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. 2023 Mar 13;5(5):211-220.
doi: 10.1096/fba.2022-00141. eCollection 2023 May.

Targeting XBP1 mRNA splicing sensitizes glioblastoma to chemotherapy

Amiee Dowdell  1   2 , Mark Marsland  1   2 , Sam Faulkner  1   2 , Craig Gedye  2   3   4 , James Lynam  2   3   4 , Cassandra P Griffin  2   3   5 , Joanne Marsland  1   2 , Chen Chen Jiang  1   2 , Hubert Hondermarck  1   2
Affiliations

Targeting XBP1 mRNA splicing sensitizes glioblastoma to chemotherapy

Amiee Dowdell et al. FASEB Bioadv. .

Abstract

Glioblastoma (GBM) is the most frequent and deadly primary brain tumor in adults. Temozolomide (TMZ) is the standard systemic therapy in GBM but has limited and restricted efficacy. Better treatments are urgently needed. The role of endoplasmic reticulum stress (ER stress) is increasingly described in GBM pathophysiology. A key molecular mediator of ER stress, the spliced form of the transcription factor x-box binding protein 1 (XBP1s) may constitute a novel therapeutic target; here we report XBP1s expression and biological activity in GBM. Tumor samples from patients with GBM (n = 85) and low-grade glioma (n = 20) were analyzed by immunohistochemistry for XBP1s with digital quantification. XBP1s expression was significantly increased in GBM compared to low-grade gliomas. XBP1s mRNA showed upregulation by qPCR analysis in a panel of patient-derived GBM cell lines. Inhibition of XBP1 splicing using the small molecular inhibitor MKC-3946 significantly reduced GBM cell viability and potentiated the effect of TMZ in GBM cells, particularly in those with methylated O6-methylguanine-DNA methyl transferase gene promoter. GBM cells resistant to TMZ were also responsive to MKC-3946 and the long-term inhibitory effect of MKC-3946 was confirmed by colony formation assay. In conclusion, this data reveals that XBP1s is overexpressed in GBM and contributes to cancer cell growth. XBP1s warrants further investigation as a clinical biomarker and therapeutic target in GBM.

Keywords: XBP1s; cancer biomarkers; glioblastoma; therapeutic targets.

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Conflict of interest statement

None of the authors have declared a conflict of interest.

Figures

FIGURE 1
FIGURE 1
XBP1s expression is increased in GBM compared to low grade gliomas. Immunohistochemical detection of spliced XBP1, representative pictures are shown for (A) grade 1, (B) grade 2, (C) grade 3 gliomas, and (D) GBM. (E) Digital quantification of XBP1s staining intensities according to grouped pathological subtypes: grade 1–3 (h‐score = 31.03, IQR 1.23–58.81) vs GBM (h‐score = 44.01, IQR 0.62–123.10). Scale bar = 30 μm. Data are expressed as individual values with medians. (F) ROC analysis for XBP1s in GBM patient samples. (G) The Kaplan–Meier survival analysis for patients with high (≥median) versus low (t test (dichotomous) or one way ANOVA (multiple comparisons) tests. XBP1s, X‐box binding protein 1, spliced. ROC, receiver operating characteristic.
FIGURE 2
FIGURE 2
Inhibition of XBP1 splicing decreases GBM cell viability. (A) XBP1s mRNA level was detected by qPCR in HA (non‐tumorigenic human astrocytes) cells and the following human GBM cell lines: U87MG, A172, BAH1, HW1, SB2b, RKI1, SJH1, WK1, RN1, MN1 and PB1. (B) GBM cell viability was studied in a panel of GBM cell lines treated with the inhibitor of XBP1 splicing MKC‐3496 (10 μM) for 72 h. (C) Correlation analysis of XBP1s mRNA and cell viability upon MKC‐3946 treatment in a panel of GBM cell lines. (D) SiRNA knockdown of XBP1 was assessed by qPCR. (E) Cell viability was studied in a panel of GBM cell lines transfected with XBP1 siRNAs for 72 h. Data shown are the mean ± SD of three independent experiments. *p < 0.05, **p < 0.01, ***p < 0.001, Student's t‐test or ANOVA. XBP1s, X‐box binding protein 1, spliced.
FIGURE 3
FIGURE 3
Inhibition of XBP1 splicing potentiates the efficacy of TMZ in GBM cells. Cell viability was studied in a panel of standard GBM cell lines U87MG and A172 (A), patient‐derived GBM cell lines with methylated MGMT gene promoter BAH1, HW1, SB2b and RKI1 (B) and unmethylated MGMT gene promoter SJH1, WK1, RN1, MN1, PB1 and HA cells (C) treated with MKC‐3496 (10 μM), TMZ (50 μM) and combination MKC‐3946 + TMZ for 72 h. (D) Colony formation assay of U87MG, BAH1 and SJH1 cells treated with MKC‐3496 (10 μM), TMZ (50 μM), and MKC‐3946 + TMZ. (E) The XBP1s mRNA level was detected by qPCR in U87MG, BAH1, and SJH1 cells treated with MKC‐3496 (10 μM), TMZ (50 μM), and MKC‐3946 + TMZ for 24 h. Data shown are the mean ± SD of three independent experiments. *p < 0.05, **p < 0.01, ***p < 0.001, Student's t‐test or ANOVA. XBP1, X‐box binding protein 1, TMZ, temozolomide.
FIGURE 4
FIGURE 4
TMZ‐resistant GBM cells are sensitive to XBP1s inhibition. The patient‐derived GBM cells BAH1 (MGMT methylated cell line) were exposed to TMZ for 3 months to generate a TMZ‐resistant subline BAH1.TMZ. (A) Representative pictures of BAH1 and BAH1.TMZ cells. (B) Cell viability assay was studied in BAH1 and BAH1.TMZ cells treated with MKC‐3946 (10 μM), TMZ (50 μM), and MKC‐3946 + TMZ for 72 h. (C) Colony formation assay of BAH1 and BAH1.TMZ cells treated with MKC‐3946 (10 μM), TMZ (50 μM), and MKC‐3946 + TMZ. (D) The XBP1s mRNA level was detected by qPCR in BAH1 and BAH1.TMZ cells treated with MKC‐3946 (10 μM), TMZ (50 μM), and MKC‐3946 + TMZ. Data shown are the mean + SD of three independent experiments. *p < 0.05, **p < 0.01, ***p < 0.001, ****p < 0.0001, Student's t‐test or ANOVA. XBP1, X‐box binding protein 1, TMZ, temozolomide.
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