Institute of Bioinformatics, International Technology Park, Bangalore, Bangalore, 560066, India.
Manipal Academy of Higher Education, Manipal, 576104, India.
Gastric Cancer. 2020 Sep;23(5):796-810. doi: 10.1007/s10120-020-01062-8. Epub 2020 Apr 24.
Phosphorylation is an important regulatory mechanism of protein activity in cells. Studies in various cancers have reported perturbations in kinases resulting in aberrant phosphorylation of oncoproteins and tumor suppressor proteins.
In this study, we carried out quantitative phosphoproteomic analysis of gastric cancer tissues and corresponding xenograft samples. Using these data, we employed bioinformatics analysis to identify aberrant signaling pathways. We further performed molecular inhibition and silencing of the upstream regulatory kinase in gastric cancer cell lines and validated its effect on cellular phenotype. Through an ex vivo technology utilizing patient tumor and blood sample, we sought to understand the therapeutic potential of the kinase by recreating the tumor microenvironment.
Using mass spectrometry-based high-throughput analysis, we identified 1,344 phosphosites and 848 phosphoproteins, including differential phosphorylation of 177 proteins (fold change cut-off ≥ 1.5). Our data showed that a subset of differentially phosphorylated proteins belonged to splicing machinery. Pathway analysis highlighted Cdc2-like kinase (CLK1) as upstream kinase. Inhibition of CLK1 using TG003 and CLK1 siRNA resulted in a decreased cell viability, proliferation, invasion and migration as well as modulation in the phosphorylation of SRSF2. Ex vivo experiments which utilizes patient's own tumor and blood to recreate the tumor microenvironment validated the use of CLK1 as a potential target for gastric cancer treatment.
Our data indicates that CLK1 plays a crucial role in the regulation of splicing process in gastric cancer and that CLK1 can act as a novel therapeutic target in gastric cancer.
磷酸化是细胞内蛋白质活性的重要调节机制。在各种癌症的研究中，已有研究报道激酶的改变导致癌蛋白和肿瘤抑制蛋白的异常磷酸化。
在本研究中，我们对胃癌组织和相应的异种移植样本进行了定量磷酸化蛋白质组学分析。利用这些数据，我们进行了生物信息学分析，以鉴定异常信号通路。我们进一步在胃癌细胞系中对上游调节激酶进行了分子抑制和沉默，并验证了其对细胞表型的影响。通过利用患者肿瘤和血液样本的体外技术，我们试图通过重建肿瘤微环境来了解激酶的治疗潜力。
使用基于质谱的高通量分析，我们鉴定了 1344 个磷酸化位点和 848 个磷酸化蛋白，包括 177 个蛋白的差异磷酸化（倍数变化截止值≥1.5）。我们的数据表明，一部分差异磷酸化蛋白属于剪接机制。通路分析突出了 CDC2 样激酶（CLK1）作为上游激酶。使用 TG003 和 CLK1 siRNA 抑制 CLK1 导致细胞活力、增殖、侵袭和迁移减少，以及 SRSF2 磷酸化的调节。利用患者自身肿瘤和血液重建肿瘤微环境的体外实验验证了 CLK1 作为胃癌治疗潜在靶点的作用。
我们的数据表明，CLK1 在胃癌中剪接过程的调节中起关键作用，CLK1 可以作为胃癌的一个新的治疗靶点。
