CEMIP负向调控GRAF1蛋白稳定性促进结肠癌转移的作用机制研究

Metastasis is one of the leading causes of death from advanced colon cancer. Identifying the involved metastatic biomarkers and clarifying the regulation mechanisms are of great importance for targeting tumor metastasis. In our previous research, we discovered that CEMIP showed higher expression in metastatic tissues compared with the paired primary tissues. Further research indicated that CEMIP was upregulated in colon cancer, and positively correlated with tumor metastasis and poorer outcomes. Moreover, we proved that up-regulation of CEMIP promoted metastasis of colon cancer cells in vitro and in vivo. However, the mechanism by which CEMIP promoted metastasis of colon cancer had not been elucidated. Notably, we identified the interaction between CEMIP and GRAF1 by co-immunoprecipitation and mass spectrometry. Meanwhile, we found that CEMIP led to the degradation and downregulation of GRAF1, and influenced the ubiquitylation of K454 on GRAF1. Moreover, our results indicated that CEMIP was positively correlated with the activation of ERK1/2. Many researches demonstrated that GRAF1 was regarded as a tumor suppressor, and repressed the activity of Cdc42. while Cdc42 as a molecular switch, was proved to activate the downstream PKA1/MAPK signaling pathway. Therefore, we deduce that CEMIP promotes the metastasis of colon cancer through negatively regulating the stability of GRAF1, which upregulates the activity of Cdc42 and activates PKA1/MAPK signaling pathway. In the current research, we will (i) identify the molecular mechanism of protein interaction, (ii) perform in vitro and in vivo experiments, to reveal the mechanism by which CEMIP negatively regulate the stability of GRAF1 and the subsequent biological effects, and to clarify the correlation between CEMIP and GRAF1 in colon cancer and the potential clinical significance. This project will provide a new theoretical foundation for not only the research but also the clinical prevention and treatment of colon cancer metastasis.

肿瘤转移是晚期结肠癌患者死亡的主要原因之一。我们已证实：CEMIP在结肠癌中高表达，与转移和预后不良密切相关，且具有促进结肠癌转移的生物学功能，但具体机制尚不明确。我们预实验发现：CEMIP与GRAF1相互作用，调控GRAF1蛋白的降解，使其蛋白表达水平下调；且CEMIP能影响GRAF1的K454发生泛素化。此外，CEMIP表达与ERK1/2活化呈正相关。研究证明GRAF1抑制Cdc42酶活性，而后者可激活PKA1/MAPK信号通路。因此，我们推测：CEMIP负向调控GRAF1蛋白稳定性，影响其抑制下游Cdc42酶及PKA1/MAPK信号通路活性的生物学功能，从而促进结肠癌转移。本研究拟通过蛋白分子机制及体内外实验，阐明CEMIP负向调控GRAF1蛋白稳定性的作用机制及其生物学意义，分析CEMIP与GRAF1在结肠癌中的相关性及其临床意义，以期为结肠癌转移的研究与临床防治提供新的理论依据。

肿瘤转移是导致结肠癌治疗失败及死亡的主要原因。申请人前期报道CEMIP为调控结肠癌转移的关键因素，但相关机制尚未完全阐明。本研究发现CEMIP通过作用于抑癌蛋白GRAF1的SH3结构域从而介导GRAF1的泛素化和降解。进一步的研究发现CEMIP作为支架蛋白桥接泛素连接酶MIB1与GRAF1，促进GRAF1降解，继而激活下游CDC42/MAPK信号途径并诱导EMT发生，最终促进结肠癌细胞的侵袭及迁移。重要的是，体内外实验也进一步证实干扰CDC42可显著抑制CEMIP诱发结肠癌细胞转移的能力。本研究揭示了CEMIP激活CDC42/MAPK信号通路促进结肠癌转移的分子网络机制，提示CEMIP及其下游分子信号途径可能是防治结肠癌转移理想的分子靶点。