Niraj Babu1,2, Sneha M Pinto1,3, Manjusha Biswas4, Tejaswini Subbannayya1,4, Manoj Rajappa4, Sonali V Mohan1,2, Jayshree Advani1, Pavithra Rajagopalan1, Gajanan Sathe1, Nazia Syed1, Vinod D Radhakrishna4, Oliyarasi Muthusamy4, Sanjay Navani5, Rekha V Kumar6, Gopal Gopisetty7, Thangarajan Rajkumar7, Padhma Radhakrishnan8, Saravanan Thiyagarajan4, Akhilesh Pandey1,2,9,10,11, Harsha Gowda1,2,12, Pradip Majumder13, Aditi Chatterjee14,15,16. 1. Institute of Bioinformatics, International Technology Park, Bangalore, Bangalore, 560066, India. 2. Manipal Academy of Higher Education, Manipal, 576104, India. 3. Center for Systems Biology and Molecular Medicine, Yenepoya (Deemed To Be University), Mangalore, 575018, India. 4. Mitra Biotech, Bangalore, 560100, India. 5. Lab Surgpath, Mumbai, 400034, India. 6. Department of Pathology, Kidwai Memorial Institute of Oncology, Bangalore, 560029, India. 7. Department of Molecular Oncology, Cancer Institute (WIA), Chennai, 600020, India. 8. Mitra Biotech, Woburn, MA, 01801, USA. 9. Department of Laboratory Medicine and Pathology, Mayo Clinic, Rochester, MN, 55905, USA. 10. Center for Individualized Medicine, Mayo Clinic, Rochester, MN, 55905, USA. 11. Center for Molecular Medicine, National Institute of Mental Health and Neurosciences (NIMHANS), Hosur Road, Bangalore, 560029, India. 12. Cancer Precision Medicine, QIMR Berghofer, Royal Brisbane Hospital, Brisbane, QLD, 4029, Australia. 13. Mitra Biotech, Woburn, MA, 01801, USA. pradipkmajumder@gmail.com. 14. Institute of Bioinformatics, International Technology Park, Bangalore, Bangalore, 560066, India. aditixchatterjee@gmail.com. 15. Manipal Academy of Higher Education, Manipal, 576104, India. aditixchatterjee@gmail.com. 16. Mitra Biotech, Bangalore, 560100, India. aditixchatterjee@gmail.com.
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.
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 patienttumor 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.
Entities:
Keywords:
Biomarker; PDX in vivo models; Phosphoserine/threonine; Spliceosome complex; Targeted therapy
Authors: Dario Righelli; Crescenzo D'Alterio; Caterina Ieranò; Maria Napolitano; Luigi Portella; Giuseppina Rea; Federica Auletta; Sara Santagata; Anna Maria Trotta; Giuseppe Guardascione; Federica Liotti; Nella Prevete; Piera Maiolino; Antonio Luciano; Antonio Barbieri; Annabella Di Mauro; Cristin Roma; Riziero Esposito Abate; Fabiana Tatangelo; Roberto Pacelli; Nicola Normanno; Rosa Marina Melillo; Stefania Scala Journal: J Immunother Cancer Date: 2022-03 Impact factor: 13.751