Zhuang Xiang, Lv Mengxin, Zhong Zhenyu, Zhang Luyu, Jiang Rong, Chen Junxia
Department of Cell Biology and Genetics, Chongqing Medical University, Chongqing, 400016, China.
The First Clinical College, Chongqing Medical University, Chongqing, 400016, China.
J Exp Clin Cancer Res. 2016 Aug 30;35(1):130. doi: 10.1186/s13046-016-0408-x.
Integrin-linked kinase (ILK) is a multifunctional adaptor protein which is involved with protein signalling within cells to modulate malignant (cancer) cell movement, cell cycle, metastasis and epithelial-mesenchymal transition (EMT). Our previous experiment demonstrated that ILK siRNA inhibited the growth and induced apoptosis of bladder cancer cells as well as increased the expression of Ribonuclease inhibitor (RI), an important cytoplasmic protein with many functions. We also reported that RI overexpression inhibited ILK and phosphorylation of AKT and GSK3β. ILK and RI gene both locate on chromosome 11p15 and the two genes are always at the adjacent position of same chromosome during evolution, which suggest that ILK and RI could have some relationship. However, underlying interacting mechanisms remain unclear between them. Here, we postulate that RI might regulate ILK signaling pathway via interacting with ILK.
Co-immunoprecipitation, GST pull-down and co-localization under laser confocal microscope assay were used to determine the interaction between ILK and RI exogenously and endogenously. Furthermore, we further verified that there is a direct binding between the two proteins by fluorescence resonance energy transfer (FRET) in cells. Next, The effects of interplay between ILK and RI on the key target protein expressions of PI3K/AKT/mTOR signaling pathway were determined by western blot, immunohistochemistry and immunofluorescence assay in vivo and in vitro. Finally, the interaction was assessed using nude mice xenograft model.
We first found that ILK could combine with RI both in vivo and in vitro by GST pull-down, co-immunoprecipitation (Co-IP) and FRET. The protein levels of ILK and RI revealed a significant inverse correlation in vivo and in vitro. Subsequently, The results showed that up-regulating ILK could increase cell proliferation, change cell morphology and regulate cell cycle. We also demonstrated that the overexpression of ILK remarkably promoted EMT and expressions of target molecules of ILK signaling pathways in vitro and in vivo. Finally, we found that ILK overexpression significantly enhanced growth, metastasis and angiogenesis of xenograft tumor; Whereas, RI has a contrary role compared to ILK in vivo and in vitro.
Our findings, for the first time, directly proved that the interplay between ILK and RI regulated EMT via ILK/PI3K/AKT signaling pathways for bladder cancer, which highlights the possibilities that ILK/RI could be valuable markers together for the therapy and diagnosis of human carcinoma of urinary bladder.
整合素连接激酶（ILK）是一种多功能衔接蛋白，参与细胞内的蛋白质信号传导，以调节恶性（癌）细胞的运动、细胞周期、转移和上皮-间质转化（EMT）。我们之前的实验表明，ILK siRNA抑制膀胱癌细胞的生长并诱导其凋亡，同时增加核糖核酸酶抑制剂（RI）的表达，RI是一种具有多种功能的重要细胞质蛋白。我们还报道，RI的过表达抑制ILK以及AKT和GSK3β的磷酸化。ILK和RI基因均位于11号染色体p15区域，在进化过程中这两个基因始终位于同一条染色体的相邻位置，这表明ILK和RI可能存在某种关系。然而，它们之间潜在的相互作用机制仍不清楚。在此，我们推测RI可能通过与ILK相互作用来调节ILK信号通路。
采用免疫共沉淀、GST下拉实验以及激光共聚焦显微镜下的共定位实验来确定ILK与RI在细胞外和细胞内的相互作用。此外，我们通过细胞内的荧光共振能量转移（FRET）进一步验证了这两种蛋白之间存在直接结合。接下来，通过蛋白质免疫印迹法、免疫组织化学和免疫荧光实验在体内和体外确定ILK与RI之间的相互作用对PI3K/AKT/mTOR信号通路关键靶蛋白表达的影响。最后，使用裸鼠异种移植模型评估这种相互作用。
我们首先通过GST下拉实验、免疫共沉淀（Co-IP）和FRET发现ILK在体内和体外均能与RI结合。ILK和RI的蛋白水平在体内和体外均呈现出显著的负相关。随后，结果表明上调ILK可增加细胞增殖、改变细胞形态并调节细胞周期。我们还证明，ILK的过表达在体外和体内均显著促进EMT以及ILK信号通路靶分子的表达。最后，我们发现ILK的过表达显著增强异种移植瘤的生长、转移和血管生成；而在体内和体外，RI与ILK的作用相反。
我们的研究结果首次直接证明，ILK与RI之间的相互作用通过ILK/PI3K/AKT信号通路调节膀胱癌的EMT，这突出了ILK/RI可能共同成为人类膀胱癌治疗和诊断有价值标志物的可能性。
