Article
PLEKHG5/RAB26 signalling regulates expression of the chemotherapy resistance gene MGMT in human glioblastoma
Der PLEKHG5/RAB26-Signalweg reguliert die Expression des Chemotherapie-Resistenzgens MGMT im humanen Glioblastom
Search Medline for
Authors
Published: | June 26, 2020 |
---|
Outline
Text
Objective: Pleckstrin homology containing family member 5 (PLEKHG5) is an exchange factor for the RAS-related protein RAB26 and was described as a target of the tumor necrosis factor α (TNFα)-protective pathway. PLEKHG5-signaling might play a key role in cancer through NF-κB activation and TNFα has been implicated in cancer chemoresistance. Expression of the DNA repair gene O-6-methylguanine-DNA methyltransferase (MGMT) by glioblastoma stem cells (GSCs) likely underlies chemoresistance of glioblastoma multiforme (GBM). Here we investigate a potential regulatory interplay between TNFα, PLEKHG5, RAB26 and MGMT in GBM and GSCs.
Methods: The CRISPR/Cas9-system and lentiviral transduction were used for gene knockout and gene transfer in human U251-MG cells. Cellular morphology was investigated using phalloidin/rhodamine staining of the cytoskeleton and digital fluorescence-based image analysis. GSCs were isolated from primary GBM tissues (n = 4) in the presence of EGF and bFGF-2 for selection of potential GSCs after enzymatic digestion of the primary GBM tissue. GSCs were positive for the stem cell markers nestin, CD133 and CD44 on protein level. The corresponding tumor tissues, isolated GSCs, U251-MG wildtype- and the U251-MG PLEKHG5-/- cells were investigated for MGMT, PLEKHG5 and RAB26 proteins using immunohisto- and immunocytochemistry. TNFα-induced cell death was analyzed in U251-MG PLEKHG5+/+ and PLEKHG5-/- cells employing a cellular apoptosis assay.
Results: We generated homozygous PLEKHG5 U251-MG knockout cells validated on the genomic DNA level and by negative PLEKHG5 immunocytochemistry. PLEKHG5, RAB26 and MGMT protein expression was detected in human glioma tissue and primary GCSs. In comparison with the wildtype, U251-MG PLEKHG5-/- cells showed a significantly compressed cell shape and decreased size (mean cell size, 2D fluorescence microscopy: 3285 ± 161 µm2 vs. 804 ± 67 µm2, p < 0.001). Expression of a constitutively active RAB26 variant led to a phenotypic rescue of the PLEKHG5 knockout and to specific overexpression of MGMT. TNFα mediated cell death was significantly increased in PLEKHG5-/- cells compared to PLEKHG5+/+ cells (30 min TNF exposure: 60.9 ± 4.6 % vs. 35.0 ± 3.9 % and 90 min: 88.1 ± 7.6 % vs. 54.6 ± 5.1 %, p < 0.05).
Conclusion: Our results suggest MGMT as a novel target of PLEKHG5/RAB26 signaling and thus provide additional insights into the regulation of chemotherapy resistance in GBM.