’, which is also present in the catalytic metalloproteinase domain of MMP members. For an up-to-date and authoritative list of all ADAMs, refer to the website of Dr Judith White at the University of Virginia (see http://www.people.virginia.edu/%7Ejw7g/Table_of_the_ADAMs.html). Among these ADAM proteins, proteinase activities have been demonstrated for ADAM8, -9, -10, -12, -15, -17, -19, -28 and -33 (Table 1). One of the major functions of ADAMs is shedding of membrane proteins. ADAM17 has been most extensively examined and is known to release soluble TNF-α from its membrane precursor, thus called TACE.(3) ADAM9, ADAM10 and ADAM17 can cleave amyloid precursor protein at the α-secretase processing site.(6) ADAM9 is implicated in the ectodomain shedding of membrane-anchored proHB-EGF.(7) ADAM12 also acts as a sheddase for proHB-EGF.(8) We have recently reported that ADAM28 cleaves IGFBP-3 at a site between the Arg97–Ala98 bond present in the central domain of this molecule.(9) Like MMPs, some ADAMs can also degrade ECM: ADAM10 cleaves type IV collagen,(10) ADAM12 digests gelatin, type IV collagen and fibronectin,(11) and ADAM15 cleaves type IV collagen and gelatin in vitro.(12)
The ADAMTS group comprises 19 members, all of which are proteinase type (Table 2). Information about their substrates and biological functions remains limited, but ADAMTS1, -2, -3, -4, -5, -8, -9 and -15 are ECM-degrading protein- ases. ADAMTS1, -4, -5, -8, -9 and -15 cleave specific Glu–X bonds (where X is most often Ala or Glu) of the core protein of aggrecan.(13,14) Due to their aggrecan-degrading activity, ADAMTS4 and ADAMTS5 are called aggrecanase-1 and aggrecanase-2, respectively,(15,16) and brevian and versican are also cleaved by ADAMTS4.(17,18) It is well known that ADAMTS 13 is a von Willebrand factor-cleaving proteinase, and its mutation causes thrombotic thrombocytopenic purpura.(19)
Regulation of ADAM activity
ADAM activities can be regulated by several mechanisms, such as gene expression, intracytoplasmic and pericellular regulation, zymogen activation and inhibition by inhibitors, although the regulation mechanisms are not completely understood.
Gene expression. The levels of ADAM17 mRNA expression show a 90% reduction in the lungs of knockout Foxm1 mice(20) indicating that foxm1, a transcription factor, may regulate the expression of ADAM17 in vivo. Expression of ADAM8 is induced via interferon-regulating factor 1 by treatment of the granular cells with TNF-α.(21) Sung et al. recently reported that intracellular reactive oxygen species and hydrogen peroxide, generated by cell stress, are inducers of ADAM9 expression.(22) In addition, the expression of ADAM12 is upregulated in hepatic stellate cells treated with TGF-β(23) and this effect is via phosphatidylinositol-3 kinase and MAPK kinase activities.
Intracytoplasmic and pericellular regulation. The intracytoplasmic tails of most ADAM contain putative recognition motifs for signaling proteins and adaptors.(3) Thus, it is plausible that these proteins could participate in the regulation of metalloproteinase activity or subcellular localization of ADAM. TPA-induced ADAM activation has been widely established. In the TPA-stimulated processing of HB-EGF, PKCδ is involved.(7) PKCδ is considered to directly associate with and phosphorylate the cytoplasmic domain of ADAM9, and then HB-EGF shedding occurs. Similarly, TPA-stimulated shedding of the nerve growth factor receptor by ADAM17 appears to involve phosphorylation of the ADAM17 cytoplasmic tail.(24) Concerning subcellular localization of ADAM, PKCɛ is known to induce ADAM12 translocation to the cell surface depending on catalytic activity of PKCɛ.(25) In addition, extracellular signal-regulated kinase-dependent phosphorylation of Thr735 in ADAM17 molecules induces translocation of ADAM17 to the cell surface.(26) It is possible to speculate that modification of the intracytoplasmic tail domains of ADAMs causes their conformational changes, leading to activation of the ADAMs through cleavage of their prodomains. It is also possible that interaction with associated proteins relocates ADAMs to specialized regions of the membranes resulting in clustering of ADAM molecules, enabling them to interact with specific substrates. However, these possibilities remain to be elucidated by further work.
Zymogen activation. Some ADAM members such as ADAM12 and ADAM17 have the furin-recognition site (RXXR sequence) at the end of the propeptide domain, and proprotein convertases such as furin can activate these proADAMs intracellularly.(27) ADAMTS4 is also activated intracellularly through processing of the propeptide by furin. However, the activation mechanism of other ADAMs without furin-recognition sites is not well known. We have reported that MMP-7 processes proADAM28 into the active form.(9) Although MMP-7 may not be only an activator for proADAM28, the data suggest a possible activation cascade of proADAMs by the action of MMPs. In addition to removal of the propeptide, secondary processing takes place and this may be important for some ADAMs to exhibit their full activity, especially in ADAMTS members. The C-terminal spacer region is cleaved with MT4-MMP(28) and truncation of the spacer region appears to result in full proteinase activity of ADAMTS4. The C-terminal fragment of ADAMTS4 is also reported to function as a competitor for the activity of the mature enzyme.(16) In addition, ADAMTS1 is cleaved within its spacer region by MMP-2, MMP-8 and MMP-15, leading to two distinct active forms.(29) However, it is not known whether these secondary cleavages are essential to many ADAM species. Thus, further studies are definitely needed to clarify the activation mechanism of ADAM species.
Inhibition of activities by inhibitors. The activities of ADAMs can be inhibited by TIMPs, which were originally cloned as endogenous inhibitors of MMPs.(30) They are composed of four different members of 21–28 kDa with 40–50% sequence homology (i.e. TIMP-1, -2, -3 and -4). TIMP-1, -2, -3 and -4 all inhibit MMP activity by binding in a 1 : 1 molar ratio to form tight, non-covalent complexes, although TIMP-1 does not inhibit MT-MMPs efficiently.(30) In contrast to universal inhibition of MMPs by TIMPs, the activities of ADAMs are inhibited mainly by TIMP-3. TIMP-3 efficiently inhibits the activity of ADAM10,(31) ADAM12,(32) ADAM17,(31) ADAM28(9) and ADAM33,(33) as well as ADAMTS4 and ADAMTS5,(34) although some inhibitory activity of TIMP-1, TIMP-2 and TIMP-4 to ADAM10,(31) ADAM17,(6) ADAM28(9) and/or ADAM33(33) have also been reported. Based on the data of the constructed mutants of TIMP-3 that disrupt the interaction with MMP but not ADAM17, the inhibition mechanism of TIMP-3 for ADAM17 is considered to be different to that for MMP,(35) although the N-terminal subdomain of loops 1 through 3 is essential to the inhibition of both MMP and ADAM17. In contrast, ADAM8, ADAM9 and ADAM19 are not inhibited by any TIMP.(36,37) Because of the limited inhibitor activity to ADAM and tissue distribution of TIMP, it is plausible that besides TIMP, there must be inhibitor molecules to ADAM. Actually, our recent study demonstrated that ADAMTS4 is inhibited through interaction with fibronectin.(38) Further studies on inhibition mechanism of ADAM activities by new molecules are necessary.
Several synthetic inhibitors have been developed. The catalytic activity of ADAM8 and ADAM19 is sensitive to the hydroxamic acid-type inhibitor BB94 (Batimastat) (IC50 for ADAM8 = 50 nM), which was developed for inhibition of MMP.(37,39) The most efficient inhibitor compound for ADAM9 is CGS27023 (Kj = 1 nM).(40) Asakura et al.(8) have selected KB-R7785 as one of the most potent inhibitors for HB-EGF shedding by ADAM12 from over 2000 metalloproteinase inhibitors. KB-R7785 also completely inhibits ADAM28 activity at 1 µM.(9) Inhibitors selective to each ADAM member have been developed and the inhibitory activity of GI254023X to ADAM10 is more than 100-fold higher than that to ADAM17.(41) In addition, INCB3619 inhibits both ADAM17 and ADAM10 300-fold more efficiently (IC50 for ADAM17 = 14 nM and IC50 for ADAM10 = 22 nM) than ADAM9 and ADAM33.(42)
Expression and functions of ADAM in cancers
There are many reports showing that members of the ADAM family are overexpressed in human cancers. These include the expression of ADAM8, ADAM9, ADAM10, ADAM12, ADAM15, ADAM17, ADAM19, ADAM28, ADAMTS1, ADAMTS4 and ADAMTS5 (Table 3). Although most of the reports describe the expression without functional analyses, expression and the possible roles of each ADAM species in cancers can be reviewed as follows.
Table 3. Expression of a disintegrin and metalloproteinases (ADAMs) in human cancers and their possible functions
ADAM8 (CD156/MS2). Ishikawa et al. screened genes encoding transmembrane/secretory proteins that are upregulated in lung cancers by cDNA microarrys, and found that ADAM8 is specifically overexpressed in most cancer tissues and elevated in serum samples from the patients(43) (Table 3). They also showed that transfection of ADAM8 into tumor cells enhances the invasive activity. Overexpression of ADAM8 has also been reported in human renal cell carcinomas (Table 3). Furthermore, ADAM8 is highly regulated in human primary brain tumors such as astrocytomas, and the expression levels and activity are associated with invasiveness.(44) These reports suggest that ADAM8 is involved in tumor cell migration and invasion.
ADAM9 (MDC9, Meltrin γ). ADAM9 is reportedly expressed in an active form at higher levels in breast cancers with lymph node metastasis than in those without metastasis, and correlates positively with HER-2/neu protein levels.(45) Similarly, ADAM9 is overexpressed in several cancers such as pancreatic cancer, stomach cancer, skin melanoma and hepatocellular carcinoma (Table 3). A recent experimental study using a mouse model has demonstrated that ADAM9 contributes to prostate carcinogenesis by cleaving EGF receptor ligands and the receptor for fibroblast growth factor.(46) ADAM9, secreted by activated stromal cells, is known to induce colon carcinoma cell invasion in vitro through binding to α6β4 and α2β1 integrins.(47) In addition, ADAM9 enhances cell adhesion and invasion of non-small cell lung carcinoma cells via modulation of α3β1 integrin and sensitivity to growth factors, and thus promotes brain metastasis of the carcinoma cells.(48) Therefore, it is suggested that ADAM9 plays a role in tumorigenesis, invasion and metastasis through modulation of growth factor activity and integrin function.
ADAM10 (Kuzbanian, MADM). Overexpression of ADAM10 appears to promote the growth of oral squamous cell carcinoma and gastric carcinoma, as downregulation of its expression with antisense oligonucleotides or treatment with anti-ADAM10 antibodies reduces proliferation of the carcinoma cells.(49,50) ADAM10-mediated L1 release is reported to enhance tumor dissemination by increasing cell migration in ovarian and uterine carcinomas.(51) L1 is also involved in the motility and invasion of lymphoma, lung carcinoma and melanoma cells, where ADAM10 seems to be a major L1-sheddase in these tumor cell lines.(52,53) ADAM10 is also overexpressed in leukemia and prostate cancer (Table 3).
ADAM12 (Meltrin α, MCMP, MLTN). Our group has examined the mRNA expression of 13 different ADAM species with putative metalloproteinase activity in human astrocytic tumors by reverse transcription–polymerase chain reaction, and found that ADAM12m is predominantly expressed in an active form in glioblastomas.(54) In that study, processing of proHB-EGF, a substrate of ADAM12m, was observed in glioblastoma tissues depending on ADAM12m expression levels, and was inhibited by treatment of the glioblastomas with ADAM inhibitor (KB-R7785), suggesting that ADAM12m plays a key role in the proliferation of glioblastoma cells through shedding of HB-EGF. The cysteine-rich domain of ADAM12 is known to support tumor cell adhesion through syndecans, which triggers signaling events and leads to β1 integrin-dependent cell spreading.(4,55) Roy et al. have disclosed that the ADAM12 levels in urine correlate with breast cancer progression, suggesting the possibility that ADAM12 may be a diagnostic marker for breast cancers and their progression.(11) They also report that ADAM12 cleaves various ECM molecules including gelatin, type IV collagen and fibronectin, suggesting a potential role for this enzyme in ECM digestion in cancer invasion and metastasis. ADAM12 expressed by carcinoma cells accelerates breast tumor progression through induction of stromal cell apotosis.(56) ADAM12 is also upregulated in cancers of stomach, liver and colon (Table 3). These data suggest that ADAM12 functions as a sheddase, adhesion molecule and ECM-degrading proteinase, and is involved in cancer progression.
ADAM15 (MDC15, Metargidin). Lung carcinoma tissues and cell lines frequently express ADAM15, but the expression has no significant correlation with tumor stage or degree of differentiation.(57) Expression of ADAM15 is upregulated in various cancers of the breast, prostate, stomach and lung (Table 3), and treatment of carcinoma cell lines with anti-ADAM15 antibodies reduces cell proliferation.(50,58) However, the recombinant disintegrin domain of human ADAM15 is reported to be a potent intrinsic inhibitor of angiogenesis, tumor growth and metastasis.(59) Similarly, ADAM15 is shown to decrease integrin αvβ3/vitronectin-mediated ovarian cancer cell adhesion and motility in an RGD-dependent fashion.(60) Thus, the data on the role of ADAM15 in cancers are inconsistent.
ADAM17 (TACE). ADAM17 is overexpressed in cancers of the breast, ovary, kidney, colon and prostate (Table 3). Interestingly, data showing that inhibition of ADAM17-mediated shedding of proTGF-α reduces the size of xenografts in nude mice suggest that ADAM17 is a target for tumorigenesis.(61) Treatment of breast cancer cell lines with anti-ADAM17 antibodies leads to a decrease in cell proliferation.(58)
ADAM19 (Meltrin β, FKSG34). ADAM19 is highly expressed in human primary brain tumors (Table 3), and the expression and activity are associated with invasiveness.(44) ADAM19 is also highly expressed in renal cell carcinomas (Table 3), but the functions of ADAM19 in cancers remain to be elucidated.
ADAM28 (MDC-Lm, MDC-Ls). We have recently reported that among the 11 different ADAM species with putative metalloproteinase activity, membrane-anchored ADAM28m and secreted-type ADAM28s are expressed predominantly in non-small cell lung carcinomas, showing positive correlations with cancer cell proliferation and lymph node metastasis.(62) In addition, we have demonstrated that both ADAM28m and ADAM28s are selectively overexpressed by carcinoma cells in human invasive breast carcinomas.(63) In that study, our in vitro and in vivo experiments have shown that inhibition or downregulation of ADAM28 activity by an ADAM inhibitor or ADAM28 small interfering RNA attenuates cell proliferation and IGF-I-induced cell signaling through decreased IGFBP-3 degradation. Based on these data, ADAM28 is considered to promote breast carcinoma cell proliferation through enhanced bioavailability of IGF-I (Fig. 2). Our preliminary study using yeast two-hybrid screening has shown that ADAM28s binds to PSGL-1 and promotes leukocyte rolling adhesion on endothelial cells (Shimoda H, Okada Y, unpublished data). Because some prostate carcinoma cell lines express PSGL-1,(64) studies on the role of ADAM28s in cancer metastasis through interaction with PSGL-1 are now under way in our laboratory. The mRNA expression level of ADAM28 is also increased in renal cell carcinomas (Table 3). ADAM28 may play a key role in cancer cell proliferation and metastasis.
ADAMTS1 (METH1, KIAA1346). Liu et al. have reported that full-length ADAMTS1 promotes pulmonary metastasis of murine mammary carcinoma (TA3) and Lewis lung carcinoma cells by stimulating tumor cell proliferation, survival and invasion, and tumor angiogenesis through shedding of HB-EGF and amphiregulin precursors.(65) However, they have suggested that ADAMTS1 fragments are likely to inhibit tumor metastasis by negatively regulating HB-EGF and amphiregulin. ADAMTS1 is also highly upregulated in breast cancers with elevated metastatic activity (Table 3).
ADAMTS4 and ADAMTS5 (aggrecanase-1 and aggrecanase-2). ADAMTS4 and ADAMTS5 are upregulated in proliferating glioblastoma cells and these proteinases may contribute to their invasive potential.(66) In addition, our study has demonstrated that ADAMTS5 is overexpressed in glioblastomas and cleaves brevican,(67) suggesting that ADAMTS5 may play a role in glioma cell invasion.
In this article, we have reviewed the characteristics of ADAMs, regulation of their activity and their involvement in cancer cell proliferation and progression. Figure 3 summarizes recent information about ADAM in cancer biology. First, ADAMs are synthesized as inactive proADAMs, which may be activated by the action of furin or MMPs or by other unknown pathways. Second, a main role of ADAMs is shedding of growth factors such as TGF-α and HB-EGF, and this processing may alter signaling on the surfaces of cancer cells, resulting in enhanced cell proliferation by autocrine and paracrine mechanism. In addition, sheddase activity of ADAMs may be regulated by cell signaling such as through the PKC and MAPK pathway. Third, ADAMs function as adhesion molecules through binding to integrins or syndecans via their disintegrin and cysteine-rich domains, which might help the cell to digest the substrates. Fourth, ADAMs may indirectly regulate cell proliferation signals through integrins. Fifth, the role of ADAMs in cancer development and metastasis may be linked to their proteinase activity to other unknown membrane-anchored molecules, which may be cytokines, chemokines and their receptors. Like MMPs, ADAMs can cleave ECM molecules and thus cancer cells invade readily and adhere to new locations to establish a secondary site of growth. However, there are still many unanswered questions about the regulation and function of ADAMs. The development of cancer models such as transgenic mice overexpressing ADAM species or selective inhibitors for each ADAM will help to determine the role of ADAMs in carcinogenesis and cancer progression in vivo.