Am J Cancer Res. 2011;1(6):716-25. Epub 2011 Jun 1.
The RET receptor tyrosine kinase is a member of the cadherin superfamily and plays a pivotal role in cell survival, differentiation and proliferation. Currently, 12 ret/ptc chimeric oncogenes, characterized by the fusion between the intracellular domain of RET and different activating genes, which can cause ligand-independent dimerization and constitutive activation, have been described. β-catenin is usually involved in the maintenance of cell-to-cell adhesion and mediates the Wnt/β-catenin pathway important during embryogenesis and in cellular malignant transformation. Recently, a novel mechanism of RET-mediated function through the β-catenin pathway has been reported in multiple endocrine neoplasia type 2 and in sporadic thyroid carcinomas. Here, we investigated the effects of the ZD6474, a small molecule RET-inhibitor, on RET/β-catenin interaction. We confirmed the ZD6474 mediated-inhibition of recombinant RET kinase and of growth of cells expressing RET/PTC. Interestingly, we firstly observed reduced cellular mobility and changed morphology of TPC1 treated cells suggesting that RET-inhibitor could affect β-catenin cellular distribution as resulted in its co-immunoprecipitation with E-cadherin. We further investigated this hypothesis showing that TPC1 treated cells displayed predominantly β-catenin cytosolic localization. Surprisingly, RET and β-catenin co-immunoprecipitated in both ZD6474-treated and untreated TPC1 cells, suggesting that RET/β-catenin interaction might not be affected by RET kinase inactivation. All together these results suggest that RET kinase activation is crucial for β-catenin stabilization (pY654), localization and its signaling pathway activation but not for β-catenin/RET physical interactions, in human papillary thyroid carcinomas. In conclusion, ZD6474, by inhibiting RET kinase, down-modulates β-catenin pathway leading its recruitment to the membrane by E-cadherin.
RET 受体酪氨酸激酶是钙粘蛋白超家族的成员，在细胞存活、分化和增殖中发挥关键作用。目前，已经描述了 12 种 ret/ptc 嵌合癌基因，其特征是 RET 的细胞内结构域与不同的激活基因融合，导致配体非依赖性二聚化和组成性激活。β-连环蛋白通常参与维持细胞间粘附，并介导 Wnt/β-连环蛋白通路，该通路在胚胎发生和细胞恶性转化中很重要。最近，据报道，RET 通过 β-连环蛋白通路介导功能的一种新机制存在于多发性内分泌肿瘤 2 型和散发性甲状腺癌中。在这里，我们研究了小分子 RET 抑制剂 ZD6474 对 RET/β-连环蛋白相互作用的影响。我们证实 ZD6474 介导的重组 RET 激酶抑制和表达 RET/PTC 的细胞生长。有趣的是，我们首先观察到 TPC1 处理的细胞的细胞迁移减少和形态改变，表明 RET 抑制剂可能影响β-连环蛋白的细胞分布，导致其与 E-钙粘蛋白共免疫沉淀。我们进一步研究了这一假设，结果表明 TPC1 处理的细胞显示出主要的β-连环蛋白细胞浆定位。令人惊讶的是，RET 和β-连环蛋白在 ZD6474 处理和未处理的 TPC1 细胞中均能共免疫沉淀，这表明 RET/β-连环蛋白相互作用可能不受 RET 激酶失活的影响。所有这些结果表明，在人甲状腺乳头状癌中，RET 激酶的激活对于β-连环蛋白的稳定（pY654）、定位及其信号通路的激活至关重要，但对于β-连环蛋白/RET 的物理相互作用则不重要。总之，ZD6474 通过抑制 RET 激酶，下调β-连环蛋白通路，使其通过 E-钙粘蛋白募集到膜上。
