Orphan G protein-coupled receptor GPR137 regulates ferroptosis by targeting the Wnt/β-catenin pathway in sonic hedgehog-medulloblastoma

Introduction

Medulloblastoma (MB) is one of the most common intracerebral malignancies; it occurs most often in children, with an annual incidence of approximately 0.74/100,000, and 30% of cases presenting as metastatic intracranial and leptomeningeal tumors (1,2). Currently, surgical resection remains the most important treatment method. Under the guidance of tumor risk classification and molecular pathological characteristics, adjuvant radiotherapy and chemotherapy with different protocols are employed (3-5). The most recent World Health Organization (WHO) classification of central nervous system (CNS) tumors (WHO CNS 2021 5th edition) divides MBs into four molecular subgroups: Wnt-activated, sonic hedgehog (SHH)-activated and tumor protein 53 (TP53) wild-type, SHH-activated and TP53 mutant, and non-Wnt/non-SHH (previously Groups 3 and 4) (4,6,7). These findings suggest that MBs have different origins (8), but the regulation of these aberrant gene pathways remains unclear. Although significant progress has been made in treatment, many challenges still remain.

Ferroptosis is an iron-dependent form of programmed cell death. The main mechanism by which ferroptosis occurs involves phospholipid molecules containing unsaturated fatty acids that undergo lipid peroxidation within the cell membrane system under the catalysis of iron overload or ester oxygenase, leading to cell death (9). Ferroptosis is regulated by multiple signaling pathways [such as the Adenosine 5’-monophosphate (AMP)-activated protein kinase (AMPK) (10), Hippo (11), and Wnt (12) pathways] and is involved in regulating tumor cell proliferation, chemotherapy resistance, and radiotherapy resistance. Therefore, ferroptosis is considered a potential target for cancer treatment. Recent studies have shown that ferroptosis also occurs in MBs (13), but the underlying mechanism requires further study.

GPR137, as a G protein-coupled receptor (GPCR), plays an important role in the occurrence and development of various tumors (14). GPR137 can promote the proliferation, migration, and invasion of gastric cancer cells by inhibiting the Hippo signaling pathway and activating the transcriptional activity of YAP/TAZ (15). It can also affect the proliferation of colorectal cancer cells by influencing the Wnt signaling pathway, thereby affecting tumor growth (16). GPR137 can also affect the proliferation and metastasis of various tumors, such as hepatocellular carcinoma, glioblastoma, and bladder cancer, although the specific mechanisms remain unknown (17). Previous studies have shown that GPR137 is widely expressed in the CNS, but its role is still unclear (18). There are few reports on whether GPR137 is involved in the proliferation and metastasis of MB. Similarly, there is no literature reporting whether GPR137 regulates tumor proliferation and metastasis by participating in the ferroptosis process.

We have previously analyzed dataset GSE148389 and found that GPR137 is expressed in all four subtypes of MB, with the highest expression in the SHH subtype. We further analyzed GPR137 expression in different cell lines using the Human Protein Atlas (HPA) database and found that Daoy, representing the SHH subtype, also exhibits high expression levels (Appendix 1; Figure S1). In this study, we investigated the changes in SHH-MB cell proliferation, the Wnt signaling pathway, and ferroptosis after GPR137 was knocked down in Daoy MB cells and further investigated whether the changes in ferroptosis could be reversed after Wnt signaling was upregulated. These experiments were performed to elucidate the role of GPR137 in regulating ferroptosis through the Wnt signaling pathway in SHH-MB. We present this article in accordance with the MDAR reporting checklist (available at https://tcr.amegroups.com/article/view/10.21037/tcr-2025-1298/rc).

Methods

Cell culture

The human MB cell line Daoy was purchased from Procell Life Science & Technology (Wuhan, China) and maintained in minimum essential medium Eagle (PM150410; Procell Life Science & Technology, Wuhan, China) supplemented with 10% fetal bovine serum (FBS; Gibco, Life Technologies, Grand Island, NY, USA). The cells were cultured at 37 ℃ in a 5% carbon dioxide (CO₂) humid atmosphere.

Reagents and antibodies

Erastin (T1765) and ferrostatin-1 (T6500) were purchased from Topscience (Shanghai, China). Anti-GPX4 (67763-1-Ig, 1:1,000), anti-GPR137 (11929-1-AP, 1:1,000), and anti-β-catenin (51067-2-AP, 1:1,000) antibodies were purchased from Abcam (Proterintech, Wuhan, China). The tubulin (Cat. No. BS1699) antibody was purchased from Bioworld Technology (Louis Park, MN, USA).

Cell infection

Human GPR137 5 short hairpin RNA (shRNA) and β-catenin-overexpressing lentivirus were purchased from Orbitalgene (Xi’an, China). Cells were incubated with shGPR137 or β-catenin-overexpressing lentivirus for 48 h at an MOI of 30. The cells were then selected by treatment with 1 µg/ml puromycin and cultured for experiments.

Cell viability assay

Cell viability was assessed with the Cell Counting Kit-8 (CCK-8) (C0005, Top-Bio, Shanghai, China). Log-phase cells were detached with trypsin, seeded into 96-well plates at the required density, and subjected to the indicated treatments. Thereafter, 100 µL of fresh medium containing 10 µL CCK-8 reagent was added to each well, gently mixed, and the plates were incubated for 2 h at 37 ℃ under 5% CO₂. Absorbance at 450 nm was then read on a Thermo Scientific Varioskan Flash microplate reader.

Cell death assay

Cultures subjected to the various treatments were first detached with EDTA-free trypsin and collected. The cells were then pelleted and washed twice with PBS to eliminate any residual medium. Next, the pellet was resuspended in 100 µL binding buffer, mixed with 5 µL propidium iodide (PI) staining solution (ST1569, Beyotime Biotechnology, Shanghai, China), and incubated for 5 min at room temperature in the dark. Finally, samples were acquired on a CytoFLEX flow cytometer (Beckman Coulter, Brea, CA, USA) and analyzed with CytExpert software (Beckman Coulter).

Colony formation assay

A single-cell suspension was prepared and seeded into 6-well plates at 500 cells per well; the plates were gently swirled to ensure even distribution. After incubation at 37 ℃ with 5% CO₂ for 1–2 weeks until macroscopic colonies appeared, the medium was aspirated and cells were fixed with methanol. The fixative was then discarded, sufficient Giemsa working solution was added to cover the colonies, and, following staining, excess dye was gently rinsed away with running water.

Western blot

Radioimmunoprecipitation assay (RIPA) lysis buffer was used to prepare cell lysates. A total of 10–20 µg of protein was loaded and separated on sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE) gels. Cell lysates were prepared using RIPA lysis buffer. On SDS-PAGE gels, a total of 10–20 µg of protein was added and separated. Following the transfer of fractionated proteins to nitrocellulose membranes, the membranes were blocked in 5% nonfat milk for 2 hours and probed with primary antibodies overnight. Before the immunoblots were hybridized with antibodies, they were sliced. After three 5-minute washes with Tris Buffered Saline (TBS) containing 0.1% Tween 20, the immunoblots were incubated for two hours with a secondary antibody coupled with horseradish peroxidase. The blots were produced in enhanced chemiluminescent reagent (WBKLS0500, Millipore, Sigma, California, USA), washed four times for five minutes each with TBS containing 0.1% Tween 20, and viewed using an image analyzer (Quantity One, Bio-Rad, Hercules, USA).

Quantitative reverse transcription-polymerase chain reaction (qRT-PCR)

Total ribonucleic acid (RNA) was isolated from tissues via a TRIzol Tiangen Reagent Kit (DP419, TIANGEN, Beijing, China). Then, 1 µg of complementary DNA (cDNA) was added to the corresponding kit (AG11728, Accurate Biology, Changsha, China), and a qRT-PCR kit (AG11701, Accurate Biology, China) was used with a Bio-Rad CFX Opus 96 Real-Time PCR System (CFX96) real-time PCR detection system (Bio-Rad, Hercules, USA). The following PCR primers were used:

Human β-catenin, 5-GTCTGCCAAGTGGGTGGTAT-3 (forward) and
5-TGATGTCTTCCCTGTCACCA-3 (reverse);
Human c-jun, 5-CAGGTGGCACAGCTTAAACA-3 (forward) and
5-TGAGTTGGCACCCACTGTTA-3 (reverse);
Human c-myc, 5-TCCACCTCCAGCTTGTACCT-3 (forward) and
5-GCTGTCGTTGAGAGGGTAGG-3 (reverse);
Human cyclin D1, 5-AAGTGCGAGGAGGAGGTCTT-3 (forward) and
5-AGGAAGCGGTCCAGGTAGTT-3 (reverse);
Human Axin2, 5-AGCCAAAGCGATCTACAAAAGG-3 (forward) and
5-AAGTCAAAAACATCTGGTAGGCA-3 (reverse);
Human actin, 5-GAGCGCGGCTACAGCTT-3 (forward) and
5-TCCTTAATGTCACGCACGATTT-3 (reverse).

The mRNA expression levels of the target genes were calculated via the comparative Ct method with the 2^−ΔΔCt method, and the results are presented as the fold change compared with those of the Ctrl group.

Wound healing

Log-phase cells were trypsinized to obtain a single-cell suspension and seeded at 5×10⁵ cells per well in 12-well plates. After incubation at 37 ℃ under 5% CO₂ until a confluent monolayer formed, a straight scratch was made across the center of each well with a sterile 200 µL pipette tip held at a constant angle. The wells were gently washed three times with sterile PBS to remove detached cells and debris, and the medium was then replaced with maintenance medium containing 1% FBS. The plates were returned to the incubator for an additional 48 h. Phase-contrast images of the same marked fields were captured at 0 and 48 h using an inverted microscope at 100× magnification. Scratch width was measured with ImageJ, and the migration rate was calculated as [(width at 0 h − width at 48 h)/width at 0 h] × 100%. Each experimental group was performed in triplicate to ensure reproducibility.

Reactive oxygen species (ROS) analysis

2,7-Dichlorodihydrofluorescein diacetate (DCFH-DA, Sigma, D6883) was used to monitor intracellular ROS in real time. Log-phase cells were seeded at an appropriate density and allowed to adhere before the desired treatments were applied. The medium was then aspirated and replaced with 10 µM DCFH-DA freshly dissolved in serum-free medium. After 30 min of incubation at 37 ℃ under 5% CO₂ in the dark, cells were gently washed three times with ice-cold PBS to remove extracellular probe. Adherent cells were detached with trypsin and resuspended in PBS, whereas suspension cells were collected by centrifugation and resuspended directly. A minimum of 10,000 viable events per sample were acquired on a CytoFLEX flow cytometer (Beckman Coulter) and analyzed with CytExpert software (Beckman Coulter).

Malondialdehyde (MDA) and glutathione (GSH) assays

The relative MDA concentration in the cells was assessed via an MDA assay kit (S0131, Beyotime Biotechnology) strictly following the protocol instructions provided with the kit. The GSH concentration in the cells was measured with a GSH assay kit (S0053, Beyotime Biotechnology) following the protocol instructions provided with the kit.

4-hydroxynonenal (4-HNE) assay

The concentration of 4-HNE was assessed using enzyme-linked immunosorbent assay (ELISA) kits (CSB-E16214h, Cusabio, Wuhan, China) in accordance with the manufacturer’s instructions.

Iron assay

FerroOrange staining was used to measure the intracellular Fe²⁺ level. After seeding 1×10⁵ cells per well into 12-well plates, the cells were incubated with the specified treatments at 37 ℃ in an incubator with 5% CO₂; 1 µM FerroOrange (F347, DOJINDO, Kumamoto, Japan) probes were introduced during the final 30 minutes of incubation. Following two Hank’s Balanced Salt Solution (HBSS) washes, the cells were resuspended in HBSS. After passing the labeled cells through a cell strainer, the samples were examined using CytExpert software on a CytoFLEX flow cytometer (Beckman Coulter).

Mitochondrial iron assay

To determine the mitochondrial iron levels, cells were washed with HBSS and loaded with 5 µM Mito-FerroGreen (M489, DOJINDO, Japan) and MitoTracker^® Deep Red FM (M22426, Thermo Fisher Scientific, MA, USA). The cells were examined using a confocal laser scanning microscope (LSM800, Carl Zeiss, Oberkochen, Germany) with a 40× oil immersion objective after being incubated for 30 minutes at 37 ℃ and cleaned with HBSS. The ImageJ software was used to measure the fluorescence intensity.

Statistical analysis

Every experiment was conducted at least three times. The means ± standard deviations are used to display the data. Each figure legend includes a description of every statistical analysis that was done. Using GraphPad Prism 8, the relevant statistical tests were used to get statistical P values. P<0.05 was deemed significant for every test (ns, not significant; *, P<0.05; **, P<0.01; ***, P<0.001).

Results

Knockdown of GPR137 suppresses the tumor characteristics of Daoy cells

GPR137 has been reported to be involved in the regulation of tumor cell proliferation. To determine whether GPR137 regulates Daoy cell proliferation, we treated Daoy cells with a GPR137 shRNA lentivirus to knock down GPR137 expression and then detected the proliferation rate (Figure 1A,1B). The proliferation rate of shGPR137 Daoy cells was significantly lower than that of control cells. Moreover, the cell death of shGPR137 cells was greater than that of control cells (Figure 1C), which suggested that GPR137 knockdown inhibited the proliferation of Daoy cells and induced cell death. In addition, GPR137 depletion significantly inhibited the colony formation ability of Daoy cells (Figure 1D). Wound healing data revealed that GPR137 depletion also suppressed the migration of Daoy cells (Figure 1E). These results indicate that GPR137 depletion suppressed the tumor characteristics of Daoy cells.

Figure 1 GPR137 deletion suppressed the tumor properties of Daoy cells. (A) Protein expression of GPR137 in control and shGPR137 groups. (B) Cell viability in control and shGPR137 groups. (C) Cell death measurement of control and shGPR137 groups. (D) Clone formation capability of control and shGPR137 groups was detected by crystal violet staining (imaged with a 1× objective). (E) Migration of Daoy cells in control and shGPR137 groups was detected by wound healing assay under bright field (imaged with a 4× objective). Daoy cells were stably transfected with lentivirus carrying a scrambled shRNA or GPR137 shRNA. Data are represented as mean ± SD of biological triplicates. **, P<0.01; ***, P<0.001 by unpaired, two-tailed Student’s t-tests. OD, optical density; SD, standard deviation.

Knockdown of GPR137 enhances Daoy cell sensitivity to ferroptosis.

One essential and unique characteristic of cancer is resistance to cell death. Iron-dependent lipid peroxides build up during ferroptosis, a new type of iron-dependent cell death. Next, we looked into whether GPR137 removal increased the ferroptosis sensitivity of Daoy cells. We applied varying amounts of the ferroptosis inducer erastin to Daoy cells. The data revealed that GPR137 depletion increased cell sensitivity to erastin-induced ferroptosis (Figure 2A). To further confirm this, GPR137-depleted cells were then treated with erastin and the ferroptosis inhibitor ferrostatin-1 (Figure 2B). We found that erastin-induced cell death was significantly blocked by ferrostatin-1.

Figure 2 GPR137 deletion enhances Daoy cells sensitivity to ferroptosis. (A) CCK-8 assay was used to analyze the responses of GPR137 deletion Daoy cells to erastin. (B) Cell death measurement showing the response of GPR137 deletion Daoy cells to erastin (10 µM)/± ferrostatin-1 (1 µM). (C) MDA production. (D) 4-HNE level. (E) ROS production. (F) GSH levels were measured in GPR137 deletion Daoy cells with or without erastin. (G) Protein expression of GPX4 and xCT in GPR137 deletion Daoy cells with or without erastin. (H) ferrous iron levels in the cell and (I) mitochondria were stained by Mitotracker Red and Mitotracker Green probes in GPR137 deletion Daoy cells with or without erastin (imaged with a 4× objective). Data are presented as the mean ± SD of three independent experiments. The P values were calculated by two-way ANOVA. *, P<0.05; **, P<0.01; ***, P<0.001. ANOVA, analysis of variance; CCK-8, Cell Counting Kit-8; GSH, glutathione; MDA, malondialdehyde; ROS, reactive oxygen species; SD, standard deviation.

Ferroptosis is driven by the lethal accumulation of lipid peroxides. The main and most researched byproduct of polyunsaturated fatty acid peroxidation is MDA (19), On the other hand, 4-HNE, a crucial byproduct of lipid peroxidation, damages cells by creating adducts with proteins (20). Thus, after erastin treatment, we examined lipid peroxides in GPR137-depleted cells (Figure 2C,2D). MDA and 4-HNE levels were higher in the erastin-treated group than in the control group, but they were significantly higher in GPR137-depleted cells after erastin treatment. Additionally, we used DCFH-DA, a ROS detector (21), and discovered that following erastin therapy, GPR137 depletion considerably raised the ROS level (Figure 2E).When combined, these findings suggest that GPR137 deficiency enhances cellular lipid peroxidation and increases ferroptosis susceptibility in Daoy cells. GSH is an antioxidant that lowers intracellular ROS levels and shields cells from oxidative stress through the GSH/GSSG cycle (22). The data revealed that GPR137 depletion significantly reduced the GSH level in cells after erastin treatment, indicating that GPR137 depletion reduced the level of antioxidants in the erastin-treated cells (Figure 2F). The xCT/GPX4 regulatory axis is a well-established pathway that mediates ferroptosis; therefore, we examined the expression of these proteins in Daoy cells (23). GPR137 depletion significantly suppressed the expression of xCT and GPX4 at the protein level in the erastin-treated cells (Figure 2G). In addition, further investigation revealed that the accumulation of intracellular Fe²⁺ induced by erastin was further increased by GPR137 depletion (Figure 2H). As mitochondria are the main source of ROS in cells, we measured the level of mitochondrial labile iron in the erastin-treated cells using the fluorophore Mito-FerroGreen (Figure 2I). The data revealed that GPR137-depleted cells contained more mitochondrial labile iron than did wild type (WT) cells treated with erastin. These data suggest that GPR137 depletion accelerates ferroptosis by decreasing the intracellular activity of GSH and increasing the oxidative activity of labile iron.

Knockdown of GPR137 downregulates the Wnt signaling pathway

Recently, GPR137 was shown to regulate tumor cell proliferation through the Wnt signaling pathway. In addition, in gastric cancer, Wnt/β-catenin signaling confers ferroptosis resistance by targeting GPX4 (12). Thus, we assumed that GPR137 depletion accelerated ferroptosis through downregulation of the Wnt signaling pathway. To investigate this, we first detected the activity of the Wnt signaling pathway after GPR137 depletion. Compared with those in the control group, the mRNA expression levels of β-catenin, c-jun, c-myc, cyclin D1 and Axin2 were significantly lower in the shGPR137 group (Figure 3A). Furthermore, the protein level of β-catenin showed the same trend (Figure 3B). These results suggest that the Wnt signaling pathway is potentially involved in the regulation of ferroptosis via GPR137 in Daoy cells.

Figure 3 GPR137 deletion downregulated Wnt signaling pathway. (A) mRNA expressions of Wnt signaling pathway (β-catenin, c-jun, c-myc, cyclin D1, and Axin2) in control and shGPR137 groups. (B) Protein level of β-catenin in control and shGPR137 groups. Data are represented as mean ± SD of biological triplicates. *, P<0.05; **, P<0.01; ***, P<0.001 by unpaired, two-tailed Student’s t-tests. SD, standard deviation.

β-catenin overexpression reverses the effect of GPR137 knockdown on ferroptosis

To further confirm the hypothesis that GPR137 depletion accelerates ferroptosis through downregulation of the Wnt signaling pathway in Daoy cells, we overexpressed β-catenin in shGPR137 Daoy cells. GPR137 depletion significantly increased the degree of cell death induced by erastin, and β-catenin overexpression reversed these changes (Figure 4A-4C). Moreover, the accumulation of lipid peroxides induced by GPR137 depletion, as indicated by the levels of MDA, 4-HNE, ROS and GSH, was attenuated by β-catenin overexpression (Figure 4D-4G). β-catenin overexpression also reversed the downregulation of xCT/GPX4 protein expression caused by GPR137 depletion (Figure 4H). Accordingly, labile iron from both the cells and the mitochondria was induced by GPR137 depletion, and this effect was significantly alleviated by β-catenin overexpression (Figure 4I,4J). These data demonstrated that GPR137 depletion accelerated ferroptosis via downregulation of the Wnt signaling pathway in Daoy cells.

Figure 4 β-catenin overexpression reversed the effect of GPR137 deletion on ferroptosis. (A) shGPR137 Daoy cells were stably transfected with lentivirus carrying β-catenin overexpression vector. (B) Cell viability of GPR137 deletion + β-catenin overexpression Daoy cells with erastin treatment. (C) Cell death measurement showing the response of GPR137 deletion + β-catenin overexpression Daoy cells to erastin (10 µM)/± ferrostatin-1 (1 µM). (D) MDA production. (E) 4-HNE level. (F) ROS production. (G) GSH levels were measured in GPR137 deletion + β-catenin overexpression Daoy cells with or without erastin. (H) Protein expression of GPX4 and xCT in GPR137 deletion + β-catenin overexpression Daoy cells with or without erastin. (I) Ferrous iron levels in the cell and (J) mitochondria were stained by Mitotracker Red and Mitotracker Green probes in GPR137 deletion + β-catenin overexpression Daoy cells with or without erastin (imaged with a 4× objective). Data are presented as the mean ± SD of three independent experiments. The P values were calculated by two-way ANOVA. *, P<0.05; **, P<0.01; ***, P<0.001; ns, not significant. ANOVA, analysis of variance; GSH, glutathione; MDA, Malondialdehyde; OE, overexpression; ROS, reactive oxygen species; SD, standard deviation.

Discussion

The mortality of children with CNS tumors has surpassed that of children with leukemia, and now ranks first. MB is the most common malignant tumor of the nervous system in children, and its mechanism of occurrence and development is still unclear. The current treatment employs a multimodal approach, primarily consisting of surgical resection followed by adjuvant radiotherapy and chemotherapy. Despite significant progress in multimodal treatment, which has led to an overall survival rate of around 70% for children over the age of 3 years, the risk of recurrence still remains (24). At the same time, radiotherapy and chemotherapy can also bring some long-term toxicities, including neurocognitive impairment, endocrine disorders, and hearing loss (25). Previous studies have shown that different molecular subtypes of MB have different origins and involve different molecular pathways, resulting in significant differences in their clinical prognoses (6-8). Multiple studies have indicated that the ferroptosis pathway is associated with the prognosis and treatment of MB. Through RNA sequencing analysis, it has been found that the ferroptosis transcriptional program is associated with the prognosis of MB patients (24). The use of ferroptosis inducers can induce ferroptosis in MB cell lines (26). In Group 3 MB (G3-MB), RNF126 regulates ferroptosis by ubiquitinating FSP1, thereby affecting tumor proliferation and metastasis (27). At the same time, inducing ferroptosis can also enhance the radiotherapy sensitivity of SHH-MB cells (13).

Orphan GPCRs are a group of GPCRs without clear ligands that play important roles in midgut development (28), tumor regulation (29), and pain (30), but their mechanisms are not clear. Kaafarani et al. (31) reported that SLC3A2 (a component of the ferroptosis protein xCT) can directly bind to orphan GPCRs (GPR27, GPR85, and GPR173), suggesting that orphan GPCRs may regulate ferroptosis. Recent studies have shown that GPR137, an orphan GPCR, participates in the regulation of various tumors through pathways such as the PI3K/AKT (32) and Hippo pathways (15). However, there are no reports on whether GPR137 regulates tumor proliferation through ferroptosis.

The Wnt/beta-catenin pathway has been shown to regulate ferroptosis in several types of tumors. According to Wang et al. (12), the β-catenin/TCF4 transcription complex directly binds to the promoter region of GPX4 and induces its expression, thereby suppressing ferroptotic cell death; activation of the Wnt/beta-catenin pathway attenuated cellular lipid ROS production and subsequently inhibited ferroptosis in gastric cancer cells. Tang et al. (33) demonstrated that high expression of USP8 activated Wnt/beta-catenin signaling and inhibited ferroptosis in hepatocellular carcinoma. Additionally, another group reported that the LGR4-Wnt/β-catenin pathway could directly regulate the transcription of SLC7A11, which inhibits ferroptosis in colorectal cancer. Herein, we found that the expression of GPX4 and SLC7A11 was decreased when GPR137 was knocked down via the inhibition of Wnt/β-catenin signaling in Daoy cells (34).

In this study, we first observed that GPR137 depletion decreased the proliferation and invasion ability of Daoy cells. Adding erastin (a ferroptosis inducer) to shGPR137 Daoy cells significantly increased cell death, which was reversed by the ferroptosis inhibitor ferrostatin-1. Moreover, we also found that the expression levels of 4-HNE, MDA, and ROS were significantly increased in shGPR137 Daoy cells, indicating significant lipid peroxidation and cell death after GRP137 depletion, which manifested as increased sensitivity to ferroptosis. To verify the effect of GPR137 on ferroptosis, we found that after erastin was added to shGPR137 Daoy cells, the expression of xCT and GPX4 was significantly downregulated, and the amount of active Fe²⁺ in the mitochondria increased. These results indicated that GPR137 depletion in Daoy cells led to lipid peroxidation, increased levels of reactive Fe²⁺, and elevated ROS levels. This represented a typical ferroptotic state. These results confirmed that GRP137 depletion increased the sensitivity of Daoy cells to ferroptosis.

We also examined changes in the Wnt pathway after GPR137 was knocked down. Compared with those in the control group, the mRNA expression levels of β-catenin, c-jun, c-myc, cyclin D1, and Axin2 were significantly lower in the shGPR137 group, and β-catenin showed the same trend at the protein level. Therefore, GRP137 could regulate ferroptosis through the Wnt pathway in Daoy cells. To verify this hypothesis, we overexpressed β-catenin in shGPR137 Daoy cells and found that it could effectively reverse various indicators related to ferroptosis in Daoy cells.

Although this study reported for the first time that knocking down GPR137 could increase ferroptosis sensitivity in Daoy MB cells and preliminarily suggested that ferroptosis might be accomplished through the Wnt pathway. However, there are still some limitations: (I) this study only used the Daoy cell line as an in vitro model for SHH subgroup MB, without validation in other SHH subgroup cell lines, animal models, or patient-derived tumor tissues. In the future, it is necessary to construct conditional gene knockout or overexpression animal models to confirm the physiological and pathological significance of GPR137 in regulating ferroptosis at the organismal level. (II) Although the study results suggest that GPR137 may become a therapeutic target, there is a lack of correlation analysis between GPR137 expression in clinical samples and patient prognosis, as well as ferroptosis markers. In the future, it is necessary to analyze the correlation between GPR137 expression levels and patient prognosis, molecular subtypes, and ferroptosis markers in MB patient tissues to confirm its clinical relevance. Further validation of its potential as a new targeted therapy strategy for SHH-MB is also needed.

Conclusions

In summary, this study shows that deletion of the orphan GPCR GPR137 in Daoy MB cells can promote cell sensitivity to ferroptosis through the Wnt pathway. These findings provide a new strategy for the targeted therapy of SHH-MB.

Acknowledgments

None.

Footnote

Reporting Checklist: The authors have completed the MDAR reporting checklist. Available at https://tcr.amegroups.com/article/view/10.21037/tcr-2025-1298/rc

Data Sharing Statement: Available at https://tcr.amegroups.com/article/view/10.21037/tcr-2025-1298/dss

Peer Review File: Available at https://tcr.amegroups.com/article/view/10.21037/tcr-2025-1298/prf

Funding: This study was supported by the Natural Science Foundation of Fujian Province (Nos. 2022J011066 and 2023J011316), Fujian Province Key Clinical Specialty Construction Project Pediatric Neurosurgery of Fujian Children’s Hospital [MWYZ(2023) No.1163], and Clinical Research Center for Pediatric Neurodevelopmental Disease of Fujian Province (No. 2022Y2016).

Conflicts of Interest: All authors have completed the ICMJE uniform disclosure form (available at https://tcr.amegroups.com/article/view/10.21037/tcr-2025-1298/coif). The authors have no conflicts of interest to declare.

Ethical Statement: The authors are accountable for all aspects of the work in ensuring that questions related to the accuracy or integrity of any part of the work are appropriately investigated and resolved.

Open Access Statement: This is an Open Access article distributed in accordance with the Creative Commons Attribution-NonCommercial-NoDerivs 4.0 International License (CC BY-NC-ND 4.0), which permits the non-commercial replication and distribution of the article with the strict proviso that no changes or edits are made and the original work is properly cited (including links to both the formal publication through the relevant DOI and the license). See: https://creativecommons.org/licenses/by-nc-nd/4.0/.

References

1. Pui CH, Gajjar AJ, Kane JR, et al. Challenging issues in pediatric oncology. Nat Rev Clin Oncol 2011;8:540-9. [Crossref] [PubMed]
2. Siegel RL, Miller KD, Jemal A. Cancer statistics, 2015. CA Cancer J Clin 2015;65:5-29. [Crossref] [PubMed]
3. Kumar V, Kumar V, McGuire T, et al. Challenges and Recent Advances in Medulloblastoma Therapy. Trends Pharmacol Sci 2017;38:1061-84. [Crossref] [PubMed]
4. Cotter JA, Hawkins C, Medulloblastoma WHO. 2021 and Beyond. Pediatr Dev Pathol 2022;25:23-33. [Crossref] [PubMed]
5. Orr BA. Pathology, diagnostics, and classification of medulloblastoma. Brain Pathol 2020;30:664-78. [Crossref] [PubMed]
6. Louis DN, Perry A, Wesseling P, et al. The 2021 WHO Classification of Tumors of the Central Nervous System: a summary. Neuro Oncol 2021;23:1231-51. [Crossref] [PubMed]
7. Okonechnikov K, Joshi P, Sepp M, et al. Mapping pediatric brain tumors to their origins in the developing cerebellum. Neuro Oncol 2023;25:1895-909. [Crossref] [PubMed]
8. Smith KS, Bihannic L, Gudenas BL, et al. Unified rhombic lip origins of group 3 and group 4 medulloblastoma. Nature 2022;609:1012-20. [Crossref] [PubMed]
9. Jiang X, Stockwell BR, Conrad M. Ferroptosis: mechanisms, biology and role in disease. Nat Rev Mol Cell Biol 2021;22:266-82. [Crossref] [PubMed]
10. Feng S, Yuan Y, Lin Z, et al. Low-dose hypomethylating agents cooperate with ferroptosis inducers to enhance ferroptosis by regulating the DNA methylation-mediated MAGEA6-AMPK-SLC7A11-GPX4 signaling pathway in acute myeloid leukemia. Exp Hematol Oncol 2024;13:19. [Crossref] [PubMed]
11. Gu Y, Wu S, Fan J, et al. CYLD regulates cell ferroptosis through Hippo/YAP signaling in prostate cancer progression. Cell Death Dis 2024;15:79. [Crossref] [PubMed]
12. Wang Y, Zheng L, Shang W, et al. Wnt/beta-catenin signaling confers ferroptosis resistance by targeting GPX4 in gastric cancer. Cell Death Differ 2022;29:2190-202. [Crossref] [PubMed]
13. Zhou H, Wang Y, Wu M, et al. FANCD2 deficiency sensitizes SHH medulloblastoma to radiotherapy via ferroptosis. J Pathol 2024;262:427-440. [Crossref] [PubMed]
14. Li Y, Yi Z, Li X, et al. Research Progress of GPR137 in Malignant Tumors: A Review. Onco Targets Ther 2025;18:545-58. [Crossref] [PubMed]
15. Li L, Tang J, Cao B, et al. GPR137 inactivates Hippo signaling to promote gastric cancer cell malignancy. Biol Direct 2024;19:3. [Crossref] [PubMed]
16. Mager LF, Koelzer VH, Stuber R, et al. The ESRP1-GPR137 axis contributes to intestinal pathogenesis. Elife 2017;6:e28366. [Crossref] [PubMed]
17. Szabo L, Morey R, Palpant NJ, et al. Erratum to: Statistically based splicing detection reveals neural enrichment and tissue-specific induction of circular RNA during human fetal development. Genome Biol 2016;17:263. [Crossref] [PubMed]
18. Vanti WB, Nguyen T, Cheng R, et al. Novel human G-protein-coupled receptors. Biochem Biophys Res Commun 2003;305:67-71. [Crossref] [PubMed]
19. Tsikas D. Assessment of lipid peroxidation by measuring malondialdehyde (MDA) and relatives in biological samples: Analytical and biological challenges. Anal Biochem 2017;524:13-30. [Crossref] [PubMed]
20. Zhong H, Yin H. Role of lipid peroxidation derived 4-hydroxynonenal (4-HNE) in cancer: focusing on mitochondria. Redox Biol 2015;4:193-9. [Crossref] [PubMed]
21. Eruslanov E, Kusmartsev S. Identification of ROS using oxidized DCFDA and flow-cytometry. Methods Mol Biol 2010;594:57-72. [Crossref] [PubMed]
22. Liu Q, Ding X, Xu X, et al. Tumor-targeted hyaluronic acid-based oxidative stress nanoamplifier with ROS generation and GSH depletion for antitumor therapy. Int J Biol Macromol 2022;207:771-83. [Crossref] [PubMed]
23. Lee N, Carlisle AE, Peppers A, et al. xCT-Driven Expression of GPX4 Determines Sensitivity of Breast Cancer Cells to Ferroptosis Inducers. Antioxidants (Basel) 2021;10:317. [Crossref] [PubMed]
24. Desterke C, Fu Y, Bonifacio-Mundaca J, et al. Ferroptosis Transcriptional Regulation and Prognostic Impact in Medulloblastoma Subtypes Revealed by RNA-Seq. Antioxidants (Basel) 2025;14:96. [Crossref] [PubMed]
25. Jackson K, Packer RJ. Recent Advances in Pediatric Medulloblastoma. Curr Neurol Neurosci Rep 2023;23:841-8. [Crossref] [PubMed]
26. Segui F, Daher B, Gotorbe C, et al. Revealing the Ferroptotic Phenotype of Medulloblastoma. J Vis Exp 2024;
27. Xie W, Wang J, Tian S, et al. RNF126-mediated ubiquitination of FSP1 affects its subcellular localization and ferroptosis. Oncogene 2024;43:1463-75. [Crossref] [PubMed]
28. Vieira Contreras F, Auger GM, Müller L, et al. The adhesion G-protein-coupled receptor mayo/CG11318 controls midgut development in Drosophila. Cell Rep 2024;43:113640. [Crossref] [PubMed]
29. Liang K, Guo Z, Zhang S, et al. GPR37 expression as a prognostic marker in gliomas: a bioinformatics-based analysis. Aging (Albany NY) 2023;15:10146-67. [Crossref] [PubMed]
30. Xu C, Wang Y, Ni H, et al. The role of orphan G protein-coupled receptors in pain. Heliyon 2024;10:e28818. [Crossref] [PubMed]
31. Kaafarani A, Darche-Gabinaud R, Bisteau X, et al. Proximity Interactome Analysis of Super Conserved Receptors Expressed in the Brain Identifies EPB41L2, SLC3A2, and LRBA as Main Partners. Cells 2023;12:2625. [Crossref] [PubMed]
32. Zhang LQ, Yang SQ, Qu XD, et al. GRP137 promotes cell proliferation and metastasis through regulation of the PI3K/AKT pathway in human ovarian cancer. Tumori 2018;104:330-7. [Crossref] [PubMed]
33. Tang J, Long G, Xiao L, et al. USP8 positively regulates hepatocellular carcinoma tumorigenesis and confers ferroptosis resistance through β-catenin stabilization. Cell Death Dis 2023;14:360. [Crossref] [PubMed]
34. Zheng H, Liu J, Cheng Q, et al. Targeted activation of ferroptosis in colorectal cancer via LGR4 targeting overcomes acquired drug resistance. Nat Cancer 2024;5:572-89. [Crossref] [PubMed]