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Background: The expression levels of CT10 regulator of kinase (Crk) and Crk-like (CrkL) are elevated in many human cancers, including glioblastoma (GBM) and diffuse intrinsic pontine glioma (DIPG). GBM and DIPG are both highly aggressive brain tumors derived from glial cells. Elevation of Crk and CrkL contributes to poor prognosis, and they have been proposed as therapeutic targets for GBM. We recently demonstrated that Crk and CrkL play essential overlapping roles in GBM cell migration. Here we have investigated if Crk and CrkL play similar roles in GBM and DIPG cells.

Methods: We induced gene knockdown of Crk, CrkL, or both in vitro in a human GBM cell line, U-118MG, and a human DIPG cell line, SF8628 by electroporating with small interfering RNAs (siRNAs). Then we determined the respective, quantitative contributions of Crk and CrkL to cellular phenotypes. Impedance-based, real-time measurements of tumor cell adhesion, migration, and invasion were performed using the xCELLigence Real-Time Cell Analyzer (Agilent).

Results: The combined use of specific and potent Crk and CrkL siRNAs induced effective knockdown of CrkII, CrkI, and CrkL in GBM and DIPG cells. Crk knockdown did not affect cell morphology or proliferation in both GBM and DIPG cells. On the other hand, CrkL knockdown caused shrinkage of cells and inhibition of cell migration and adhesion in both cell lines. In both GBM and DIPG cells, Crk/CrkL double knockdown resulted in more pronounced morphological alterations and robust inhibition of proliferation and adhesion. Furthermore, Crk/CrkL double knockdown completely blocked cell migration and invasion in both cell lines.

Conclusion/Significances: These results demonstrate both the predominant role of CrkL and the essential overlapping functions of Crk and CrkL in GBM and DIPG cells. Our study indicates that migration and invasion of GBM and DIPG cells depends entirely on Crk and CrkL. Our results suggest that inhibition of Crk and CrkL activity may suppress invasion of glioma into healthy brain tissues. Impedance-based, real-time measurement of glioma cell migration represents a robust assay for monitoring Crk and CrkL activities.

Support: Tom Keaveny Endowed Fund for Pediatric Cancer Research (to TP), Masonic Cancer Alliance Partners Advisory Board grants from Children’s Mercy Hospital (CMH) and the University of Kansas Cancer Center (KUCC) (to TP), and Natalie’s A.R.T. Foundation (to TP).

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Essential Overlapping Functions Of Crk And Crkl In Glioblastoma And Diffuse Intrinsic Pontine Glioma Cells