Abstract 2647: Essential roles of CRK and CRKL in glioblastoma cell migration and invasion
Authors: Piyanka Hettiarachchi, Neka Large, Taeju Park
Journal: Cancer Research (2025)

Abstract The dysregulation of CT10 regulator of kinase (CRK) and CRK-like (CRKL) expression has been implicated in various human cancers, notably glioblastoma, in which their elevated levels are associated with poor prognosis. Although CRK and CRKL have been proposed as therapeutic targets for invasive cancers, the mechanism underlying the contribution of CRK and CRKL to tumorigenesis was unclear. Previously, we induced transient knockdown of CRK, CRKL, or both in the U-118MG human glioblastoma cell line and demonstrated the requirement of CRK and CRKL for glioblastoma cell migration and invasion. CRKL knockdown induced cell shrinkage and inhibited cell proliferation, adhesion, and invasion, while CRK knockdown exhibited little or modest inhibitory effects. Combined knockdown of both CRK and CRKL knockdown resulted in pronounced morphological alterations and a complete inhibition of cell migration and invasion, highlighting their overlapping roles. To establish this finding as a generally accepted proof of principle for various glioblastoma cells and prepare mutant glioblastoma cell lines for orthotopic xenograft mouse models, we induced transient and stable knockdown of CRK/CRKL in LN-229 and U-87MG glioblastoma cell lines, which are proven to form tumors with high efficacy upon orthotopic transplantation. The gene knockdown of CRK and CRKL using siRNAs demonstrated substantial decreases in CRK and CRKL protein levels in LN-229 cells, as confirmed by Western blot analyses. We utilized the xCELLigence real-time cell analysis system to analyze the effects of CRK/CRKL knockdown on cell migration, adhesion, and invasion. CRKL knockdown caused modest inhibition of cell proliferation and substantial inhibition of cell adhesion, migration, and invasion. CRK/CRKL double knockdown resulted in pronounced morphological alterations, more robust inhibition of proliferation, and complete inhibition of cell adhesion, migration, and invasion. Stable knockdown of CRK and CRKL using lentiviral infection of shRNAs showed similar results. The results suggest similar responses of both U-118MG and LN-229 cells to CRK and CRKL knockdown. The similar and consistent outcomes between the two glioblastoma cell lines underscore the critical requirement of CRK and CRKL in glioblastoma cell migration and invasion. In addition to testing the cellular functions of CRK/CRKL in U-87MG cells, we plan to transplant the glioblastoma cells with stable knockdown of CRK/CRKL to immunodeficient mouse brains and study the requirement of CRK and CRKL for in vivo tumor formation and diffuse invasion of glioblastoma into surrounding brain tissues. These in vitro and in vivo studies will establish CRK and CRKL as therapeutic targets for blocking diffuse invasion of glioblastoma in the brain. Citation Format: Piyanka Hettiarachchi, Neka Large, Taeju Park. Essential roles of CRK and CRKL in glioblastoma cell migration and invasion [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2025; Part 1 (Regular Abstracts); 2025 Apr 25-30; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2025;85(8_Suppl_1):Abstract nr 2647.