Phosphoproteomic analysis identifies CLK1 as a novel therapeutic target in gastric cancer

Abstract

Background 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. Methods 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. Results 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. Conclusions 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. Keywords Phosphoserine/threonine • Spliceosome complex • Targeted therapy • Biomarker • PDX in vivo models List of abbreviations CLK Cdc2-like kinase PDX Patient-derived xenografts IHC Immunohistochemistry TMT Tandem Mass Tag bRPLC Basic pH reverse phase chromatography HCD Higher energy collision dissociation IPA Ingenuity pathway analysis FBS Fetal bovine serum Electronic supplementary material The online version of this article (

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“…Phosphopeptide enrichment using TiO 2 was done as discussed previously, 21 flow‐through of phospho Fe‐NTA enrichment was first dried in a vacuum concentrator then resuspended in 2,5‐dihydroxybenzoic acid (DHB) solution (5% 2,5‐dihydroxybenzoic acid, 80% ACN, 3% TFA, and HPLC grade). TiO 2 beads (GL Science 5020‐75010) first dehydrated, then resuspended in 5% DHB solution and incubated at room temperature for 15 min on the rotator.…”

Identification of Prognostic Alternative Splicing Signature in Gastric Cancer

“…3C). CLK1 had been considered as a novel therapeutic target in GC through phosphoproteomic analysis, and facilitated the proliferation, migration and invasion of GC cells via modulation of the phosphorylation of SRSF2 28 . The regulatory network in our study also reveals that CLK1 activates the RI event of SRSF2, and generates the spliced variants which favors the prognosis.…”

“…3C). CLK1 had been considered as a novel therapeutic target in GC through phosphoproteomic analysis, and facilitated the proliferation, migration and invasion of GC cells via modulation of the phosphorylation of SRSF2 28 .…”

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“…In vitro assays have revealed that gastric cancer cells treated with TG003 or CLK1 siRNA to inhibit the activity of CLK1 show reduced proliferation, invasion, and migration. Thus, CLK1 can be developed as a novel therapeutic target in gastric cancer by employing a personalized tumor explant culture system [95] . Similarly, the inhibition of SRPK1 could simultaneously influence a variety of oncogenic processes, including angiogenesis, apoptosis, proliferation, migration, invasion, and metastasis.…”