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Publication Date

5-2023

Abstract

Background: Glioblastoma and diffuse intrinsic pontine glioma (DIPG) are high-grade gliomas, and their diffuse invasion into neighboring healthy brain tissues contributes to recurrence and dismal treatment outcomes. The expression levels of CT10 regulator of kinase (Crk) and Crk-like (CrkL) are elevated in many human cancers, including glioblastoma and DIPG. Elevation of Crk and CrkL leads to poor prognosis, and they have been proposed as therapeutic targets for glioblastoma. Recently we used gene knockdown to demonstrate that Crk and CrkL are required for the motility of glioblastoma and DIPG cells. Here we used synthetic messenger RNA (mRNA) to investigate the effects of Crk and CrkL overexpression on the motility of glioblastoma and DIPG cells.

Methods: We synthesized mRNA in vitro and electroporated a human glioblastoma cell line, U-118MG, and a human DIPG cell line, SF8628 with mRNA to induce overexpression of Crk and CrkL. Then we determined the respective, quantitative contributions of Crk and CrkL to cellular functions. Impedance-based, real-time measurements of tumor cell adhesion, migration, and invasion were performed using the xCELLigence Real-Time Cell Analyzer (Agilent).

Results: Transfection with CrkII, CrkI, or CrkL mRNA led to an mRNA concentration-dependent expression of exogenous CrkII, CrkI, and CrkL in glioblastoma and DIPG cells. Crk and CrkL overexpression stimulated cell migration for both cell lines, and the stimulation correlated with the Crk and CrkL protein levels. On the other hand, the effect of Crk and CrkL overexpression on cell morphology or proliferation was minimal or modest for both glioblastoma and DIPG cells.

Conclusion/Significances: Our results demonstrate that Crk and CrkL overexpression increases the motility of high-grade glioma cells. Together with our previous study using gene knockdown, these results indicate that the protein levels of Crk and CrkL are critical regulators of glioma cell motility. Our study suggests that the increased motility of the tumor cells may accelerate the infiltration of glioma cells to healthy brain tissues, leading to poor prognosis for Crk/CrkL-overexpressing patients. Therefore, Crk and CrkL may be crucial therapeutic targets for blocking diffuse invasion of high-grade gliomas to improve the efficacy of the standard of care.

Support: 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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Overexpression of Crk and CrkL Increases the Motility of High-Grade Glioma Cells

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