A Disintegrin And Metalloprotease 9 Research Articles

Therapeutic restoration of miR-126-3p as a multi-targeted strategy to modulate the liver tumor microenvironment.

Impaired natural killer (NK) cell-mediated antitumor responses contribute to the growth of liver tumors. Expression of a disintegrin and metalloprotease 9 (ADAM9) increases shedding of membrane-bound major histocompatibility complex class I chain-related protein A and results in evasion from NK cell-mediated cytolysis. ADAM9 is also involved in angiogenesis and tumor progression and is a target of miR-126-3p, a tumor suppressor that is downregulated and alters tumor cell behavior in the liver and other cancers. We evaluated the restoration of miR-126-3p and modulation of the miR-126-3p/ADAM9 axis as a therapeutic approach to simultaneously enhance NK cell-mediated cytolysis while targeting both tumor cells and their microenvironment. Precursor miRNAs were loaded into milk-derived nanovesicles to generate therapeutic vesicles (therapeutic milk-derived nanovesicles) for the restoration of functional miR-126-3p in recipient cancer cells. Administration of therapeutic milk-derived nanovesicles increased miR-126-3p expression and reduced ADAM9 expression in target cells and was associated with an increase in membrane-bound major histocompatibility complex class I chain-related protein A. This enhanced NK cell cytolysis in adherent tumor cells and in multicellular tumor spheroids while also impairing angiogenesis and modulating macrophage chemotaxis. Moreover, IV administration of therapeutic milk-derived nanovesicles with adoptive transfer of NK cells reduced tumor burden in orthotopic hepatocellular cancer xenografts in mice. A directed RNA therapeutic approach can mitigate NK cell immune evasion, reduce angiogenesis, and alter the tumor cell phenotype through the restoration of miR-126-3p in liver tumor cells. The pleiotropic effects elicited by this multi-targeted approach to modulate the local tumor microenvironment support its use for the treatment of liver cancer.

ADAM9 deubiquitination induced by USP22 suppresses proliferation, migration, invasion, and epithelial-mesenchymal transition of trophoblast cells in preeclampsia

IntroductionThe dysregulation of deubiquitination has been shown to affect the development of pre-eclampsia (PE). A disintegrin and metalloprotease 9 (ADAM9) plays roles in diverse physiological contexts, including PE. Here, this study aimed to investigate whether ADAM9 regulated trophoblast cell dysfunction through ubiquitin-specific protease 22 (USP22) deubiquitinase-mediated deubiquitination during PE. MethodsLevels of genes and proteins were tested via qRT-PCR and western blotting assays. Cell proliferation, migration, and invasion were detected using cell counting kit-8, 5-ethynyl-2′-deoxyuridine (EdU), flow cytometry, transwell and wound healing assays, respectively. Epithelial-mesenchymal transition related markers were assayed using western blotting. Proteins between USP22 and ADAM9 were identified by co-immunoprecipitation assay. ResultsADAM9 was highly expressed in PE patients, functionally, ADAM9 overexpression weakened the proliferation, migration, invasion, and EMT progression in trophoblast cells. Mechanistically, the deubiquitinase USP22 removed ubiquitination on ADAM9 and maintained its stability. Forced expression of USP22 also suppressed the proliferation and mobility in trophoblast cells. Moreover, the regulatory effects of USP22 on trophoblast cells were reversed by ADAM9 silencing. In addition, USP22 interacted with ADAM9 to regulate the activation of Wnt/β-catenin pathway. DiscussionADAM9 was deubiquitinated and stabilized by USP22 and then suppressed the proliferation, migration, invasion, and EMT progression in trophoblast cells, indicating a new pathway of USP10/RUNX1 axis in PE process.

ADAM9: A regulator between HCMV infection and function of smooth muscle cells.

Lots of epidemiological and clinical studies have shown that human cytomegalovirus (HCMV) is related to the pathogenesis of atherosclerosis. Released by inflammatory cells and vascular smooth muscle cell (VSMCs), metalloproteinases are observed in many pathological vessel conditions, including atherosclerosis and restenosis.This study was designed to investigate the effect of HCMV infection on the expression of metalloproteinases and their involvements in the HCMV-induced functional changes of VSMCs.Differential metalloproteinase after HCMV infection was assayed using reverse transcription-polymerase chain reaction (RT-PCR) microarray. The most significant increased a disintegrin and metalloprotease 9 (ADAM9) was chosen to investigate the mechanism of its specific increase after infection using the treatment of UV-irradiated replication-deficient HCMV, HCMV-infected cell lysate filters or Foscarnet. The function of proliferation, migration, production of inflammatoty factors and phenotypic transformation were determined by using cell counting kit-8, transwell, Enzyme-linked immunosorbent assay, RT-quantitative PCR(qPCR) and Western blot, respectively. Moreover, the effect of ADAM9 deficiency on HCMV replication was also determined using RT-qPCR and immunofluorescence.The expression levels of 6 genes were upregulated and 14 genes were downregulated at different time points after HCMV infection. Among these, the expression level of ADAM9 increased most significantly at each time point and the abnormal expression of ADAM9 might be induced by the early gene products of HCMV. Further studies found that ADAM9 promoted the proliferation, the migration, the production of inflammatory factors and the transit from the contractile phenotype (decreased ACTA2 expression) to the synthetic phenotype (increased osteopontin [OPN] expression). Knockdown theADAM9expression could rescue the decreased ACTA2 expression, but has no effect on OPN expression. ADAM-9 deficiency didn't affect the replication of HCMV.The findings of our study suggest that HCMV infection changed VSMC function through ADAM9 expression, which may contribute to the understanding of the underlying pathological mechanisms of HCMV-induced atherosclerosis.

MicroRNA-21 Derived from Exosomes of Bone Marrow Mesenchymal Stem Cells (BMSCs) Alleviates Pancreatic Cancer by Downregulation of a Disintegrin and Metalloprotease 9 (ADAM9)

We aimed to explore underlying mechanism by which microRNA-21 (miR-21) derived from bone marrow mesenchymal stem cells (BMSCs) extracted exosomes (exo) in pancreatic cancer (PC). Bioinformatics analysis identified candidate miRNAs and target mRNAs in PC those were verified by luciferase reporter assay. BMSCs and exo were isolated and co-cultivated with PC cells. PC cells were then treated with plasmids loaded with miR-21 or a disintegrin and metalloprotease 9 (ADAM9), followed by detection of invasion, metastasis and apoptosis through Transwell assay and flow cytometry. MiR-21 was downregulated in PC tissues and cells, while ADAM9 was upregulated and positively correlated with poor prognosis. Overexpression of miR-21 restrained the capacities of proliferation, invasion and migration of PC cells by inhibiting ADAM9 expression. Specific inhibitor GW4869 reduced release of exo and declined miR-21 expression. Treatment with BMSC-exo containing miR-21 suppressed the malignant characteristics of cancer cells. MiR-21 derived from exo of BMSCs inhibited PC progression by ADAM9 down-regulation, providing insight into novel strategy against PC.

Retraction: miR-126a-3p induces proliferation, migration and invasion of trophoblast cells in preeclampsia-like rats through inhibiting a disintegrin and metalloprotease 9(ADAM9).

Retraction: miR-126a-3p induces proliferation, migration and invasion of trophoblast cells in preeclampsia-like rats through inhibiting a disintegrin and metalloprotease 9(ADAM9).

Stabilization of ADAM9 by N-\u03b1-acetyltransferase 10 protein contributes to promoting progression of androgen-independent prostate cancer

N-α-Acetyltransferase 10 protein (Naa10p) was reported to be an oncoprotein in androgen-dependent prostate cancer (PCa; ADPC) through binding and increasing transcriptional activity of the androgen receptor (AR). PCa usually progresses from an androgen-dependent to an androgen-independent stage, leading to an increase in the metastatic potential and an incurable malignancy. At present, the role of Naa10p in androgen-independent prostate cancer (AIPC) remains unclear. In this study, in silico and immunohistochemistry analyses showed that Naa10 transcripts or the Naa10p protein were more highly expressed in primary and metastatic PCa cancer tissues compared to adjacent normal tissues and non-metastatic cancer tissues, respectively. Knockdown and overexpression of Naa10p in AIPC cells (DU145 and PC-3M), respectively, led to decreased and increased cell clonogenic and invasive abilities in vitro as well as tumor growth and metastasis in AIPC xenografts. From the protease array screening, we identified a disintegrin and metalloprotease 9 (ADAM9) as a potential target of Naa10p, which was responsible for the Naa10p-induced invasion of AIPC cells. Naa10p can form a complex with ADAM9 to maintain ADAM9 protein stability and promote AIPC’s invasive ability which were independent of its acetyltransferase activity. In contrast to the Naa10p-ADAM9 axis, ADAM9 exerted positive feedback regulation on Naa10p to modulate progression of AIPC in vitro and in vivo. Taken together, for the first time, our results reveal a novel cross-talk between Naa10p and ADAM9 in regulating the progression of AIPC. Disruption of Naa10p–ADAM9 interactions may be a potential intervention for AIPC therapy.

ADAM9 Mediates Triple-Negative Breast Cancer Progression via AKT/NF-κB Pathway.

Upregulation of a disintegrin and metalloprotease 9 (ADAM9) is correlated with progression of cancers, such as prostate, bladder, and pancreatic cancers. However, its role in triple-negative breast cancer (TNBC) is still unclear. Our study aimed to investigate whether ADAM9 is upregulated and promoted the aggressiveness in TNBC. Breast cancer cell lines and patient specimens were used to evaluate the ADAM9 expression by western blotting and immunohistochemistry staining, respectively. Compared with the non-TNBC, ADAM9 expression was significantly increased in TNBC cells and TNBC patient specimens. Based on the data acquired from public databases, the correlation between ADAM9 expression and breast cancer patient survival was analyzed by Kaplan-Meier method. It was shown that ADAM9 overexpression was significantly correlated with poorer survival in patients with TNBC. Furthermore, ADAM9 in TNBC cells was knocked down by small interference RNA and then studied by the MTT/colony formation assay, wound healing assay and transwell invasion assay on the cell proliferation, migration, and invasion, respectively. We found that inhibiting ADAM9 expression suppressed TNBC cell proliferation, migration, and invasion by lowering the activation of AKT/NF-κB pathway. Our results demonstrated that ADAM9 is an important molecule in mediating TNBC aggressiveness and may be a potential useful therapeutic target in TNBC treatment.

MiR-126a-3p induces proliferation, migration and invasion of trophoblast cells in pre-eclampsia-like rats by inhibiting A Disintegrin and Metalloprotease 9.

The present study aimed to investigate the underlying mechanism of miR-126a-3p in the proliferation, migration and invasion of trophoblast cells in pre-eclampsia-like rats by targeting A Disintegrin and Metalloprotease 9 (ADAM9). First, the interaction between miR-126a-3p and ADAM9 was confirmed via biochemical assays. Placental tissues and trophoblast cells were then obtained. RNA in situ hybridization was performed in order to detect miR-126a-3p expression in the placenta. Subsequently, a series of biological assays, including reverse transcription-quantitative PCR (RT-qPCR), Western blotting, MTT assay, apoptosis assay, cell cycle assay, wound healing assay and transwell assay were adopted in order to determine the cell proliferation, cell cycle distribution, apoptotic rate, and migration and invasion of trophoblast cells in each group. The results revealed that miR-126a-3p was down-regulated in the placenta of pre-eclampsia-like rats. In vivo experiments’ results indicated that miR-126a-3p could inhibit ADAM9 expression, and induce cyclin D1, Matrix metalloproteinase (MMP) 2 (MMP-2), MMP-9 expression. MTT, apoptosis and cell cycle assay results revealed that trophoblast cells transfected with miR-126a-3p mimic or si-ADAM9 exhibited higher proliferative activity and a lower apoptotic rate compared with the blank group (all P<0.05). The wound healing assay and transwell assay results confirmed that, compared with the blank group, the migration and invasion ability of trophoblast cells in the miR-126a-3p mimic group and small interfering RNA (siRNA)-ADAM9 group were significantly increased (all P<0.05). Conversely, miR-126a-3p inhibitor treatment revealed the opposite effect (all P<0.05). In conclusion, the present study demonstrated that miR-126a-3p could enhance proliferation, migration and invasion, but decrease the apoptosis rate of trophoblast cells in pre-eclampsia-like rats through targeting ADAM9.

1960P - ADAM9 as a target for lung cancer treatment

BackgroundSuppression of cancer metastasis is an urgent therapeutic need because metastasis is a major cause of high mortality in different types of cancers including lung cancer. Overexpression of a disintegrin and metalloprotease 9 (ADAM9), a member of the ADAM family of type I transmembrane proteins, is observed in many cancers and correlates with lung cancer brain metastasis. Since it contributes to tumorigenesis due to its ability in cleaving and releasing a number of molecules that involves cancer progression, it would be a potential target for lung cancer treatment. MethodsWe have performed the genome-wide approach to explore ADAM9-regulated genes. Moreover, we have developed small compounds as ADAM9 inhibitors to target ADAM9’s catalytic domain using virtual screening and evaluated them by inhibiting ADAM9-mediated downstream pathways. ResultsOverexpression of ADAM9 in lung cancer cells promotes tumor metastasis. Several ADAM9-mediated pathways are investigated from RNA-seq analysis. In the other hand, we have validated the potency of developed small compounds in reducing ADAM9 protease activity, cancer cell growth, and cell migration. In tumor animal models, ADAM9 inhibitors exhibited high efficacy to reduce cancer progression in animals bearing lung tumors. Notably, no liver and kidney toxicity were detected, suggesting no severe toxicity after drug treatment. ConclusionsWe demonstrate inhibition of ADAM9 activity by potential ADAM9 inhibitor provide anti-lung tumor benefits in vitro and in vivo. Notably, ADAM9 inhibitor treatment has no systemically acute toxicity in mice. Thus, targeting ADAM9 provides a potential strategy for lung cancer treatment. Legal entity responsible for the studyThe authors. FundingThe National Health Research Institutes, Taiwan. DisclosureAll authors have declared no conflicts of interest.

ADAM9 mediates the interleukin-6-induced Epithelial–Mesenchymal transition and metastasis through ROS production in hepatoma cells

Interleukin (IL)-6 has been implicated in the invasion and metastasis of hepatocellular carcinoma (HCC). However, the molecular events that mediate this process are poorly understood. Here, we showed that IL-6 promoted the epithelial–mesenchymal transition (EMT) in HCC cell lines, and upregulated a disintegrin and metalloprotease 9 (ADAM9) expression by activating the JNK signaling pathway. ADAM9 was upregulated in human HCCs which promoted HCC cell invasion and the EMT by interacting with NADPH oxidase 1 and inducing reactive oxygen species generation. Knockdown of ADAM9 inhibited the IL-6-induced EMT. Additionally, ADAM9 expression was positively correlated with IL-6 and Snail expression in human HCC specimens. Taken together, our results showed that ADAM9 is an important mediator of IL-6-induced HCC cell migration and invasion, and may provide a novel therapeutic target for HCC management.

Characterization of the catalytic properties of the membrane-anchored metalloproteinase ADAM9 in cell-based assays.

ADAM9 (A Disintegrin And Metalloprotease 9) is a membrane-anchored metalloproteinase that has been implicated in pathological retinal neovascularization and in tumor progression. ADAM9 has constitutive catalytic activity in both biochemical and cell-based assays and can cleave several membrane proteins, including epidermal growth factor and Ephrin receptor B4; yet little is currently known about the catalytic properties of ADAM9 and its post-translational regulation and inhibitor profile in cell-based assays. To address this question, we monitored processing of the membrane-anchored Ephrin receptor B4 (EphB4) by co-expressing ADAM9, with the catalytically inactive ADAM9 E > A mutant serving as a negative control. We found that ADAM9-dependent shedding of EphB4 was not stimulated by three commonly employed activators of ADAM-dependent ectodomain shedding: phorbol esters, pervanadate or calcium ionophores. With respect to the inhibitor profile, we found that ADAM9 was inhibited by the hydroxamate-based metalloprotease inhibitors marimastat, TAPI-2, BB94, GM6001 and GW280264X, and by 10 nM of the tissue inhibitor of metalloproteinases (TIMP)-3, but not by up to 20 nM of TIMP-1 or -2. Additionally, we screened a non-hydroxamate small-molecule library for novel ADAM9 inhibitors and identified four compounds that selectively inhibited ADAM9-dependent proteolysis over ADAM10- or ADAM17-dependent processing. Taken together, the present study provides new information about the molecular fingerprint of ADAM9 in cell-based assays by showing that it is not stimulated by strong activators of ectodomain shedding and by defining a characteristic inhibitor profile. The identification of novel non-hydroxamate inhibitors of ADAM9 could provide the basis for designing more selective compounds that block the contribution of ADAM9 to pathological neovascularization and cancer.

MiR-20b reduces 5-FU resistance by suppressing the ADAM9/EGFR signaling pathway in colon cancer.

Chemoresistance is a major obstacle to cancer therapy including that of colon cancer (CC). Although the dysregulation of many miRNAs has been implicated in 5-fluorouracil (5-FU) resistance in CC cells, the specific role of miR-20b in chemoresistance has not been documented. In the present study, we first determined the expression of miR-20b by RT-PCR and the levels of adisintegrin and metalloprotease9 (ADAM9) and epidermal growth factor receptor (EGFR) by western blotting in CC and adjacent non-cancerous tissues from 5-FU-sensitive or -resistant CC patients. Subsequently, 5-FU-sensitive (HCT116) and -resistant (HCT116-R) cells were obtained, and the levels of miR-20b, ADAM9 and EGFR were detected. Meanwhile, the 5-FU resistance of the cells was examined by assessing cell viability (by MTT assay) and apoptosis (by flow cytometry). After transfection of miR-20b into HCT116-R cells, drug resistance was reexamined. We then confirmed the relationship between miR-20b and ADAM9 by luciferase reporter assay. Finally, 5-FU resistance in HCT116 and HCT116-R cells was compared after transfection with miR-20b. Our results showed that miR-20b was expressed at lower levels in the 5-FU-resistant tissues and cells than in the 5-FU-sensitive tissues and cells. The opposite was the case for expression of ADAM9 and EGFR. In addition, we demonstrated that ADAM9 is a direct target of miR-20b and that miR-20b decreased the 5-FU resistance of HCT116-R cells. Our findings suggest that miR-20b reduces 5-FU resistance to induce apoptosis invitro by suppressing ADAM9/EGFR in CC cells.

Abstract 461: Microrna-126 Overexpression Rescues Impaired Efferocytosis in Diabetes

Cardiac healing after myocardial ischemia requires accumulation of macrophages at the site of injury for the clearance of apoptotic/dead cardiomyocytes, a process known as efferocytosis. Effective clearance of apoptotic cells in turn results in inflammation resolution during cardiac regeneration. Diabetes accompanied by hyperglycemia is known to compromise the function of macrophages leading to impaired inflammation resolution during cardiac regeneration, wound healing, atherosclerosis and auto-immune disorders. However, the effect of diabetes on macrophage efferocytosis and molecular mechanisms involved have not been understood so far. We found miR-126 is essential for the macrophage efferocytosis of apoptotic myocytes in high glucose conditions (HG). In-vivo, miR-126 expression was reduced and, a corresponding increase in A Disintegrin And Metalloprotease 9 (ADAM9) expression was observed in human diabetic heart failure tissues compared with the normal counterparts. In-vitro, macrophages efferocytosis was reduced in HG compared to normal glucose. Over-expression of miR-126 increased the efferocytosis in HG. Mechanistically, reduced miR-126 leads to increased expression of human ADAM9. ADAM9 in turn cleaves Mer tyrosine receptor kinase (MerTK), a proto-oncogene that plays a role in efferocytosis, leading to the formation of soluble-Mer that reduces the macrophage efferocytosis in HG conditions. This, we term as “not ready to eat” signal of the macrophages, which could be rescued by overexpressing miR-126 and termed as “ready to eat” signal of the macrophages.

Estrogen induced the expression of ADAM9 through estrogen receptor α but not estrogen receptor β in cultured human neuronal cells

Purpose of the studyTo investigate the induction of ‘a disintegrin and metalloprotease’ 9 (ADAM9) by which estrogen receptor (ER) subtypes and estrogen regulation of expression in cultured human neuronal cells. MethodsSH-SY5Y cells were treated with E2 and the expression of ADAM9 were detected by RT-PCR and Western blot analysis. The peGFP-C3-ERα and peGFP-C3-ERβ vectors were constructed and transfected into SH-SY5Y cells. Then, the mRNA and protein expression level of ADAM9 were detected by RT-PCR and Western blot analyses with the treatment of E2. E2, the ERα agonist PPT, the ERβ agonist DPN and the non-specific ER antagonist ICI 182,780 were added, and the expression of ADAM9 protein and transcriptional activities of ADAM9 promoter were detected by Western blot and luciferase assays, respectively. ResultsE2 induced ADAM9 expression with a time-dependent manner in SHSY5Y cells, reaching the peak at 24h. peGFP-C3-ERα transfection significantly increased ADAM9 expression in the presence of E2, whereas peGFP-C3-ERβ had no effect. The E2-induced overexpression of ADAM9 protein was downregulated by ICI 182,780. PPT significantly increased ADAM9 protein expression, whereas DPN had no effect. The E2-induced increase in the transcriptional activities of the ADAM9 promoter was downregulated by ICI 182,780. PPT significantly increased the transcriptional activities of the ADAM9 promoter, whereas DPN had no effect. ConclusionsActivation of ERα but not ERβ increases ADAM9 expression in cultured human neuronal cells, implicating that ERα-activation functions in the direct mechanism underlying the estrogen-mediated preventative effects against Aβ production and further reduction in the risk of Alzheimer's disease.

Abstract 3533: ADAM9 regulates microRNAs expression for lung cancer metastasis

Abstract Lung cancer is the leading cause of cancer death worldwide, and brain metastasis is a major cause of morbidity and mortality in lung cancer. Currently, it is not clear which molecules or what mechanisms mediate lung cancer brain metastasis. To explore this question, we compared our established brain-metastatic lung cancer sublines with their parental cancer cells, and identified ADAM9 (a disintegrin and metalloprotease 9) was overexpressed in metastatic sublines. Knocking down ADAM9 expression in lung cancer cells also showed significant reduction of lung cancer brain metastasis in vitro and in vivo. Furthermore, using a genome-wide approach to screen ADAM9-related mRNAs and microRNAs involved in brain metastasis, we identified that CDH2, encoding N-cadherin, was up-regulated by ADAM9 in brain-metastatic lung cancer sublines, and the 3′UTR of CDH2 contains the predicted binding sites of several microRNAs that were down-regulated in ADAM9-overexpressed cancer cells. Luciferase assays and western blot analysis showed that CDH2 is a target gene of microRNAs. The up-regulation of microRNAs, in turn, reduced the expression of its target gene and further inhibited the migration ability of aggressive lung adenocarcinoma cells. This study revealed that ADAM9 activates CDH2 through the release of microRNA inhibition of CDH2 in lung adenocarcinoma and plays a role in lung cancer metastasis. Citation Format: Yuh-Pyng Sher, Li-Ju Wang, Mong-Hsun Tsai, Ting-Ting Kuo, Eric Y. Chuang, Liang-Chuan Lai. ADAM9 regulates microRNAs expression for lung cancer metastasis. [abstract]. In: Proceedings of the 105th Annual Meeting of the American Association for Cancer Research; 2014 Apr 5-9; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2014;74(19 Suppl):Abstract nr 3533. doi:10.1158/1538-7445.AM2014-3533

MiR-126 inhibits cell growth, invasion, and migration of osteosarcoma cells by downregulating ADAM-9.

Osteosarcoma (OS) has become one of the most common primary malignant tumors in the children and adolescents with a poor prognosis mainly due to high metastasis. A disintegrin and metalloprotease 9 (ADAM-9) plays a role in tumorigenesis, invasion, and metastasis in several tumors. miR-126 has been reported to be downregulated in OS tumor. However, the involvement of ADAM-9 in the pathology of OS and the relationship between miR-126 and ADAM-9 in OS cells remain unclear. In this study, using quantitative reverse-transcribed PCR (qRT-PCR) analysis on 37 pairs of OS tumors and matched adjacent normal bone tissues, we found that ADAM-9 is significantly upregulated, while miR-126 is downregulated in human OS tumors. Association analysis revealed that upregulation of ADAM-9 and downregulation of miR-126 are significantly involved in advanced clinical stage development and distant metastasis. Luciferase reporter assay revealed that miR-126 could directly target ADAM-9 3' untranslated region (UTR) and inhibit its expression in U2OS and MG-63 cells. Functional experiments revealed that downregulating ADAM-9 by miR-126 inhibited cellular growth, invasion, and migration in U2OS and MG-63 cells. In rescue experiments, restored ADAM-9 expression attenuated miR-126-mediated suppression, while knockdown of ADAM-9 by small interfering RNA (siRNA) represented similar results with miR-126-mediated tumor suppression in U2OS cells. Taken together, our data indicated that miR-126 inhibits cell growth, invasion, and migration of OS cells by downregulating ADAM-9.

ADAM9 decreases in castration resistant prostate cancer and is a prognostic factor for overall survival.

A disintegrin and metalloprotease 9 (ADAM9) is a membrane-anchored enzyme which is considered to be involved in some diseases including tumor. However, the role of ADAM9 in castration resistant prostate cancer (CRPC) is not clear. This study aimed to explore the different expressions on protein and messenger RNA (mRNA) level of ADAM9 between hormonal sensitive prostate cancer (HSPC) and CRPC tissue, and find the correlation with prognosis. Clinicopathologic characteristics of 106 HSPC and 76 CRPC cases were collected. The ADAM9 expressions were analyzed using immunohistochemistry. ADAM9 mRNA of 32 additional cases (16 HSPC and 16 CRPC patients) were analyzed via quantitative real-time polymerase chain reaction (RT-PCR). The prediction values of variables for overall survival (OS) of CRPC patients were analyzed using Cox regression. ADAM9 protein expression was significantly downregulated in CRPC compared with HSPC tissue (31.6% vs. 81.1%, P < 0.001). The relativity transcription level of ADAM9 mRNA was 0.45 for CRPC tissue and 1.0 for HSPC tissue (P = 0.002). In the CRPC group, patients with low ADAM9 protein expression were significantly associated with shorter OS than patients with high expression (38.6 months vs. 57.8 months, hazard rate (HR) = 2.638, P = 0.023). ADAM9 expression was low in CRPC, correlated with poor prognosis and might be involved in the succession from HSPC to CRPC by various functions.

Design and characterization of a novel cellular prion-derived quenched fluorimetric substrate of α-secretase

Under normal conditions, the cellular prion protein (PrP c) undergoes a proteolytic attack between amino acids 111 and 112 which gives rise to the N-terminal secreted N1 fragment and its C-terminal membrane-tethered counterpart C1. Importantly, this cleavage precludes the integrity of the neurotoxic 106–126 sequence. Here, we describe an original and reliable assay based on a quenched fluorimetric substrate (JMV2770) encompassing the 111/112 sequence of PrP c. In whole brain homogenate, the JMV2770-hydrolysing activity is optimal at neutral pH and sensitive to the metalloprotease inhibitor BB3103 but not to acidic and serine protease blockers. JMV2770 is efficiently cleaved by intact HEK293 cells and fibroblasts in culture, consistent with an hydrolysis by a typical ectoprotease. Overexpressions of α-secretases a disintegrin and metalloprotease-9 (ADAM9), ADAM10 or TACE (ADAM17) in human cells increase BB3103-sensitive JMV2770 hydrolysis, while invalidation of ADAM10 and TACE or reduced expression of ADAM9 by an antisense approach significantly reduced its cleavage. Finally, analysis of JMV2770 hydrolysis following transient transfection of ADAM10 or ADAM9 cDNA in ADAM10 −/− fibroblasts allowed to confirm our previous data establishing that ADAM9 does not behave as a genuine α-secretase but rather acts as an important upstream regulator of ADAM10 activity.

Overexpression of ADAM9 enhances growth factor-mediated recycling of E-cadherin in human colon cancer cell line HT29 cells

Growth factor-mediated stimulation of epithelial cells induces the disassembly of E-cadherin-mediated cell–cell adhesion. We found that overexpression of a disintegrin and metalloprotease 9 (ADAM9) enhanced growth factor-mediated induction of endocytosis and dynamic recycling of E-cadherin in HT29 human colon cancer cells. In addition, ubiquitination and degradation of E-cadherin were reduced in these cells. ADAM9 constitutively interacted with E-cadherin, and the two proteins co-localized at the plasma membrane of HT29 cells. Administration of a metalloprotease inhibitor or overexpression of an ADAM9 mutant lacking metalloprotease activity attenuated growth factor-dependent endocytosis and recycling of E-cadherin as well as scattering of HT29 cells. These results suggest that the metalloprotease activity of ADAM9 mediates growth factor-induced endocytosis and dynamic recycling of E-cadherin and prevents E-cadherin degradation.

The disintegrin domain of ADAM9: a ligand for multiple beta1 renal integrins.

Renal tubular epithelial cells in all nephron segments express a distinct member of the metalloprotease-disintegrin family, ADAM9 (a disintegrin and metalloprotease 9), in a punctate basolateral distribution co-localized to the beta1 integrin chain [Mahimkar, Baricos, Visaya, Pollock and Lovett (2000) J. Am. Soc. Nephrol. 11, 595-603]. Discrete segments of the nephron express several defined beta1 integrins, suggesting that ADAM9 interacts with multiple renal integrins and thereby regulates epithelial cell-matrix interactions. Intact ADAM9 and a series of deletion constructs sequentially lacking the metalloprotease domain and the disintegrin domain were assembled as chimaeras with a C-terminal GFP (green fluorescent protein) tag. Stable expression of the ADAM9/GFP protein on the surface of HEK-293 cells (human embryonic kidney 293 cells) significantly decreased adhesion to types I and IV collagen, vitronectin and laminin, but had little effect on adhesion to fibronectin. Expression of the disintegrin/cysteine-rich/GFP construct yielded a similar, but more marked pattern of decreased adhesion. Expression of the cysteine-rich/GFP construct had no effect on adhesion, indicating that the disintegrin domain was responsible for the competitive inhibition of cell-matrix binding. To define the specific renal tubular beta1 integrins interacting with the ADAM9 disintegrin domain, a recombinant GST (glutathione S-transferase)-disintegrin protein was used as a substrate in adhesion assays in the presence or absence of specific integrin-blocking antibodies. Inclusion of antibodies to alpha1, alpha3, alpha6, alphav and beta1 blocked adhesion of HEK-293 cells to GST-disintegrin protein. Immobilized GST-disintegrin domain perfused with renal cortical lysates specifically recovered the alpha3, alpha6, alphav and beta1 integrin chains by Western analysis. It is concluded that ADAM9 is a polyvalent ligand, through its disintegrin domain, for multiple renal integrins of the beta1 class.