Inhibition of the Phospholipase Cε-c-Jun N-Terminal Kinase Axis Suppresses Glioma Stem Cell Properties

Abstract

Glioma stem cells (GSCs), the cancer stem cells of glioblastoma multiforme (GBM), contribute to the malignancy of GBM due to their resistance to therapy and tumorigenic potential; therefore, the development of GSC-targeted therapies is urgently needed to improve the poor prognosis of GBM patients. The molecular mechanisms maintaining GSCs need to be elucidated in more detail for the development of GSC-targeted therapy. In comparison with patient-derived GSCs and their differentiated counterparts, we herein demonstrated for the first time that phospholipase C (PLC)ε was highly expressed in GSCs, in contrast to other PLC isoforms. A broad-spectrum PLC inhibitor suppressed the viability of GSCs, but not their stemness. Nevertheless, the knockdown of PLCε suppressed the survival of GSCs and induced cell death. The stem cell capacity of residual viable cells was also suppressed. Moreover, the survival of mice that were transplanted with PLCε knockdown-GSCs was longer than the control group. PLCε maintained the stemness of GSCs via the activation of JNK. The present study demonstrated for the first time that PLCε plays a critical role in maintaining the survival, stemness, and tumor initiation capacity of GSCs. Our study suggested that PLCε is a promising anti-GSC therapeutic target.

Keywords: brain tumor initiating cell; c-Jun N-terminal kinase; glioma initiating cell; phospholipase Cε.

Figure 1

PLCε is highly expressed in glioma stem cells (GSCs), but not in differentiated GSCs. GSCs (GS-Y01, GS-Y03, and TGS01; Stem) and isogenic differentiated GSCs (Diff) were evaluated by immunoblot analyses (a) or RT-PCR (b) for the indicated proteins and mRNAs. Representative images of two (a) or three (b) biological replicates are shown. The numbers below the Western blot images show the relative band intensities after each band was quantified by densitometry and normalized by the GAPDH value. Graphs indicate the quantification of PCR analyses by densitometry. The values are presented as the means ± SDs of the triplicate samples of an independent experiment. * p < 0.05 vs. Stem by the Student’s t-test.

Figure 2

Genetic silencing of PLCε suppresses cell viability. GSCs (GS-Y01, GS-Y03, and TGS01) were transiently transfected with siRNA against PLCE1. The cells were then subjected to a cell viability assay using WST-8 (a), Western blot analyses (b), or a propidium iodide (PI) uptake assay (c). The values represent the means + SDs of the triplicate samples of a representative experiment. Similar results were obtained from two independent biological replicates. * p < 0.05 vs. siControl-transfected cells by the Student’s t-test. Representative fluorescence images of are shown at Supplemental Figure S2.

Figure 3

Genetic silencing of PLCε by siRNA suppresses the stemness of GSCs. (a) GSCs were transiently transfected with siRNA against PLCE1 or control siRNA. After 4 days, the transfected cells were subjected to flow cytometric analyses for the cell surface expression of CD133. The graphs show the means + SDs of triplicate samples. Representative flow cytometric histograms are shown at Supplemental Figure S3. (b) Cells that were transfected as in (a) were subjected to Western blot analyses for the expression of the indicated proteins. (c) Cells that were transfected as in (a) were subjected to sphere-forming analyses. Graphs show the means ± SDs of triplicate samples (left). Representative photographs of spheres are shown (right). Bars: 100 μm. Similar results were obtained from two independent biological replicates. * p < 0.05 vs. siControl-transfected cells by the Student’s t-test.

Figure 4

Silencing of PLCε prolongs survival in the xenograft model. GS-Y03 cells were transiently transfected with siRNA against PLCE1 or siControl for 4 days, after which the cell viability was assessed. An equal number (1 × 10⁴) of viable cells was then implanted intracranially into each nude mouse. A Kaplan–Meier analysis shows the survival curves for mice (n = 6 for each group). * p < 0.05 vs. siControl-transfected cells by the Log-rank test.

Figure 5

PLCε silencing suppresses GSC stemness by inhibiting the JNK axis. (a) GSCs (GS-Y01, GS-Y03, and TGS01) transiently transfected with siRNA against PLCE1 were subjected to Western blot analyses for the expression of the indicated proteins. (b) TGS01 and GS-Y03 cells that were transiently transfected with the activated JNK1 expression plasmid (JNK-CA) or control vector (GFP) for 24 h were transiently transfected with siRNA against PLCE1 or control siRNA for 4 days. The indicated proteins were detected by Western blotting. Similar results were obtained from two independent biological replicates. The numbers below the Western blot images show the relative band intensities after each band was quantified by densitometry and normalized by the GAPDH value.