胚胎和肿瘤组织SCF/KIT信号通路上调重要转录因子Pea3的分子机制研究

Cell migration is a very important event in the organ formation during embryonic development and the metastasis of solid tumors, in which the transcription factor Pea3 plays a key role. It has been suggested that the activation of ERK1/2, the common downstream transduction hub of RTK signaling, could clearly inhibit the ubiquitination degradation of Pea3. However, its detail mechanism has not been completely elucidated. Our preliminary results showed that the upregulation of Pea3 by ERK1/2 activation was closely related with the phosphorylation of transacetylase p300. We presumed that the phosphorylated p300 might acetylate Ubc9, the unique E2 conjugase in the process of SUMO modification, so as to block the SUMO-dependent protein degradation. Therefore, we aims to investigate the role of SCF/KIT signaling and its downstream ERK1/2 as well as p300 activation for acetylating Ubc9 and consequently inhibiting SUMO-dependent Pea3 degradation in vivo and in vitro. Furthermore, we also wish to find out the exact binding sites in Pea3 to SUMO by the way of constructing Pea3 overexpression plasmid harboring ΨKXE sequences and corresponding mutants in potential binding sites to SUMO in combination with Co-IP experiments. Our results may provide important evidences to demonstrate the regulatory mechanism of the upregulation of Pea3 in embryonic and tumor development.

细胞迁移是胚胎发育中器官形成和实体瘤细胞转移的重要事件，转录因子Pea3发挥关键调控作用。据报道RTKs信号下游共同转导枢纽ERK1/2活化具有明显抑制Pea3泛素化降解作用，但调控机制尚不清楚。我们预实验显示：ERK1/2活化增加Pea3表达与乙酰基转移酶p300磷酸化密切相关，推测磷酸化p300可能具有促进下游SUMO化修饰中唯一的E2结合酶Ubc9乙酰化，进而抑制靶蛋白的SUMO化修饰降解作用。因此，本项目拟以SCF/KIT信号及其下游ERK1/2为研究对象，在体和离体研究ERK1/2活化和p300磷酸化是否具有使Ubc9乙酰化、进而抑制Pea3的SUMO化修饰和降解作用；在此基础上，构建含不同ΨKXE序列的Pea3表达质粒和可能被SUMO化位点的点突变质粒，Co-IP实验明确SUMO与Pea3的结合位点，为阐明胚胎发育和肿瘤组织持续高表达转录因子Pea3的调控机制提供重要实验依据。

重要转录活化子ETVs家族成员在多种恶性肿瘤组织呈高表达。已知ETVs可与基质金属蛋白酶（MMPs）和诱导肿瘤细胞由上皮向间质转变（EMT）的相关基因启动子结合，促进其转录，在恶性肿瘤细胞的侵袭与转移中发挥重要作用，被认为是导致中晚期癌症患者死亡的重要因素之一，但有关ETVs在恶性肿瘤细胞持续高水平表达的调控机制尚不完全清楚。有报道提示RTKs及其下游信号分子异常活化与ETVs表达增加密切相关。因此，本项目以消化道常见的结直肠癌为研究对象，结合临床结直肠癌患者标本和结直肠癌细胞系体外培养，应用现代形态学和分子生物学技术，以SCF/KIT信号及其下游ERK为研究对象，探究上述信号分子在结直肠癌发生发展中促进ETV4持续高表达的分子机制，证实ERK分子活化通过促进ETV4的Ser73位点磷酸化，阻碍其与E3泛素连接酶COP1结合，避免其被泛素-蛋白酶体降解，引起ETV4蛋白增加；阐明了ERK/MAPK信号通路激活在组蛋白乙酰基转移酶p300活化以及Ubc9乙酰化中的调控作用，即Ubc9第65位赖氨酸（K65）位点乙酰化与ETV4的相互作用减弱，抑制ETV4的SUMO化修饰和降解。本研究结果为阐明胚胎发育和肿瘤组织持续高表达转录因子ETV4的调控机制提供新的重要实验依据，为临床ETV4表达增加的恶性肿瘤靶向治疗提供新的思路和靶点，具有潜在的应用价值和广阔的市场前景。