Medical Student Texas A&M School of Medicine Dallas, Texas, United States
Introduction: Glioblastoma multiforme (GBM) is a highly invasive primary brain cancer characterized by both single-cell and collective invasion. Our lab has demonstrated that TNF-weak like factor (TWEAK) induction of the noncanonical NF-κB pathway increases the invasiveness of GBM cells in an NF-κB-inducing kinase (NIK)-dependent manner. However, the specific roles of NIK in GBM invasion and adoption of specific invasion phenotypes is unknown.
Methods: Induction of NIK protein and mRNA expression during invasion with and without TWEAK was monitored by immunoblot and qRT-PCR. Using BT116 glioma cells expressing red fluorescent protein (RFP) under the promoter of NIK (pNIK-RFP), we investigated NIK mRNA expression during 3D sphere invasion by confocal microscopy. Chromatin immunoprecipitation (ChIP) and CrispR-Cas9 gene editing was used to examine transcriptional regulators of NIK expression.
Results: Analysis of BT116 pNIK-RFP cells revealed induction of NIK mRNA in invading cells, with the farthest invading cells exhibiting the highest RFP intensity, and thus highest expression of NIK mRNA. NIK promoter-driven RFP gene expression demonstrated that GBM cells with high NIK transcription exhibit characteristics of leader invasion cells. Quantification of RFP showed that leader cells exhibited the highest levels of NIK transcription, which increased upon TWEAK treatment. ChIP showed that NIK expression is directly regulated E2F4 and E2F5 transcription factors.
Conclusion : Transcriptional regulation of NIK expression by TWEAK promotes a leader cell phenotype and collective invasion of GBM cells, with the highest NIK mRNA expression observed in the most invasive cells. These results suggest that in addition to NIK stabilization at the protein level, inflammatory cytokines in the tumor microenvironment can promote tumor progression through transcriptional upregulation of NIK mRNA. Our findings suggest that targeting NIK at the transcriptional and posttranscriptional level may be required for attenuation of GBM invasion potential.
