Loss of Multimerin-2 and EMILIN-2 Expression in Gastric Cancer Associate with Altered Angiogenesis.

Gastric cancer is a deadly tumor and a relatively common disease worldwide. Surgical resection and chemotherapy are the main clinical options to treat this type of disease, however the median overall survival rate is limited to one year. Thus, the development of new therapies is a highly necessary clinical need. Angiogenesis is a promising target for this tumor type, however clinical trials with the use of anti-angiogenic drugs have so far not met expectations. Therefore, it is important to better characterize the expression of molecules whose expression levels may impact on the efficacy of the treatments. In this study the characteristics of the gastric tumor associated blood vessels were first assessed by endomicroscopy. Next, we analyzed the expression of Multimerin-2, EMILIN-2 and EMILIN-1, three molecules of the EMI Domain ENdowed (EDEN) protein family. These molecules play important functions in the tumor microenvironment, affecting cancer progression both directly and indirectly impinging on angiogenesis and lymphangiogenesis. All the molecules were highly expressed in the normal mucosa whereas in a number of patients their expression was altered. We consider that better characterizing the gastric tumor microenvironment and the quality of the vasculature may achieve effective patient tailored therapies.

EMILIN2 down-modulates the Wnt signalling pathway and suppresses breast cancer cell growth and migration.

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.

Multimerin-2 orchestrates the cross-talk between endothelial cells and pericytes: A mechanism to maintain vascular stability.

Tumor angiogenesis is vital for the growth and development of various solid cancers and as such is a valid and promising therapeutic target. Unfortunately, the use of the currently available anti-angiogenic drugs increases the progression-free survival by only a few months. Conversely, targeting angiogenesis to prompt both vessel reduction and normalization, has been recently viewed as a promising approach to improve therapeutic efficacy. As a double-edged sword, this line of attack may on one side halt tumor growth as a consequence of the reduction of nutrients and oxygen supplied to the tumor cells, and on the other side improve drug delivery and, hence, efficacy. Thus, it is of upmost importance to better characterize the mechanisms regulating vascular stability. In this context, recruitment of pericytes along the blood vessels is crucial to their maturation and stabilization. As the extracellular matrix molecule Multimerin-2 is secreted by endothelial cells and deposited also in juxtaposition between endothelial cells and pericytes, we explored Multimerin-2 role in the cross-talk between the two cell types. We discovered that Multimerin-2 is an adhesion substrate for pericytes. Interestingly, and consistent with the notion that Multimerin-2 is a homeostatic molecule deposited in the later stages of vessel formation, we found that the interaction between endothelial cells and pericytes promoted the expression of Multimerin-2. Furthermore, we found that Multimerin-2 modulated the expression of key cytokines both in endothelial cells and pericytes. Collectively, our findings posit Multimerin-2 as a key molecule in the cross-talk between endothelial cells and pericytes and suggest that the expression of this glycoprotein is required to maintain vascular stability.

Deregulated expression of Elastin Microfibril Interfacer 2 (EMILIN2) in gastric cancer affects tumor growth and angiogenesis.

Gastric cancer is a frequent human tumor and often a lethal disease. Targeted therapy for gastric carcinomas is far behind vis-à-vis other solid tumors, primarily because of the paucity of cancer-driving mutations that could be efficiently and specifically targeted by current therapy. Thus, there is a need to discover actionable pathways/proteins and new diagnostic and prognostic biomarkers. In this study, we explored the role of the extracellular matrix glycoprotein EMILIN2, Elastin Microfibril Interfacer 2, in a cohort of gastric cancer patients. We discovered that EMILIN2 expression was consistently suppressed in gastric cancer and high expression levels of this glycoprotein were linked to abnormal vascular density. Furthermore, we found that EMILIN2 had a dual effect on gastric carcinoma cells: on one hand, it decreased tumor cell proliferation by triggering apoptosis, and on the other hand, it evoked the production of a number of cytokines involved in angiogenesis and inflammation, such as IL-8. Collectively, our findings posit EMILIN2 as an important onco-regulator exerting pleiotropic effects on the gastric cancer microenvironment.

Multimerin-2 maintains vascular stability and permeability.

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.

TIMP-1 is Overexpressed and Secreted by Platinum Resistant Epithelial Ovarian Cancer Cells.

Epithelial Ovarian Cancer (EOC) is the most lethal gynecological cancer in developed countries, and the development of new strategies to overcome chemoresistance is an awaited clinical need. Angiogenesis, the development of new blood vessels from pre-existing vasculature, has been validated as a therapeutic target in this tumor type. The aim of this study is to verify if EOC cells with acquired resistance to platinum (PT) treatment display an altered angiogenic potential. Using a proteomic approach, we identified the tissue inhibitor of metalloproteinases 1 (TIMP-1) as the only secreted factor whose expression was up-regulated in PT-resistant TOV-112D and OVSAHO EOC cells used as study models. We report that TIMP-1 acts as a double-edged sword in the EOC microenvironment, directly affecting the response to PT treatment on tumor cells and indirectly altering migration and proliferation of endothelial cells. Interestingly, we found that high TIMP-1 levels in stage III-IV EOC patients associate with decreased overall survival, especially if they were treated with PT or bevacizumab. Taken together, these results pinpoint TIMP-1 as a key molecule involved in the regulation of EOC PT-resistance and progression disclosing the possibility that it could be used as a new biomarker of PT-resistance and/or therapeutic target.

The ablation of the matricellular protein EMILIN2 causes defective vascularization due to impaired EGFR-dependent IL-8 production affecting tumor growth.

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.

The angiostatic molecule Multimerin 2 is processed by MMP-9 to allow sprouting angiogenesis.

Angiogenesis is a crucial process occurring under physiological and pathological conditions, including cancer. The development of blood vessels is tightly regulated by a plethora of cytokines, endothelial cell (EC) receptors and extracellular matrix (ECM) components. In this context, we have shown that Multimerin 2 (MMRN2), an ECM molecule specifically secreted by ECs, exerts angiostatic functions by binding VEGFA and other pro-angiogenic cytokines. Here, we demonstrate that during angiogenic stimuli MMRN2 mRNA levels significantly decrease. Furthermore, we provide evidence that MMRN2 is processed by matrix metalloproteinases (MMPs) including MMP-9 and, to a lesser degree, by MMP-2. This proteolytic cleavage correlates with an increased migration of ECs. Accordingly, MMRN2 down-regulation is associated with an increased number of EC pseudopodia at the migrating front and this effect is attenuated using specific MMP-9 inhibitors. The down-modulation of MMRN2 occurs also in the context of tumor-associated angiogenesis. Immunofluorescence performed on tumor sections indicate a broad co-localization of MMP-9 and MMRN2, suggesting that the molecule may be extensively remodeled during tumor angiogenesis. Given the altered expression in tumors and the key role of MMRN2 in blood vessel function, we postulate that analyses of its expression may serve as a marker to predict the efficacy of the treatments. In conclusion, these data further support the role of MMRN2 as a key molecule regulating EC function and sprouting angiogenesis.

Dissecting the CD93-Multimerin 2 interaction involved in cell adhesion and migration of the activated endothelium.

The glycoprotein CD93 has recently been recognized to play an important role in the regulation of the angiogenic process. Moreover, CD93 is highly expressed in the endothelial cells of tumor blood vessel and faintly expressed in the non-proliferating endothelium. Much evidence suggests that CD93 mediates adhesion in the endothelium. Here we identify Multimerin 2 (MMRN2), a pan-endothelial extracellular matrix protein, as a specific ligand for CD93. We found that CD93 and MMRN2 are co-expressed in the blood vessels of various human tumors. Moreover, disruption of the CD93-MMRN2 interaction reduced endothelial cell adhesion and migration, making the interaction of CD93 with MMRN2 an ideal target to block pathological angiogenesis. Model structures and docking studies served to envisage the region of CD93 and MMRN2 involved in the interaction. Site-directed mutagenesis identified different residue hotspots either directly or indirectly involved in the binding. We propose a molecular model in which the coiled-coil domain of MMRN2 is engaged by F238 of CD93. Altogether, these studies identify the key interaction surfaces of the CD93-MMRN2 complex and provide a framework for exploring how to inhibit angiogenesis by hindering the CD93-MMRN2 interaction.

MULTIMERIN2 binds VEGF-A primarily via the carbohydrate chains exerting an angiostatic function and impairing tumor growth.

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.