Satsuki Mochizuki

ADAMs in cancer cell proliferation and progression

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)

Targeted deletion or pharmacological inhibition of MMP-2 prevents cardiac rupture after myocardial infarction in mice

MMPs are implicated in LV remodeling after acute myocardial infarction (MI). To analyze the role of MMP-2, we generated MI by ligating the left coronary artery of MMP-2-KO and WT mice, the latter of which were administered orally an MMP-2-selective inhibitor or vehicle (TISAM). The survival rate was significantly higher in MMP-2-KO and TISAM-treated mice than in control WT mice. The main cause of mortality in control WT mice was cardiac rupture, which was not observed in MMP-2-KO or TISAM-treated mice. Control WT mice, but not MMP-2-KO or TISAM-treated mice, showed activation of the zymogen of MMP-2, strong gelatinolytic activity, and degradation of ECM components, including laminin and fibronectin, in the infarcted myocardium. Although infarcted cardiomyocytes in control WT mice were rapidly removed by macrophages, the removal was suppressed in MMP-2-KO and TISAM-treated mice. Macrophage migration was induced by the infarcted myocardial tissue from control WT mice and was inhibited by treatment of macrophages with laminin or fibronectin peptides prior to migration assay. These data suggest that inhibition of MMP-2 activity improves the survival rate after acute MI by preventing cardiac rupture and delays post-MI remodeling through a reduction in macrophage infiltration.

Regulatory Role of Dendritic Cells in Postinfarction Healing and Left Ventricular Remodeling

Background— Inflammation and immune responses are integral components in the healing process after myocardial infarction. We previously reported dendritic cell (DC) infiltration in the infarcted heart; however, the precise contribution of DC in postinfarction healing is unclear. Methods and Results— Bone marrow cells from CD11c-diphtheria toxin receptor/green fluorescent protein transgenic mice were transplanted into lethally irradiated wild-type recipient mice. After reconstitution of bone marrow–derived cells, the recipient mice were treated with either diphtheria toxin (DC ablation) or vehicle (control), and myocardial infarction was created by left coronary ligation. CD11c+ green fluorescent protein–positive DCs expressing CD11b and major histocompatibility complex class II were recruited into the heart, peaking on day 7 after myocardial infarction in the control group. Mice with DC ablation for 7 days showed deteriorated left ventricular function and remodeling. The DC-ablated group demonstrated enhanced and sustained expression of inflammatory cytokines such as interleukin-1&bgr;, interleukin-18, and tumor necrosis factor-&agr;, prolonged extracellular matrix degradation associated with a high level of matrix metalloproteinase-9 activity, and diminished expression level of interleukin-10 and endothelial cell proliferation after myocardial infarction compared with the control group. In vivo analyses revealed that DC-ablated infarcts had enhanced monocyte/macrophage recruitment. Among these cells, marked infiltration of proinflammatory Ly6Chigh monocytes and F4/80+ CD206− M1 macrophages and, conversely, impaired recruitment of anti-inflammatory Ly6Clow monocytes and F4/80+ CD206+ M2 macrophages in the infarcted myocardium were identified in the DC-ablated group compared with the control group. Conclusions— These results suggest that the DC is a potent immunoprotective regulator during the postinfarction healing process via its control of monocyte/macrophage homeostasis.

Hyaluronan inhibits expression of ADAMTS4 (aggrecanase-1) in human osteoarthritic chondrocytes

Background: Intra-articular injection of hyaluronan (HA) has been suggested to have a disease-modifying effect in osteoarthritis, but little is known about the possible mechanisms. Objective: To investigate the effects of HA species of different molecular mass, including 800 kDa (HA800) and 2700 kDa (HA2700), on the expression of aggrecanases (ie, ADAMTS species), which play a key role in aggrecan degradation. Methods: The effects of HA species on the expression of ADAMTS1, 4, 5, 8, 9 and 15 in interleukin 1α (IL1α)-stimulated osteoarthritic chondrocytes were studied by reverse transcription PCR and real-time PCR. Expression of ADAMTS4 protein and aggrecanase activity and signal transduction pathways of IL1, CD44 and intracellular adhesion molecule 1 (ICAM1) were examined by immunoblotting. Results: IL1α treatment of chondrocytes induced ADAMTS4, and HA800 and HA2700 significantly decreased IL1α-induced expression of ADAMTS4 mRNA and protein. IL1α-stimulated aggrecanase activity in osteoarthritic chondrocytes was reduced by treatment with HA2700 or transfection of small interfering RNA for ADAMTS4. A similar result was obtained when HA2700 was added to explant cultures of osteoarthritic cartilage. HA2700 neither directly inhibited nor bound to ADAMTS4. Downregulation of ADAMTS4 expression by HA2700 was attenuated by treatment of IL1α-treated chondrocytes with antibodies to CD44 and/or ICAM1. The increased phosphorylation of IL1 receptor-associated kinase-1 and extracellular signal-regulated protein kinase1/2 induced by the IL1α treatment was downregulated by enhanced IRAK-M expression after HA2700 treatment. Conclusion: These data suggest that HA2700 suppresses aggrecan degradation by downregulating IL1α-induced ADAMTS4 expression through the CD44 and ICAM1 signalling pathways in osteoarthritic chondrocytes.

ADAM28 Is Overexpressed in Human Breast Carcinomas: Implications for Carcinoma Cell Proliferation through Cleavage of Insulin-like Growth Factor Binding Protein-3

A disintegrin and metalloproteinases (ADAMs) are involved in various biological events including cell adhesion, cell fusion, membrane protein shedding, and proteolysis. In the present study, our reverse transcription-PCR analysis showed that among the 12 different ADAM species with a putative metalloproteinase motif, prototype membrane-anchored ADAM28m and secreted-type ADAM28s are selectively expressed in human breast carcinoma tissues. By real-time quantitative PCR, their expression levels were significantly higher in carcinomas than in nonneoplastic breast tissues. In situ hybridization, immunohistochemistry, and immunoblotting analyses indicated that ADAM28 is predominantly expressed in an active form by carcinoma cells within carcinoma tissues. A direct correlation was observed between mRNA expression levels and proliferative activity of the carcinoma cells. Treatment of ADAM28-expressing breast carcinoma cells (MDA-MB231) with insulin-like growth factor-I (IGF-I) increased cell proliferation, cleavage of IGF binding protein (IGFBP)-3, as well as IGF-I cell signaling; these processes were all significantly inhibited by treatment with ADAM inhibitor or anti-ADAM28 antibody. Down-regulation of ADAM28 expression in MDA-MB231 cells with small interfering RNA significantly reduced cell proliferation, IGFBP-3 cleavage, and growth of xenografts in mice. In addition, cleavage of IGFBP-3 in breast carcinoma tissues was correlated with ADAM28 expression levels and inhibited by treatment with ADAM inhibitor or anti-ADAM28 antibody. These results show that ADAM28 is overexpressed in an activated form in human breast carcinoma cells and suggest that ADAM28 is involved in cell proliferation through enhanced bioavailability of IGF-I released from the IGF-I/IGFBP-3 complex by selective IGFBP-3 cleavage in human breast carcinomas.

Increased RANKL expression is related to tumour migration and metastasis of renal cell carcinomas

Receptor activator of NF‐κB ligand (RANKL) and its receptor, receptor activator of NF‐κB (RANK), play a key role in osteoclastogenesis, and osteoprotegerin (OPG) acts as a decoy receptor for RANKL. We investigated the role of the RANKL–RANK–OPG system in renal cell carcinomas (RCCs), which frequently metastasize to bones. Real‐time quantitative PCR revealed that RANKL mRNA expression was higher in clear cell RCCs than in papillary and chromophobe RCCs. Similarly, RANKL protein expression level in clear cell RCCs was higher than that in papillary and chromophobe RCCs, showing positive correlations with the primary tumour stage and distant metastasis. There was no significant association between the expression level of RANK, OPG and histological subtypes of RCC. RANKL and RANK expression was observed in metastatic RCCs in the bone and other organs, suggesting that they play a role in metastasis to the bone and other organs. Recombinant RANKL protein stimulated migration of a clear cell RCC cell line, Caki‐1, in vitro, and this enhanced migration was inhibited by the administration of recombinant OPG protein. Furthermore, multivariate Cox analysis revealed that elevated RANKL and RANK expression with low‐OPG expression was a significant and independent predictor of recurrence, bone metastasis and a poor prognosis. These data suggest that the RANKL–RANK–OPG system is involved not only in the bone metastasis of RCCs but also in metastasis to other organs through the stimulation of cancer cell migration. Copyright

ADAM‐12 (meltrin α) is involved in chondrocyte proliferation via cleavage of insulin‐like growth factor binding protein 5 in osteoarthritic cartilage

OBJECTIVE ADAMs are a gene family of multifunctional proteins. We undertook this study to determine which ADAM species is up-regulated in osteoarthritic (OA) cartilage and to examine its pathobiologic function. METHODS Expression of the 13 different metalloproteinase-type ADAMs was screened by reverse transcription-polymerase chain reaction (PCR), and expression levels of prototype membrane-anchored ADAM-12 (ADAM-12m) were determined by real-time PCR. ADAM-12m expression in articular cartilage was examined by in situ hybridization, immunohistochemistry, and immunoblotting. Chondrocytes were used for functional analyses of ADAM-12m. RESULTS ADAM-12m was selectively expressed in 87% of OA cartilage, and the expression level was significantly higher in OA cartilage than in normal cartilage. In situ hybridization showed that OA chondrocytes were responsible for the expression. ADAM-12m was immunolocalized on the membranes of OA chondrocytes, and its immunoreactivity correlated directly with the Mankin score and with degrees of chondrocyte cloning and proliferation. Immunoblotting analysis of OA chondrocytes demonstrated an active form of ADAM-12m. ADAM-12m expression in OA chondrocytes was selectively enhanced by transforming growth factor beta (TGFbeta), which also induced chondrocyte proliferation and degradation of insulin-like growth factor binding protein 5 (IGFBP-5). TGFbeta-induced chondrocyte proliferation was inhibited by suppression of IGF-1 signaling. In addition, TGFbeta-induced chondrocyte proliferation, chondrocyte cloning in agarose gel culture, and digestion of IGFBP-5 were inhibited with ADAM inhibitor, anti-ADAM-12 antibody, and small interfering RNA for ADAM-12. CONCLUSION These data suggest a novel function of ADAM-12m in chondrocyte proliferation and cloning in OA cartilage through enhanced bioavailability of IGF-1 from the IGF-1-IGFBP-5 complex by selective IGFBP-5 digestion.

High efficacy of third generation EGFR inhibitor AZD9291 in a leptomeningeal carcinomatosis model with EGFR-mutant lung cancer cells.

Leptomeningeal carcinomatosis (LMC) remarkably decreases the quality of life of EGFR-mutant lung cancer patients. In contrast to the lesions outside the central nervous system (CNS), molecular mechanisms of EGFR tyrosine kinase inhibitor (TKI) resistance in CNS lesions including LMC are largely unknown. In this study, we established an in vivo imaging model for LMC with EGFR mutant lung cancer cell lines harboring an exon 19 deletion in EGFR and evaluated the effect of first generation EGFR-TKIs, erlotinib, second generation afatinib, and third generation AZD9291. In PC-9/ffluc model, erlotinib treatment slowed the development of LMC. Importantly, treatment with afatinib or AZD9291 apparently delayed the development of LMC. Moreover, treatment with a higher dose of AZD9291, also associated with inhibited phosphorylation of EGFR downstream molecule S6, regressed LMC refractory to the aforementioned EGFR-TKI treatments. These observations suggest that the third generation EGFR-TKI AZD9291 may be an effective treatment for first or second generation EGFR-TKI resistant LMC caused by EGFR-mutant lung cancer.

Binding of ADAM28 to P-selectin glycoprotein ligand-1 enhances P-selectin-mediated leukocyte adhesion to endothelial cells

ADAMs (a disintegrin and metalloproteinases) are a recently discovered gene family of multifunctional proteins with the disintegrin-like and metalloproteinase domains. To analyze the biological functions of ADAM28, we screened binding molecules to secreted-type ADAM28 (ADAM28s) by the yeast two-hybrid system and identified P-selectin glycoprotein ligand-1 (PSGL-1). Binding between the disintegrin-like domain of ADAM28s and the extracellular portion of PSGL-1 was determined by yeast two-hybrid assays, binding assays of the domain-specific recombinant ADAM28s species using PSGL-1 stable transfectants and leukocyte cell lines expressing native PSGL-1 (HL-60 cells and Jurkat cells), and co-immunolocalization and co-immunoprecipitation of the molecules in these cells. Incubation of HL-60 cells with recombinant ADAM28s enhanced the binding to P-selectin-coated wells and P-selectin-expressing endothelial cells. In addition, intravenous injection of ADAM28s-treated HL-60 cells increased their accumulation in the pulmonary microcirculation and alveolar spaces in a mouse model of endotoxin-induced inflammation. These data suggest a novel function that ADAM28s promotes PSGL-1/P-selectin-mediated leukocyte rolling adhesion to endothelial cells and subsequent infiltration into tissue spaces through interaction with PSGL-1 on leukocytes under inflammatory conditions.

ADAM28 as a target for human cancers.

ADAM28 is a member of the ADAM (a disintegrin and metalloproteinase) gene family and consists of two isoforms, prototype membrane-type form and short secreted form. The metalloproteinase domain of ADAM28 has the zinc-binding consensus sequence, and ADAM28 exhibits catalytic activity to a few substrates such as insulin-like growth factor binding protein-3. The disintegrin domain interacts with integrins alpha4beta1, alpha4beta7 and alpha9beta1. In human non-small cell lung carcinomas and breast carcinomas, ADAM28 is overexpressed predominantly by carcinoma cells, and the expression correlates with carcinoma cell proliferation and lymph node metastasis. In this review we present our data on the activation of proADAM28, the tissue localization in human cancers and the interaction molecules, and discuss the regulation of ADAM28 activity and gene expression, the functions of ADAM28 in human cancers and the possibility of ADAM28 as a target for cancers.