Investigating the role of NF-κB/RelA in hypoxia induced stemness and EMT characteristics using NF-κB overexpression and CRISPR/Cas9- knockout models of glioblastoma cells.


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Samna Sagadevan1,Ben Smith1,Vinodh Kannappan1,Sathishkumar Kurusamy1,Karim Azar1,Weiguang Wang1
1University of Wolverhampton

Abstract

Background

 Despite decades of research the therapeutic outcome of Glioblastoma Multiforme (GBM) remains dismal. No contemporary chemotherapeutic regimen is effective. Recent understanding of the molecular mechanisms behind chemoresistance has focussed on a small population (~1%) of GBM cancer stem cells (GCSCs) which promotes therapeutic resistance and local invasion of GBM cells. Evidence indicate that intra-tumoral hypoxia drives CSC phenotypes in solid tumours via epithelial-to-mesenchymal transition (EMT). We have previously demonstrated that hypoxic GBM cells displayed activation of stemness and EMT related markers along with increased invasiveness and resistance to temozolomide (TMZ).

NF-κB a key transcription factor is highly up-regulated in hypoxia-induced CSCs, but the insights on how NF-κB coordinates the stemness, EMT and chemoresistance still remain obscure. In this study we used U373MG GBM cell line model with ectopic overexpression of NF-κB-p65 and CRISPR-Cas9 NF-κB-p65 knock out in parallel to investigate the pivotal role played by NF-kB pathway in hypoxia-induced activation of EMT pathway, CSC features, upregulation of anti-apoptotic signalling, resistance to TMZ, migration and invasion characteristics.  

Method

Cell culture, stable transfection, NF-kB overexpression, CRISPR Cas9 knock out, CSC markers, real-time PCR, western blot, MTT, migration and matrigel invasion assay.

Results

NFκB p65 overexpressed GBM cells showed increased expression of CSC markers such as ALDH, CD133, sox2, oct4 and nanog and were highly resistant to TMZ. Whereas, the p65 knock out cells were not resistant to TMZ and showed decreased expression of CSC features under both normoxic and hypoxic conditions. Furthermore, p65 overexpressed cells displayed a typical EMT signature and enhanced migration and invasion characteristics, but the p65 knock out cells had decreased EMT, migration and invasion characteristics.

Conclusion

Together the results demonstrate the central role played by NF-kB p65 in hypoxia induced EMT and CSCs and would lead to the development of drugs that can target the GCSCs.