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Thus, it can be speculated that exogenous ADAMTS may interact with fibulin-2 produced by A cells and thereby inducing the anti-tumor effect observed.

Associated Data

A previous report also supported the pro-tumor activity of this metalloprotease since its exogenous expression potentiated invasive capacity of the human placental choriocarcinoma cell line JEG-3 [ 32 ]. Interestingly, an enhanced stromal collagen-1 deposition correlates with breast cancer metastasis [ 38 ]. In this scenario and in the absence of fibulin-2, ADAMTS could contribute to tumor progression by increasing the migratory capacity of breast tumor cells on collagen By contrast, several studies have highlighted the anti-tumor role of this metalloprotease, including the phenotypic characterization of Adamts12 -deficient mouse [ 31 ].

For instance, thrombospondin type-1 motifs at carboxy-terminal region of the protease influence its antimetastatic potential. However this enzyme undergoes proteolytic processing events that could modify this effect [ 29 ]. Thus, this metalloprotease was initially catalogued as a tumor-suppressor factor due to the angio-inhibitory properties related to the thrombospondin type-1 domains located at the carboxy-terminal region [ 39 ].

Nonetheless, ADAMTS-1 can also generate anti-angiogenic peptides through the digestion of thrombospondin-1[ 41 ] or endorse the recruitment of fibroblasts involved in tumor growth, an effect also associated to an increased deposition of collagen-1 [ 42 ]. Moreover, ADAMTS-1 interacts with other member of the fibulin family, fibulin-1 [ 34 ], which in this case acts as a cofactor of the metalloprotease. We explored the possibility that fibulin-2 could be a regulatory factor of the metalloprotease activity.

Given that ADAMTS displays low aggrecanase activity in vitro [ 29 ], we evaluated the digestion of a peptide corresponding to the human aggrecan interglobular domain. However, our results indicated that the presence of fibulin-2 did not potentiate the cleavage of this peptide by ADAMTS Despite of this, it cannot be ruled out that this fibulin could act as an enhancer or inhibitor of ADAMTS enzymatic activity in other circumstances considering the limited number of known in vivo substrates for this enzyme.

Immunohistochemical staining revealed that ADAMTS was mainly detected in the stroma of grade 1 breast carcinomas, similar to what had been previously reported in colorectal cancer [ 30 ]. Staining pattern of fibulin-2 showed an equivalent situation.

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However, both proteins are hardly detected in grade 2 and 3 breast carcinomas. Thus, it can be speculated that a potential interaction between these two proteins may happen in less aggressive breast cancers and that this interaction could form part of a stromal response aimed to block cancer progression. In consequence, pro or anti-tumor activities of ADAMTS could also be influenced by factors other than interaction with fibulin For instance, these interactions may underlie the molecular mechanisms by which some tumor-protective metalloproteases exert their anti-tumor activities.

Additionally, tumor susceptibility assays using mice lacking both Adamts12 and Fbln2 genes would be necessary to understand in depth the significance of this interaction not only in breast cancer but also in tumors from different origins. The Matchmaker Two-hybrid System and a human fetal kidney library prey vectors containing a sequence enconding amino acids of the GAL4 protein AD were purchased from Clontech.

Both bait and prey vectors were introduced into Saccharomyces cerevisiae AH cells and selection of double transformants was performed on synthetic minimal medium lacking leucine, tryptophan and histidine. Identification of the prey inserts was performed by direct sequencing. Anti-actin antibody was from Sigma-Aldrich. Immunoreactive proteins were visualized using HRP-peroxidase labeled anti-rabbit or anti-mouse secondary antibody and the ECL detection system Pierce.

Relative expression levels were quantified and normalized to actin levels within the same blot using the Image J software. MCF-7 cells extract producing fibulin-2 was used as a negative control. As indicated, different amounts of fibulin-2 were added to the reaction.

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Peptide degradation was examined by western blot using an anti-His antibody Quiagen. For MCF-7 cells, 2. Cells that reached the lower surface were stained with crystal violet. At least three independent experiments were performed with triplicates for each condition. Cells were counted in eight randomly selected microscopic fields. Briefly, 1,25 x 10 4 cells were seeded per well and migration was monitored by time-lapse microscopy using a Zeiss Axio Observer Microscopy. Experiments were performed with triplicates and covered area was quantified at different times using Image J.

After 7 days, mammospheres were collected and enzymatically dissociated as previously described [ 43 ]. Mammospheres formation was monitored microscopically daily to ensure they derived from single cells and not from aggregates. Number of mammospheres was determined after 7 days culture. As a control, the opposite flank in each animal was injected with identical number of control MCF-7 cells transfected with an empty vector. Tumor growth was monitored weekly as previously described [ 29 ].

For estrogen supplementation, pellets of 1. Mice were housed under specific pathogen-free conditions and following the guidelines of the Committee on Animal Experimentation of the Universidad de Oviedo, Asturias, Spain. Slides were processed for indirect peroxidase immunohistochemistry in the following way: Selected slides were counterstained with haematoxylin.

The intensity of immunostaining was evaluated by two independent observers directly upon microscope and was scored as absent, slight, moderate and strong. Study methodologies were conformed to the standards set by the Declaration of Helsinki and were approved by the Ethics Committee of the Universidad de Oviedo-Principado de Asturias Spain. To assess the effect of ADAMTS and fibulin-2 on breast cancer prognosis, survival probability was determined using the data available at www. Results were represented as Kaplan-Meier long-rank test survival plots.

Statistical analysis were carried out using the GraphPad Prism 5. The occurrence of significant differences was determined with the Student-Welch t test.

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National Center for Biotechnology Information , U. Journal List Oncotarget v. Published online Jan Esteban , 7 Alvaro J. Obaya , 2, 7 and Santiago Cal 1, 2. Author information Article notes Copyright and License information Disclaimer. Santiago Cal , se.

Received Dec 12; Accepted Jan This is an open-access article distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited. This article has been cited by other articles in PMC. Associated Data Supplementary Materials oncotargets Abstract Balance between pro-tumor and anti-tumor effects may be affected by molecular interactions within tumor microenvironment.

Open in a separate window. Survival analysis To assess the effect of ADAMTS and fibulin-2 on breast cancer prognosis, survival probability was determined using the data available at www. Statistical analysis Statistical analysis were carried out using the GraphPad Prism 5. Click here to view. Acknowledgments We thank Dr. Interactions of the complement system with molecules of extracellular matrix: Vong S, Kalluri R.

The role of stromal myofibroblast and extracellular matrix in tumor angiogenesis. Nat Rev Mol Cell Biol.

A comparative analysis of the fibulin protein family. Biochemical characterization, binding interactions, and tissue localization. Molecular evolution of the fibulins: The proteoglycans aggrecan and Versican form networks with fibulin-2 through their lectin domain binding. TM14 is a new member of the fibulin family fibulin-7 that interacts with extracellular matrix molecules and is active for cell binding. Fibrillin-1 and fibulin-2 interact and are colocalized in some tissues.

Binding of the G domains of laminin alpha1 and alpha2 chains and perlecan to heparin, sulfatides, alpha-dystroglycan and several extracellular matrix proteins. The dual role of fibulins in tumorigenesis. Fibulin-5 suppresses lung cancer invasion by inhibiting matrix metalloproteinase-7 expression. Fibulin-3 is associated with tumour progression and a poor prognosis in nasopharyngeal carcinomas and inhibits cell migration and invasion via suppressed AKT activity.

Cell Mol Life Sci.

Fibulin-2 binds to the short arms of laminin-5 and laminin-1 via conserved amino acid sequences. Binding of mouse and human fibulin-2 to extracellular matrix ligands. Integrin-binding and cell-adhesion studies of fibulins reveal a particular affinity for alpha IIb beta 3.

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Anti-angiogenic and tumor-suppressive roles of candidate tumor-suppressor gene, Fibulin-2, in nasopharyngeal carcinoma. Epigenetic analysis of childhood acute lymphoblastic leukemia. Loss of fibulin-2 expression is associated with breast cancer progression. Identification, characterization, and intracellular processing of ADAM-TS12, a novel human disintegrin with a complex structural organization involving multiple thrombospondin-1 repeats.

New and paradoxical roles of matrix metalloproteinases in the tumor microenvironment. The ADAMTS12 metalloprotease gene is epigenetically silenced in tumor cells and transcriptionally activated in the stroma during progression of colon cancer. Higher sensitivity of Adamtsdeficient mice to tumor growth and angiogenesis.

Dysregulated expression of adamalysin-thrombospondin genes in human breast carcinoma.

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Site-specific inductive and inhibitory activities of MMP-2 and MMP-3 orchestrate mammary gland branching morphogenesis. Remodeling and homeostasis of the extracellular matrix: Estradiol and fibulin-1 inhibit motility of human ovarian- and breast-cancer cells induced by fibronectin. La viajera del tiempo Spanish Edition. Perdida en el tiempo Spanish Edition. Historia de dos almas Spanish Edition. La Princesa del Vikingo: Capricho del destino Spanish Edition.

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