KIAA1199通过PP2A/Stathmin途径介导微管失稳促进结肠癌细胞转移的机制研究

Metastasis is one of the leading causes of death from advanced colon cancer. However, the mechanism of metastasis has not been elucidated. 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 KIAA1199, a cell-migration inducing protein, showed higher expression in metastatic tissues compared with the paired primary tissues, was upregulated in colon cancer and positively correlated with tumor metastasis, and predicted poorer outcomes. Moreover, we indicated that down-regulation of KIAA1199 suppressed migration and invasion of colon cancer cells in vitro, and inhibited metastasis of colon cancer cells in vivo. However, the mechanisms by which KIAA1199 promotes metastasis in colon cancer need to be further explored. In our previous research, we identified the interaction between KIAA1199 and PP2A by co-immunoprecipitation and mass spectrometry. Meanwhile, we found that PP2A also bound to stathmin, a microtubule-associated protein. Moreover, our results indicated that the expression of phosphorylated stathmin increased while KIAA1199 expression was down-regulated. PP2A was reported to induce microtubule depolymerization by regulating dephosphorylation of stathmin, which was closely associated with metastasis of tumor cells. Therefore, we deduce that KIAA1199 might promote metastasis of colon cancer cells through microtubule destabilization regulated by PP2A/Stathmin pathway, which has not been reported. In the current research, we will (i) perform in vitro experiments, (ii) establish the orthotopic transplantation tumor and metastasis model of human colon cancer in NOD/SCID mice, and (iii) identify protein interaction and the molecular mechanism, to elucidate the mechanism by which KIAA1199 promotes metastasis of colon cancer cells, and to reveal the inhibiting effect of paclitaxel on the function of KIAA1199. This project will provide a new theoretical foundation for the 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，其具体机制尚不明确。我们前期通过基因芯片筛选发现，KIAA1199在结肠癌转移灶中的表达明显高于原发灶；临床分析显示其高表达是影响结肠癌转移和预后不良的因素之一；体内外研究表明该蛋白可促进结肠癌细胞转移，但其机制尚不清楚。我们预实验发现KIAA1199与蛋白磷酸酶PP2A结合，PP2A同时也与微管相关蛋白Stathmin结合，且敲低KIAA1199可增加磷酸化Stathmin蛋白表达。据报道非磷酸化Stathmin导致微管解聚，与肿瘤细胞转移密切相关。因此，我们推测KIAA1199通过PP2A/Stathmin途径介导微管失稳从而促进结肠癌细胞转移。本研究拟通过体外实验、小鼠原位瘤转移模型、蛋白相互作用及分子机制等研究，阐明KIAA1199促进结肠癌细胞转移的作用机制，并揭示紫杉醇对KIAA1199功能的抑制作用，为治疗结肠癌转移提供研究基础。

肿瘤转移是晚期结肠癌患者死亡的主要原因之一，也是结肠癌临床治疗的难题，其具体机制尚不明确。本课题组前期发现KIAA1199高表达是影响结肠癌转移和预后不良的因素之一，且该蛋白具有促进结肠癌转移的功能，但其作用机制尚不清楚。在本研究中，我们发现下调KIAA1199可抑制小鼠原位结肠癌发生器官转移，进一步验证了KIAA1199促进结肠癌转移的功能。为了探索KIAA1199促进结肠癌转移的作用机制，我们通过免疫共沉淀和质谱分析发现KIAA1199与蛋白磷酸酶PP2A相结合。PP2A是细胞中主要的丝氨酸/苏氨酸磷酸酶，可调控细胞信号通路中的关键蛋白分子脱磷酸化，从而调节细胞周期、细胞迁移及细胞存活等生物学行为。我们通过Pulldown实验和磷酸酶活性检测，发现KIAA1199通过其C-末端与PP2A结合，可增强PP2A的磷酸酶活性；并发现PP2A抑制剂可抑制KIAA1199/PP2A复合物的磷酸酶活性，也可抑制KIAA1199促进结肠癌细胞侵袭迁移的能力。另外我们还通过免疫共沉淀发现Stathmin是KIAA1199/PP2A复合物的下游底物。Stathmin是一种微管相关蛋白，以磷酸化与非磷酸化形式存在于细胞中；该蛋白在多种肿瘤中高表达，并与肿瘤转移及预后不良相关。本研究发现上调KIAA1199可降低磷酸化Stathmin蛋白表达，并调节其亚细胞再分布；临床资料显示，在结肠癌组织中KIAA1199与磷酸化Stathmin蛋白（Ser-16）的表达呈负相关；并且下调Stathmin表达可抑制KIAA1199促进结肠癌细胞侵袭迁移的能力。以上数据表明PP2A/Stathmin途径在KIAA1199促进结肠癌转移过程中起关键作用。微管由α/β-tubulin 异二聚体构成，非磷酸化Stathmin可与tubulin异二聚体结合，阻止微管聚合，导致微管失稳。在本研究中，我们发现上调KIAA1199可降低乙酰化tubulin蛋白表达，导致微管失稳；并且证实紫杉醇可抑制KIAA1199促进结肠癌细胞体外侵袭迁移和体内转移的功能。综上所述，本研究首次阐明KIAA1199通过PP2A/Stathmin途径介导微管失稳促进结肠癌细胞转移的作用机制，并揭示紫杉醇对KIAA1199促进结肠癌转移功能的抑制作用。因此，KIAA1199有可能成为预测结肠癌转移的生物标标记物和结肠癌治疗的新靶点。