The ECM molecule EMILIN2: a dual role in the tumor microenvironment

EMILIN2 is an extracellular matrix molecule belonging to the EDEN (EMI Domain ENdowed) protein family. The protein is characterized by the cysteine-rich EMI domain at the N-terminus, the hallmark of the family, \u3b1-helical domains at the C-terminus, a proline-rich motif adjacent to a collagenous stalk preceding the globular gC1q domain. Here we report that this molecule exerts a double function in the tumor microenvironment, simultaneously directly acting on cancer cells and, indirectly on the tumor vasculature. First, based on sequence homology with the Cysteine Rich Domain (CRD) of the Frizzled receptors, we hypothesized that EMILIN2 could affect Wnt signaling activation and we demonstrated a direct interaction with the Wnt1 ligand. As a consequence, EMILIN2 negatively affects the viability, migration and tumourigenic potential of MDA-MB-231 breast cancer cells in a number of 2D and 3D in vitro assays. Moreover, in vivo experiments show that the ectopic expression of EMILIN2, as well as treatment with the recombinant protein, significantly reduce tumor growth in nude mice. Second, based on our previously published evidences that treatment with EMILIN2 enhanced angiogenesis, we sought to corroborate these findings and shed light on the molecular mechanisms involved. EMILIN2 challenged ECs display an increased expression of IL-8 as assessed by a protein array, ELISA, Real Time and Western blot analysis. The induction of IL-8 production occurs through activation of EGFR following interaction with EMILIN2 and a consequent activation of the Jak2/STAT3 pathway. Similarly, EMILIN2 induces expression of IL-8 also by fibroblast, the major source of this molecule and EMILIN2 itself. Accordingly, manipulation of EMILIN2 expression by fibroblast lead to significant changes in EC behavior when challenged with the conditioned media derived from these cells in terms of both viability and random motility. Manipulation of EMILIN2 expression by fibroblast leads also to an altered EC spouting from spheroids in a 3D co-culture setting. In line with our findings the pro-angiogenic function of EMILIN2, which was tested in a number of in vitro, ex vivo and in vivo assays, was blocked by Cetuximab, an anti-EGFR monoclonal antibody. Taken together these results suggest that the expression of EMILIN2 in the microenvironment may significantly affect the growth and development of the tumor