Raphin-1 mediates the survival and sensitivity to radiation of pediatric-type diffuse high-grade glioma via phosphorylated eukaryotic initiation factor 2α-dependent and -independent processes

Abstract

The primary treatment for fatal pediatric-type diffuse high-grade glioma (PED-DHGG) which harbor the H3K27M or H3G34R/V mutation is radiation, but it provides only short-term relief. Inhibitors of phosphorylated eIF2α (PeIF2α) phosphatase-namely raphin-1 and salubrinal-decrease survival of PED-DHGG cell lines and sensitize them to radiation. However, although both drugs increase PeIF2α, they have different effects on common targets and different targets altogether. Here, we aimed to identify PeIF2α-phosphatase-dependent and PeIF2α-phosphatase-independent molecular targets. Raphin-1 but not salubrinal, decreased the level of BiP and CReP and increased that of DR5, in an ISRIB-independent manner. Raphin-1 induced similar changes in MEF^S51A cells and in irradiated PED-DHGG, suggesting a PeIF2α-independent contribution to raphin-1's radiosensitizing effect. Importantly, while the expression of [S51D] eIF2α decreased the survival of PED-DHGG and both raphin-1 and salubrinal decreased the survival of MEF^WT cells, only raphin-1 decreased the survival of mutant MEF^S51A cells. Our results suggest that the sensitivity of PED-DHGG to raphin-1 is mediated by both PeIF2α-dependent and PeIF2α-independent processes. Elucidating these processes could reveal targets for the development of drugs to overcome radiotherapy resistance of PED-DHGG.

Keywords: GRP78/BiP; eIF2α phosphorylation; pediatric‐type diffuse high‐grade glioma; radiation; raphin‐1; salubrinal.

Fig. 1

Raphin‐1 but not salubrinal leads to a sustained decrease in the cellular level of CReP and BiP. SU‐DIPG‐VI and KNS‐42 cells (A and B, respectively) were treated with the specified concentrations of either raphin‐1 or salubrinal for the indicated time and processed for western blot analysis as described in Section 2. Numbers at the bottom of the autoradiograms indicate treatment‐dependent changes in the level of the proteins and equal loading control (Ponceau). The experiments were reproduced several times with similar results. The number of determinations for each experimental condition was as follows: SU‐DIPG‐VI – 3 h: R1 n = 7, Sal n = 3, 24 h: R1 CReP n = 14, BiP n = 13, Sal n = 5, 48 h: R1 n = 8, Sal n = 5; KNS‐42 – 3 h: n = 2, 24 h: n = 4, 48 h: n = 2. R1‐raphin‐1, Sal‐salubrinal.

Fig. 2

Raphin‐1 decreases CReP and BiP levels and cell survival via PeIF2α‐independent manner. (A) Transfection with CReP siRNA is associated with decreased BiP level. (B) The effect of ISRIB on the level of CReP and BiP in raphin‐1‐treated cells. (E) the effect of raphin‐1 and salubrinal on protein expression in MEF‐WT and MEF‐S51A‐eIF2a. (A, B, E) SU‐DIPG‐VI and MEF cells were treated with the indicated concentrations of either raphin‐1 or salubrinal for the indicated time and processed for western blot analysis as described in Section 2. Numbers at the bottom of the autoradiograms indicate treatment‐dependent changes in the level of the proteins and equal loading control (Ponceau). The number of determinations for each experimental condition were as follows: A: n = 3, B: n = 3, E: n = 2. R1‐raphin‐1, Sal—salubrinal. (C, D) The effect of raphin‐1 and salubrinal on the survival of MEF‐WT and MEF‐S51A‐eIF2a. MEF cells were plated in triplicates and treated with raphin‐1 and salubrinal as described in Section 2. Values are mean survival (%) ± SD. The significance of the differences between treatments and control were determined using an unpaired Student t‐test – *P < 0.05, **P < 0.005.

Fig. 3

Mechanisms that underlie the effect of raphin‐1 on CReP and BiP level. SU‐DIPG‐VI cells were treated with the specified concentrations of drugs and for the indicated time, then processed for western blot analysis as described in Section 2. Numbers at the bottom of the autoradiograms indicate treatment‐dependent changes in the level of the proteins and equal loading control (Ponceau). The number of determinations for each experimental condition was as follows: A: n = 2, B: n = 2, C: n = 2. R1‐raphin‐1, CHI—cycloheximide, CB‐5083—VCP/P97 inhibitor, ZVD—pan‐caspase inhibitor z‐vad‐fmk.

Fig. 4

Raphin‐1 and salubrinal differ in their effect on UPR downstream effectors. (A–D) SU‐DIPG‐VI and KNS‐42 cells were treated with the indicated concentrations of raphin‐1 and salubrinal for 24 h. RNA was extracted and treatment‐induced changes in DR5 and XBP1s mRNA were evaluated by qRT‐PCR as described in Section 2. Data are mean relative quantification (RQ) ± SD of two independent experiments. The significance of the differences between treatments and control was determined using an unpaired Student t‐test—*P < 0.05, **P < 0.005. (E–G) SU‐DIPG‐VI and KNS‐42 cells were treated with the specified concentrations of drugs and for the indicated time and processed for western blot analysis as described in Section 2. Numbers at the bottom of the autoradiograms indicate treatment‐dependent changes in the level of the proteins and equal loading control (Ponceau). The number of determinations for each experimental condition was as follows: E: DR5 n = 5, XBP1s n = 2, F: n = 4, G: n = 2, H: n = 3. R1‐raphin‐1, Sal‐salubrinal.

Fig. 5

The effect of HA15, a specific inhibitor of BiP, on raphin‐1's downstream targets. (A, B) SU‐DIPG‐VI and KNS‐42 cells were plated in triplicates and treated with HA15 as described in Section 2. Values are mean survival (%) or cell death (%) ± SD of two independent experiments. The significance of the differences between treatments and control were determined using an unpaired Student t‐test—**P < 0.005. (C, D) SU‐DIPG‐VI and KNS‐42 cells were treated with the indicated concentrations of HA15 for 24 h and processed for western blot analysis as described in Section 2. Numbers at the bottom of the autoradiograms indicate treatment‐dependent changes in the level of the proteins and equal loading control (Ponceau). The number of determinations for each experimental condition were as follows: C: n = 2, D: n = 2.

Fig. 6

Raphin‐1 and ONC201 share downstream effectors in PED‐DHGG. (A, B) SU‐DIPG‐VI and KNS‐42 cells were plated in triplicates and treated with ONC201 as described in Section 2. Values are mean survival (%) or cell death (%) ± SD of two independent experiments. The significance of the differences between treatments and control was determined using an unpaired Student t‐test—*P < 0.05, **P < 0.005. (C, D) SU‐DIPG‐VI and KNS‐42 cells were treated with the indicated concentrations of raphin‐1, salubrinal, and ONC201 for 24 h and processed for western blot analysis as described in Section 2. Numbers at the bottom of the autoradiograms indicate treatment‐dependent changes in the level of the proteins and equal loading control (Ponceau). The number of determinations for each experimental condition was as follows: C: n = 3, D: n = 2. R1‐raphin‐1, Sal‐salubrinal, Onc‐ONC201. (E) SU‐DIPG‐VI cells were treated for 48 h with 15 μm raphin‐1, fixed, and processed for transmission electron microscopy, as described in Section 2. Bar = 500 nm, arrows point to mitochondria in control and raphin‐1‐treated cells. In control untreated cells, 20% of 400 counted mitochondria exhibited a decrease in their content, compared with 90% of 215 counted mitochondria in raphin‐1‐treated cells.

Fig. 7

Raphin‐1 increases the sensitivity of PED‐DHGG to radiation. (A, B) SU‐DIPG‐VI and KNS‐42 cells were plated in triplicates and treated with raphin‐1 and radiation as described in Section 2. Values are mean survival (%) or cell death (%) ± SD of two independent experiments. The significance of the differences between treatments and control was determined using an unpaired Student t‐test – *P < 0.05, **P < 0.005. (C) SU‐DIPG‐VI cells were treated with 15 μm raphin‐1 and ionizing irradiation for 24 h and processed for western blot analysis as described in Section 2. Numbers at the bottom of the autoradiograms indicate treatment‐dependent changes in the level of the proteins and equal loading control (Ponceau). The experiment was reproduced once with similar results (n = 2). R1‐raphin‐1, Gy‐gray.

Fig. 8

Raphin‐1 increases sensitivity of PED‐DHGG via PeIF2α‐independent mechanisms. (A) Both raphin‐1 and salubrinal inhibit PeIF2α phosphatase and increase the cellular level of PeIF2α. However, the affinity of raphin‐1 to CReP‐PP1 is higher than to GADD34‐PP1, as indicated by the thicker truncated line. (B) Despite the similar effect on increased PeIF2α, both drugs manifest different effect on shared molecular targets and different targets altogether. Raphin‐1 alters the conformation of CReP and reduces its level, a phenomenon that can lead to a decreased level of BiP. Reduced level of BiP leads to an increased level of DR5. (C) Experiments with ISRIB and MEF‐S51A show that the differential effect of raphin‐1 on downstream effectors is PeIF2α‐independent. (D) Unlike salubrinal, raphin‐1 can decrease PED‐DHGG survival by activating PeIF2α‐independent processes. (E) Raphin‐1 triggers PeIF2α‐independent processes that mediate its radiosensitizing effect on PED‐DHGG. = Indicates no change in protein level.