Hyperplasia suppressor gene inhibits the progression of malignant meningioma via the Wnt/β-catenin signaling pathway

Introduction

Meningiomas constitute the most common primary tumors arising within the intracranial space, representing roughly 30–40% of all central nervous system neoplasms (1). Although most meningiomas exhibit benign behavior, a portion displays clearly aggressive growth tendencies, high recurrence rates, and poor clinical outcomes (2,3). The malignant subtype accounts for only 1–3% of all cases yet is distinguished by rapid expansion and extensive infiltration into surrounding brain tissue, making it particularly challenging to manage (4,5). Even with current multimodal treatment approaches—comprising maximal surgical removal, postoperative radiotherapy, and systemic chemotherapeutic strategies—patients with high-grade lesions still face poor prognoses, with 5-year survival rates ranging from 50% to 70%. These limitations highlight an urgent demand to clarify the molecular drivers of malignant transformation and to identify effective therapeutic targets (6,7).

The hyperplasia suppressor gene (HSG), also identified as mitofusin 2 (Mfn2), is a versatile polypeptide integrated into the mitochondrion’s outer envelope, where it coordinates diverse biological processes (8,9). Initially recognized for its role in suppressing vascular smooth muscle cell proliferation, HSG has since been established in maintaining mitochondrial dynamics, particularly mitochondrial fusion, bioenergetics, and cell-fate decisions (10,11). A growing body of evidence suggests that HSG exerts tumor-suppressive functions across multiple cancer types, such as lung, breast, and pancreatic malignancies (12-14). Its overexpression (OE) has been associated with reduced cancer cell growth, enhanced apoptosis, and diminished metastatic potential, largely through modulation of pathways including PI3K/Akt and Ras/Raf/ERK (15,16). Conversely, diminished HSG levels are frequently correlated with enhanced tumor aggressiveness and poor clinical outcomes. Despite these findings, the detailed expression pattern and mechanistic relevance of HSG in malignant meningioma are yet to be clarified.

The Wnt/β-catenin signaling cascade represents an evolutionarily conserved system essential for orchestrating embryonic morphogenesis and maintaining physiological stability in mature tissues (17). Aberrant activation of this pathway is a hallmark of many cancers, where it promotes uncontrolled cell proliferation, survival, and invasion (18). A key molecular event in canonical Wnt signaling is the stabilization and nuclear localization of β-catenin, enabling its cooperation with transcriptional regulators to drive oncogenic gene expression, including targets such as c-Myc and Cyclin D1 (19,20). Several studies have implicated dysregulated Wnt/β-catenin signaling in the progression of meningiomas. As an illustrative example, immunohistochemical analyses frequently reveal prominent β-catenin nuclear localization in advanced meningioma cases, demonstrating a significant correlation with elevated tumor proliferation rates and enhanced invasive behavior (21,22). Furthermore, various non-coding RNAs and signaling molecules have been found to promote meningioma malignancy by activating this pathway (2,23). Glycogen synthase kinase 3β (GSK3β), a key negative regulator in the Wnt pathway, is often inactivated in tumors, leading to β-catenin stabilization (24). These observations suggest that modulating the Wnt/β-catenin axis could offer a viable direction for developing new therapeutic strategies.

Considering that HSG exerts inhibitory effects in multiple cancers and that the Wnt/β-catenin cascade is pivotal in driving meningioma progression, we proposed that these two factors may be mechanistically connected. It is plausible that HSG exerts its anti-tumor effects in malignant meningioma by negatively regulating the Wnt/β-catenin signaling cascade. Emerging evidence in cancer research points to significant functional interplay between mitochondrial dynamics and nuclear signaling networks, although the underlying molecular details continue to be incompletely characterized. This study sought to elucidate the biological contribution of HSG in malignant meningioma and to define its putative tumor-suppressive mechanism via the Wnt/β-catenin signaling cascade. We present this article in accordance with the MDAR reporting checklist (available at https://tcr.amegroups.com/article/view/10.21037/tcr-2026-1-0204/rc).

Methods

Cell culture and reagents

The human malignant meningioma cell lines IOMM-Lee and CH-157MN were sourced from Shanghai Institute of Cell Research (Shanghai, China). Cell cultures were propagated in DMEM containing 10% fetal bovine serum (FBS). All cultures were kept in 37 ℃ with 5% CO₂. The Wnt/β-catenin pathway activator, SKL2001, was purchased from MedChemExpress (HY-101085, MCE, USA).

Construction of HSG OE vector and transfection

The full-length coding sequence of the human HSG gene was retrieved from the National Center for Biotechnology Information (NCBI) database. Following polymerase chain reaction (PCR) amplification of the HSG sequence, the product was inserted into a pCDNA3.1(+) plasmid via standard molecular cloning techniques. Both the constructed HSG-pCDNA3.1(+) recombinant plasmid and the negative control empty vector [Nc-pCDNA3.1(+)] were confirmed through DNA sequencing. For loss-of-function experiments, small interfering RNAs (siRNAs) specifically targeting HSG (si-HSG) and a negative control siRNA (si-NC) were synthesized by GenePharma (Shanghai, China). For transfection experiments, IOMM-Lee cells were plated in 6-well plates and allowed to grow to about 80% density. Plasmid and siRNA delivery were carried out with Lipofectamine^® 3000 Transfection Reagent (Invitrogen, Carlsbad, CA, USA). Transfection efficiency was confirmed by quantitative real-time polymerase chain reaction (qRT-PCR) at 24, 48, and 72 hours post-transfection to determine the optimal time point for subsequent experiments.

Experimental groups

For the initial characterization of HSG function, cells were separated into three distinct treatment groups—(I) control group: non-transfected cells maintained in standard medium; (II) OE-NC group: cells receiving the empty Nc-pCDNA3.1(+) vector; (III) OE-HSG group: cells transfected with HSG-pCDNA3.1(+) to achieve HSG OE. For the loss-of-function experiments, a separate set of groups was established—(I) control group: non-transfected cells; (II) si-NC group: cells transfected with negative control siRNA; (III) si-HSG group: cells transfected with HSG-targeted siRNA. For the experiments designed to determine whether stimulating the Wnt/β-catenin cascade could counteract the impacts of HSG OE, an additional set of groups was established: (I) OE-NC group; (II) OE-HSG group; (III) OE-HSG + SKL2001 group: cells transfected with HSG-pCDNA3.1(+) and subsequently treated with 40 µM SKL2001 for 24 hours.

Cell viability assay

The Cell Counting Kit-8 (CCK-8, CK04, Dojindo, Japan) was employed to assess cellular proliferation as per the supplier’s manual. IOMM-Lee cells from respective experimental conditions were dispensed into 96-well plates at 2×10³ cells per well. After a 24-hour culture period, each well received 10 µL of CCK-8 reagent and was kept at 37 °C for another 2 hours. Absorbance readings at 450 nm were collected with a BioTek microplate reader. Viability measurements were adjusted to control levels and reported in percentage form. Each experimental setup was conducted in triplicate, with six replicate wells per group.

Colony formation assay

IOMM-Lee cells after transfection were distributed at 500 cells per well into 6-well dishes and allowed to grow for 14 days, during which the medium was renewed every third day. At the endpoint, after fixation in 4% paraformaldehyde (PFA) for 15 minutes, the colonies were stained with 0.1% crystal violet for an additional 10 minutes. Only clusters composed of more than 50 cells were considered valid colonies and subsequently counted. Each experimental set was repeated three times with three internal replicates per group.

Wound healing assay

Cellular migration capacity was determined by performing a scratch assay. Once transfected cells reached full confluence in 6-well plates, a linear wound was created with a sterile 200-µL tip. Loose cell debris was washed off with PBS, and the cells were subsequently cultured in low-serum Dulbecco’s Modified Eagle Medium (DMEM) [1% fetal bovine serum (FBS)]. Photographs of the wound were taken immediately after scratching and again at 24 hours. ImageJ software (NIH, Bethesda, MD, USA) was used to calculate wound closure. All experiments were repeated three times.

Transwell invasion assay

To assess invasive behavior, Matrigel-coated Transwell inserts (8-µm pore size; Corning, USA) were used. Transfected cells (5×10⁴) in serum-free DMEM were placed in the top chamber, whereas the bottom chamber contained 10% FBS-DMEM to generate a chemotactic gradient. After 24 hours, remaining non-invaded cells on the upper membrane surface were gently wiped away. Cells that had penetrated the Matrigel and adhered to the lower surface were fixed with 4% PFA and stained with 0.1% crystal violet. Invasive cells were counted in five randomly selected fields at 200× magnification using ImageJ. Each assay was carried out in triplicate.

Apoptosis and cell cycle analysis

Apoptosis was detected with an Annexin V-FITC/propidium iodide (PI) kit (BD Biosciences). Cells were detached, chilled in PBS, and suspended in binding buffer at 1×10⁶ cells/mL. A 100-µL sample was stained with Annexin V-FITC and PI (5 µL each) during a 15-minute dark incubation at ambient temperature. After adding 400 µL binding buffer, samples were analyzed within 1 hour using a BD FACSCalibur cytometer, and apoptotic rates were calculated with FlowJo software. Cell-cycle profiles were evaluated concurrently on the same flow cytometer, using samples prepared through the corresponding pretreatment workflow.

qRT-PCR

Cellular RNA was purified via TRIzol reagent (Invitrogen, Carlsbad, CA, USA) and quantified on a NanoDrop 2000 system (Thermo Fisher, USA). Using 1 µg of RNA, complementary DNA (cDNA) was synthesized with a Takara kit (Japan). qRT-PCR was then conducted with SYBR Green Master Mix (Applied Biosystems, USA). Relative transcript levels were normalized against glyceraldehyde-3-phosphate dehydrogenase (GAPDH) and computed utilizing the 2^−ΔΔCt algorithm. The primers for HSG, β-catenin, c-Myc, Cyclin D1, andGSK3β are summarized in Table 1.

Western blot analysis

Cell lysates were generated using ristocetin-induced platelet aggregation (RIPA) buffer containing protease inhibitors, and protein concentrations were measured with a bicinchoninic acid (BCA) kit. Equal protein amounts were loaded for sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE) and transferred onto polyvinylidene fluoride (PVDF) membranes. Membranes, pre-blocked for 1 hour in 5% milk/Tris-buffered saline with Tween 20 (TBST), were subsequently incubated with primary antibodies at 4 °C throughout the night. The following antibodies were used: anti-GAPDH (1:5,000, Proteintech, 60004-1-Ig), anti-HSG/Mfn2 (1:1,000, Abcam, ab56889), anti-Bcl-2 (1:2,000, Abcam, ab182858), anti-Bax (1:20,000, Abcam, ab32503), anti-cleaved caspase-3 (1:5,000, Abcam, ab32351), anti-MMP-2 (1:1,000, Abcam, ab92536), anti-MMP-7 (1:1,000, Abcam, ab207299), anti-MMP-9 (1:5,000, Abcam, ab76003), anti-β-catenin (1:1,000, Abcam, ab32572), anti-c-Myc (1:1,000, Abcam, ab32072), anti-Cyclin D1 (1:10,000, Abcam, ab134175), anti-GSK3β (1:2,500, Proteintech, 82061-1-RR), and anti-phospho-GSK3β (Ser9) (1:5,000, Proteintech, 67558-1-PBS), anti-LC3B (1:1,000, Abcam, ab192890), and anti-p62/SQSTM1 (1:1,000, Abcam, ab109012). After TBST rinsing, membranes were exposed to HRP-linked secondary antibodies (anti-rabbit, 1:2,000, Abcam ab6789; anti-mouse, 1:2,000, Abcam ab150077) for 1 hour. Protein signals were developed using an enhanced chemiluminescence system (Millipore, USA) and recorded with a Bio-Rad ChemiDoc imager. Densitometric analysis was performed using ImageJ, with GAPDH as the loading control. All assays were repeated three times.

Statistical analysis

Each experiment was conducted in triplicate, and quantitative data are shown as mean ± SD. GraphPad Prism 10.1.2 (San Diego, CA) was utilized for statistical evaluations. Between-group comparisons employed Student’s t-test, whereas multiple-group differences were determined using one-way analysis of variance (ANOVA) with Tukey’s adjustment. Values with P<0.05 were regarded as statistically meaningful.

Results

HSG OE suppresses proliferative and metastatic behaviors in IOMM-Lee cells

To assess the impact of HSG on malignant meningioma cells, a stable OE-HSG model was established in IOMM-Lee cells (Figure 1). Real-time PCR (RT-qPCR) results indicated a significant upregulation of HSG messenger RNA (mRNA) in the OE-HSG group relative to both comparison groups (P<0.001; Figure 1A). CCK-8 assays revealed that HSG OE markedly reduced cell viability relative to the control groups (P<0.001; Figure 1B). Wound-healing experiments further demonstrated that OE-HSG cells exhibited a significantly lower healing rate at 24 h compared with controls (P<0.001; Figure 1C,1E), indicating a reduction in migratory capability. Similarly, Transwell analyses revealed that HSG upregulation significantly diminished invasive cell numbers (P<0.001; Figure 1D,1F). Overall, these data establish that elevated HSG expression inhibits proliferation, migration, and invasion in malignant meningioma cells.

Figure 1 Functional impact of HSG OE on proliferative, migratory, invasive and apoptotic activities in IOMM-Lee cells. (A) HSG transcript levels following transfection were quantified by RT-qPCR. (B) Cellular viability was measured using CCK-8 assays. (C) Wound-healing assay showing representative images of migratory ability at 0 and 24 h (magnification ×). (D) Cell invasion potential was examined via Transwell assays (stained). (E,F) Statistical analysis of wound closure rates and counts of invading cells. (G) Representative flow cytometry plots of apoptotic cells. (H) Statistical analysis of apoptosis rates. (I) Western blot and densitometric quantification of apoptosis-related proteins (Bax, Bcl-2, and caspase-3). (J) Flow cytometric analysis of cell cycle distribution in each group. Data are presented as mean ± SD (n=3). Statistical analysis was performed using one-way ANOVA followed by Tukey’s post-hoc test. ***, P<0.001; ns, not significant. ANOVA, analysis of variance; CCK-8, Cell Counting Kit-8; HSG, hyperplasia suppressor gene; mRNA, messenger RNA; NC, negative control; OE, overexpression; RT-qPCR, real-time polymerase chain reaction; SD, standard deviation.

HSG OE promotes apoptosis and alters cell-cycle distribution

The mechanisms underlying the observed growth inhibition were subsequently investigated. Flow cytometric assessment indicated a significant rise in apoptotic populations in OE-HSG cells relative to both comparison groups (P<0.001; Figure 1G,1H), indicating that HSG OE promotes apoptosis in IOMM-Lee cells. Consistent with this trend, Western blotting demonstrated elevated levels of pro-apoptotic Bax and cleaved caspase-3, accompanied by a reduction in the Bcl-2 (P<0.001; Figure 1I), supporting the enhancement of apoptosis. Additionally, OE-HSG expression produced a clear redistribution of cell-cycle phases (P<0.001; Figure 1J), characterized by more cells arrested in G0/G1 and fewer advancing to the S phase. This indicates that increased HSG expression triggers both apoptotic signaling and G0/G1-phase arrest. Overall, HSG-mediated growth suppression appears to result from both apoptosis activation and interruption of cell-cycle progression.

HSG limits IOMM-Lee cells aggressiveness via Wnt/β-catenin cascade inhibition

To explore whether the inhibitory effects of HSG on malignant meningioma progression are mediated through the Wnt/β-catenin cascade, we examined changes in its downstream mediators. Immunoblotting revealed that forced HSG expression sharply diminished the levels of MMP2, MMP7, and MMP9 relative to control and OE-NC groups (P<0.001; Figure 2A), indicating a suppression of extracellular matrix degradation and invasion-related factors. Parallel qRT-PCR measurements showed that overexpressing HSG led to strong reductions in β-catenin, c-Myc, and Cyclin D1 transcripts (P<0.001), while GSK3β mRNA expression remained unchanged (P>0.05; Figure 2B). Western blotting confirmed reduced β-catenin, c-Myc, and Cyclin D1 levels and a decline in phosphorylation of GSK3β, while total GSK3β levels remained stable (P<0.01; Figure 2C). These observations collectively suggest that HSG exerts its inhibitory effects on malignant meningioma cells by dampening Wnt/β-catenin cascade activation through enhanced β-catenin turnover and repression of downstream oncogenic targets.

Figure 2 HSG suppresses Wnt/β-catenin signaling, downregulates invasion-associated proteins, and induces autophagy in IOMM-Lee cells. (A) Protein expression levels of MMP2, MMP7, and MMP9 were detected by Western blot after HSG OE. (B) Transcript abundances of β-catenin, c-Myc, Cyclin D1, and GSK3β were analyzed using RT-qPCR. (C) Western blot analysis and quantification of Wnt/β-catenin pathway-related proteins, including β-catenin, c-Myc, Cyclin D1, GSK3β, and p-GSK3β. (D) Western blot analysis and quantification of autophagy markers LC3-I, LC3-II, and p62. Data are presented as mean ± SD (n=3). Statistical analysis was performed using one-way ANOVA followed by Tukey’s post-hoc test. *, P<0.05; **, P<0.01; ***, P<0.001; ns, not significant. ANOVA, analysis of variance; HSG, hyperplasia suppressor gene; mRNA, messenger RNA; NC, negative control; OE, overexpression; RT-qPCR, real-time polymerase chain reaction; SD, standard deviation.

Furthermore, because of the known interplay between mitochondrial dynamics and cellular stress responses, we evaluated autophagy markers. Western blot analysis demonstrated that HSG OE significantly increased the LC3-II/LC3-I ratio and concurrently decreased the protein expression of p62 (P<0.001; Figure 2D). These observations suggest that in addition to inducing apoptosis, HSG promotes autophagic flux in malignant meningioma cells. Collectively, these findings suggest that HSG exerts its inhibitory effects by dampening Wnt/β-catenin cascade activation and simultaneously engaging autophagic and apoptotic pathways.

Validation of HSG tumor-suppressive effects in CH-157MN cell line

To ensure that the observed effects were not limited to a single cell line, we validated our principal findings in a second malignant meningioma cell line, CH-157MN. Consistent with the results in IOMM-Lee cells, HSG OE in CH-157MN cells significantly suppressed cell viability and clonogenic growth (P<0.01; Figure 3B,3C). Furthermore, HSG OE induced a robust increase in the apoptosis rate (P<0.001; Figure 3D) and caused cell cycle arrest at the G0/G1 phase (Figure 3F). Western blot analysis in this validation cell line similarly showed a pro-apoptotic shift (increased Bax, decreased Bcl-2) and a marked downregulation of Wnt/β-catenin pathway components (β-catenin, c-Myc, and Cyclin D1) (P<0.001; Figure 3E). These data confirm that the tumor-suppressive function of HSG is consistent across different malignant meningioma models.

Figure 3 Validation of HSG tumor-suppressive effects in CH-157MN cells. (A) HSG mRNA expression levels in CH-157MN cells following transfection. (B) Cell viability measured by CCK-8 assay. (C) Representative images and quantification of colony formation (stained). (D) Flow cytometric analysis of apoptosis rates. (E) Western blot analysis and quantification of Bax, Bcl-2, β-catenin, c-Myc, and Cyclin D1. (F) Flow cytometric analysis of cell cycle distribution. Data are presented as mean ± SD (n=3). **, P<0.01; ***, P<0.001; ns, not significant. CCK-8, Cell Counting Kit-8; HSG, hyperplasia suppressor gene; mRNA, messenger RNA; NC, negative control; OE, overexpression; SD, standard deviation.

Knockdown of endogenous HSG promotes malignant phenotypes and activates Wnt/β-catenin signaling

To further substantiate the tumor-suppressive role of endogenous HSG, we performed loss-of-function experiments using siRNA. Following the successful knockdown of HSG in IOMM-Lee cells, we observed that targeted depletion of HSG significantly promoted cell survival (P<0.001; Figure 4A). Moreover, Transwell assays demonstrated a significant enhancement in the invasive capacity of the cells (P<0.001; Figure 4B). Molecularly, the depletion of HSG led to the upregulation of key Wnt/β-catenin signaling proteins, including β-catenin, c-Myc, and Cyclin D1 (P<0.001; Figure 4C). These loss-of-function results corroborate our OE findings, confirming that endogenous HSG restrains the aggressive behavior of malignant meningioma cells by suppressing the Wnt/β-catenin pathway.

Figure 4 Knockdown of endogenous HSG promotes malignant phenotypes and activates Wnt/β-catenin signaling. (A) HSG mRNA expression was confirmed by RT-qPCR after siRNA transfection, and cell viability was assessed by CCK-8 assay. (B) Representative images and quantitative analysis of the Transwell invasion assay (stained). (C) Western blot analysis and corresponding quantification of Wnt/β-catenin signaling pathway proteins, including β-catenin, c-Myc, and Cyclin D1. Data are presented as mean ± SD (n=3). ***, P<0.001; ns, not significant. CCK-8, Cell Counting Kit-8; HSG, hyperplasia suppressor gene; mRNA, messenger RNA; RT-qPCR, real-time polymerase chain reaction; SD, standard deviation; si-HSG, siRNA targeting HSG; si-NC, siRNA targeting negative control; siRNA, small interfering RNA.

Reactivation of Wnt/β-catenin signaling counteracts the antitumor effects mediated by HSG in IOMM-Lee cells

To confirm the causal link between Wnt/β-catenin pathway inhibition and the tumor-suppressive functions of HSG, a pathway agonist, SKL2001, was utilized in rescue experiments. In HSG-overexpressing cells, SKL2001 treatment partially restored the clonogenic capacity (P<0.001; Figure 5A). Similarly, the impaired migratory and invasive abilities of these cells were significantly attenuated following Wnt pathway activation (P<0.001; Figure 5B-5E). Furthermore, SKL2001 treatment markedly counteracted the pro-apoptotic effect of HSG (P<0.001; Figure 5F) and reversed the G0/G1 cell cycle arrest (Figure 5G). Overall, these results confirm that HSG exerts its growth-suppressive and pro-apoptotic actions by dampening Wnt/β-catenin signaling, and that pharmacological reactivation of this pathway can negate the tumor-suppressive influence of HSG in malignant meningioma cells.

Figure 5 Restoring Wnt/β-catenin pathway activity negates the tumor-inhibitory influence exerted by HSG on IOMM-Lee cells. (A) Assessment of clonogenic growth potential in IOMM-Lee cells after manipulation of HSG expression and pharmacologic activation with SKL2001. Representative images of crystal violet-stained colonies and quantitative analysis of colony formation rate are shown. (B) Assessment of IOMM-Lee cell migration at baseline and 24 h using a wound-healing assay. (C) Transwell invasion assay analyzing invasive potential in each group (stained). (D,E) Statistical evaluation of wound-healing progression (D) and cell invasion counts (E). (F) Flow cytometric analysis of apoptosis with representative scatter plots and quantification of apoptotic rates. (G) Flow cytometric assessment of cell-cycle distribution and statistical comparison of the proportions of cells in G0/G1, S, and G2/M phases. Data are expressed as mean ± SD (n=3). Statistical analysis was performed using one-way ANOVA followed by Tukey’s post-hoc test. ***, P<0.001; &&& P<0.001 vs. OE-NC group (G). ANOVA, analysis of variance; HSG, hyperplasia suppressor gene; NC, negative control; OE, overexpression; SD, standard deviation.

Reactivation of the Wnt/β-catenin axis abolishes the suppressive influence of HSG on the signaling network

Finally, the direct impact of Wnt pathway activation on the molecular effectors downstream of HSG was examined. HSG OE led to substantial reductions in MMP2, MMP7, and MMP9 protein levels, as confirmed by Western blotting, whereas SKL2001 treatment reversed these effects (P<0.001; Figure 6A). Furthermore, RT-qPCR together with Western blot analyses further revealed that HSG significantly downregulated β-catenin, c-Myc, and Cyclin D1 levels while increasing the p-GSK3β/GSK3β ratio (P<0.01), indicating inhibition of Wnt/β-catenin signaling. SKL2001 restored β-catenin signaling activity, counteracting HSG-induced suppression (P<0.001; Figure 6B,6C). Overall, the evidence demonstrates that HSG inhibits malignant meningioma progression by interfering with Wnt/β-catenin signaling cascade.

Figure 6 Restoration of Wnt/β-catenin signaling mitigates HSG-induced inhibition of pathway components and invasion-related protein expression in IOMM-Lee cells. (A) Protein expression patterns of MMP2, MMP7, and MMP9 as determined by Western blot and quantitative analysis. (B) RT-qPCR assessment of β-catenin, c-Myc, Cyclin D1, and GSK3β transcript levels. (C) Western blot analysis and quantitative evaluation of β-catenin, c-Myc, Cyclin D1, GSK3β, and p-GSK3β protein levels. Data are presented as mean ± SD (n=3). Statistical analysis was performed using one-way ANOVA followed by Tukey’s post-hoc test. ***, P<0.001; ns, not significant. ANOVA, analysis of variance; HSG, hyperplasia suppressor gene; mRNA, messenger RNA; NC, negative control; OE, overexpression; RT-qPCR, real-time polymerase chain reaction; SD, standard deviation.

Discussion

Malignant meningioma represents a formidable clinical challenge, characterized by aggressive growth, high recurrence rates, and limited therapeutic efficacy despite multimodal treatment approaches (1). The 5-year survival rate for World Health Organization (WHO) grade III meningiomas remains disappointingly low at approximately 50–70%, emphasizing the critical demand for alternative molecular strategies (25,26). Our work identifies HSG as a previously unrecognized tumor suppressor in malignant meningioma, capable of triggering apoptotic responses, imposing cell-cycle arrest, and diminishing cellular invasiveness. What distinguishes HSG from conventional tumor suppressors is its unique subcellular localization at the outer mitochondrial membrane, which positions it at the interface between mitochondrial bioenergetics and nuclear transcriptional regulation (27). Further investigation revealed that these suppressive effects depend on the blockade of the Wnt/β-catenin cascade, offering a promising mechanistic foundation for future targeted therapies.

The anti-proliferative effect of HSG observed in our study aligns with its established tumor-suppressive role in multiple cancer types. In lung adenocarcinoma, increasing HSG levels has been demonstrated to curb cell proliferation while activating apoptotic pathways in studies conducted in cultured cells and animal models (28,29). Similarly, in breast cancer, reduced HSG expression correlates with poor prognosis and enhanced tumor angiogenesis (30). Our data extend these observations to malignant meningioma, in which an increase in HSG levels led to a pronounced decline in both cellular viability and clonogenic growth capacity. The induction of G0/G1 phase arrest observed in our experiments is particularly noteworthy, as this effect coincided with substantial suppression of Cyclin D1, an essential driver of progression from G1 to S phase (31). Considering that Cyclin D1 is transcriptionally regulated as a classic effector of the Wnt/β-catenin cascade, its suppression by HSG provides a direct mechanistic link between mitochondrial dynamics and cell cycle regulation (32). Notably, similar regulatory mechanisms involving mitochondrial dynamics and Wnt/β-catenin signaling have been implicated in other central nervous system malignancies. In glioblastoma, for instance, mitochondrial fusion-fission balance has been shown to influence tumor cell migration and therapeutic resistance, and aberrant Wnt signaling is a well-established driver of tumor stemness and progression (33). These parallels suggest that HSG-mediated suppression of the Wnt/β-catenin pathway may represent a conserved anti-tumor mechanism across neuro-oncological contexts. At the same time, this connection is further supported by recent evidence demonstrating that mitochondrial fusion proteins, including HSG, can influence nuclear gene expression through retrograde signaling pathways (11,34).

Beyond cell cycle control, our study reveals that HSG promotes apoptosis through the intrinsic mitochondrial pathway. The coordinated rise in Bax and cleaved caspase-3, accompanied by diminished Bcl-2 levels, reflects a transition toward an apoptosis-favoring cellular environment induced by HSG (35). This observation matches the recognized positioning of HSG at the outer mitochondrial membrane, where it regulates mitochondrial dynamics and integrity (36). Disturbance of mitochondrial function represents a classical mechanism leading to Cytochrome C liberation and the subsequent initiation of caspase-dependent apoptosis (37,38). Interestingly, our evaluation of autophagy markers revealed an increased LC3-II/LC3-I ratio and decreased p62 levels upon HSG OE, indicating that HSG also induces autophagic processes. The concurrent induction of apoptosis and autophagy suggests a dual mechanism of cell death or stress response. Mitophagy, a selective form of autophagy that removes damaged mitochondria, has been implicated in restraining early tumor development by targeting and eliminating major intracellular sources of reactive oxygen radicals (39). Whether HSG-mediated apoptosis involves mitophagy or other mitochondrial quality control mechanisms warrants further investigation.

The invasive capacity of malignant meningioma is a primary determinant of patient prognosis and therapeutic resistance (40,41). Our findings demonstrate that HSG significantly suppresses both migration and invasion, which was associated with a profound downregulation of matrix metalloproteinases MMP2, MMP7, and MMP9. These enzymes are critical for extracellular matrix degradation, a prerequisite for tumor cell dissemination and invasion (42,43). High-grade meningiomas commonly exhibit increased MMP expression, which correlates with heightened invasiveness and a greater likelihood of recurrence (44). By reducing MMP levels, HSG appears to interfere with the extracellular matrix-modifying capacity of tumor cells, ultimately restricting their local invasive behavior. This anti-invasive effect is consistent with observations in pancreatic cancer, as HSG has been shown to inhibit angiogenesis, reduce lipid droplet accumulation, and attenuate tumor aggressiveness in gastric, clear cell renal carcinomas, and cutaneous head and neck melanomas (45-47). The consistency of these observations across multiple malignancies underscores the possibility that HSG functions as a tumor suppressor with broad applicability.

Mechanistically, this anti-invasive activity is further supported by the finding that Wnt/β-catenin signaling—an established driver of meningioma progression—functions as a critical downstream component under the control of HSG. Aberrant Wnt/β-catenin activation, including nuclear β-catenin accumulation, has been associated with poor outcomes in high-grade meningiomas (48). Here, elevated HSG levels diminished overall β-catenin expression and significantly lowered the levels of its downstream effectors, notably c-Myc and Cyclin D1. Given that β-catenin/TCF transcription complexes directly upregulate MMP2, MMP7, and MMP9, inhibition of this pathway by HSG provides a plausible explanation for the observed reduction in invasive capacity (49). Moreover, HSG diminished inhibitory phosphorylation of GSK3β at Ser9, suggesting enhanced GSK3β activity and promoting β-catenin degradation (50). As GSK3β inactivation is a hallmark of Wnt pathway activation, our findings indicate that HSG may reinforce the β-catenin destruction complex, thereby suppressing a broader pro-invasive transcriptional program. While a strong regulatory relationship between HSG and Wnt/β-catenin signaling is evident, how a mitochondrial outer-membrane protein modulates a pathway that largely functions in the cytoplasm and nucleus remains incompletely elucidated. One plausible explanation involves mitochondrial retrograde signaling, whereby changes in mitochondrial function or morphology can modulate nuclear gene expression and signaling pathways (51). Mitochondria are known to influence cellular calcium homeostasis, reactive oxygen species production, and metabolic flux, all of which can impact Wnt signaling (52,53). Additionally, HSG has been reported to interact with signaling molecules such as Ras and Akt, suggesting that it may function as a signaling scaffold that integrates mitochondrial status with oncogenic pathways (54,55). Future studies employing proteomics and protein-protein interaction analyses will be essential to delineate the direct or indirect molecular connections between HSG and the β-catenin destruction complex.

Several limitations of this study merit discussion. First, to ensure the robustness of our findings and address potential cell-line specific biases, we validated the principal results in a second malignant meningioma cell line, confirming that the tumor-suppressive role of HSG is broadly applicable. Furthermore, loss-of-function experiments via HSG knockdown demonstrated opposing effects—enhancing proliferation and invasion—which further substantiates its role as an endogenous tumor suppressor. However, validation in in vivo models such as orthotopic xenografts or patient-derived xenografts is still necessary to confirm the translational relevance of our findings (56). Second, the clinical significance of HSG expression in human meningioma specimens remains to be determined. A comprehensive analysis of HSG expression across a large cohort of meningioma patients, stratified by WHO grade, would be invaluable in establishing HSG as a prognostic biomarker. Further investigations may refine patient stratification to identify those with maximal therapeutic responsiveness to HSG-based interventions. Beyond Wnt/β-catenin signaling, our findings suggest HSG’s functional repertoire extends to modulating mitochondrial metabolism, redox homeostasis, and autophagic flux. A more holistic understanding of HSG’s multifaceted roles in meningioma biology will require integrative approaches combining transcriptomics, proteomics, and metabolomics. Additionally, the reliance on conventional two-dimensional cell cultures limits the evaluation of tumor-stroma interactions and the influence of the extracellular matrix. Future investigations utilizing advanced three-dimensional models, such as patient-derived meningioma organoids or sphere cultures, will be essential to validate the biological relevance of HSG within a more complex and physiologically representative tumor microenvironment. From a clinical perspective, the identification of the HSG-Wnt/β-catenin axis highlights its significant translational potential. Complementary therapeutic strategies—such as restoring HSG activity through gene therapy or small-molecule activators, or achieving concerted suppression of Wnt signaling using pathway-specific inhibitors—may yield innovative treatment paradigms. These approaches could offer new hope for managing this highly aggressive intracranial tumor, particularly for patients resistant to conventional therapies.

This study defines HSG as a functionally significant tumor suppressor in malignant meningioma. Our findings delineate a mechanism underlying its anti-oncogenic effects: HSG activation coordinately restricts proliferative, migratory, and invasive phenotypes while stimulating apoptosis and cell cycle arrest, primarily through negative regulation of Wnt/β-catenin transduction. These results provide new insights into meningioma biology and highlight the therapeutic potential of modulating this pathway. Complementary therapeutic strategies that either restore HSG activity or achieve concerted suppression of Wnt signaling may yield innovative treatment paradigms for this aggressive intracranial tumor.

Conclusions

Acknowledgments

None.

Footnote

Reporting Checklist: The authors have completed the MDAR reporting checklist. Available at https://tcr.amegroups.com/article/view/10.21037/tcr-2026-1-0204/rc

Data Sharing Statement: Available at https://tcr.amegroups.com/article/view/10.21037/tcr-2026-1-0204/dss

Peer Review File: Available at https://tcr.amegroups.com/article/view/10.21037/tcr-2026-1-0204/prf

Funding: This study was supported by the Yichang Science and Technology Innovation Fund (No. A24-2-028).

Conflicts of Interest: All authors have completed the ICMJE uniform disclosure form (available at https://tcr.amegroups.com/article/view/10.21037/tcr-2026-1-0204/coif). The authors have no conflicts of interest to declare.

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