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. 2019 Sep 24:5:137.
doi: 10.1038/s41420-019-0216-0. eCollection 2019.

The splicing FK506-binding protein-51 isoform plays a role in glioblastoma resistance through programmed cell death ligand-1 expression regulation

Paolo D'Arrigo #  1 , Marina Digregorio #  2 , Simona Romano  1 , Martina Tufano  1 , Anna Rea  1 , Felix Hausch  3 , Matthias Dedobbeleer  2 , Vincenza Vigorito  1 , Salvatore Russo  1 , Michael Bauder  3 , Bernard Rogister  2 , Maria Fiammetta Romano  1
Affiliations

The splicing FK506-binding protein-51 isoform plays a role in glioblastoma resistance through programmed cell death ligand-1 expression regulation

Paolo D'Arrigo et al. Cell Death Discov. .

Abstract

Gliomas aberrantly express programmed cell death ligand-1 (PD-L1), which has a pivotal role in immunoevasion. The splicing isoform of FKBP5, termed FKBP51s, is a PD-L1 foldase, assisting the immune checkpoint molecule in maturation and expression on the plasma membrane. The concept that PD-L1 supports tumor-intrinsic properties is increasingly emerging. The aim of the present work was to confirm the pro-tumoral effect of PD-L1 on human glioma cell survival, stemness capacity and resistance, and to address the issue of whether, by targeting its foldase either chemically or by silencing, the aggressive tumor features could be attenuated. PD-L1-depleted glioma cells have a reduced threshold for apoptosis, while PD-L1 forced expression increases resistance. Similar results were obtained with FKBP51s modulation. The ability of PD-L1 to counteract cell death was hampered by FKBP51s silencing. PD-L1 expression was particularly high in glioma cells with a cancer-stem-cell profile. Moreover, PD-L1 sustained the spheroid formation capability of glioma cells. Targeting of FKBP51s by small-interfering RNA (siRNA) or the specific inhibitor SAFit2, reduced the number of formed spheroids, along with PD-L1 expression. Finally, in an orthotopic mouse model of glioblastoma, daily treatment with SAFit2 significantly reduced tumor PD-L1 expression, and tumor growth. In treated mice, caspase-3 activation and reduced vimentin expression were observed in excised tumors. In conclusion, targeting of FKBP51s hampers PD-L1 and its pro-tumoral properties, thereby affecting the self-renewal and growth capacities of glioblastoma cells in vitro and in vivo.

Keywords: Apoptosis; CNS cancer.

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Conflict of interest statement

Conflict of interestThe authors declare that they have no conflict of interest.

Figures

Fig. 1
Fig. 1. PD-L1 regulates glioma cell apoptosis.
a Analysis by western blot assay of PD-L1 and FKBP51s expression levels in D54MG cells, silenced for FKBP51s, using different siRNAs (siFKBP51sUTR1, siFKBP51sUTR2, and siFKBP51s) or PD-L1. The blot also shows caspase-7 levels recognized in its inactive (procaspase) and active forms. On the bottom of the panel, means and standard deviations of cell-death values of D54MG cells silenced for FKBP51s and PD-L1, are shown. Columns indicate the percentage of hypodiploid cells (N = 4). b Western blot assay of PD-L1 and FKBP51s levels in U251MG cells silenced for FKBP51s and PD-L1. On the bottom of the panel, means and standard deviations of cell-death values of U251MG cells silenced for FKBP51s and PD-L1, are shown. Columns indicate the percentage of hypodiploid cells (N = 4). c Graphical representation of means and standard deviations of cell-death values in PD-L1-depleted D54MG and U251MG cells. Twenty-four hours after transfection with PD-L1 siRNA, cells were treated with etoposide. They were then harvested after a further 24 h and analyzed for PI incorporation via flow cytometry. Columns indicate the percentage of hypodiploid cells (N = 3). d Flow-cytometric histograms of PD-L1 expression in D54MG and U251MG cells, cultured for 6 h with etoposide. On the right, a graphical representation is given of values (mean and standard deviation) from three different experiments. e Western blot assay of PD-L1 levels in GBM cells cultured with etoposide. f Measurement by qPCR of FKBP51s transcript level in cells treated or not treated with etoposide, for 6 h (N = 4). g Western blot assay of exogenous PD-L1 levels in D54MG and U251MG cells cultured in the absence or the presence of etoposide. h Graphical representation of means and standard deviations of cell-death values in PD-L1 D54MG and U251MG glioma cells. Twenty-four hours after transfection with EV or PD-L1-GFP, cells were treated with etoposide. They were then harvested after a further 24 h and analyzed for PI incorporation via flow cytometry. Columns indicate the percentage of hypodiploid cells. Each experiment was performed at least three times and in triplicate
Fig. 2
Fig. 2. FKBP51s regulates glioma cell death.
a Western blot assay of FKBP51s levels in D54MG and in U251MG silenced for FKBP51s. Cells were treated with FKBP51s siRNA or NSRNA for 24 h. Then, some of the cells were harvested for lysate preparation and some cells were treated with etoposide. After a further 24 h, cell death was measured. Representative flow-cytometric histograms of PI incorporation are shown along with graphical representations of means and standard deviations of cell-death values (N = 3). b Western blot assays of exogenous FKBP51s levels and graphical representations of means and standard deviations of cell-death values in D54MG and U251MG cells transfected with FKBP51s with EV- or FKBP51s-carrying plasmids (N = 3). Cells were treated with etoposide at 24 h from transfection. After a further 24 h, cell death was measured by flow cytometry (N = 3). c Western blot assays of the exogenous PD-L1-GFP level in D54MG and U251MG cells, silenced or not silenced for FKBP51s. Silencing of FKBP51s produced a decrease in PD-L1-GFP level in both cell lines. d D54MG and U251MG cells transfected with EV, PD-L1-GFP + NSRNA and PD-L1-GFP + FKBP51s siRNA, were treated with etoposide. After 24 h, cell death was measured using flow cytometry. FKBP51s depletion reduced the antiapoptotic effect of exogenous PD-L1. Each experiment was performed at least three times and in triplicate
Fig. 3
Fig. 3. PD-L1 sustains GBM capacity to form spheroids.
a Immunoblot of p-Akt and p-S6K1 in TM-GBM and SVZ-GBM cells. Levels of phospho-enzymes were higher in SVZ-GBM cells than in TM-GBM cells. b Expression of PD-L1 and CD133 in TM-GBM and SVZ-GBM cells. Levels of both proteins were higher in SVZ-GBM cells than in TM-GBM cells. c Flow cytometry measurements of PD-L1 expression in TM-GBM and SVZ-GBM cells. Graph columns represent mean fluorescence intensities with related p-values (N = 3). A representative histogram is shown in overlay. d RT-qPCR results for mRNA levels of PD-L1 in TM-GBM and SVZ-GBM cells. Graph columns represent normalized quantification relative to TM-GBM cells (N = 3). e Representative images of formed spheroids taken using an optical microscope and spheroid counts in TM-GBM and SVZ-GBM cultures (scale bar represents 100 μm). Graph columns represent spheroid numbers relative to TM-GBM cells (N = 3). A western blot of Sox-2 levels shows increased levels of the stemness marker in the spheroids (+), in comparison with adherent cells (−). f Quantification using flow cytometry of PD-L1 levels, in TM-GBM-formed spheroids and SVZ-GBM-formed spheroids. Graph columns represent MFI (N = 3). g Spheroid formation assay with TM-GBM and SVZ-GBM cells silenced or not silenced for PD-L1. Graph columns represent the relative spheroid numbers after a 4-day culture. NSRNA-treated cells were used as the reference sample. Each experiment was performed at least three times and in triplicate
Fig. 4
Fig. 4. FKBP51s regulates PD-L1 expression and spheroid formation in TM-GBM and SVZ-GBM cells.
a Immunoblot of PD-L1 and FKBP51s levels in cells silenced for FKBP51s. Canonical FKBP51 confirmed the specificity of the silencing. b Flow cytometry analysis of PD-L1 expression in cells, silenced or not silenced for FKBP51s. Graph columns represent MFI using non-silenced cells as the reference sample (N = 3). c Spheroid assay with cells from TM-GBM and SVZ-GBM cells, silenced or not silenced for FKBP51s. Representative images of formed spheroids are shown. Graph columns represent the relative spheroid numbers, after a 4-day culture, using NSRNA-treated cells as the reference sample (N = 3). d Spheroid assay with TM-GBM cells and SVZ-GBM cells transfected with EV, PD-L1-GFP+ NSRNA, and PD-L1-GFP + FKBP51s siRNA. FKBP51s depletion reduced the spheroid formation stimulated by exogenous PD-L1 (scale bar represents 250μm) (N = 3). e Fluorescent microscopy visualization of ectopic PD-L1-GFP in the spheroid assay (scale bar represents 50 μm). FKBP51s depletion reduced spheroid numbers and the extent of fluorescence. f Western blot assay of FKBP51s levels in FKBP51s-silenced spheroids. Each experiment was performed at least three times and in triplicate
Fig. 5
Fig. 5. SAFit2 decreases PD-L1 expression and spheroid formation and impairs in vitro growth of TM-GBM and SVZ-GBM cells.
a Cytometry analysis of PD-L1 expression in cells treated with SAFit2 for 12 h. Graph columns represent mean fluorescence intensities using cells with Dimethyl sulfoxide (DMSO) as the reference sample (N = 3). b Spheroid assay with cells treated with SAFit2 or the vehicle. Representative pictures of formed spheroids are shown along graph columns for the relative spheroid number measured after a 4-day culture. DMSO-treated cells were used as the reference sample (N = 3). c Ki67 expression measured by immunofluorescence in cells treated with SAFit2 or the vehicle (scale bar represents 25 μm). Graph columns represent the percentage of Ki67-positive cells, relative to the control (N = 6). d Kinetics of counts of TM-GBM and SVZ-GBM cells treated with SAFit2 or the vehicle. SAFit2 significantly reduced cell counts (N = 4)
Fig. 6
Fig. 6. SAFit2 impairs GBM growth in vivo.
a Graphic representation of tumor volumes formed in 16 mice treated daily with vehicle and 16 mice treated with SAFit2. b Luciferase assay, performed on mice treated or not treated with SAFit2, 2 weeks after in-brain injection with GFP+ luc+ U87MG cells. Three mice from each group are shown before starting the treatment and after 10 days (left). Graph columns represent mean values of signal intensities from the two groups, before and after treatment (right) (N = 16). c Expression of active caspase-3 by immunofluorescence on brain slices (scale bar represents 50 μm). Box plots represent the values of GFP+ cleaved Casp3+ cells measured using ImageJ software, from seven mice treated with vehicle and seven mice treated with SAFit2. d Expression of PD-L1 determined by immunofluorescence on brain slices (scale bar represents 50 μm). Box plots represent the values of GFP+ PD-L1+ cells measured using ImageJ software, from nine mice treated with vehicle and nine mice treated with SAFit2
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