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

Abstract

ADAM 12 is a member of a family of disintegrincontaining 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 ϳ68-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. ADAMs1 (a disintegrin 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 (1-3). 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) (4). ADAM 12-S is an active protease known to cleave IGF...

Expression of a disintegrin and metalloprotease (ADAM and ADAMTS) enzymes in human non-small-cell lung carcinomas (NSCLC)

“…We demonstrate in the present paper that cultured BZR lung-derived cancer cell lines expressed ADAM-12 mRNA, while 16-HBE and BEAS-2B cells derived from normal epithelial cells did not express significant levels of ADAM-12 mRNA. Others have reported an overexpression of ADAM-12 in liver carcinomas (Le Pabic et al, 2003), and its levels in urine from patients have been correlated with survival in breast cancer (Roy et al, 2004). These data, taken together with the faint expression of ADAM-12 mRNA levels in healthy lung extracts, indicate that tumour cells are probably the main producer of ADAM-12 in our experimental conditions.…”

Emerging roles of ADAM and ADAMTS metalloproteinases in cancer

“…ADAM-9, -15, -17 mRNA levels are higher in breast cancer surgical samples whereas ADAM-10 mRNA levels are not modulated [132,143]. ADAM-12, as previously stated in the "proliferation and apoptosis" section of this review, is an apoptosis-modulating gene which is upregulated in human breast cancer tissues whereas nonmalignant breast lesions express very low amounts of the proteinase.…”

“…Several ADAMs and ADAMTSs are found to be modulated in breast cancer patients [66,132,143]. ADAM-9, -15, -17 mRNA levels are higher in breast cancer surgical samples whereas ADAM-10 mRNA levels are not modulated [132,143].…”

“…Accordingly, the overexpression of soluble ADAM-12 lacking the cytoplasmic tail (secreted splice variant of ADAM-12) accelerates the development of tumour by delaying tumour cell apoptosis [91]. It is worth noting that urine ADAM-12 might be a potentially important non-invasive biomarker in breast cancer since levels are enhanced in breast cancer patients [143].…”

Expression profiling of metalloproteinases and tissue inhibitors of metalloproteinases in normal and degenerate human achilles tendon

“…ADAM-12 has been demonstrated to cleave insulin-like growth factor binding proteins 3 and 5, pro-heparin-binding epidermal growth factor, and the ECM components gelatin, type IV collagen, and fibronectin (43)(44)(45)(46), and may therefore be significant in painful tendinopathy, both as a regulator of cytokine activity and as a mediator of ECM degradation. ADAM-12 is also reported to support cell attachment and influence cell spreading and migration (47)(48)(49).…”