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. 2014 Jan 30;5(2):363-74.
doi: 10.18632/oncotarget.1620.

EFEMP1 Induces γ-secretase/Notch-mediated Temozolomide Resistance in Glioblastoma

Free PMC article

EFEMP1 Induces γ-secretase/Notch-mediated Temozolomide Resistance in Glioblastoma

Lotte Hiddingh et al. Oncotarget. .
Free PMC article


Glioblastoma is the most common malignant primary brain tumor. Temozolomide (TMZ) is the standard chemotherapeutic agent for this disease. However, intrinsic and acquired TMZ-resistance represents a major obstacle for this therapy. In order to identify factors involved in TMZ-resistance, we engineered different TMZ-resistant glioblastoma cell lines. Gene expression analysis demonstrated that EFEMP1, an extracellular matrix protein, is associated with TMZ-resistant phenotype. Silencing of EFEMP1 in glioblastoma cells resulted in decreased cell survival following TMZ treatment, whereas overexpression caused TMZ-resistance. EFEMP1 acts via multiple signaling pathways, including γ-secretase-mediated activation of the Notch pathway. We show that inhibition of γ-secretase by RO4929097 causes at least partial sensitization of glioblastoma cells to temozolomide in vitro and in vivo. In addition, we show that EFEMP1 expression levels correlate with survival in TMZ-treated glioblastoma patients. Altogether our results suggest EFEMP1 as a potential therapeutic target to overcome TMZ-resistance in glioblastoma.


Figure 1
Figure 1. Identification of EFEMP1 as differentially expressed transcript in TMZ-resistant glioblastoma cells
A, Hs683, U87, and LNZ308 glioblastoma cells (WT) and TMZ-resistant subclones (R1 and R2) were analyzed for TMZ sensitivity by automated cell counting at four days after TMZ treatment. B, number of differentially expressed transcripts overlapping between resistant subclones Rl and R2 of each individual glioblastoma cell line (left) and number of overlapping transcripts among the resistant glioblastoma subclones (right). C, transcripts that were differentially expressed in at least five out of six TMZ-resistant glioblastoma subclones are shown in heatmap format. D, the top-6 transcripts overexpressed in all six resistant subclones were validated by qRT-PCR. Shown are averages, error bars indicate SD. *p<0.05 t test.
Figure 2
Figure 2. EFEMP1 induces TMZ-resistance in glioblastoma cells
A, EFEMP1 mRNA expression levels of Hs683-EFEMP1, Hs683-WT and Hs683-R1 cells as determined by qRT-PCR. B, Hs683-EFEMP1, Hs683-WT, and Hs683-R1 cells treated with TMZ and cell viability measured after four days. C, representative images of the effect of EFEMP1 overexpression in Hs683 cells on TMZ sensitivity as measured by clonogenic assays. Size bar, 5 mm. D, quantification of C. E, knock down of EFEMP1 using siEFEMP1 or siCTRL in Hs683-Rl cells 48 hrs after transfection as determined by qRT-PCR. F, cell viability of cells depicted in E upon TMZ treatment as determined by a clonogenic assay. Shown are averages, error bars indicate SD. *p<0.05 t test.
Figure 3
Figure 3. EFEMP1 induces Notch signaling in glioblastoma cells
A, schematic overview of EFEMP1 activation of EGFR [19] and Notch [22] signaling. MAGP-1/2, CCN3, YB-1, DLL, and Jagged are alternative Notch ligands [–48, 50, 51]. B, mRNA expression of the Notch-induced genes HES1 and HEY1 in the TMZ-resistant glioblastoma cells as determined by qRT-PCR. C, cell viability analysis of Hs683-WT and Hs683-Rl glioblastoma cells four days after treatment with TMZ and DAPT (25 μM). D, HES1 expression after treatment with clinically available GSIs R04929097, BMS-708163, LY450139, and MK-0752 as determined by qRT-PCR. E, cell viability analysis of Hs683-WT and Hs683-R1 four days after treatment with TMZ and RO4929097 (50 μM). Shown are averages, error bars indicate SD. *p<0.05 t test.
Figure 4
Figure 4. RO4929097 and TMZ treatment of glioblastoma cells in vitro
A, cell viability analysis of U87-WT and U87-R1 cells four days after treatment with RO4929097 (30 μM) and TMZ (100 μM). B, similar as A for a panel of primary glioblastoma cell lines. C, MGMT methylation status and MGMT protein expression of the individual primary glioblastoma cell lines used in B. Shown are averages, error bars indicate SD. *p<0.05 t test.
Figure 5
Figure 5. RO4929097 and TMZ treatment of orthotopic Hs683-Fluc glioblastoma in vivo
A, mean Fluc activity values of different treatment groups measured over time using a CCD camera. Arrows indicate five consecutive treatment days starting at day six after Hs683-Fluc cells injection in the striatum of nude mice. B, Fluc activity values of individual mice per treatment group at the start of treatment (day 6) and six days after treatment had finished. C, representative Fluc bioluminescence images of treatment groups at different time points after injection of cells (day 0). Shown are averages, error bars indicate SEM. The p values indicate two-sided t-test.
Figure 6
Figure 6. EFEMP1 expression correlates to TMZ treatment efficacy and survival in glioblastoma patients
A, EFEMP1 expression levels in multiple publicly available glioblastoma datasets as visualized by using R2 (R2.amc.n1). Glioblastoma datasets (red) [–34] were compared to normal brain (grey) [27]. [Number of patients] included in dataset are shown. B, survival of glioblastoma patients with high- (red and green), or low (blue and purple) EFEMP1 expression was plotted for TMZ-treated (n=82, upper panel) or non-treated (n=70, lower panel) patients ( C, EFEMP1 mRNA expression levels in patient samples upon clinical progression following TMZ treatment (post-TMZ), compared to matching samples biopsied at diagnosis (pre-TMZ). Numbers (#) represent individual glioblastoma patients. Expression levels were normalized to expression in non-neoplastic brain tissue. Shown are averages, error bars indicate SD. *p<0.05 t test.

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