Institute of Enzymology, Research Centre for Natural Sciences, 1117 Budapest, Hungary.
Doctoral School of Biology, Institute of Biology, ELTE Eötvös Loránd University, 1117 Budapest, Hungary.
Int J Mol Sci. 2023 Oct 13;24(20):15136. doi: 10.3390/ijms242015136.
Colon cancer is a leading cause of death worldwide. Identification of new molecular factors governing the invasiveness of colon cancer holds promise in developing screening and targeted therapeutic methods. The Tyrosine Kinase Substrate with four SH3 domains (TKS4) and the CD2-associated protein (CD2AP) have previously been linked to dynamic actin assembly related processes and cancer cell migration, although their co-instructive role during tumor formation remained unknown. Therefore, this study was designed to investigate the TKS4-CD2AP interaction and study the interdependent effect of TKS4/CD2AP on oncogenic events. We identified CD2AP as a novel TKS4 interacting partner via co-immunoprecipitation-mass spectrometry methods. The interaction was validated via Western blot (WB), immunocytochemistry (ICC) and proximity ligation assay (PLA). The binding motif of CD2AP was explored via peptide microarray. To uncover the possible cooperative effects of TKS4 and CD2AP in cell movement and in epithelial-mesenchymal transition (EMT), we performed gene silencing and overexpressing experiments. Our results showed that TKS4 and CD2AP form a scaffolding protein complex and that they can regulate migration and EMT-related pathways in HCT116 colon cancer cells. This is the first study demonstrating the TKS4-CD2AP protein-protein interaction in vitro, their co-localization in intact cells, and their potential interdependent effects on partial-EMT in colon cancer.
结肠癌是全球主要的死亡原因之一。鉴定新的分子因素来控制结肠癌的侵袭性有望开发出筛查和靶向治疗方法。酪氨酸激酶底物 4（TKS4）和 CD2 相关蛋白（CD2AP）先前与动态肌动蛋白组装相关过程和癌细胞迁移有关，尽管它们在肿瘤形成过程中的共同指导作用仍不清楚。因此，本研究旨在研究 TKS4-CD2AP 相互作用，并研究 TKS4/CD2AP 对致癌事件的相互依赖效应。我们通过共免疫沉淀-质谱方法鉴定 CD2AP 为 TKS4 的新相互作用伙伴。通过 Western blot（WB）、免疫细胞化学（ICC）和邻近连接分析（PLA）验证了相互作用。通过肽微阵列探索了 CD2AP 的结合基序。为了揭示 TKS4 和 CD2AP 在细胞运动和上皮-间充质转化（EMT）中的可能协同效应，我们进行了基因沉默和过表达实验。我们的结果表明，TKS4 和 CD2AP 形成支架蛋白复合物，可调节 HCT116 结肠癌细胞的迁移和 EMT 相关途径。这是首次在体外证明 TKS4-CD2AP 蛋白-蛋白相互作用、它们在完整细胞中的共定位以及它们对结肠癌部分 EMT 的潜在相互依赖效应的研究。
