EphB3酪氨酸低磷酸化状态的调控机制及其对非小细胞肺癌间质-上皮转化的影响

The Eph/ephrin signaling plays an important role in tumorigensis. In the previous study, we discovered dual functions of EphB3 in NSCLC (Ji et al, Cancer Research, 2011; Li et al, Nature Communicaions, 2012). The expression of EphB3 was upregulated in NSCLC tissues, and knockdown of EphB3 significantly inhibited cell survival in vitro, as well as tumorigenesis and metastasis in vivo, which was independent of the kinase activity of EphB3 (Ji et al, Cancer Research, 2011). The subsequent study revealed the dual functions of EphB3 kinase activity. Despite the high expression level of EphB3 in NSCLC, the level of EphB3 phosphotylation is very low. Consistent with this observation, stimulation of EphB3 recepter overexpressed in NSCLC cells by purified ligand resulted in effective inhibition of cell migration and induced mesenchymal-epithelial transition (MET). Therefore, the phosphotylation of EphB3 is critical for its function. However, it remains largely unknown how the low phosphorylation level of EphB3 in NSCLC is maintained, as well as the related application in NSCLC therapy. In this project, we plan to clarify the relationship between EphB3 phosphorylation and the MET of NSCLC, reveal the mechanisms underlying the regulation of EphB3 phosphorylation by RACK1/PTPmu, and investigate the effect of the cognate ligand of EphB3, ephrin-B1/2-Fc in NSCLC therapy. This research will improve our understanding of the development of NSCLC, and may provide new therapeutic targets for NSCLC.

我们前期研究结果表明EphB3受体酪氨酸的磷酸化状态决定其在非小细胞肺癌发生发展过程中的双重作用 (Ji et al, Cancer Research, 2011; Li et al, Nature Communicaions, 2012)。EphB3对非小细胞肺癌发生发展的促进作用不依赖于其激酶活性，激活EphB3受体磷酸化抑制细胞的迁移和转移能力，并诱导细胞形态发生间质-上皮转化。但是，关于EphB3在肺癌细胞中维持低磷酸化状态及其诱导间质-上皮转化的分子机制仍不清楚。在本项目中，我们拟深入研究EphB3的磷酸化状态与非小细胞肺癌肿瘤细胞间质-上皮转化的相关性，揭示RACK1/PTPmu调控EphB3磷酸化状态的分子机制，探索EphB3的配体蛋白在非小细胞肺癌治疗中的意义。我们的研究将加深对非小细胞肺癌发生发展机制的认识，为非小细胞肺癌的治疗提供新的靶点。

在本项目的研究中，主要完成了以下四个方面的工作：（1）在肺癌临床样本、原代组织和小鼠模型中揭示了EphB3磷酸化，RACK1和PTPmu的表达水平，以及肿瘤相关成纤维细胞（cancer-associated fibroblast，CAF）中ephrinB1/B2的表达之间的相关性，并分析了它们的表达模式及与肺癌进程之间的相关性。（2）利用多种体内外模型，发现调控CAF中的ephrinB1对肺癌细胞的迁移和转移具有显著影响，但是对成瘤的影响不明显。（3）检测并且分析了EphB3、RACK1和PTPmu之间的相互作用，发现三个分子存在于同一个复合物中，其相互作用随配体刺激而增强。激活的EphB3可以磷酸化RACK1，促使其招募PTPmu对EphB3进行去磷酸化，从而促进了肺癌细胞的迁移和转移。（4）在genetic mouse model中研究了EphB3的磷酸化对小鼠肺癌发生发展的影响。此外，在裸鼠模型中探索了ephrinB1-Fc对肺癌转移的治疗潜能。.本项目的研究有助于进一步认识EphB3在肺癌转移中的作用和机制，及其对肿瘤微环境的影响，并为肺癌转移提供了潜在的治疗策略。