CRK/CRKL-Dependent Transcriptional Programs Regulate Glioblastoma Cell Motility
Presenter Status
Post-Doctorial Research
Abstract Type
Basic Research
Primary Mentor or Principal Investigator
Taeju Park
Presentation Type
Poster
Start Date
21-5-2026 12:00 PM
End Date
21-5-2026 1:00 PM
Abstract Text
Background
Glioblastoma is a highly aggressive and invasive brain tumor characterized by diffuse cell migration that severely limits the effectiveness of current therapies. The adaptor proteins CRK and CRKL are key regulators of cytoskeletal signaling and have been shown to play essential, overlapping roles in glioblastoma cell migration and invasion. We previously demonstrated that CRK and CRKL play essential overlapping roles in migration and invasion of cultured glioblastoma cells and that glioblastoma cell motility positively correlates with CRK/CRKL gene dosage. However, the downstream transcriptional programs mediating CRK/CRKL-dependent cell motility remain poorly defined.
Objectives/Goal
The goal of this study is to identify and characterize downstream transcriptional targets of CRK and CRKL that regulate glioblastoma cell migration and invasion, with the long-term objective of uncovering novel therapeutic targets to limit tumor invasiveness.
Methods/Design
To identify candidate downstream molecules of CRK and CRKL, we performed RNA sequencing in U-118MG human glioblastoma cells following CRKI, CRKII, or CRKL overexpression, as well as CRKL single knockdown and CRK/CRKL double knockdown. Comparative transcriptomic analyses have been performed to identify pro-migratory genes and anti-migratory genes downstream of CRK and CRKL. Functional annotation and pathway enrichment analyses were used to identify signaling pathways associated with differentially expressed genes. Selected candidate genes were prioritized for validation based on predicted roles in cell motility and cytoskeletal regulation.
Results
We identified pro-migratory genes that were commonly upregulated in glioblastoma cells overexpressing CRKI, CRKII, or CRKL and commonly downregulated in glioblastoma cells with CRKL knockdown and CRK/CRKL double knockdown. We also identified anti-migratory genes that were commonly downregulated upon CRKI/CRKII/CRKL overexpression and commonly upregulated upon CRKL knockdown and CRK/CRKL double knockdown. Pathway analyses revealed significant enrichment of cytoskeletal regulation, tight junction signaling, and cell motility related pathways. From these differentially expressed gene sets, we selected DSCAM and KIRREL-3 as top pro-migratory candidate genes and ATF7-NPFF as a top anti-migratory candidate gene for validation. We also selected cytoskeleton-related, small GTPase-regulating candidate genes for validation. Differential expressions of these genes upon CRK/CRKL knockdown and overexpression are being validated by quantitative PCR and Western blot analysis. Candidate genes that demonstrate pro- or anti-migratory activity will be further evaluated using stable gene modulation in orthotopic glioblastoma xenograft models to assess their effects on tumor growth and invasion in vivo.
Conclusions
These findings define novel CRK/CRKL-dependent transcriptional programs that regulate glioblastoma cell motility. Ongoing functional validation studies will determine the roles of candidate genes in glioblastoma migration and invasion in vitro and in vivo. Together, this work advances our understanding of glioblastoma invasiveness and may identify new molecular targets for therapeutic intervention.
CRK/CRKL-Dependent Transcriptional Programs Regulate Glioblastoma Cell Motility
Background
Glioblastoma is a highly aggressive and invasive brain tumor characterized by diffuse cell migration that severely limits the effectiveness of current therapies. The adaptor proteins CRK and CRKL are key regulators of cytoskeletal signaling and have been shown to play essential, overlapping roles in glioblastoma cell migration and invasion. We previously demonstrated that CRK and CRKL play essential overlapping roles in migration and invasion of cultured glioblastoma cells and that glioblastoma cell motility positively correlates with CRK/CRKL gene dosage. However, the downstream transcriptional programs mediating CRK/CRKL-dependent cell motility remain poorly defined.
Objectives/Goal
The goal of this study is to identify and characterize downstream transcriptional targets of CRK and CRKL that regulate glioblastoma cell migration and invasion, with the long-term objective of uncovering novel therapeutic targets to limit tumor invasiveness.
Methods/Design
To identify candidate downstream molecules of CRK and CRKL, we performed RNA sequencing in U-118MG human glioblastoma cells following CRKI, CRKII, or CRKL overexpression, as well as CRKL single knockdown and CRK/CRKL double knockdown. Comparative transcriptomic analyses have been performed to identify pro-migratory genes and anti-migratory genes downstream of CRK and CRKL. Functional annotation and pathway enrichment analyses were used to identify signaling pathways associated with differentially expressed genes. Selected candidate genes were prioritized for validation based on predicted roles in cell motility and cytoskeletal regulation.
Results
We identified pro-migratory genes that were commonly upregulated in glioblastoma cells overexpressing CRKI, CRKII, or CRKL and commonly downregulated in glioblastoma cells with CRKL knockdown and CRK/CRKL double knockdown. We also identified anti-migratory genes that were commonly downregulated upon CRKI/CRKII/CRKL overexpression and commonly upregulated upon CRKL knockdown and CRK/CRKL double knockdown. Pathway analyses revealed significant enrichment of cytoskeletal regulation, tight junction signaling, and cell motility related pathways. From these differentially expressed gene sets, we selected DSCAM and KIRREL-3 as top pro-migratory candidate genes and ATF7-NPFF as a top anti-migratory candidate gene for validation. We also selected cytoskeleton-related, small GTPase-regulating candidate genes for validation. Differential expressions of these genes upon CRK/CRKL knockdown and overexpression are being validated by quantitative PCR and Western blot analysis. Candidate genes that demonstrate pro- or anti-migratory activity will be further evaluated using stable gene modulation in orthotopic glioblastoma xenograft models to assess their effects on tumor growth and invasion in vivo.
Conclusions
These findings define novel CRK/CRKL-dependent transcriptional programs that regulate glioblastoma cell motility. Ongoing functional validation studies will determine the roles of candidate genes in glioblastoma migration and invasion in vitro and in vivo. Together, this work advances our understanding of glioblastoma invasiveness and may identify new molecular targets for therapeutic intervention.
Comments
Restricted to Title/Author List/Abstract only as requested by primary author
Poster Board Number: 39