Skip to main page content
U.S. flag

An official website of the United States government

Dot gov

The .gov means it’s official.
Federal government websites often end in .gov or .mil. Before sharing sensitive information, make sure you’re on a federal government site.

Https

The site is secure.
The https:// ensures that you are connecting to the official website and that any information you provide is encrypted and transmitted securely.

Access keys NCBI Homepage MyNCBI Homepage Main Content Main Navigation
. 2016 Apr 28;7(4):e2209.
doi: 10.1038/cddis.2016.102.

CD95 maintains stem cell-like and non-classical EMT programs in primary human glioblastoma cells

M Drachsler  1 , S Kleber  1 , A Mateos  1 , K Volk  1 , N Mohr  1 , S Chen  1 , B Cirovic  2 , J Tüttenberg  3 , C Gieffers  4 , J Sykora  4 , C R Wirtz  5 , W Mueller  6 , M Synowitz  7   8 , A Martin-Villalba  1
Affiliations

CD95 maintains stem cell-like and non-classical EMT programs in primary human glioblastoma cells

M Drachsler et al. Cell Death Dis. .

Erratum in

Abstract

Glioblastoma (GBM) is one of the most aggressive types of cancer with limited therapeutic options and unfavorable prognosis. Stemness and non-classical epithelial-to-mesenchymal transition (ncEMT) features underlie the switch from normal to neoplastic states as well as resistance of tumor clones to current therapies. Therefore, identification of ligand/receptor systems maintaining this privileged state is needed to devise efficient cancer therapies. In this study, we show that the expression of CD95 associates with stemness and EMT features in GBM tumors and cells and serves as a prognostic biomarker. CD95 expression increases in tumors and with tumor relapse as compared with non-tumor tissue. Recruitment of the activating PI3K subunit, p85, to CD95 death domain is required for maintenance of EMT-related transcripts. A combination of the current GBM therapy, temozolomide, with a CD95 inhibitor dramatically abrogates tumor sphere formation. This study molecularly dissects the role of CD95 in GBM cells and contributes the rational for CD95 inhibition as a GBM therapy.

PubMed Disclaimer

Figures

Figure 1
Figure 1
CD95 is a prognostic biomarker in GBM patients and linked to stem cell and EMT gene expression patterns. (a) CD95 expression in TCGA GBM samples compared with unmatched non-tumor control tissue (Wilcoxon test). (b) CD95 expression compared between distinct GBM subtypes in the TCGA data set (Wilcoxon test with Holm correction). (c) Overall survival in TCGA GBM patients stratified according to CD95 expression. (d) Progression-free survival in TCGA GBM patients stratified according to CD95 expression (log-rank test). (e) Overall survival in TCGA GBM patients grouped by subtype (log-rank test). (f) TCGA data set genes ranked according to signal-to-noise ratios for CD95high/CD95int versus CD95low expression groups. (positive values (red): higher expression in the CD95high/int group; negative values (blue): higher expression in the CD95low group). (g) GSEA for enrichment of an adult NSC gene signature in the ranked TCGA gene data set. (h) GSEA for enrichment of an EMT gene signature in the ranked TCGA gene data set
Figure 2
Figure 2
CD95high GBM cells co-express known GSC markers. (a) CD95 expression in seven patient tumor samples analyzed by flow cytometry. (b–e) Co-expression of known GSC markers CD44 (n=7), Integrin α6 (ITGA6; n=7), CD15 (n=3) and CD133 (n=7) within the patient samples (Friedman test). (f–k) Correlation of CD95 expression and known GCS markers in the TCGA GBM data set. (n=519 patients, R: Pearson's correlation coefficient, P: probability for no or negative correlation)
Figure 3
Figure 3
CD95high GBM cells display superior self-renewal in vitro and are highly tumorigenic in vivo. (a–e) Sphere formation by FACS-sorted CD95high and CD95neg cells freshly isolated from five patient tumor samples plated at 500 cells per well (t-test). (f) Representative gating strategy for cell sorting. (g–i) Sphere formation by CD95high and CD95neg cells freshly isolated from three tumor samples plated under limiting-dilution conditions. Estimated stem cell frequencies: (L) CD95neg: 1 : 110, CD95high: 1 : 1 (limiting dilution not reached); (M) CD95neg: 1 : 771, CD95high: 1 : 163; (N) CD95neg: 1 : 122, CD95high: 1 : 30. (j) 1 × 103 unsorted, CD95neg or CD95high cells were injected into the striatum of Fox Chase SCID beige mice (n=5 for unsorted and CD95neg, n=6 for CD95high). (k) Tumor growth was monitored by T2-weighted MRI. (l) Tumor volumes at the end of the MRI observation period 22 weeks after injection (ANOVA). (m) Kaplan–Meier curve showing the survival of the animals after the MRI observation period. In accordance with the observed tumor growth, CD95high-injected animals show the highest mortality
Figure 4
Figure 4
Sphere formation is dependent on CD95/CD95L signaling. (a–c) Sphere formation by freshly isolated cells from three patient tumor samples treated with DMSO (control), APG101 (APG), TMZ or a combination of both (n≥12 per conditions, overall comparison: one-way ANOVA, pairwise comparison: Tukey's multiple comparison test). (d–k) CD95L-ELISA for patient-derived cell culture supernatants (d–g) or lysates (h–k) treated with DMSO (Co) or TMZ (two sided t-test with Holm correction)
Figure 5
Figure 5
Expression of CD95 and CD95L is increasing in recurrent GBM. (a) Anti-CD95 and -CD95L immunohistochemistry (IHC) of primary and matching relapsing tumors (representative images, scale bar: 200 μm). (b) Scoring results for anti-CD95 IHC of 27 primary and relapsing tumors. (Wilcoxon test). (c) Individual relative CD95 scores (relapse score – primary tumor score) for the 27 patients showing an overall increase of CD95 expression in the relapsing tumors (average relative score=0.70, 95% Cl: 0.14–1.27). (d) Scoring results for anti-CD95L IHC of 27 primary and relapsing tumors. (Wilcoxon test). (e) Individual relative CD95L scores for 27 patients showing an overall increase in CD95L expression in the relapsing tumors (average relative score=0.73, 95% CI: 0.13–1.33)
Figure 6
Figure 6
Tyrosine phosphorylation of CD95 recruits P85 and SFK and induces ncEMT. (a) PLA for tyrosine phosphorylation of the CD95 receptor in patient-derived CD95neg GBM cells transduced with a CD95 wild-type (CD95-WT) or CD95 mutant (tyrosine-to-alanine, CD95-mut) lentivirus stimulated with PBS (Co) or CD95L-T4. (b and c) Quantification of PLA results (t-test). (d and e) IP for CD95 and P-Tyrosine GSCs in naive, CD95-WT and CD95-mut GBM cells stimulated with CD95L-T4. Blots were probed with anti-P85 (regulatory PI3K subunit), anti-Sfk, anti-CD95 or anti-P-Tyrosine antibodies, respectively. (f) Expression of 62 genes expected to be upregulated during ncEMT measured by qPCR array in four FACS sorted as well as in a lentivirally transduced patient samples. Heat map displays log10 (CD95high or CD95wt)−log10 (CD95neg or CD95mut) values
See this image and copyright information in PMC

Similar articles

See all similar articles

Cited by

See all "Cited by" articles

References

    1. Chen J, Li Y, Yu T-S, McKay RM, Burns DK, Kernie SG et al. A restricted cell population propagates glioblastoma growth after chemotherapy. Nature 2012; 488: 522–526. - PMC - PubMed
    1. Driessens G, Beck B, Caauwe A, Simons BD, Blanpain C. Defining the mode of tumour growth by clonal analysis. Nature 2012; 488: 527–530. - PMC - PubMed
    1. Schepers AG, Snippert HJ, Stange DE, van den Born M, van Es JH, van de Wetering M et al. Lineage tracing reveals Lgr5+ stem cell activity in mouse intestinal adenomas. Science 2012; 337: 730–735. - PubMed
    1. Mani SA, Guo W, Liao M-J, Eaton EN, Ayyanan A, Zhou AY et al. The epithelial-mesenchymal transition generates cells with properties of stem cells. Cell 2008; 133: 704–715. - PMC - PubMed
    1. Scheel C, Eaton EN, SH-J Li, Chaffer CL, Reinhardt F, Kah K-J et al. Paracrine and autocrine signals induce and maintain mesenchymal and stem cell states in the breast. Cell 2011; 145: 926–940. - PMC - PubMed

MeSH terms