MULTIMERIN2 (MMRN2), also known as Endoglyx-1, is an extracellular matrix glycoprotein whose function has so far remained elusive. Given its specific localization in tight association with the endothelium we hypothesized that this protein could modulate neo-angiogenesis. By multiple assays we showed that MMRN2 significantly impaired endothelial cell (EC) migration and organization of a functional vessel network. The interaction of ECs with MMRN2 induced a striking impairment of VEGFR1 and VEGFR2 activation. We focused our attention on VEGFR2, a chief regulator of angiogenesis, and clarified that MMRN2 interfered with the VEGF/VEGFR2 axis through a direct binding with VEGF-A. This novel interaction was assessed in several assays and the affinity was estimated (KdB50 nM). We next questioned whether the anti-angiogenic properties of MMRN2 could impair tumor growth. Although overexpression of MMRN2 by HT1080 cells did not affect their growth and apoptotic rate in vitro, it remarkably affected their growth in vivo. In fact, MMRN2-positive cells failed to efficiently grow and form well-vascularized tumors; a similar outcome was observed following treatment of established tumors with a MMRN2 adenoviral construct. Tumor-section immunostaining revealed a strong co-localization of VEGF-A with the ectopically expressed MMRN2. These novel findings suggest that VEGF may be sequestered by MMRN2 and be less available for the engagement to the receptors. Taken together these results highlight MMRN2 as a crucial player in the regulation of EC function, neoangiogenesis and hence tumor growth. We hypothesize that secreted and deposited MMRN2 may function as a homeostatic barrier halting the sprouting of novel vessels, and suggest that these studies may embody the potential for the development of novel tools for cancer treatment.

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 β-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.

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.

Probe-based Confocal Laser Endomicroscopy (pCLE) is a novel imaging technique for gastrointestinal endoscopy providing in vivo microscopy at subcellular resolution. It offers the possibility to analyze neoangiogenesis and vessel density in vivo. Angiogenetic switch is essential in cancer progression. Aim of the paper was to review the use of this imaging tool to analyze colorectal and gastric cancers vascularization in vivo. The aim is to provide the possibility of combining diagnostic evidences with vascularization and molecular profile to evaluate the efficacy of an antiangiogenic treatment in association with conventional therapy. pCLE can be considered a revolutionary method for real-time assessment of changes in vascularization pattern in this tumors and it may open the possibility to address the use of anti-angiogenic therapy in order to improve the outcome of the treatment.