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Matrix Biol ; 2019 Aug 15.
Artículo en Inglés | MEDLINE | ID: mdl-31422156


Multimerin-2 is an extracellular matrix glycoprotein and member of the elastin microfibril interface-located (EMILIN) family of proteins. Multimerin-2 is deposited along blood vessels and we previously demonstrated that it regulates the VEGFA/VEGFR2 signaling axis and angiogenesis. However, its role in modulating vascular homeostasis remains largely unexplored. Here we identified Multimerin-2 as a key molecule required to maintain vascular stability. RNAi knockdown of Multimerin-2 in endothelial cells led to cell-cell junctional instability and increased permeability. Mechanistically cell-cell junction dismantlement occurred through the phosphorylation of VEGFR2 at Tyr951, activation of Src and phosphorylation of VE-cadherin. To provide an in vivo validation for these in vitro effects, we generated Multimerin-2-/- (Mmrn2-/-) mice. Although Mmrn2-/- mice developed normally and displayed no gross abnormalities, endothelial cells displayed cell junctional defects associated with increased levels of VEGFR2 phospho-Tyr949 (the murine counterpart of human Tyr951), impaired pericyte recruitment and increased vascular leakage. Of note, tumor associated vessels were defective in Mmrn2-/- mice, with increased number of small and often collapsed vessels, concurrent with a significant depletion of pericytic coverage. Consequently, the Mmrn2-/- vessels were less perfused and leakier, leading to increased tumor hypoxia. Chemotherapy efficacy was markedly impaired in Mmrn2-/- mice and this was associated with poor drug delivery to the tumor xenografts. Collectively, our findings demonstrate that Multimerin-2 is required for proper vessel homeostasis and stabilization, and unveil the possibility to utilize expression levels of this glycoprotein in predicting chemotherapy efficacy.

EBioMedicine ; 46: 79-93, 2019 Aug.
Artículo en Inglés | MEDLINE | ID: mdl-31303496


BACKGROUND: Metastatic colorectal cancer (CRC) remains a deadly disease. Identifying locally advanced CRC patients with high risk of developing metastasis and improving outcome of metastatic CRC patients require discovering master regulators of metastasis. In this context, the non-coding part of the human genome is still largely unexplored. METHODS: To interrogate the non-coding part of the human genome and disclose regulators of CRC metastasis, we combined a transposon-based forward genetic screen with a novel in vitro assay, which forces cells to grow deprived of cell-substrate and cell-cell contacts (i.e. forced single cell suspension assay - fSCS). FINDINGS: We proved that fSCS selects CRC cells with mesenchymal and pro-metastatic traits. Moreover, we found that the transposon insertions conferred CRC cells resistance to fSCS and thus metastatic advantage. Among the retrieved transposon insertions, we demonstrated that the one located in the 3'UTR of BTBD7 disrupts miR-23b::BTBD7 interaction and contributes to pro-metastatic traits. In addition, miR-23b and BTBD7 correlate with CRC metastasis both in preclinical experiments and in clinical samples. INTERPRETATION: fSCS is a simple and scalable in vitro assay to investigate pro-metastatic traits and transposon-based genetic screens can interrogate the non-coding part of the human genome (e.g. miRNA::target interactions). Finally, both Btbd7 and miR-23b represent promising prognostic biomarkers and therapeutic targets in CRC. FUND: This work was supported by Marie Curie Actions (CIG n. 303877) and Friuli Venezia Giulia region (Grant Agreement n°245574), Italian Association for Cancer Research (AIRC, MFAG n°13589), Italian Ministry of Health (GR-2010-2319387 and PE-2016-02361040) and 5x1000 to CRO Aviano.

Proteínas Adaptadoras Transductoras de Señales/genética , Neoplasias Colorrectales/genética , Neoplasias Colorrectales/patología , Regulación Neoplásica de la Expresión Génica , MicroARNs/genética , Interferencia de ARN , Comunicación Celular , Línea Celular Tumoral , Proliferación Celular , Transición Epitelial-Mesenquimal/genética , Matriz Extracelular/metabolismo , Pruebas Genéticas , Humanos , Metástasis de la Neoplasia , Estadificación de Neoplasias
Oncogene ; 37(25): 3399-3414, 2018 06.
Artículo en Inglés | MEDLINE | ID: mdl-29483644


EMILIN2 is an extracellular matrix constituent playing an important role in angiogenesis; however, the underlying mechanism is unknown. Here we show that EMILIN2 promotes angiogenesis by directly binding epidermal growth factor receptor (EGFR), which enhances interleukin-8 (IL-8) production. In turn, IL-8 stimulates the proliferation and migration of vascular endothelial cells. Emilin2 null mice were generated and exhibited delayed retinal vascular development, which was rescued by the administration of the IL-8 murine ortholog MIP-2. Next, we assessed tumor growth and tumor-associated angiogenesis in these mice. Tumor cell growth in Emilin2 null mice was impaired as well as the expression of MIP-2. The vascular density of the tumors developed in Emilin2 null mice was prejudiced and vessels perfusion, as well as response to chemotherapy, decreased. Accordingly, human tumors expressing high levels of EMILIN2 were more responsive to chemotherapy. These results point at EMILIN2 as a key microenvironmental cue affecting vessel formation and unveil the possibility to develop new prognostic tools to predict chemotherapy efficacy.

Glicoproteínas/metabolismo , Glicoproteínas/fisiología , Interleucina-8/metabolismo , Melanoma Experimental/irrigación sanguínea , Melanoma Experimental/patología , Neovascularización Patológica/patología , Animales , Apoptosis , Biomarcadores de Tumor/genética , Biomarcadores de Tumor/metabolismo , Proliferación Celular , Receptores ErbB/genética , Receptores ErbB/metabolismo , Femenino , Glicoproteínas/genética , Humanos , Interleucina-8/genética , Masculino , Melanoma Experimental/metabolismo , Ratones , Ratones Endogámicos C57BL , Ratones Noqueados , Neovascularización Patológica/genética , Neovascularización Patológica/metabolismo , Ratas , Ratas Endogámicas F344 , Células Tumorales Cultivadas , Ensayos Antitumor por Modelo de Xenoinjerto
Int J Mol Sci ; 17(11)2016 Nov 01.
Artículo en Inglés | MEDLINE | ID: mdl-27809279


The extracellular matrix (ECM) is a complex network of proteins, glycoproteins, proteoglycans, and polysaccharides. Through multiple interactions with each other and the cell surface receptors, not only the ECM determines the physical and mechanical properties of the tissues, but also profoundly influences cell behavior and many physiological and pathological processes. One of the functions that have been extensively explored is its impingement on angiogenesis. The strong impact of the ECM in this context is both direct and indirect by virtue of its ability to interact and/or store several growth factors and cytokines. The aim of this review is to provide some examples of the complex molecular mechanisms that are elicited by these molecules in promoting or weakening the angiogenic processes. The scenario is intricate, since matrix remodeling often generates fragments displaying opposite effects compared to those exerted by the whole molecules. Thus, the balance will tilt towards angiogenesis or angiostasis depending on the relative expression of pro- or anti-angiogenetic molecules/fragments composing the matrix of a given tissue. One of the vital aspects of this field of research is that, for its endogenous nature, the ECM can be viewed as a reservoir to draw from for the development of new more efficacious therapies to treat angiogenesis-dependent pathologies.

Microambiente Celular/fisiología , Matriz Extracelular/fisiología , Neovascularización Patológica/fisiopatología , Neovascularización Fisiológica/fisiología , Animales , Citocinas/metabolismo , Matriz Extracelular/metabolismo , Humanos , Péptidos y Proteínas de Señalización Intercelular/metabolismo , Modelos Biológicos , Neovascularización Patológica/metabolismo , Unión Proteica
Oncotarget ; 7(2): 2022-37, 2016 Jan 12.
Artículo en Inglés | MEDLINE | ID: mdl-26655500


Angiogenesis is a key process occurring under both physiological and pathological conditions and is a hallmark of cancer. We have recently demonstrated that the extracellular matrix (ECM) molecule MULTIMERIN2 exerts an angiostatic function through the binding to VEGF-A. In this study we identify the region of the molecule responsible for the binding and demonstrate that the interaction involves the carbohydrate chains. MULTIMERIN2 interacts with other VEGF-A isoforms and VEGF family members such as VEGF-B, -C, -D and PlGF-1 suggesting that the molecule may function as a reservoir for different cytokines. In response to VEGF-A165, we show that MULTIMERIN2 impairs the phosphorylation of VEGFR2 at both Y1175 and Y1214 residues, halts SAPK2/p38 activation and negatively affects endothelial cell motility. In addition, MULTIMERIN2 and its active deletion mutant decrease the availability of the VEGFR2 receptor at the EC plasma membrane. The ectopic expression of MULTIMERIN2 or its active deletion mutant led to a striking reduction of tumor-associated angiogenesis and tumor growth. In conclusion, these data pinpoint MULTIMERIN2 as a key angiostatic molecule and disclose the possibility to develop new prognostic tools and improve the management of cancer patients.

Antígenos de Superficie/metabolismo , Carbohidratos/química , Fibrosarcoma/prevención & control , Células Endoteliales de la Vena Umbilical Humana/metabolismo , Glicoproteínas de Membrana/metabolismo , Neovascularización Patológica/prevención & control , Factor A de Crecimiento Endotelial Vascular/metabolismo , Animales , Apoptosis , Movimiento Celular , Proliferación Celular , Femenino , Fibrosarcoma/irrigación sanguínea , Fibrosarcoma/patología , Técnica del Anticuerpo Fluorescente , Glicosilación , Humanos , Técnicas para Inmunoenzimas , Ratones , Ratones Endogámicos BALB C , Ratones Desnudos , Fosforilación , ARN Mensajero/genética , Reacción en Cadena en Tiempo Real de la Polimerasa , Reacción en Cadena de la Polimerasa de Transcriptasa Inversa , Resonancia por Plasmón de Superficie , Células Tumorales Cultivadas , Ensayos Antitumor por Modelo de Xenoinjerto
J Pathol ; 232(4): 391-404, 2014 Mar.
Artículo en Inglés | MEDLINE | ID: mdl-24374807


EMILIN2 is an extracellular matrix (ECM) protein that exerts contradictory effects within the tumour microenvironment: it induces apoptosis in a number of tumour cells, but it also enhances tumour neo-angiogenesis. In this study, we describe a new mechanism by which EMILIN2 attenuates tumour cell viability. Based on sequence homology with the cysteine-rich domain (CRD) of the Frizzled receptors, we hypothesized that EMILIN2 could affect Wnt signalling activation and demonstrate direct interaction with the Wnt1 ligand. This physical binding leads to decreased LRP6 phosphorylation and to the down-modulation of ß-catenin, TAZ and their target genes. As a consequence, EMILIN2 negatively affects the viability, migration and tumourigenic potential of MDA-MB-231 breast cancer cells in a number of two- and three-dimensional in vitro assays. EMILIN2 does not modulate Wnt signalling downstream of the Wnt-Frizzled interaction, since it does not affect the activation of the pathway following treatment with the GSK3 inhibitors LiCl and CHIR99021. The interaction with Wnt1 and the subsequent biological effects require the presence of the EMI domain, as there is no effect with a deletion mutant lacking this domain. Moreover, in vivo experiments show that the ectopic expression of EMILIN2, as well as treatment with the recombinant protein, significantly reduce tumour growth and dissemination of cancer cells in nude mice. Accordingly, the tumour samples are characterized by a significant down-regulation of the Wnt signalling pathway. Altogether, these findings provide further evidence of the complex regulations governed by EMILIN2 in the tumour microenvironment, and they identify a key extracellular regulator of the Wnt signalling pathway.

Neoplasias de la Mama/metabolismo , Movimiento Celular , Proliferación Celular , Glicoproteínas/metabolismo , Vía de Señalización Wnt , Proteína Wnt1/metabolismo , Animales , Neoplasias de la Mama/genética , Neoplasias de la Mama/patología , Línea Celular Tumoral , Supervivencia Celular , Regulación hacia Abajo , Femenino , Regulación Neoplásica de la Expresión Génica , Glicoproteínas/genética , Células HEK293 , Humanos , Proteína-6 Relacionada a Receptor de Lipoproteína de Baja Densidad/metabolismo , Ratones , Ratones Desnudos , Mutación , Invasividad Neoplásica , Fosforilación , Unión Proteica , Dominios y Motivos de Interacción de Proteínas , Factores de Tiempo , Factores de Transcripción/metabolismo , Transfección , Carga Tumoral , Microambiente Tumoral , Proteína Wnt1/genética , beta Catenina/metabolismo