A Disintegrin And Metalloprotease 8 Research Articles

A Novel Class of Human ADAM8 Inhibitory Antibodies for Treatment of Triple-Negative Breast Cancer.

New targeted treatments are urgently needed to improve triple-negative breast cancer (TNBC) patient survival. Previously, we identified the cell surface protein A Disintegrin And Metalloprotease 8 (ADAM8) as a driver of TNBC tumor growth and spread via its metalloproteinase and disintegrin (MP and DI) domains. In proof-of-concept studies, we demonstrated that a monoclonal antibody (mAb) that simultaneously inhibits both domains represents a promising therapeutic approach. Here, we screened a hybridoma library using a multistep selection strategy, including flow cytometry for Ab binding to native conformation protein and in vitro cell-based functional assays to isolate a novel panel of highly specific human ADAM8 dual MP and DI inhibitory mAbs, called ADPs. The screening of four top candidates for in vivo anti-cancer activity in an orthotopic MDA-MB-231 TNBC model of ADAM8-driven primary growth identified two lead mAbs, ADP2 and ADP13. Flow cytometry, hydrogen/deuterium exchange-mass spectrometry (HDX-MS) and alanine (ALA) scanning mutagenesis revealed that dual MP and DI inhibition was mediated via binding to the DI. Further testing in mice showed ADP2 and ADP13 reduce aggressive TNBC characteristics, including locoregional regrowth and metastasis, and improve survival, demonstrating strong therapeutic potential. The continued development of these mAbs into an ADAM8-targeted therapy could revolutionize TNBC treatment.

Human eosinophils constitutively express a unique serine protease, PRSS33

BackgroundEosinophils play important roles in asthma, especially airway remodeling, by producing various granule proteins, chemical mediators, cytokines, chemokines and proteases. However, protease production by eosinophils is not fully understood. In the present study, we investigated the production of eosinophil-specific proteases/proteinases by transcriptome analysis. MethodsHuman eosinophils and other cells were purified from peripheral blood by density gradient sedimentation and negative/positive selections using immunomagnetic beads. Protease/proteinase expression in eosinophils and release into the supernatant were evaluated by microarray analysis, qPCR, ELISA, flow cytometry and immunofluorescence staining before and after stimulation with eosinophil-activating cytokines and secretagogues. mRNAs for extracellular matrix proteins in human normal fibroblasts were measured by qPCR after exposure to recombinant protease serine 33 (PRSS33) protein (rPRSS33), created with a baculovirus system. ResultsHuman eosinophils expressed relatively high levels of mRNA for metalloproteinase 25 (MMP25), a disintegrin and metalloprotease 8 (ADAM8), ADAM10, ADAM19 and PRSS33. Expression of PRSS33 was the highest and eosinophil-specific. PRSS33 mRNA expression was not affected by eosinophil-activating cytokines. Immunofluorescence staining showed that PRSS33 was co-localized with an eosinophil granule protein. PRSS33 was not detected in the culture supernatant of eosinophils even after stimulation with secretagogues, but its cell surface expression was increased. rPRSS33 stimulation of human fibroblasts increased expression of collagen and fibronectin mRNAs, at least in part via protease-activated receptor-2 activation. ConclusionsActivated eosinophils may induce fibroblast extracellular matrix protein synthesis via cell surface expression of PRSS33, which would at least partly explain eosinophils' role(s) in airway remodeling.

Patient-Derived Induced Pluripotent Stem Cells Revealed ADAM8/CD156 As a Novel Marker of TKI-Resistant Chronic Myeloid Leukemia Cells

Since the emergence of tyrosine kinase inhibitors (TKIs), long-term survival of patients with chronic myelogenous leukemia (CML) has been improved. However, those TKIs have not fully succeeded in curing CML, mainly due to TKI-resistant CML stem cells. CML stem cells are often difficult to analyze because they represent an extremely minor population of CML cells. To overcome this obstacle, we established integration-free induced pluripotent stem cells (iPSCs) from bone marrow (BM) cells of two patients with CML in chronic phase (CML-CP) and obtained CML pre-hematopoietic progenitor cells (CML-pre-HPCs), immature hematopoietic cells phenotypically defined by CD34+/CD45-/CD43+ cells.In semisolid and liquid cultures, CML-pre-HPCs recapitulated the principal features of CML stem cells, multi-potency and the resistance against imatinib. Gene expression enrichment analysis for CML-pre-HPCs demonstrated that several gene sets, including those related to the maintenance of hematopoietic stem cells were enriched. In addition, we found that a disintegrin and metalloprotease 8 (ADAM8), also known as CD156, was highly enhanced in CML-pre-HPCs and the expression level of ADAM8 was even increased after the treatment of imatinib in vitro. To address the significance of ADAM8 in CMP-CP patient, we evaluated purified ADAM8+ cells by fluorescence-activated cell sorting (FACS) in primary samples.First, FACS analysis found that ADAM8+ cells were enriched more in BM samples of patient with newly diagnosed CML-CP than normal or other types of leukemias among CD34+ fraction. ADAM8+ cells were enriched in CD34+/CD38- fraction compaered to CD34+/38+ fraction in BM of CML-CP patients, indicating that ADMA8+ cells represent immature hematopietic cells. In cell viability assays, ADAM8+/CD38+ CML cells in newly diagnosed CML-CP patient enhibited imatinib-resistance and imatinib-induced apoptosis in vitro was strongly suppressed in ADAM8+ CML cells compared to ADAM8- cells. Even in CD34+/CD38+ fraction, which was previously known as TKI-sensitive fraction, ADAM8+ cells exhibited TKI-resistance in both cell viability and apoptosis assay, indicating that ADAM8 would be a useful marker of TKI-resistant CML cells.Finally, to evaluate the significance of ADAM8 as a marker of TKI-resistant CML cells in vivo, we measured the frequency of CML cells in BM samples of CML-CP patients who had achieved major or complete molecular response (MMR; n = 2 or CMR; n = 1) after the administration of TKIs by limiting dilution analysis. In CML patients with MMR, CML cells remained in ADAM8+ cells at higher frequency in spite of steep decline of CML cells in ADAM8- cells The frequency of CML cells was as high in CD34+/CD38+/ADAM8+ fraction as in CD34+/38- CML stem cell fraction. Even in a patient with CMR, residual CML cells were detected in ADAM8+ population among CD34+/CD38+ fraction, whereas CML cells were undetectable in ADAM8- population.In conclusion, we have established a powerful platform with CML-iPSCs to investigate the pathophysiology of TKI-resistant CML stem cells. Using this platform, we have identified ADAM8 as a novel marker of TKI-resistant CML cells. CD34+/CD38+/ADAM8+ fraction, as well as CD34+/CD38- fraction, was an important population that defines residual CML cells even in CML-CP patients with deep molecular response after the treatment of TKI. ADAM8 would become an attractive candidate of novel therapeutic targets against TKI-resistant CML cells. DisclosuresKataoka:Yakult: Honoraria; Boehringer Ingelheim: Honoraria; Kyowa Hakko Kirin: Honoraria.

WITHDRAWN: A disintegrin and metalloprotease 8 (ADAM8) expression correlates with prognosis in solid tumors: A systematic review and meta-analysis

// Qianna Jin 1 , Jiaqing Cao 2 , Nan He 3,4 , Xin Jin 4 , Tao Liu 4 , Xiaoming Lu 3,4 , Kaixiong Tao 3 and Guobin Wang 3 1 Department of Radiology, Union Hospital, Tongji Medical College, Huazhong University of Science and Technology, Wuhan 430022, China 2 Department of Gastrointestinal Surgery, the Second Affiliated Hospital of Nanchang University, Nanchang 330006, China 3 Department of Gastrointestinal Surgery, Union Hospital, Tongji Medical College, Huazhong, University of Science and Technology, Wuhan, China 4 Cancer Center, Union Hospital, Tongji Medical College, Huazhong University of Science and Technology, Wuhan, China Correspondence to: Nan He, email: hero_nan@163.com Keywords : ADAM8; solid tumor; prognosis; survival; meta-analysis Received: March 01, 2017     Accepted: August 29, 2017     Epub: January 05, 2018 Abstract A disintegrin and metalloprotease 8 (ADAM8) expression appears to be predictive of prognosis in various solid tumors, though the evidence is not yet conclusive. We therefore performed a meta-analysis to explore the relationship between ADAM8 expression and prognosis in patients with solid tumors. Relevant publications were searched in several widely used databases, and 12 studies (1722 patients) were included in the meta-analysis. Pooled odds ratios (ORs) and 95% confidence intervals (CIs) were calculated to evaluate the strength of the association between ADAM8 expression and prognosis. Associations between ADAM8 expression and prognosis were observed for 3-year overall survival (OR = 3.09, 95% CI = 2.33 to 4.10, P < 0.00001) and 5-year overall survival (OR = 4.15, 95% CI = 3.14 to 5.48, P < 0.00001). Similar results were observed when disease free survival (DFS) were analyzed, 3-year DFS (OR = 2.47, 95% CI = 1.66 to 3.69, P < 0.00001) and 5-year DFS (OR = 3.30, 95% CI = 2.29 to 4.74, P < 0.00001). In conclusion, elevated ADAM8 expression is associated with poor prognosis in most solid tumors. ADAM8 is a valuable biomarker for prognosis prediction and a promising therapeutic target in human solid tumors.

Upregulation of a disintegrin and metalloprotease 8 is associated with progression and prognosis of patients with gastric cancer

A disintegrin and metalloprotease 8 (ADAM8) is involved in the tumorigenesis of several types of solid tumors. However, its exact role in gastric cancer (GC) remains unclear. The aim of this study was to evaluate the clinical significance of ADAM8 in GC and to explore its biological effects on gastric carcinogenesis. In this study, quantitative reverse transcription-polymerase chain reaction, Western blotting, and immunohistochemical staining analysis revealed that ADAM8 messenger RNA expression was significantly upregulated in GC tissues compared with noncancerous tissues (P = 0.004), and that positive ADAM8 expression is much more common in tumor tissues compared with normal tissues (P < 0.001) and is correlated with T stage (P = 0.036), N stage (P = 0.048), vessel invasion (P = 0.002), and a shorter patient overall survival (P = 0.024). In vitro assay indicated that ADAM8 overexpression promoted cell growth and increased migration and invasion abilities by decreasing the p-p38/p-extracellular regulated protein kinases (p-ERK) ratio. In conclusion, ADAM8 promotes GC cell proliferation and invasion, and its expression is positively correlated with poor survival, indicating that it might be a promising target in GC therapy.

Neutralization of ADAM8 ameliorates liver injury and accelerates liver repair in carbon tetrachloride-induced acute liver injury

Although some studies have described the function of ADAM8 (a disintegrin and metalloprotease 8) related with rheumatoid arthritis, cancer and asthma, etc., the concrete role of ADAM8 in acute liver injury is still unknown. So mice respectively received anti-ADAM8 monoclonal antibody (mAb) of 100 μg/100 μl, 200 μg/100 μl or 300 μg/100 μl in PBS or PBS pre-injection. Then acute liver injury was induced in the mice by intraperitoneal (i.p.) injection of carbon tetrachloride (CCl₄). Serum AST and ALT level, Haematoxylin-eosin (H&E) staining, the expression level of vascular endothelial growth factor (VEGF), cytochrome P450 1A2 (CYP1A2) and proliferating cell nuclear antigen (PCNA) were detected in the mice after CCl4 administration. Our results showed that anti-ADAM8 mAb pre-injection could effectively lower AST and ALT levels (P < 0.05 or P < 0.01) and reduce liver injury (P < 0.05 or P <0.01), induce the expression of VEGF, CYP1A2 and PCNA (P <0.05 or P < 0.01) in dose-dependent manner compared with the control mice which received PBS pre-injection. In summary, our study suggested that ADAM8 might promote liver injury by inhibiting the proliferation of hepatocytes, angiogenesis and affecting the metabolism function of liver during acute liver injury induced by CCl₄. Anti-ADAM8 mAb injection might be suitable as a potential method for acute liver injury therapy.

ADAM8 in Asthma. Friend or Foe to Airway Inflammation?

Airway inflammation has been suggested as the pathological basis in asthma pathogenesis. Recruitment of leukocytes from the vasculature into airway sites is essential for induction of airway inflammation, a process thought to be mediated by a disintegrin and metalloprotease 8 (ADAM8). However, there is an apparent controversy about whether ADAM8 helps or hampers transmigration of leukocytes through endothelium in airway inflammation of asthma. This review outlines the current contradictory concepts concerning the role of ADAM8 in airway inflammation, particularly focusing on the recruitment of leukocytes during asthma, and attempts to bridge the existing experimental data on the basis of the functional analysis of different domains of ADAM8 and their endogenous processing in vivo. We suggest a possible hypothesis for the specific mechanism by which ADAM8 regulates the transmigration of leukocytes to explain the disparity existing in current studies, and we also raise some questions that require future investigations.

Prognostic and clinical implication of a disintegrin and metalloprotease 8 expression in pediatric medulloblastoma

AimTo investigate the expression patterns and clinical value of a disintegrin and metalloprotease 8 (ADAM8) in pediatric medulloblastoma. MethodsWe evaluated the expression of ADAM8 mRNA and protein in pediatric medulloblastoma tissues by Quantitative RT-PCR, Western blot analysis and Immunohistochemical staining, respectively. The correlation of ADAM8 immunostaining with clinical-pathological features of medulloblastoma patients and its prognostic relevance were further determined. ResultsADAM8 mRNA and protein were both most highly expressed in medulloblastoma tissues when compared with normal cerebellum tissues (both P<0.001). According to the immunohistochemisty analysis, we found statistically significant correlations of high ADAM8 protein expression with advanced metastatic stage (P=0.01), aggressive histopathological type (P=0.006), as well as with undifferentiated tumor (P=0.02). We further determined a statistically significant association between ADAM8 protein expression and clinical outcome of medulloblastoma patients, with higher expression levels of ADAM8 associating with a worse overall survival (P=0.02). ConclusionOur results provide the first evidence that the up-regulation of ADAM8 in medulloblastoma tissues might be correlated with the advanced tumor progression and poor prognosis of patients with this disease. Detection of ADAM8 expression might facilitate the prognostic assessment and improve the therapeutic strategy for medulloblastoma patients.

Upregulation of a Disintegrin and Metalloprotease 8 Influences Tumor Metastasis and Prognosis in Patients with Osteosarcoma

To investigate the clinicopathological and prognostic value of a disintegrin and metalloprotease 8 (ADAM8) in osteosarcoma. ADAM8 expression in osteosarcoma tissues was examined by immunohistochemistry in 69 patients. ADAM8 was positively expressed in 61 of 69 (88.4%) osteosarcoma specimens with cytoplasmic staining, and also increased in the specimens with recurrence (P = 0.008) and metastasis (P = 0.002). Patients with strong ADAM8 expression had significantly poorer overall survival (OS) and disease-free survival (DFS) (both P < 0.001) when compared with the patients with the weak expression of ADAM8. On multivariate analysis, ADAM8 expression was found to be an independent prognostic factor for both OS (P < 0.001) and DFS (P < 0.001). Our results suggest for the first time that ADAM8 might be applied as a novel marker for the prediction of recurrence and metastasis potency and a significant indicator of poor prognosis for patients with osteosarcoma.

Overexpression of a disintegrin and metalloprotease 8 in human gliomas is implicated in tumor progression and prognosis

A disintegrin and metalloprotease 8 (ADAM8) has been shown to be expressed in various cancer types, and its expression was associated with advanced progression of several tumors. However, little is known about ADAM8 in human gliomas. Therefore, we here evaluated the correlation of ADAM8 expression with the clinicopathological features and prognosis in the patients with gliomas. Immunohistochemistry and western blot were used to investigate the expression of ADAM8 protein, respectively, in 128 patients with gliomas. The expression levels of ADAM8 in glioma tissues were significantly higher (P = 0.002) than those in non-neoplastic brain tissues according to the immunohistochemistry analysis. In addition, a high level of ADAM8 expression was significantly more common in glioma tissues with advanced grade than those with low grade (P = 0.01), which were in line with the results of western blot analysis (P = 0.01). Moreover, the increased expression of ADAM8 was significantly correlated with low Karnofsky performance score (KPS) (P = 0.008), frequent intra-tumor necrosis (P = 0.01), and poor overall survival (P = 0.008). Furthermore, multivariate analysis identified the expression levels of ADAM8 (P = 0.01) and intra-tumor necrosis (P = 0.03) to be independent prognostic factors. These findings suggest for the first time that ADAM8 is frequently overexpressed in human gliomas and is closely associated with poor clinical outcome.

Possible Early Prediction of Preterm Birth by Determination of Novel Proinflammatory Factors in Midtrimester Amniotic Fluid

Interferon-gamma-inducible T cell-alpha chemoattractant (ITAC) is a chemokine, directing activated T lymphocytes toward sites of inflammation. ADAM-8 (A disintegrin and metalloprotease-8) is a glycoprotein expressed in cells promoting inflammation. Elastase, a protease targeting at the degradation of intra- or extracellular proteins, is inhibited by secretory leukocyte proteinase inhibitor (SLPI), which protects against microbial invasion. Adhesion molecules (soluble intercellular adhesion molecule--sICAM-1 and soluble vascular cell adhesion molecule-sVCAM--1) serve as markers of inflammation or tissue damage. We hypothesized that elevated midtrimester amniotic fluid concentrations of above substances, and decreased levels of SLPI could possibly be useful predictors of asymptomatic intra-amniotic inflammation and/or infection, eventually resulting in preterm labor and delivery. The study involved 312 women undergoing midtrimester amniocentesis. Thirteen cases, progressing to preterm delivery (<37 weeks), were matched with 21 controls (delivering >37 weeks) for age, parity, and gestational age at amniocentesis. Amniotic fluid levels of the above substances were measured by enzyme-linked immunosorbent assay (ELISA). Only amniotic fluid ITAC and ADAM-8 levels were significantly higher (P=0.005 and P < 0.02, respectively) in women delivering at <37 weeks than at >37 weeks. SLPI concentrations significantly increased in women going into labor without ruptured membranes irrespective of pre- or term delivery (P < 0.007, P < 0.001, respectively) and correlated with elastase (r=0.508, P < 0.002). In conclusion, elevated midtrimester amniotic fluid levels of ITAC and ADAM-8 could predict occult infections/inflammations, possibly resulting in preterm birth.

Elevated Mid-Trimester Amniotic Fluid ADAM-8 Concentrations as a Potential Risk Factor for Preterm Delivery

To determine during mid-trimester amniocentesis if elevated concentrations of ADAM-8 (A Disintegrin And Metalloprotease 8) and/or cortisol can recognize women at risk for spontaneous preterm delivery. The study involved 312 women who underwent mid-trimester amniocentesis. Thirteen patients, who progressed to preterm delivery, were matched with 21 controls for age, parity, gestational age at amniocentesis, and year of amniocentesis. ADAM-8 and cortisol levels were measured by enzyme-linked immunosorbent assay and radioimmunoassay, respectively. ADAM-8 mean amniotic fluid concentrations were significantly higher in women with preterm delivery than in women delivering at term (mean 1213.9 [SE 96.7] pg/mL [range, 780 to 1854 pg/mL] vs mean 937.2 [SE 50.3] pg/mL [range, 486 to 1508 pg/mL], P < .02). Amniotic fluid ADAM-8 concentrations higher than 1149 pg/mL had the highest specificity and odds ratio (OR) in the identification of the women with increased risk for preterm delivery (sensitivity 61.5%; specificity 81.7%; OR, 9.6 [95% confidence interval (CI), 1.8 to 50.3]). Women with preterm delivery had suggestively higher amniotic fluid concentrations of cortisol (mean 1.3 [SE 0.2] microg/dL [range, 0.4 to 2.2 microg/dL]) than women delivering at term (mean 1.0 [SE 0.09] microg/dL [range, 0.6 to 1.7 microg/dL], P < .07). Furthermore, cortisol levels were positively correlated with ADAM-8 levels (Spearman's r = .418, P < .014). Elevated mid-trimester amniotic fluid ADAM-8 concentrations possibly are a risk factor for preterm delivery, particularly if ADAM-8 levels are greater than 1149 pg/mL. Potential intrauterine inflammation is also associated with suggestively increased amniotic fluid cortisol levels.

Metalloprotease‐disintegrin ADAM8: Expression analysis and targeted deletion in mice

ADAM8 (a disintegrin and metalloprotease 8, also referred to as MS2/CD156a) is a membrane-anchored metalloprotease that was first identified in a macrophage cell line and has been implicated in neurodegenerative diseases. Here, we evaluated the expression of ADAM8 during mouse development and generated mice lacking ADAM8 (Adam8-/- mice). During early mouse development, ADAM8 is expressed by maternal cells in the decidua and by trophoblast derivatives of the embryo but not in the derivatives of the inner cell mass. At later stages, prominent expression of ADAM8 is seen in the embryo proper, in the gonadal ridge, thymus, developing cartilage and bone, brain and spinal cord, and in the mesenchyme in close proximity to the branch point between the jugular vein and developing lymphatic vessels. Examination of Adam8-/- mice, however, revealed no major defects in these or other structures during development or in adult tissues and no evident pathological phenotypes.