Download Full Text (853 KB)
Background: The expression levels of CT10 regulator of kinase (Crk) and Crk-like (CrkL) are elevated in many human cancers, including glioblastoma (GBM), and are believed to contribute to poor prognosis. Although Crk and CrkL have been proposed as therapeutic targets in these tumors, the lack of a reliable, quantitative assay to measure Crk and CrkL activity has hindered development of inhibitors.
Methods: Here, we knocked down Crk, CrkL, or both using small interfering RNAs (siRNAs) in a human GBM cell line, U-118MG, to determine the respective, quantitative contributions of Crk and CrkL to cellular phenotypes.
Results: The combined use of specific and potent Crk and CrkL siRNAs induced effective knockdown of CrkII, CrkI, and CrkL. Whereas Crk knockdown did not affect cell morphology, proliferation, adhesion, or invasion, CrkL knockdown caused shrinkage of cells and inhibition of cell proliferation, adhesion, and invasion. Crk/CrkL double knockdown resulted in more pronounced morphological alterations and more robust inhibition of proliferation, adhesion, and invasion. Furthermore, Crk/CrkL double knockdown completely blocked cell migration, and this effect was rescued by transient overexpression of CrkL but not of Crk. Quantification of protein levels indicated that CrkL is expressed more abundantly than CrkII and CrkI in U-118MG cells.
Conclusions/Significances: These results demonstrate both the predominant role of CrkL and the essential overlapping functions of Crk and CrkL in U-118MG cells. Furthermore, our study indicates that migration of U-118MG cells depends entirely on Crk and CrkL. Thus, impedance-based, real-time measurement of tumor cell migration represents a robust assay for monitoring Crk and CrkL activities.
Support: This work was supported by Tom Keaveny Endowed Fund for Pediatric Cancer Research (to TP) and by a MCA Partners Advisory Board grant from Children’s Mercy Hospital (CMH) and the University of Kansas Cancer Center (KUCC) (to TP).
Park, Taeju; Large, Neka; and Curran, Tom, "Quantitative Assessment Of Glioblastoma Cell Phenotypes Establishes Cell Migration As A Robust Readout Of CRK AND CRKL Activity" (2021). Research at Children's Mercy Month 2021. 33.