Department of Translational Research, Experimental Oncology Division 2, CRO, Aviano, Italy.
J Pathol. 2014 Mar;232(4):391-404. doi: 10.1002/path.4316.
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.
EMILIN2 是一种细胞外基质 (ECM) 蛋白，它在肿瘤微环境中发挥着矛盾的作用：它在许多肿瘤细胞中诱导细胞凋亡，但也增强了肿瘤新生血管生成。在这项研究中，我们描述了一种 EMILIN2 降低肿瘤细胞活力的新机制。基于与卷曲受体（Frizzled receptors）的富含半胱氨酸结构域（CRD）的序列同源性，我们假设 EMILIN2 可能影响 Wnt 信号激活，并证明与 Wnt1 配体的直接相互作用。这种物理结合导致 LRP6 磷酸化减少，β-catenin、TAZ 及其靶基因下调。因此，EMILIN2 在多种二维和三维体外实验中，负调控 MDA-MB-231 乳腺癌细胞的活力、迁移和致瘤潜力。EMILIN2 不会调节 Wnt 信号通路在 Wnt-Frizzled 相互作用之后的下游信号，因为它不会影响用 GSK3 抑制剂 LiCl 和 CHIR99021 处理后通路的激活。与 Wnt1 的相互作用及其随后的生物学效应需要 EMI 结构域的存在，因为缺乏该结构域的缺失突变体没有影响。此外，体内实验表明，EMILIN2 的异位表达以及重组蛋白的治疗显著减少了裸鼠中肿瘤的生长和癌细胞的扩散。因此，肿瘤样本的 Wnt 信号通路明显下调。总之，这些发现为 EMILIN2 在肿瘤微环境中的复杂调控提供了进一步的证据，并确定了 Wnt 信号通路的关键细胞外调节剂。
