IRE1 endoribonuclease signaling promotes myeloid cell infiltration in glioblastoma

Abstract

Background: Intrinsic or environmental stresses trigger the accumulation of improperly folded proteins in the endoplasmic reticulum (ER), leading to ER stress. To cope with this, cells have evolved an adaptive mechanism named the unfolded protein response (UPR) which is hijacked by tumor cells to develop malignant features. Glioblastoma (GB), the most aggressive and lethal primary brain tumor, relies on UPR to sustain growth. We recently showed that IRE1 alpha (referred to IRE1 hereafter), 1 of the UPR transducers, promotes GB invasion, angiogenesis, and infiltration by macrophage. Hence, high tumor IRE1 activity in tumor cells predicts a worse outcome. Herein, we characterized the IRE1-dependent signaling that shapes the immune microenvironment toward monocytes/macrophages and neutrophils.

Methods: We used human and mouse cellular models in which IRE1 was genetically or pharmacologically invalidated and which were tested in vivo. Publicly available datasets from GB patients were also analyzed to confirm our findings.

Results: We showed that IRE1 signaling, through both the transcription factor XBP1s and the regulated IRE1-dependent decay controls the expression of the ubiquitin-conjugating E2 enzyme UBE2D3. In turn, UBE2D3 activates the NFκB pathway, resulting in chemokine production and myeloid infiltration in tumors.

Conclusions: Our work identifies a novel IRE1/UBE2D3 proinflammatory axis that plays an instrumental role in GB immune regulation.

Keywords: ER stress; IRE1; chemokines; glioblastoma; inflammation.

Figure 1.

Impact of IRE1 on myeloid recruitment to GB in vitro and in vivo. (A) Hierarchical clustering of TCGA GB patients based on high/low IRE1 activity was confronted to immune markers for MM, MDM, MG, PMN, and T cells. UP (n) and P values denote the proportion of signature genes that were found upregulated between the groups (n = number of genes) and the estimated 2-sided directional P-value of test, respectively. (B) Total immune infiltrate of GB (n = 82) and grade II/III (n = 8/14) glioma analyzed by flow cytometry using anti-CD45 antibodies. ^***P < .001 according to unpaired t-test compared to GB. (C) Percentage of specific GB infiltrated leukocytes populations analyzed by flow cytometry using anti-CD45/CD11b antibodies. Tumors were classified according to IRE1 activity (high/blue in red/blue) (n = 31). (D) Total immune infiltrate of GB classified according to IRE1 activity (n = 14/7) and analyzed by flow cytometry using anti-CD45 antibodies. Tumors were classified using transcriptome analysis according to IRE1 signature. ^***P < .001 according to unpaired t-test compared to tumors with high IRE1 activity. (E) Deeper characterization of immune subtypes was performed combining specific markers that is MM markers CD14, CD64 (for MDM/MMG), CD163 (for MDM), PMN markers CD15 and CD66B, and T marker CD3. Tumors were classified according to IRE1 activity (high/low, n = 10/5). ns: not significant, ^*P < .05 and ^**P < .01 according to unpaired t-test compared to tumors with high IRE1 activity. (F) and (G) Freshly isolated Mo and PMN were placed in Boyden chambers toward fresh medium (–), conditioned media from U87 (par.), U87 DN (DN), RADH87 (par.), RADH87 cells overexpressing IRE1 (WT) or Q780* (Q*) (n = 3, mean ± SD). ns: not significant, ^*P < .05, ^**P < .01, ^****P < .0001 according to unpaired t-test compared to media from parental. (H) Representative immunohistological analysis of myeloid infiltration in GB resected from GL261 and CT2A-implanted mice treated with plug with IRE1 inhibitor MKC8866 (for GL261) and B2-1 (for CT2A), respectively. MM and PMN were detected with anti-IBA1 and anti-Ly6G antibodies, respectively. Scale bar 100 µm. (I) Semi-quantitative analysis of IBA1 and Ly6G staining from (H). P values from unpaired t-test compared to CTR; ns: not significant.

Figure 2.

IRE1-mediated synthesis of myeloid-attracting chemokines. (A) Hierarchical clustering of TCGA GB patients based on high/low IRE1 activity (n = 264/275) confronted to chemokines expression involved in myeloid chemoattraction. P values obtained using unpaired t-test comparing IRE1 high vs low tumors. (B) Chemokines mRNA expression in TCGA GB tumors with high/low MM and PMN infiltration determined according to CD14 or CD15 levels, respectively. CD14/CD15 high/low groups were determined using the median of the marker mRNA expression as cut-off (high/low n = 263/263). P values according to unpaired t-test comparing chemokines mRNA expression between CD14/CD15 high vs low tumors. (C) Correlation between chemokines secretion and Mo/PMN migration toward tumor conditioned media from different GB lines (n = 7 and 20 for Mo and PMN, respectively). R square and P values of the slopes were calculated using Pearson correlation coefficient analysis between chemokines secretion and myeloid attraction; ns: not significant and ^*P < .05. (D) Myeloid migration assay was performed as described in Figure 1, in the presence of SB225002, a CXCR2 antagonist (n = 3 and 4 for Mo and PMN, respectively, mean ± SD). ns: not significant and ^**P < .01 according to unpaired t-test compared to DMSO. (E) Quantification of myeloid-attracting chemokines mRNA abundance using RT-qPCR in parental U87 and RADH87 cells (–) transiently overexpressing TRAP-Nck (TRAP), IRE1-Nck (DN), IRE1-Q780* (Q) (n = 4). P values according to an ANOVA test comparing the 4 conditions. (F) Quantification of chemokines mRNA expression using RT-qPCR in parental U87 (par.), U87 DN (stably overexpressing IRE1-Nck), parental RADH87 or RADH87 cells stably overexpressing wild-type (WT) or Q780* (Q*) IRE1 (n = at least 6, mean ± SD). ns: not significant, ^**P < .01, ^***P < .001, ^****P < .0001 according to unpaired t-test compared to parental.

Figure 3.

IRE1/XBP1-dependent regulation of UBE2D3. (A) IRE1, XBP1s, and RIDD signatures were confronted to NFκB signaling gene signature using the TCGA GB dataset (IRE1 high/low n = 264/275; XBP1s high/low n = 261/210; and RIDD high/low n = 285/249). P values obtained with unpaired t-test comparing IRE1, XBP1s, and RIDD high vs low tumors. (B) Venn diagram of the intersection of XBP1s target genes identified by ChIPseq with RIDD targets. (C) Association of NFκB signature with NCSTN, UBE2D3, and UFM1 low/high GB from TCGA. NCSTN, UBE2D3, and UFM1 high/low groups were determined using the median of the mRNA expression as cut-off (high/low n = 263/263). P values obtained from unpaired t-test comparing NCSTN, UBE2D3, and UFM1 high vs low tumors. (D) UBE2D3 mRNA expression in TCGA GB categorized according to their IRE1, XBP1s, and RIDD signatures (IRE1 high/low n = 258/265; XBP1s high/low n = 261/210; and RIDD high/low n = 252/258). P values obtained with unpaired t-test comparing IRE1, XBP1s, and RIDD high vs low tumors. (E) UBE2D3 mRNA expression in XBP1s low/high TCGA GB (RNAseq dataset; high/low n = 80/86). P value obtained from unpaired t-test comparing XBP1s high vs low tumors. (F) Quantitation of UBE2D3 mRNA expression using RT-qPCR in U87 and RADH87 cells silenced for XBP1 (n = 7 and 4 for U87 and RADH87, respectively). ns: not significant according to unpaired t-test compared to control. (G) Western blot analysis of UBE2D3 in parental (NT), control (siCTR) and XBP1-silenced (siXBP1) U87 and RADH87 cells. Actin (ACT) was used as loading control. Data are representative of 3 biological replicates (see Supplementary Figure S4F). (H) Quantification of UBE2D3 mRNA expression by RT-qPCR in cells with active (parental U87 and RADH87 (par)) and inactive IRE1 signaling (U87 DN and RADH87 Q780*) (n = 4, mean ± SD). ^*P < .05 according to unpaired t-test compared to parental. (I) Quantitation of UBE2D3 expression with RT-qPCR in U87 DN and RADH87 Q* cells and transfected with XBP1s (n = 3). ^*P < .05, ^***P < .001 according to unpaired t-test comparing CTR to XBP1s conditions. (J) Putative XBP1s binding sites on human UBE2D3 promoter regions analyzed with MatInspector and TFBIND. (K) and (L) Gel shift assays performed on 4 putative XBP1s binding sites using U87 nuclear extracts after XBP1s overexpression (K). Validation of putative binding sites h1, h2, and h3 using gradual amounts of unlabeled probes used in competition assay (L).

Figure 4.

IRE1/RIDD-dependent regulation of UBE2D3. (A) Western blot analysis of UBE2D3 in U87 cells treated with MKC8866 (MKC) under basal or ER stress condition using thapsigargin (Tg, 50 nM). P value obtained using an ANOVA test. (B) Western blot analysis of IRE1 and UBE2D3 in U87 cells silenced for UBE2D3 and UBE2D3 overexpressing RADH87 cells under basal or ER stress condition using tunicamycin (Tm, 1 µg/mL). (C) Western blot analysis of IRE1 and UBE2D3 in UBE2D3 overexpressing U87 cells silenced for SYVN1 under basal or ER stress condition using Tm. (D) Schematic representation of IRE1 regulation of UBE2D3 expression with a retro-control loop involving SYVN1.

Figure 5.

Impact of UBE2D3 on NFκB activation and chemokines synthesis. (A) and (B) Western blot analysis of NFκB, phospho-NFκB, IκB, and phospho-IκB in control (EV) and transiently (U87) or stably (RADH87) UBE2D3 overexpressing cells (A); and after MIB1 downregulation (siMIB1) (B). UBE2D3 overexpression was checked. (C) Chemokines mRNA expression in TCGA GB specimens categorized according to UBE2D3 expression. UBE2D3 high/low (red/blue, n = 263/263) groups were determined using the median of the mRNA expression as cut-off. P values obtained from unpaired t-test comparing UBE2D3 high vs low tumors. (D) Quantification of chemokines expression using RT-qPCR in control (CTR) U87, parental (par.) RADH87, transient U87 and stable RADH87 cells overexpressing UBE2D3 (n = 3, mean ± SD). ^*P < .05, ^**P < .01, ^***P < .001, ^****P<.0001 according to unpaired t-test compared to control. (E) Quantification of chemokines expression using RT-qPCR in U87 (EV) or UBE2D3 overexpressing cells treated with 5 µM JSH-23 (n = 3, mean ± SD). ^*P < .05, ^**P < .01, ^****P < .0001 according to unpaired t-test compared to control. (F) Myeloid migration (Mo and PMN) was performed as described in Figure 1, toward media conditioned by U87 control (CTR) and UBE2D3 overexpressing cells (n = 3, mean ± SD). ^*P < .05 and ^***P < .001 according to unpaired t-test compared to control. (G) Schematic representation of UBE2D3 impact on inflammatory response in GB.

Figure 6.

Impact of UBE2D3 overexpression on inflammation in vivo. (A) UBE2D3 protein overexpression in 5 UBE2D3 transfected GL261 stable lines. UBE2D3 protein level was measured with anti-Flag antibodies. (B) Left panel: brain sections from mice injected with GL261 control (CTR) or GL261_UBE2D3 cells analyzed for vimentin expression. Scale bar 1 mm. Right panel: tumor volume in control and UBE2D3 overexpressing (oe) group (n = 3 and 10, mean ± SD). ns: not significant according to unpaired t-test compared to control. (C)–(E) Left panel: Representative immunohistological NFκB expression (n = 7/15, mean ± SD) (C), macrophages/microglia infiltration (n = 18, mean ± SD) (D) and neutrophils infiltration (n = 24, mean ± SD) (E) in GL261 control or GL261_UBE2D3 tumors detected by anti-NFκB, anti-IBA1 and anti-Ly6G antibodies, respectively. Scale bar 100 µm. Right panel: semi-quantitative analyses of NFκB (C), IBA1 (D) and Ly6G staining (E) in control and GL261_UBE2D3 tumors. ^*P < .05, ^**P < .01 according to unpaired t-test compared to control. (F) UBE2D3 protein silencing in 2 GL261 stable lines transfected with shube2d3 construct. UBE2D3 protein level was measured using anti-ube2d3 antibodies. (G) Left panel: brain sections from mice injected with GL261 shCTR or shube2d3 GL261 cells analyzed for vimentin expression. Scale bar 1 mm. Right panel: tumor volume in shCTR and shube2d3 group (n = 4, mean ± SD). ^*P < .05 according to unpaired t-test comparing to control. (H) Mouse survival of mouse-bearing parental, shCTR and shube2d3 GL261 cells. ^***P < .001 according to unpaired t-test comparing to parental. (I) UBE2D3 expression in LGG and GB (mean; P value according to unpaired t-test compared to GB); and its impact on patients’ survival. (J) Schematic representation of IRE1/UBE2D3 axis in the regulation of pro-tumoral inflammation.