Leucine-rich repeats and immunoglobulin-like domains 3 suppresses hypoxia-induced vasculogenic mimicry in glioma by promoting the ubiquitination and degradation of Snail2

TL;DR

LRIG3 suppresses hypoxia-induced vasculogenic mimicry in glioma by promoting the breakdown of Snail2 through ubiquitination.

Contribution

LRIG3's novel role in suppressing hypoxia-induced VM via Snail2 ubiquitination is identified.

Findings

LRIG3 expression inversely correlates with VM density in hypoxic glioma regions.

LRIG3 overexpression inhibits glioma cell invasion and tube formation under hypoxia.

LRIG3 promotes Snail2 ubiquitination and proteasomal degradation, suppressing VM.

Abstract

Leucine-rich repeats and immunoglobulin-like domains 3 (LRIG3) functions as a tumor suppressor in glioma. Although our previous study demonstrated that LRIG3 inhibited angiogenesis via the PI3K/AKT/VEGFA pathway under normoxia, its impact on glioma vascularization under hypoxia remains elusive. Vasculogenic mimicry (VM), an alternative form of neovascularization, plays a pivotal role in glioma progression, particularly within hypoxic tumor microenvironments. This study aimed to investigate the effects of LRIG3 on hypoxia-induced VM in glioma and to elucidate the underlying molecular mechanisms.

The effects of LRIG3 on VM were evaluated in vitro using tube formation and 3D spheroid invasion assays. Histological analysis of intracranial xenografts and glioblastoma specimens was performed to assess LRIG3's impact on glioma vascularization in vivo. The underlying mechanisms were investigated using western blot, quantitative real-time PCR (qRT-PCR), and ubiquitination assays.

LRIG3 expression was inversely correlated with VM density in the central hypoxic regions of both xenografts and glioblastoma specimens. Under hypoxia, LRIG3 overexpression inhibited the invasion and tube formation capacities of glioma cells, whereas its knockdown promoted these activities. Mechanistically, LRIG3 suppressed VM phenotypes by downregulating Snail2 at the post-translational level, rather than affecting VEGFA. LRIG3 promoted the ubiquitination of Snail2, leading to its proteasomal degradation and destabilization under hypoxia.

LRIG3 inhibits hypoxia-induced VM in glioma by facilitating the proteasomal degradation of Snail2 via ubiquitination.

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Full text

Background

Gliomas are the most prevalent type of primary brain tumor in adults, with glioblastoma (GBM) representing the most aggressive and lethal subtype, characterized by extensive vascular proliferation and profound invasiveness.1 A key challenge in treating high-grade gliomas is their complex and aberrant vascularization, which supports rapid tumor growth and is driven by key features of the tumor microenvironment, such as hypoxia.2 Beyond conventional angiogenesis, gliomas can form an alternative tumor-perfusing network through vasculogenic mimicry (VM), a process where highly plastic tumor cells create functional, matrix-rich tubular structures that are independent of endothelial cells.3 The presence of VM is a hallmark of increased malignancy and is associated with higher tumor grade and poorer patient outcomes.4 Notably, this process is particularly prominent within the hypoxic and necrotic core of tumors, highlighting a close association between the hypoxic microenvironment and the formation of VM.5, 6

The epithelial-mesenchymal transition (EMT), a key developmental process often reactivated during tumorigenesis, plays a pivotal role in tumor progression, invasion, and metastasis. This process involves a phenotypic switch where cells downregulate epithelial markers, such as E-cadherin, while upregulating mesenchymal markers like N-cadherin, driven by a cohort of key transcription factors including Snail, Twist, and ZEB family members. The tumor microenvironment plays a critical role in governing the activity of these transcription factors. Within high-grade gliomas, the pervasive state of hypoxia is a particularly potent signal that triggers EMT, a process largely driven by the stabilization of hypoxia-inducible factor-1α (HIF-1α).7 Accumulating evidence now highlights the pivotal contribution of EMT to the formation of VM in various cancers, including glioma.8

The Leucine-rich repeats and immunoglobulin-like domains 3 (LRIG3) protein, a member of the LRIG family of single-pass transmembrane proteins, is commonly downregulated in human cancers and is suggested as a tumor suppressor.9 In glioma, previous studies have shown that LRIG3 inhibits cell proliferation and invasion by modulating the EGFR and MET signaling pathways.10 Our recent work further demonstrated that under normoxic conditions, LRIG3 suppresses tumor-induced angiogenesis by targeting VEGFA through the PI3K/AKT pathway.11 However, the function of LRIG3 within the hypoxic tumor microenvironment, a hallmark of glioma progression, remains unknown. Given that hypoxia drives distinct and aggressive vascular programs, determining how LRIG3 contributes to glioma vascularization in this altered context is of critical importance.

In the present study, we investigated the role of LRIG3 in regulating glioma vascularization under hypoxic conditions. We demonstrate that LRIG3 inhibits hypoxia-associated VM by targeting the EMT transcription factor Snail2. Our findings reveal that LRIG3 promotes the ubiquitination and subsequent proteasomal degradation of Snail2, thereby reversing the EMT phenotype and suppressing VM formation. This study elucidates a novel, context-dependent anti-vascularization mechanism for LRIG3 distinct from its role in normoxic angiogenesis, thereby enhancing our understanding of glioma plasticity and offering a potential new target for therapeutic intervention.

Materials and methods

Cell culture

Human glioma cell lines U251 and LN229 were purchased from the American Type Culture Collection (USA). All cell lines were cultured in Dulbecco’s modified eagle’s medium (Thermo Fisher Scientific; USA) supplemented with 10% fetal bovine serum (Hyclone; USA) and 1% penicillin/streptomycin. Cells were maintained in a humidified incubator at 37 °C with 5% CO₂ and were routinely tested for mycoplasma contamination bimonthly using the MycoAlert PLUS Kit (Lonza; USA). For hypoxia experiments, cells were transferred to a hypoxic incubator (1% O₂, 5% CO₂, and 94% N₂) for 24 h prior to subsequent assays. The hypoxia-mimetic agent, cobalt chloride (CoCl₂; Sigma-Aldrich; USA; Cat# 232696), was used at a final concentration of 200 μM where indicated. For protein stability assays, cycloheximide (CHX; Selleck Chemicals; USA; Cat# S7418) was used at 10 µg/mL and the proteasome inhibitor MG132 (Selleck Chemicals; USA; Cat# S2619) was used at 20 µM.

Generation of genetically modified glioma cell lines

To generate cell lines with stable overexpression of LRIG3, lentiviral vectors were produced as previously described.12 U251 and LN229 cells were then transduced with lentivirus encoding LRIG3, with cells transduced with an empty vector serving as a control. For transient gene knockdown, small interfering RNA (siRNA) duplexes targeting LRIG3, Snail2, or HIF-1α, along with a non-targeting control siRNA (siCtrl), were synthesized by GenePharma (Shanghai, China). Cells were transfected with 25 pmol of the indicated siRNA duplexes using Lipofectamine RNAiMAX (Thermo Fisher Scientific; USA). All siRNA sequences are listed in Supplementary Table S1.

Human tissue specimens

A total of 37 human glioma tissue samples were obtained from patients who underwent surgical resection at the Department of Neurosurgery, Tongji Hospital, between September 2018 and December 2020. Histological analysis by two independent pathologists confirmed all specimens as glioblastoma (WHO Grade IV). The clinical characteristics of the patients are summarized in Supplementary Table S2. This study was approved by the Research Ethics Committee of Tongji Hospital, Tongji Medical College, Huazhong University of Science and Technology (Serial no. TJ-IBR20181111), and written informed consent was obtained from all participants prior to their inclusion in the study.

Orthotopic xenograft models

Male BALB/c nude mice (4-5 weeks old) were used for the orthotopic xenograft model (n = 6/group). Briefly, 5×10⁵ U251 cells stably expressing LRIG3 or a vector control were stereotactically injected into the mice. Mice were monitored daily and were humanely euthanized upon the initial presentation of severe neurological symptoms or significant weight loss. Brains were then harvested for subsequent histological analysis. All animal experiments were conducted in accordance with the guidelines of the Institutional Animal Care and Use Committee and were approved by the Ethical Committee of Tongji Hospital.

Histological analysis and immunohistochemistry (IHC)

Human glioblastoma specimens and mouse brain tissues containing xenografts were fixed in 4% paraformaldehyde and embedded in paraffin. For histological analysis, 4-μm-thick sections were prepared. For IHC, sections were incubated with primary antibodies against LRIG3, VEGFA, or HIF-1α. For dual staining to distinguish VM from endothelial-lined vessels, sections were co-stained with Periodic acid-Schiff (PAS) and an anti-CD34 antibody.

Tube formation assay

Matrigel (10 mg/mL, 200 µL; Corning; USA; Cat#356234) was added to each well of a 24-well plate and allowed to polymerize for 30 min at 37 °C. Subsequently, 2×10⁴ cells suspended in 200 µL of Endothelial Cell Growth Medium-2 (Lonza; USA) were seeded onto the Matrigel layer. After a 12-h incubation under either normoxic or hypoxic conditions, the formation of capillary-like structures was observed and photographed under a microscope (Carl Zeiss; Germany) at ×100 magnification. The degree of tube formation was quantified by the number of complete tube-like structures from six random fields per well using ImageJ software (NIH, USA).

Three-dimensional (3D) spheroid invasion assay

To assess cell invasion, glioma cells were seeded into ultra-low attachment 96-well U-bottom plates (Corning; USA) at a density of 1000 cells per well and centrifuged at 100 g for 5 min to form single spheroids. After a 12-hour incubation, 150 µL of medium was removed, and 50 µL of Matrigel was gently added to each well. The plates were centrifuged at 300 g for 5 min at 4 °C to center the spheroids within the Matrigel. The Matrigel was allowed to solidify for 30 min at 37 °C, after which 100 µL of complete growth medium was added13, 14. Images of the spheroids were captured at 0, 24, 48, and 72 h using a microscope (Carl Zeiss; Germany). The total invasive area extending beyond the initial spheroid core at 72 h was quantified using ImageJ software (NIH, USA).

Western blot and ubiquitination assays

Western blot analysis was performed as previously described.11 For ubiquitination assays, cells were co-transfected with HA-tagged Ubiquitin and other indicated plasmids and treated with MG132 (20 µM) for 8 h before harvesting. Cell lysates were then subjected to immunoprecipitation (IP) with an anti-Flag antibody.15 The resulting immunoprecipitates were subsequently analyzed by western blot. The primary antibodies used in this study are listed in Supplementary Table S3.

Quantitative real-time PCR

Quantitative real-time PCR (qRT-PCR) was performed as previously described.11 Primer sequences are listed in Supplementary Table S1.

Statistical analysis

All quantitative data are presented as mean ± standard deviation (SD) from at least three independent experiments. Statistical significance between two groups was determined using a two-tailed Student’s t-test. For comparisons among multiple groups, one-way analysis of variance followed by a post-hoc test was used. All statistical analyses were performed using SPSS 18.0 software (IBM Corp.; USA). A P-value < 0.05 was considered statistically significant.

Results

LRIG3 expression is inversely correlated with VM in the central hypoxic regions of gliomas

To evaluate the impact of LRIG3 on glioma vascularization in vivo, we established an orthotopic glioma xenograft model in nude mice. IHC analysis of these xenografts revealed that in both the LRIG3-overexpressing and vector-control tumors, the central regions exhibited strong staining for the hypoxia marker HIF-1α and high levels of VEGFA expression, whereas the peripheral regions did not (Figure 1a). This indicated that LRIG3's previously known regulatory effect on VEGFA was abrogated under the hypoxic conditions of the tumor core. Dual CD34/PAS staining further demonstrated that PAS-positive, CD34-negative VM channels were predominantly located in these central hypoxic areas. Notably, overexpression of LRIG3 resulted in a significant reduction in VM density in the tumor center compared to the control group (Figure 1a-c). To assess the clinical relevance of these findings, we analyzed 37 human glioblastoma specimens. In the 12 VM-positive cases, we observed a similar trend: tumors with low LRIG3 expression displayed high VM density in their central, hypoxic regions, while tumors with high LRIG3 expression showed markedly reduced VM (Figure 1d-f). Taken together, these in vivo data demonstrated a clear inverse correlation between LRIG3 expression and VM density within the hypoxic core of gliomas.Fig. 1LRIG3 expression is inversely correlated with VM in the central regions of gliomas. (a) Representative images of immunohistochemical (IHC) staining for LRIG3, VEGFA, HIF-1α, and dual CD34/PAS in orthotopic xenografts derived from U251 cells overexpressing LRIG3 or vector-control cells. The tumor center (green boxes) and periphery (yellow boxes) are shown. Red arrowheads indicate PAS+/CD34+ endothelial-lined vessels; blue arrowheads indicate PAS+/CD34- VM channels. Scale bar, 100 µm. (b) Representative IHC staining in two human glioblastoma (GBM) specimens with low or high LRIG3 expression. (c) Quantification of VM density (number of channels per field) in the peripheral and central regions of xenografts. (d) Ratio of VM channels (PAS+/CD34-) to total vascular channels (PAS+) in xenografts. (e) Quantification of VM density in the peripheral and central regions of GBM specimens. (f) Ratio of VM channels to total vascular channels in GBM specimens. Data are represented as mean ± SD (n = 6 mice per group or n = 12 patient specimens). *, P < 0.05; **, P < 0.01; ***, P < 0.001; ns, not significant.Fig. 1

LRIG3 suppresses VM formation and glioma cell invasion under hypoxia in vitro

To investigate the function of LRIG3 in vitro, we first performed tube formation assays to directly assess its role in VM. Under hypoxic conditions, U251 and LN229 cells overexpressing LRIG3 (LRIG3 group) formed significantly fewer tube-like structures compared to their control counterparts (Vector group) (Figure 2a, b). Conversely, knockdown of LRIG3 (siLRIG3 group) enhanced the tube-forming capacity of these cells compared to the negative control (siCtrl group). In contrast, LRIG3 expression levels did not affect tube formation under normoxia. Given that cell invasiveness is a critical feature of the aggressive phenotype associated with VM, we next evaluated the effect of LRIG3 on glioma cell invasion using a 3D spheroid assay. We observed that hypoxia itself promoted the invasive capacity of glioma cells compared to normoxia (Figure 2c, d). Notably, LRIG3 overexpression suppressed glioma cell invasion, whereas LRIG3 knockdown promoted it, an effect observed under both normoxic and hypoxic conditions. Collectively, these results demonstrated that LRIG3 inhibited both the formation of VM networks and cell invasion, and its inhibitory effect on VM was specific to the hypoxic context.Fig. 2LRIG3 suppresses VM formation and glioma cell invasion under hypoxia in vitro. (a) Representative images of tube formation assays performed with U251 and LN229 cells. Cells stably expressing an LRIG3 vector (LRIG3) or an empty vector (Vector), or with siRNAs targeting LRIG3 (siLRIG3) or a negative control (siCtrl), and subsequently cultured under normoxic or hypoxic conditions. Scale bar, 100 µm. (b) Quantification of the number of tube-like structures per field from the experiments described in (a). (c) Representative images of 3D spheroid invasion assays with glioma cells treated as in (a). Scale bar, 100 µm. (d) Quantification of the relative invasion area from the experiments described in (c). Data are represented as mean ± SD from three independent experiments. *, P < 0.05; **, P < 0.01; ***, P < 0.001; ns, not significant.Fig. 2

LRIG3 reverses EMT and VM phenotypes by suppressing Snail2 expression, rather than VEGFA

To investigate the molecular mechanism underlying LRIG3-mediated VM suppression under hypoxia, we first examined its effect on VEGFA, a key angiogenic factor previously identified as a downstream target of LRIG3 under normoxia.11 Western blot analysis revealed that LRIG3 expression exhibited no significant difference between normoxic and hypoxic conditions, while VEGFA was markedly upregulated under hypoxia (Figure 3a). Consistent with our in vivo findings, overexpression of LRIG3 had no significant effect on VEGFA protein levels in glioma cells cultured under either hypoxia or CoCl₂-induced chemical hypoxia (Figure 3b-c). Notably, this lack of regulatory effect of LRIG3 on VEGFA appeared to be mediated by HIF-1α, as its knockdown partially restored the ability of LRIG3 to suppress VEGFA under hypoxia (Figure 3d). These findings suggested that LRIG3 regulated hypoxia-associated VM through a VEGFA-independent pathway.Fig. 3LRIG3 reverses EMT and VM phenotypes by suppressing Snail2 expression, rather than VEGFA. (a) Western blot analysis of LRIG3, VEGFA, and HIF-1α protein levels in U251 and LN229 cells cultured under normoxia or hypoxia. (b, c) Western blot analysis of VEGFA protein levels in glioma cells with LRIG3 overexpression or vector control, cultured under hypoxia (b) or CoCl₂-induced chemical hypoxia (c). (d) Western blot analysis of VEGFA, HIF-1α, and LRIG3 in LRIG3-overexpressing cells following transfection with control siRNA (siCtrl) or siRNA targeting HIF-1α (siHIF-1α) under hypoxia. (e) Western blot analysis of EMT markers (E-cadherin, N-cadherin), a VM marker (VE-cadherin), and key EMT-associated transcription factors in glioma cells with LRIG3 overexpression or knockdown under hypoxia. β-actin was used as a loading control. All blots shown are representative of three independent experiments.Fig. 3

Since EMT is a key process driving VM, and given that hypoxia potently induces EMT, marked by a cadherin switch in glioma, we aimed to determine whether LRIG3 could reverse this hypoxia-driven transition.16, 17 LRIG3 overexpression led to an increase in the epithelial marker E-cadherin and a decrease in the mesenchymal marker N-cadherin and the VM marker VE-cadherin (Figure 3e). Conversely, LRIG3 knockdown produced the opposite effects. To identify the downstream effector, we screened several key EMT-associated transcription factors (Twist, ZEB1, ZEB2, Snail1, and Snail2). We found that Snail2 protein levels consistently showed a strong inverse correlation with LRIG3 expression (Figure 3e). To confirm that Snail2 is the critical downstream target of LRIG3 in regulating VM, we performed phenotype reversal experiments. Specifically, knockdown of Snail2 in LRIG3-silenced cells successfully reversed the pro-VM phenotype, as demonstrated by restored E-cadherin expression, reduced N-cadherin and VE-cadherin levels (Figure S1A, B), and reversed the increased tube formation capacity induced by LRIG3 knockdown (Figure S1c, d). Collectively, these results demonstrated that LRIG3 inhibits VM in hypoxic glioma cells by reversing the EMT process through the suppression of Snail2.

LRIG3 promotes the proteasomal degradation of Snail2 via ubiquitination

To elucidate the mechanism by which LRIG3 regulates Snail2, we initially assessed whether this regulation occurred at the transcriptional level. Analysis by qRT-PCR revealed that Snail2 mRNA levels were unaffected by either the overexpression or knockdown of LRIG3 in glioma cells under hypoxia (Figure S2a, b), indicating that LRIG3 modulates Snail2 at a post-translational level. We therefore hypothesized that LRIG3 might regulate the protein stability of Snail2. To test this, we performed a CHX chase assay and found that the half-life of Snail2 was significantly shortened in LRIG3-overexpressing cells, whereas it was prolonged following LRIG3 knockdown (Figure 4a, b). Furthermore, treatment with the proteasome inhibitor MG132 abrogated LRIG3-mediated downregulation of Snail2 protein levels (Figure S2c, d). These findings confirmed that LRIG3 promoted the degradation of Snail2 through the proteasomal pathway, as MG132-mediated inhibition of proteasomal activity abolished LRIG3′s regulatory effect on Snail2 stability. Since ubiquitination is a primary mechanism for targeting proteins to the proteasome, we next investigated whether LRIG3 facilitates the ubiquitination of Snail2. Co-immunoprecipitation assays demonstrated that LRIG3 overexpression markedly enhanced the level of ubiquitinated Snail2 (Figure 4c). Taken together, these findings confirmed that LRIG3 promotes the degradation of Snail2 by facilitating its ubiquitination and subsequent proteasomal degradation.Fig. 4LRIG3 promotes the proteasomal degradation of Snail2 via ubiquitination. (a) Cycloheximide (CHX) chase assay to assess Snail2 protein stability. Glioma cells with LRIG3 overexpression or knockdown were cultured under hypoxia and treated with CHX (10 µg/mL) for the indicated time points. Snail2 protein levels were subsequently analyzed by western blot. (b) Quantification of the remaining Snail2 protein levels from (a), normalized to β-actin. (c) Ubiquitination assay. Glioma cells were co-transfected with Flag-Snail2, HA-Ubiquitin (HA-Ub), and Myc-LRIG3 as indicated. Snail2 was immunoprecipitated (IP) with an anti-Flag antibody, and ubiquitinated Snail2 was detected by immunoblotting (IB) with an anti-HA antibody. All blots shown are representative of three independent experiments. Data are represented as mean ± SD from three independent experiments. **, P < 0.01; ***, P < 0.001.Fig. 4

Discussion

Extensive and aberrant neovascularization is a hallmark characteristic of high-grade gliomas and has long been a primary therapeutic target. However, the clinical efficacy of anti-angiogenic agents that target VEGFA, such as bevacizumab, is often transient as tumors inevitably develop resistance.18 A critical mechanism underlying this therapeutic escape is the activation of alternative vascularization pathways, including vasculogenic mimicry (VM), which allows aggressive tumor cells to form their own perfusion channels independent of endothelial cells.19 This process is particularly active within the hypoxic tumor core, a key feature of glioma progression.5, 20 It is well established that the hypoxic microenvironment drives a mesenchymal transition in glioblastoma, largely through mechanisms involving HIF-1α stabilization and the induction of EMT-transcription factors such as ZEB1.17 This hypoxia-triggered EMT program is recognized to confer enhanced cellular plasticity and invasive capacity, thereby facilitating VM formation.21, 22

Our study provides new insight into this clinical challenge by revealing that LRIG3 counteracts VM through a novel, context-dependent mechanism. We demonstrate that under hypoxia, LRIG3's function diverges from its established role in regulating VEGFA, instead inhibiting VM by promoting the degradation of the key EMT transcription factor Snail2. This finding reveals a sophisticated functional plasticity for LRIG3, positioning it as a critical regulator of glioma vascularization. Our findings delineate a distinct, oxygen-dependent role for LRIG3 in suppressing glioma vascularization. Under normoxia, LRIG3 targets the PI3K/AKT/VEGFA axis to inhibit angiogenesis.11 Under hypoxia, it switches to target the Snail2 protein for ubiquitin-mediated degradation, thereby specifically disrupting the vasculogenic mimicry program. This mechanistic shift underscores LRIG3's capacity to inhibit tumor vascular networks through complementary pathways tailored to the microenvironment.

Initially identified through research on the EGFR pathway, the LRIG family of proteins are now recognized as key modulators of receptor tyrosine kinases. LRIG1, for example, is known to suppress EGFR signaling by facilitating its lysosomal degradation,9 a mechanism that has also been linked to the inhibition of the EMT process.23, 24 While LRIG3 shares structural similarities with LRIG1 and has been reported to downregulate EGFR and MET signaling,10, 25 our findings unveil a distinct regulatory axis. In the hypoxic environment of glioma, LRIG3-mediated suppression of VM is not coupled to VEGFA regulation but is instead dependent on controlling Snail2 protein stability. This observation is consistent with a report in colorectal cancer where LRIG3 was also found to inhibit Snail2, albeit through a different upstream pathway (ERK signaling).26 This parallel suggests that the LRIG3-Snail2 regulatory axis may be active in multiple cancers, with its function being dependent on the specific tumor microenvironment.

The discovery of this LRIG3-Snail2 regulatory axis holds significant translational implications. A major challenge in glioma therapy is the inevitable resistance to anti-angiogenic agents like bevacizumab, which primarily target VEGFA.27, 28 Critically, such resistance is frequently associated with an adaptive increase in alternative vascularization pathways, including VM, and a hypoxic, mesenchymal shift in the tumor phenotype.29 By demonstrating that LRIG3 deletion promotes the hypoxia-Snail2-VM axis, our work provides a mechanistic explanation for a potential aggressive, treatment-resistant phenotype. Therefore, strategies aimed at restoring LRIG3 function or mimicking its action could concurrently address both canonical angiogenesis and the alternative VM pathway, offering a promising dual-targeting strategy to mitigate therapeutic escape in glioblastoma.

Our study has several limitations that should be acknowledged. First, our investigation into the role of LRIG3 in regulating EMT under hypoxia was predicated on the paradigm that hypoxia itself is a potent inducer of EMT and associated cadherin switch in glioma. A direct comparison quantifying the changes in EMT markers between normoxia and hypoxia could have further strengthened the foundational framework of the study. Additionally, the analysis of EMT was restricted to a selection of classical markers, which may not fully represent the complexity of this process. This was a single-center study with a limited sample size, and our results require validation in larger, multi-center cohorts. While we established a functional link between LRIG3 and Snail2, the precise molecular interactions governing this relationship require further investigation. As glioma vascularization is a complex, multifactorial process, future studies are warranted to gain deeper insight into this regulatory mechanism.

Conclusion

In this study, we have delineated a mechanistic switch wherein the hypoxic microenvironment steers LRIG3 function from a canonical anti-angiogenic pathway to a distinct anti-VM pathway mediated by the targeted degradation of Snail2. This dual anti-vascularization function—suppressing angiogenesis under normoxia and VM under hypoxia—positions LRIG3 as a particularly attractive therapeutic target. Strategies designed to restore or enhance its function could therefore offer a comprehensive benefit by simultaneously inhibiting both endothelial-dependent angiogenesis and tumor cell-driven VM. Targeting LRIG3 represents a promising strategy to counteract the vascular plasticity of glioma and potentially overcome the acquired resistance to conventional VEGFA-targeted therapies.

Ethics statement

Ethical approval was granted by the Research Ethics Committee of Tongji Hospital, Tongji Medical College, Huazhong University of Science and Technology (Serial no. TJ-IBR20181111). Informed consent was obtained from all subjects involved in the study.

Funding and support

This study was supported by the Wuxi Municipal Health Commission Youth Project (Project number Q202108) and Guizhou Provincial Science and Technology Projects (Grant No. QianKeHe Foundation-ZK [2024] Gen-eral-244).

CRediT authorship contribution statement

Yang Guo: Writing – original draft, Investigation, Formal analysis, Conceptualization. Dongsheng Guo: Supervision. Ran Xu: Resources, Methodology, Funding acquisition. Wenjin Qiu: Resources, Methodology, Funding acquisition. Chenghao Peng: Writing – review & editing, Supervision, Project administration, Investigation, Formal analysis, Data curation, Conceptualization.

Declaration of interest

The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper.