ADAM 12 Cleaves Extracellular Matrix Proteins and Correlates with Cancer Status and Stage

ADAM 12 is a member of a family of disintegrin-containing metalloproteases that have been implicated in a variety of diseases including Alzheimer's disease, arthritis, and cancer. We purified ADAM 12 from the urine of breast cancer patients via Q-Sepharose anion exchange and gelatin-Sepharose affinity chromatography followed by protein identification by matrix-assisted laser desorption/ionization-time of flight mass spectrometry. Four peptides were identified that spanned the amino acid sequence of ADAM 12. Immunoblot analysis using ADAM 12-specific antibodies detected an ∼68-kDa band identified as the mature form of ADAM 12. To characterize catalytic properties of ADAM 12, full-length ADAM 12-S was expressed in COS-7 cells and purified. Substrate specificity studies demonstrated that ADAM 12-S degrades gelatin, type IV collagen, and fibronectin but not type I collagen or casein. Gelatinase activity of ADAM 12 was completely abrogated by zinc chelators 1,10-phenanthroline and EDTA and was partially inhibited by the hydroxamate inhibitor Marimastat. Endogenous matrix metalloprotease inhibitor TIMP-3 inhibited activity. To validate our initial identification of this enzyme in human urine, 117 urine samples from breast cancer patients and controls were analyzed by immunoblot. The majority of samples from cancer patients were positive for ADAM 12 (67 of 71, sensitivity 0.94) compared with urine from controls in which ADAM 12 was detected with significantly lower frequency. Densitometric analyses of immunoblots demonstrated that ADAM 12 protein levels were higher in urine from breast cancer patients than in control urine. In addition, median levels of ADAM 12 in urine significantly increased with disease progression. These data demonstrate for the first time that ADAM 12 is a gelatinase, that it can be detected in breast cancer patient urine, and that increased urinary levels of this protein correlate with breast cancer progression. They further support the possibility that detection of urinary ADAM 12 may prove useful in the development of noninvasive diagnostic and prognostic tests for breast and perhaps other cancers. ADAM 12 is a member of a family of disintegrin-containing metalloproteases that have been implicated in a variety of diseases including Alzheimer's disease, arthritis, and cancer. We purified ADAM 12 from the urine of breast cancer patients via Q-Sepharose anion exchange and gelatin-Sepharose affinity chromatography followed by protein identification by matrix-assisted laser desorption/ionization-time of flight mass spectrometry. Four peptides were identified that spanned the amino acid sequence of ADAM 12. Immunoblot analysis using ADAM 12-specific antibodies detected an ∼68-kDa band identified as the mature form of ADAM 12. To characterize catalytic properties of ADAM 12, full-length ADAM 12-S was expressed in COS-7 cells and purified. Substrate specificity studies demonstrated that ADAM 12-S degrades gelatin, type IV collagen, and fibronectin but not type I collagen or casein. Gelatinase activity of ADAM 12 was completely abrogated by zinc chelators 1,10-phenanthroline and EDTA and was partially inhibited by the hydroxamate inhibitor Marimastat. Endogenous matrix metalloprotease inhibitor TIMP-3 inhibited activity. To validate our initial identification of this enzyme in human urine, 117 urine samples from breast cancer patients and controls were analyzed by immunoblot. The majority of samples from cancer patients were positive for ADAM 12 (67 of 71, sensitivity 0.94) compared with urine from controls in which ADAM 12 was detected with significantly lower frequency. Densitometric analyses of immunoblots demonstrated that ADAM 12 protein levels were higher in urine from breast cancer patients than in control urine. In addition, median levels of ADAM 12 in urine significantly increased with disease progression. These data demonstrate for the first time that ADAM 12 is a gelatinase, that it can be detected in breast cancer patient urine, and that increased urinary levels of this protein correlate with breast cancer progression. They further support the possibility that detection of urinary ADAM 12 may prove useful in the development of noninvasive diagnostic and prognostic tests for breast and perhaps other cancers. ADAMs 1The abbreviations used are: ADAM, a disintegrin and metalloprotease; ADAM 12-L, membrane-anchored long form of ADAM 12; ADAM 12-S, secreted form of ADAM-12; ADH, atypical ductal hyperplasia; LCIS, lobular carcinoma in situ; DCIS, ductal carcinoma in situ; IBC, locally invasive breast cancer; ROC, receiver operating characteristic; MALDI-TOF, matrix-assisted laser desorption ionization time-of-flight; CHAPS, 3-[(3-cholamidopropyl)dimethylammonio]-1-propanesulfonic acid; EGF, epidermal growth factor; MMP, matrix metalloprotease; ECM, extracellular matrix; TIMP, tissue inhibitor of metalloproteinases; IGFBP, insulin-like growth factor-binding protein. (adisintegrin and metalloprotease) are a family of integral membrane and secreted glycoproteins that are related to snake venom metalloproteases and matrix metalloproteases (MMPs). These proteases are multidomain proteins composed of a prodomain, metalloprotease domain, disintegrin domain, and a cysteine-rich region, and membrane-anchored ADAMs also contain a transmembrane and cytoplasmic domain. ADAMs display diverse roles in cell surface remodeling, ectodomain shedding, regulation of growth factor availability, and in mediating cell-cell and cell-matrix interactions in both normal development and pathological states such as Alzheimer's disease, arthritis, cancer, and cardiac hypertrophy (1Buxbaum J.D. Liu K.N. Luo Y. Slack J.L. Stocking K.L. Peschon J.J. J. Biol. Chem. 1998; 273: 27765-27767Abstract Full Text Full Text PDF PubMed Scopus (839) Google Scholar, 2Chubinskaya S. Mikhail R. Deutsch A. Tindal M.H. J. Histochem. Cytochem. 2001; 49: 1165-1176Crossref PubMed Scopus (37) Google Scholar, 3Seals D.F. Courtneidge S.A. Genes Dev. 2003; 17: 7-30Crossref PubMed Scopus (897) Google Scholar). Human ADAM 12 is expressed as two alternatively spliced forms: a membrane-anchored long form (ADAM 12-L) and a shorter secreted form (ADAM 12-S) (4Gilpin B.J. Loechel F. Mattei M.G. Engvall E. Albrechtsen R. Wewer U.M. J. Biol. Chem. 1998; 273: 157-166Abstract Full Text Full Text PDF PubMed Scopus (284) Google Scholar). ADAM 12-S is an active protease known to cleave IGFBP-3 and IGFBP-5 (5Loechel F. Gilpin B.J. Engvall E. Albrechtsen R. Wewer U.M. J. Biol. Chem. 1998; 273: 16993-16997Abstract Full Text Full Text PDF PubMed Scopus (188) Google Scholar, 6Loechel F. Fox J.W. Murphy G. Albrechtsen R. Wewer U.M. Biochem. Biophys. Res. Commun. 2000; 278: 511-515Crossref PubMed Scopus (277) Google Scholar, 7Shi Z. Xu W. Loechel F. Wewer U.M. Murphy L.J. J. Biol. Chem. 2000; 275: 18574-18580Abstract Full Text Full Text PDF PubMed Scopus (168) Google Scholar). Recently, ADAM 12 has been reported to play a role in cardiac hypertrophy, cleaving membrane-bound heparin-binding-EGF in response to G protein-coupled receptor stimulation of cardiomyocytes leading to shedding of heparin-binding EGF from the cell surface and transactivation of the EGF receptor (8Asakura M. Kitakaze M. Takashima S. Liao Y. Ishikura F. Yoshinaka T. Ohmoto H. Node K. Yoshino K. Ishiguro H. Asanuma H. Sanada S. Matsumara Y. Takeda H. Beppu S. Tada M. Hori M. Higashiyama S. Nat. Med. 2002; 8: 35-40Crossref PubMed Scopus (641) Google Scholar). Increased expression of ADAM 12 mRNA and protein has been reported in breast, colon, and lung carcinoma tissue (9Iba K. Albrechtsen R. Gilpin B.J. Loechel F. Wewer U.M. Am. J. Pathol. 1999; 154: 1489-1501Abstract Full Text Full Text PDF PubMed Scopus (183) Google Scholar). In vitro, the cysteine-rich domain of ADAM 12 has been shown to support tumor cell adhesion mediated through syndecans and integrin cell surface receptors to generate signals for cell adhesion and spreading (10Iba K. Albrechtsen R. Gilpin B. Frohlich C. Loechel F. Zolkiewska A. Ishiguro K. Kojima T. Liu W. Langford J.K. Sanderson R.D. Brakebusch C. Fassler R. Wewer U.M. J. Cell Biol. 2000; 149: 1143-1155Crossref PubMed Scopus (223) Google Scholar). ADAM 12 mRNA levels are increased almost 6-fold in hepatocellular carcinoma, and its expression correlates with tumor aggressiveness and progression (11Le Pabic H. Bonnier D. Wewer U.M. Coutand A. Musso O. Baffet G. Clement B. Theret N. Hepatology. 2003; 37: 1056-1066Crossref PubMed Scopus (184) Google Scholar). MMPs, a class of matrix-degrading enzymes, play an important role in tumor growth and metastasis and are involved in the remodeling of the tumor microenvironment (12Lochter A. Sternlicht M.D. Werb Z. Bissell M.J. Ann. N. Y. Acad. Sci. 1998; 857: 180-193Crossref PubMed Scopus (104) Google Scholar, 13Kleiner D.E. Stetler-Stevenson W.G. Cancer Chemother. Pharmacol. 1999; 43: S42-S51Crossref PubMed Scopus (648) Google Scholar). Elevated levels of MMPs have been measured in body fluids such as serum and plasma of human patients and in tumor-bearing animals. More recently, we have reported that MMPs can also be detected in urine of patients with a variety of cancers including prostate, bladder, renal, and breast and serve as independent predictors of disease status (14Moses M.A. Wiederschain D. Loughlin K.R. Zurakowski D. Lamb C.C. Freeman M.R. Cancer Res. 1998; 58: 1395-1399PubMed Google Scholar). Our research has also identified an ∼125-kDa gelatinase species in urine of cancer patients as a complex of MMP-9 with neutrophil gelatinase-associated lipocalin and showed its presence in urine to be predictive of disease status (15Yan L. Borregaard N. Kjeldsen L. Moses M.A. J. Biol. Chem. 2001; 276: 37258-37265Abstract Full Text Full Text PDF PubMed Scopus (580) Google Scholar). A number of studies have confirmed these original findings in a variety of human cancers; however, the presence of other proteases as disease markers in urine has not yet been thoroughly explored. The present study is the result of a biomarker identification initiative in our group whose goal is to identify proteins present in urine of cancer patients and to determine whether their presence might be relevant to disease status. Using anion exchange and affinity chromatographic steps followed by MALDI-TOF mass spectrometry, we identified ADAM 12 in urine from breast cancer patients. This result was confirmed by Western blot analysis of urine using ADAM 12-specific antibodies. Comparative analysis of urine from breast cancer patients and healthy individuals showed not only that detection of ADAM 12 in urine is indicative of disease status but also that levels of this enzyme in urine correlate with stages of disease. The physiological substrate of ADAM 12 has not yet been determined. Given the importance of extracellular matrix degradation in tumorigenesis and metastasis we tested the ability of this metalloprotease to degrade ECM components. We found that ADAM 12 can degrade various ECM proteins including gelatin, type IV collagen, and fibronectin, suggesting a potential role for this enzyme in ECM remodeling, a hallmark of neoplastic disease. Identification of Urinary ADAM 12—Initial identification of ADAM 12 was made using 50 ml of urine (total protein ∼5 mg). Urine was concentrated by ultrafiltration using an Amicon membrane XM-50 (Millipore Corporation, Bedford, MA). The sample was diluted with 2 volumes of 50 mm Tris, pH 7.5 (buffer A) and applied to a Q-Sepharose column. After washing, a gradient of 0–400 mm NaCl in buffer A was applied. Fractions containing gelatinase activity were pooled, the NaCl concentration was adjusted to 200 mm, and the fractions were incubated with gelatin-Sepharose (Amersham Biosciences) for 16 h at 4 °C with constant shaking. After a wash step, elution was performed using a stepwise gradient of 5, 10, and 20% Me2SO, respectively, in buffer A containing 200 mm NaCl. Eluted fractions were concentrated and separated via SDS-PAGE under non-reducing conditions and stained using the Sterling rapid silver stain kit (National Diagnostics, Atlanta, GA) according to the manufacturer's instructions. Several distinct bands of ∼52, 80, 120, and 140 kDa were detected. Protein bands were excised from the gel, subjected to tryptic digest, and analyzed by MALDI-TOF mass spectrometry (Perceptive STR, Applied Biosystems, Framingham, MA) to determine the molecular weights of the proteins and for peptide mapping of the tryptic digests using a 337-nm wavelength laser for desorption and the reflectron mode of analysis. Using the MSFit search program the peptide maps generated were searched against a FASTA data base of public domain proteins constructed of protein entries in the non-redundant data base held by the NCBI and Swiss-Prot. Peptide matches identified by MSFit were filtered according to their MOWSE (molecular weight search) score, percentage of masses matched, molecular weight, and number of observations of peptides and proteins. For conservative identification of proteins at least two peptides with good scores that match a single protein are required. Expression and Purification of ADAM 12-S—The full-length human ADAM 12-S gene was cloned into a plasmid (p1151), and transfection of COS-7 cells (ATCC, CRL 1651) was conducted as described previously (5Loechel F. Gilpin B.J. Engvall E. Albrechtsen R. Wewer U.M. J. Biol. Chem. 1998; 273: 16993-16997Abstract Full Text Full Text PDF PubMed Scopus (188) Google Scholar). Expressed recombinant ADAM 12-S secreted into the conditioned medium was purified as described previously with minor modifications. Briefly, conditioned medium from transfected COS-7 cells was subjected to successive steps of SP-Sepharose and concanavalin A-Sepharose chromatography as described (6Loechel F. Fox J.W. Murphy G. Albrechtsen R. Wewer U.M. Biochem. Biophys. Res. Commun. 2000; 278: 511-515Crossref PubMed Scopus (277) Google Scholar). ADAM 12-S containing fractions from the concanavalin A-Sepharose column were pooled and after buffer exchange applied to a Vivapure H Mini Q column (VivaScience, Carlsbad, CA) pre-equilibrated with buffer A containing 0.05% CHAPS. Pure ADAM 12-S was detected in the flow-through fraction as determined by SDS-PAGE and immunoblot analysis. Substrate Specificity Studies—Gelatin-, type IV collagen-, fibronectin-, and casein-degrading activities of ADAM 12-S was measured as described previously (14Moses M.A. Wiederschain D. Loughlin K.R. Zurakowski D. Lamb C.C. Freeman M.R. Cancer Res. 1998; 58: 1395-1399PubMed Google Scholar). ADAM 12-S (1 μm) was mixed with non-reducing sample buffer and separated on a 10% polyacrylamide gel containing either 0.1% gelatin, 0.1% type IV collagen, or 0.02% fibronectin and a 12% polyacrylamide gel containing 0.1% casein. After electrophoresis, the gel was washed with 2.5% Triton X-100 for 30 min. Substrate digestion was conducted by incubating the gel in 50 mm Tris, pH 7.6, containing 5 mm CaCl2, 1 μm ZnCl2, and 0.02% NaN3 at 37 °C for 18 h. Gels were stained with 0.1% Coomassie, and bands of enzyme activity were detected as zones of clearance on a background of uniform blue staining. To determine whether ADAM 12 possessed type I collagen-degrading activity, pure enzyme (1 μm) was analyzed using a radiometric collagenase assay as described previously (16Moses M.A. Sudhalter J. Langer R. Science. 1990; 248: 1408-1410Crossref PubMed Scopus (424) Google Scholar). For inhibition assays, ADAM 12 (160 nm) was incubated with 30 μg of gelatin in assay buffer (50 mm Tris, pH 7.5) in the presence or absence of various inhibitors: 5 mm 1,10-phenanthroline, 5 mm EDTA, 1 mm Marimastat, or two different concentrations (100 and 500 nm) of either TIMP-1, TIMP-2, TIMP-3, or TIMP-4 in a final volume of 30 μl at 37 °C for 18 h. Reactions were stopped with the addition of non-reducing sample buffer, and the samples were resolved by SDS-PAGE analysis followed by Coomassie staining. Urine Sample Collection and Processing—Urine collection was performed as described previously (14Moses M.A. Wiederschain D. Loughlin K.R. Zurakowski D. Lamb C.C. Freeman M.R. Cancer Res. 1998; 58: 1395-1399PubMed Google Scholar). Samples were collected in sterile containers and immediately frozen at -20 °C. Urine was collected according to the institutional bioethical guidelines pertaining to discarded clinical material. Prior to analysis, urine was tested for the presence of blood and leukocytes using Multistix 9 urinalysis strips (Bayer, Elkhart, IN), and samples containing blood or leukocytes were excluded. Creatinine concentration of urine was determined using a commercial kit (Sigma) according to the manufacturer's protocol. Protein concentration of urine was determined by the Bradford method using bovine serum albumin as the standard. Patient Population—117 urine samples were analyzed for the presence of ADAM 12. The control group consisted of 46 women (median age ∼50 years) with no discernable disease. The breast cancer group included 71 patients diagnosed at various stages of breast cancer including atypical ductal hyperplasia (ADH), lobular carcinoma in situ (LCIS), ductal carcinoma in situ (DCIS), locally invasive breast cancer (IBC), and metastatic disease (see Fig. 3B). Western Blot Analysis—Equal amounts of proteins (20 μg) were separated by SDS-PAGE under reducing conditions. Resolved proteins were electrophoretically transferred to nitrocellulose membranes (Schleicher & Schuell, Keene, NH) and treated as described previously (15Yan L. Borregaard N. Kjeldsen L. Moses M.A. J. Biol. Chem. 2001; 276: 37258-37265Abstract Full Text Full Text PDF PubMed Scopus (580) Google Scholar). Labeled proteins were visualized with enhanced chemiluminescence (Pierce). Polyclonal antibodies against human ADAM 12, rb122 (4Gilpin B.J. Loechel F. Mattei M.G. Engvall E. Albrechtsen R. Wewer U.M. J. Biol. Chem. 1998; 273: 157-166Abstract Full Text Full Text PDF PubMed Scopus (284) Google Scholar) and S-18 (sc-16526, Santa Cruz Biotechnology), were used at 1 μg/ml and 2 μg/ml concentration, respectively. Band intensities were analyzed with UN-SCAN-IT™ (Silk Scientific, Orem, UT) software digitizer technology. Statistical Analysis—Sensitivity, specificity, false-positive rate, false-negative rate, and likelihood ratio were calculated using standard formulas. The presence of ADAM 12 was compared between cancer groups (ADH/LCIS, DCIS, IBC, and metastatic disease) and controls using logistic regression analysis. For each group, the presence of ADAM 12 is expressed as a percentage, and 95% confidence intervals were calculated based on the normal approximation. The nonlinear relationship between ADAM 12 level and breast cancer was modeled using logistic regression analysis with probability determined by maximum likelihood estimation for selected intervals of ADAM 12 (17Breslow N.E. Day N.E. Statistical Methods in Cancer Research: The Analysis of Case-control Studies. 1. International Agency for Research on Cancer, Lyon, France1980: 192-224Google Scholar). Median levels of ADAM 12 were compared between controls and cancer patients using the Kruskal-Wallis test with pairwise comparisons based on Mann-Whitney U tests, whereas mean levels were compared by analysis of variance. To evaluate the diagnostic accuracy of ADAM 12, receiver operating characteristic (ROC) curve analysis was utilized to plot all pairs of true positives and false positives for varying levels of ADAM 12. ROC curves were constructed for differentiating between all breast cancer patients and controls and separately for each cancer group. ROC curves that go up steeply from (0, 0) and reach near (0, 1) indicate a test that is almost perfect in discriminating cancer patients from controls, whereas curves close to the 45° diagonal line have low discriminatory power. Area under the curve with 95% confidence intervals were determined using the Hanley-McNeil algorithm as an index of the performance of ADAM 12 as a biomarker of disease. In addition, the area under the curve equals the probability that a randomly chosen pair of patients and controls will be correctly classified. Actual numbers of cancer patients and controls are depicted as histograms with the theoretical probability curve superimposed to illustrate the increasing likelihood of breast cancer for higher levels of ADAM 12. To account for multiple comparisons among the five independent groups (four breast cancer groups according to stage of disease and the normal control group), a conservative two-tailed criterion of p < 0.01 (0.05 divided by 5) was regarded as statistically significant. All statistical analysis was performed using the SAS statistical package (version 6.12, SAS Institute, Cary, NC). A power analysis indicated that a minimum of 13 patients in each of the breast cancer stages would provide 80% power for detecting differences in ADAM 12 level (version 5.0, nQuery Advisor, Statistical Solutions, Boston, MA). Isolation and Identification of ADAM 12—Urine from breast cancer patients was subjected to stepwise chromatography and in an effort to isolate proteases an affinity chromatography step was performed using gelatin-Sepharose. The fact that ADAM 12 bound gelatin-Sepharose beads supported our data (see below) that this enzyme has gelatin-degrading capability. The fraction collected when 20% Me2SO was applied to the gelatin-Sepharose column contained several distinct ∼52-, 80-, 120-, and 140-kDa protein bands (Fig. 1A, lane 2). MALDI-TOF analysis of the ∼120-kDa protein band (Fig. 1A, lane 2,*) identified it as a disintegrin and metalloprotease domain 12 (ADAM 12) (accession NP_003465). Four distinct peptides spanning the amino acid sequence of human ADAM 12 were identified (Fig. 1B). The high MOWSE score, percentage of masses matched, and the number of peptides identified predicted the presence of ADAM 12 in the protein band analyzed with high confidence. Peptide 1 and peptide 2 corresponded to regions of the prodomain, peptide 3 corresponded to a region in the catalytic domain, and peptide 4 corresponded to the C-terminal region of ADAM 12-L. To confirm these results, a larger cohort of urine samples including samples from breast cancer patients as well as age- and sex-matched controls was analyzed for ADAM 12. Urine samples were concentrated, normalized for protein (20 μg of total protein was used for each analysis), and subjected to Western blot analysis. In non-reduced urine samples, a polyclonal ADAM 12-specific antibody, rb122, recognized an ∼120-kDa protein band (Fig. 1C, lane 1), whereas an ∼68-kDa band was detected when the sample was reduced with β-mercaptoethanol and boiled for 5 min (Fig. 1C, lane 2). Immunoblot analysis with other ADAM 12-specific antibodies, polyclonal antibody S-18 and monoclonal antibodies 8F8 and 6E6, gave similar results (data not shown). Based on molecular mass the ∼68-kDa band was identified as the mature form of ADAM 12-S (5Loechel F. Gilpin B.J. Engvall E. Albrechtsen R. Wewer U.M. J. Biol. Chem. 1998; 273: 16993-16997Abstract Full Text Full Text PDF PubMed Scopus (188) Google Scholar). In light of the fact that the ADAM 12-specific antibody detected the mature form of the protein in urine of cancer patients the identification of two peptides from the prodomain of ADAM 12 by mass spectrometry was an interesting finding. It has been reported that the ADAM 12 prodomain remains associated with the protease even after furin cleavage (6Loechel F. Fox J.W. Murphy G. Albrechtsen R. Wewer U.M. Biochem. Biophys. Res. Commun. 2000; 278: 511-515Crossref PubMed Scopus (277) Google Scholar) under non-reducing conditions. This suggests that tryptic cleavage (an essential step preceding mass spectrometric analysis) of this mature ADAM 12-associated propeptide resulted in the detection of the two peptides from the prodomain. Although the size of the protein band detected by immunoblot analysis using ADAM 12-specific antibody is most consistent with that of mature ADAM 12-S, the polyclonal antibody rb122 recognizes the cysteine-rich region of ADAM 12, a region of shared homology in both the -L and -S forms. Therefore we cannot rule out the possibility that the band detected is a processed form of ADAM 12-L. Full-length ADAM 12-L has a reported mass of ∼120 kDa, whereas the mature (without the propeptide) membrane-anchored form is ∼90 kDa (18Cao Y. Kang Q. Zhao Z. Zolkiewska A. J. Biol. Chem. 2002; 277: 26403-26411Abstract Full Text Full Text PDF PubMed Scopus (57) Google Scholar). Although the ∼120-kDa species analyzed by mass spectrometry contained the full-length protein, immunoblot analysis indicated that the major species in urine appears to be the ∼68-kDa mature form of the enzyme. Substrate Specificity of ADAM 12—To test the ability of ADAM 12 to degrade ECM proteins, recombinant ADAM 12-S protein was expressed in COS-7 cells and purified to homogeneity as described previously (Fig. 2A). When assayed by substrate zymography, ADAM 12 could proteolytically degrade gelatin. The purified human ADAM 12-S sample consisted of an ∼68-kDa mature form of the enzyme that displayed a gelatinase activity band (Fig. 2B, lane 2). ADAM 12 also degraded fibronectin (Fig. 2B, lane 3) and type IV collagen (Fig. 2B, lane 4) but did not degrade casein (Fig. 2B, lane 5) when assayed by substrate zymography or type I collagen when tested in the radiometric collagenase assay (data not shown). The gelatin-degrading activity of ADAM 12 was used to evaluate the effect of different classes of protease inhibitors on this enzyme. Pure ADAM 12-S was incubated with gelatin (∼140-kDa protein band, Fig. 2C, lane 2) at 37 °C for either 2 or 18 h in the absence (Fig. 2C, lanes 3 and 4) or presence of various inhibitors. Disappearance of the ∼140-kDa band indicated that ADAM 12 cleaved gelatin in a time-dependent manner (Fig. 2C, lanes 3 and 4). The classical chelators of zinc metalloproteases, EDTA and 1,10-phenanthroline, inhibited gelatin degradation by ADAM 12, whereas the addition of 1 mm Marimastat resulted in partial inhibition of enzyme activity (Fig. 2C, lanes 5–7). The endogenous inhibitors TIMP-1, TIMP-2, and TIMP-4 did not inhibit the gelatinase activity of ADAM 12 even at concentrations 5-fold higher than the enzyme; however, TIMP-3 (500 nm) was an effective inhibitor of ADAM 12 activity (Fig. 2C, lanes 8–15). ADAM 12 Levels during Disease Progression—117 urine specimens were analyzed, including 46 from healthy age-matched female volunteers and 71 from patients that had been diagnosed with various stages of breast cancer. The majority of samples from cancer patients (Fig. 3A, Table I) were positive for ADAM 12 in contrast to the normal urine samples in which ADAM 12 was detected with significantly lower frequency. The frequency of detection of the ADAM 12 species was higher (94%) in the breast cancer group (67 of 71 urine samples were positive) as compared with the control group (15%) (7 of 46 were positive). Urine samples from the breast cancer patients and the control group also differed in the levels of ADAM 12 protein. An immunoblot analysis of representative urine samples from healthy individuals and patients with progressive stages of breast cancer is presented in Fig. 3B. Densitometric analysis of ADAM 12 bands indicated that levels of urinary ADAM 12 increase with progressive stage of disease. Very little or no ADAM 12 was detected in controls; slightly higher protein levels were associated with the initial stages of breast cancer, e.g. ADH/LCIS and DCIS, whereas significantly higher levels were observed in urine from patients with locally invasive and metastatic disease (Fig. 3B).Table IADAM 12 expression in breast cancer patients and controlsControlsADH/LCISDCISIBCMetastaticNo. of patients4613162913Age, years41 ± 1352 ± 657 ± 1359 ± 1255 ± 7Presence of ADAM 127 (15%)10 (78%)13 (82%)25 (86%)11 (85%)ADAM 12 level (DU)aDU, densitometric unitsMean ± S.E.12.4 ± 4.223.0 ± 6.547.0 ± 9.868.4 ± 12.985.9 ± 17.8Median014464380IQRbIQR, interquartile range0-712-3115-6720-9242-106Range0-1440-800-1406-2894-290a DU, densitometric unitsb IQR, interquartile range Open table in a new tab Statistical Analysis—Table I presents the percentage of patients with positive expression of ADAM 12 as well as the median levels for each group. The mean densitometric score for control samples (12 densitometric units) was set as threshold, and percent positive for each cancer group was calculated relative to the control group. As indicated in Fig. 4A, based on comparing percentages, there was a significantly higher proportion of individuals in each breast cancer group with expression of ADAM 12 compared with controls (all p < 0.01). Table II shows the diagnostic characteristics of ADAM 12 as a dichotomous test (based on positive or negative test result) for all patients and for each cancer stage group. For all cancer patients the sensitivity of ADAM 12 based on any level of positive expression is 0.94 (95% confidence interval, 0.86–0.98), and the specificity is 0.61 (28 of 46 controls) (95% confidence interval, 0.46–0.75). Setting a threshold value of 12 densitometric units yielded a much higher specificity of 0.85 (39 of 46 controls) for all cancer patients. Overall accuracy was 0.81 based on 95 of the 117 individuals correctly classified as cases or controls (95% confidence interval, 0.73–0.88). The calculated likelihood ratio was 2.4, which for a positive test result indicates that the presence of urinary ADAM 12 is associated with 2.4 times greater likelihood that the individual has breast cancer. Median levels of ADAM 12 for all 71 cancer patients and the 46 controls were 37 (interquartile range 14–84) and 0 (interquartile range 0–7), respectively (p < 0.001, Mann-Whitney U test). ROC curves were constructed for the various groups, including all cancer patie