Asporin promotes gastric cancer progression by regulating NRF2 ubiquitination and competitively binding to KEAP1

Introduction

Gastric cancer (GC) is among the leading causes of cancer-related deaths worldwide, with a notably high incidence in East Asia (1,2). Due to the lack of early symptoms, GC is frequently diagnosed at advanced stages, which severely limits treatment effectiveness and negatively impacts patient survival outcomes (3,4). Although significant research efforts have been made, the complex molecular mechanisms underlying GC initiation and progression remain poorly elucidated, underscoring the urgent need for targeted investigations aimed at identifying novel therapeutic targets and diagnostic biomarkers (5,6).

In this context, asporin (ASPN), a small leucine-rich proteoglycan (SLRP) localized in the extracellular matrix, has been identified as a key molecule. ASPN is known to participate in multiple critical cancer-related processes, such as autonomous growth signaling, resistance to growth inhibition, evasion of apoptosis, and promotion of metastasis (7). Increased expression of ASPN has been reported in various malignancies, including pancreatic, colorectal, prostate, bladder, and certain subtypes of breast cancer, indicating its potential as a therapeutic target (8,9). Recently, ASPN has been proposed as a promising biomarker for the early detection of GC (10). However, its precise functional role, underlying molecular mechanisms, and therapeutic potential in GC pathophysiology remain to be fully elucidated.

Our study demonstrates significantly elevated ASPN expression in GC tissues compared to adjacent non-tumorous tissues. Through a series of comprehensive in vitro and in vivo experiments, we have established that ASPN plays a crucial role in promoting GC cell proliferation, migration, and invasion. Notably, our findings reveal that ASPN modulates the KEAP1-NRF2 pathway, a key regulator of cancer progression. This pathway controls the cellular response to oxidative stress and maintains redox homeostasis, and its dysregulation is a well-documented feature of multiple cancers. ASPN stabilizes NRF2 by competing with KEAP1, thereby reducing NRF2 ubiquitination and proteasomal degradation, and enhancing pathway activity. These results indicate that ASPN functions as a key oncogenic driver and represents a promising candidate for both diagnostic and therapeutic applications in GC. Collectively, this study provides novel mechanistic insights into GC pathogenesis and underscores the potential of ASPN as a biomarker and therapeutic target. Further investigation is needed to evaluate the clinical applicability of ASPN in early diagnosis and personalized treatment approaches for GC. We present this article in accordance with the ARRIVE and MDAR reporting checklists (available at https://tcr.amegroups.com/article/view/10.21037/tcr-2025-aw-2442/rc).

Methods

Clinical samples

Twelve GC tumor samples and corresponding adjacent non-tumor tissues were obtained from patients undergoing surgical resection at Nantong First People’s Hospital. All patients were untreated and had never received any anti-cancer treatment before the operation. The study was conducted in accordance with the Declaration of Helsinki and its subsequent amendments. The study was approved by the Ethics Committee of Nantong First People’s Hospital (No. 2023KT039). Written informed consent was obtained from all participants prior to sample collection.

Constructing plasmids and culturing cells

Gene silencing was achieved using a short hairpin RNA (shRNA)-mediated strategy targeting the ASPN gene. Two specific shRNA sequences, designated as shASPN#1 (5'-GCTTACCACCAACTTTATTGG-3') and shASPN#2 (5'-GCTCTGCCAAACCCTTCTTTA-3'), were designed and cloned into lentiviral vectors according to established protocols. Lentiviral particles were generated and subsequently used to infect the human GC cell lines MKN45 and MKN28, which were obtained from the Chinese Academy of Sciences (Shanghai, China). The cells were cultured in Dulbecco’s Modified Eagle’s Medium (DMEM) supplemented with 10% fetal bovine serum (FBS; Hyclone, Logan, UT, USA) and maintained in a humidified incubator at 37 ℃ with 5% CO₂.

Western blot analysis

To evaluate protein expression levels, Western blot analysis was conducted. Tumor tissues and cultured cells were lysed using RIPA buffer, and the resulting protein extracts were separated by 10% sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE). The separated proteins were transferred onto polyvinylidene difluoride (PVDF) membranes (Millipore, Billerica, MA, USA) via electroblotting. Membranes were blocked with 5% non-fat milk and incubated overnight at 4 ℃ with primary antibodies specific to ASPN (Abcam, ab31303), KEAP1 (Abcam, ab205719), NRF2 (Abcam, ab62352), ubiquitin (Abcam, ab140601), and GAPDH (Abcam, ab8245) as a loading control. Protein bands were detected using horseradish peroxidase (HRP)-conjugated secondary antibodies and visualized with a chemiluminescence imaging system (Thermo Fisher Scientific, Waltham, MA, USA).

Immunofluorescence staining process

For detailed analysis of cellular localization, immunofluorescence staining was performed. Cells were fixed with 4% paraformaldehyde for 30 minutes at room temperature and rinsed multiple times with phosphate-buffered saline (PBS). Endogenous nonspecific binding was blocked with 1% bovine serum albumin (BSA) for 60 minutes at 37 ℃. Primary antibodies were applied and incubated overnight at 4 ℃. Following washing, cells were incubated with fluorophore-conjugated secondary antibodies for 120 minutes at room temperature in the dark. Nuclear counterstaining was performed using 4',6-diamidino-2-phenylindole (DAPI). Fluorescent signals were visualized and captured using a laser-scanning confocal microscope to assess the subcellular localization of the target proteins.

Colony formation and 5-ethynyl-2'-deoxyuridine (EdU) assays

The colony formation assay was employed to assess the long-term proliferative capacity of the cells. A total of 1,000 cells were seeded into each well of six-well plates and cultured for 14 days. Resulting colonies were fixed with methanol, stained with crystal violet, and manually counted under a light microscope. In parallel, the EdU incorporation assay was conducted to evaluate short-term cell proliferation. Cells were seeded at a density of 10,000 per well in 96-well plates in triplicate and incubated for 24 hours. EdU labeling and detection were carried out using the Cell-Light™ EdU Apollo567 In Situ Proliferation Kit (RiboBio) following the manufacturer’s protocol, and fluorescent images were captured using a fluorescence microscope.

Transwell migration and invasion assays

Cell migration and invasion were evaluated using 24-well Transwell inserts with 8-µm pore membranes. For invasion assays, the inserts were pre-coated with Matrigel (Corning, USA) to mimic the extracellular matrix. A total of 100,000 cells were suspended in serum-free DMEM and seeded into the upper chamber, while the lower chamber was filled with DMEM supplemented with 10% fetal bovine serum (FBS) as a chemoattractant. Following a 24-hour incubation period at 37 ℃ in a 5% CO₂ atmosphere, non-migratory and non-invasive cells remaining on the upper surface of the membrane were gently removed with a cotton swab. Migratory and invasive cells that had migrated or invaded through the membrane were fixed with 4% paraformaldehyde, stained with crystal violet, and quantified using a Leica inverted microscope. The experiments were performed in triplicate.

Co-immunoprecipitation (Co-IP) and ubiquitination assays

To investigate protein-protein interactions and ubiquitination events, Co-IP and ubiquitination assays were performed. In the Co-IP assay, cell lysates were incubated overnight at 4 ℃ with primary antibodies targeting ASPN, KEAP1, or an isotype control (IgG). Antibody-bound proteins were then captured using protein A/G agarose beads (Thermo Fisher Scientific, USA) following a two-hour incubation at room temperature. The immunoprecipitated complexes were subsequently analyzed by Western blotting. For the ubiquitination assay, cells transfected with shASPN or a non-targeting control shRNA (sh-NC) were treated with the protein synthesis inhibitor cycloheximide (CHX, 10 µM) for six hours. NRF2 was immunoprecipitated from cell lysates using anti-NRF2 antibodies, and its ubiquitinated forms were detected by immunoblotting with an anti-ubiquitin antibody after SDS-PAGE and membrane transfer.

Tumor xenograft experiment

To further validate the in vitro findings, an in vivo tumor xenograft model was established. Experiments were performed under a project license (No. P20260305-023) granted by the Institutional Animal Care and Use Committee of Nantong First People’s Hospital, in compliance with Nantong University national or institutional guidelines for the care and use of animals. Control shRNA group (n=5), shASPN group (n=5), or a combination of shASPN and an NRF2 overexpression vector (n=5) group was stably expressed in MKN45 and MKN28 cells. A total of 5×10⁶ cells suspended in a 1:1 mixture of Matrigel and phosphate-buffered saline (PBS) were injected subcutaneously into the flanks of 5-week-old BALB/c nude mice. Tumor growth was monitored daily, and tumor volumes were measured every two days using digital calipers, and the volume was calculated using the following formula: V = W² × L/2. At the end of the experimental period, the mice were humanely euthanized. Tumors were surgically excised, weighed, and processed for subsequent molecular and histopathological analyses.

Statistical analysis

All statistical analyses were conducted using SPSS software (Version 22.0, IBM, Armonk, NY, USA). Student’s t-test was applied for comparisons between two groups, whereas one-way analysis of variance (ANOVA) followed by Tukey’s post-hoc test was utilized for comparisons among multiple groups. A P value of less than 0.05 was considered statistically significant.

Results

ASPN expression in GC

To investigate the role of ASPN in GC, we initially assessed its mRNA expression levels in GC tissues using the GEPIA database, which integrates data from The Cancer Genome Atlas (TCGA) and the Genotype-Tissue Expression (GTEx) project. This platform provided a robust foundation for our analysis. Our results demonstrated significantly elevated ASPN mRNA expression in GC tissues compared to normal gastric tissues, as illustrated by comparative expression plots (Figure 1A,1B). This finding suggests that ASPN may play a critical role in GC pathogenesis. To further explore the clinical relevance of ASPN expression in GC, we conducted a Kaplan-Meier survival analysis. The analysis revealed a significant association between high ASPN expression and reduced overall survival in GC patients (Figure 1C, P=0.03). These results indicate that elevated ASPN levels may serve as a prognostic biomarker for adverse clinical outcomes in GC, highlighting the need for further investigation into its functional role and therapeutic potential. To validate these mRNA findings at the protein level, we performed Western blot analysis on 24 paired GC tissue samples and their corresponding adjacent non-tumorous tissues. The results consistently showed significantly higher ASPN protein expression in tumor tissues compared to normal counterparts (Figure 1D,1E, P<0.05). This protein-level validation further supports the hypothesis that ASPN overexpression is a characteristic feature of GC and may contribute to its malignant progression. Collectively, these preliminary findings suggest that ASPN exhibits oncogenic properties in GC, as evidenced by its elevated expression and association with poor patient prognosis. Further investigations are warranted to elucidate the precise molecular mechanisms by which ASPN influences GC progression and to evaluate its potential as a therapeutic target.

Figure 1 ASPN is overexpressed in GC and correlates with poor patient prognosis. (A) ASPN expression across various tumor tissues was analyzed using the TCGA database. (B) Comparison of ASPN expression in paired GC tissues and adjacent normal gastric tissues based on TCGA data. (C) Kaplan-Meier survival analysis was performed to evaluate overall survival in patients stratified by high versus low ASPN expression levels (P=0.025), revealing a significant association between elevated ASPN expression and reduced survival. (D) Western blot analysis was used to assess ASPN protein expression in tumor tissues (T) and matched adjacent normal tissues (N) from 12 GC patients. (E) The quantification of ASPN protein levels normalized to GAPDH is shown, and the statistical comparison between tumor (T) and adjacent normal (N) tissues was based on paired samples. *, P<0.05. ASPN, asporin; GC, gastric cancer; TCGA, The Cancer Genome Atlas; TPM, transcripts per million.

Biological impact of ASPN knockdown on GC cells

To further investigate the functional role of ASPN in GC, we designed two specific small hairpin RNA (shRNA) constructs, shASPN#1 and shASPN#2, to specifically target and suppress ASPN expression in the MKN45 and MKN28 GC cell lines, which are widely recognized as established in vitro models of GC. This experimental strategy enabled a precise assessment of the biological consequences of ASPN downregulation. Western blot analysis following shRNA transfection confirmed that both constructs effectively reduced ASPN protein expression levels (Figure 2A), thereby validating their suitability for subsequent functional analyses. Following successful ASPN knockdown, we performed a series of functional assays to evaluate its impact on key aspects of GC cell behavior. The EdU incorporation assay, which serves as an indicator of DNA synthesis and cell proliferation, revealed a significant reduction in the proliferation rate of ASPN-depleted cells compared to control cells (Figure 2B). Consistent with these findings, colony formation assays, which assess the capacity of single cells to form colonies in vitro, further confirmed the diminished proliferative potential upon ASPN silencing (Figure 2C). In addition to proliferation, we investigated the role of ASPN in cell migration and invasion—key determinants of tumor metastasis. Transwell assays demonstrated that ASPN depletion significantly impaired both the migratory and invasive capacities of GC cells through Matrigel-coated and uncoated membranes (Figure 3A,3B). Collectively, these results highlight ASPN as a key regulator of aggressive GC cell phenotypes, potentially by promoting cell proliferation and motility. The identification of ASPN as a functional oncogenic driver contributes to the understanding of the molecular mechanisms underlying GC progression and suggests its potential as a candidate target for therapeutic intervention.

Figure 2 Knockdown of ASPN inhibits GC cell proliferation. (A) MKN45 and MKN28 cells were transfected with two specific shRNA constructs targeting ASPN (shASPN#1 and shASPN#2) or a non-targeting negative control (sh-NC). ASPN protein expression levels were assessed by Western blot analysis. (B,C) Cell proliferation was evaluated using colony formation assay (B) and EdU incorporation assay (C) (scale bar =100 μm). Scale bar =100 µm. *, P<0.05. ASPN, asporin; EdU, 5-ethynyl-2'-deoxyuridine; GC, gastric cancer; NC, negative control.

Figure 3 Knockdown of ASPN inhibits GC cell migration and invasion. (A) Migration and (B) invasion assays were performed using Transwell chambers. MKN45 and MKN28 cells were transfected with shASPN#1 and shASPN#2 or a non-targeting negative control (sh-NC). Crystal violet staining. Scale bar =100 µm. *, P<0.05. ASPN, asporin; GC, gastric cancer.

ASPN influence on KEAP1/NRF2 signaling pathway

The KEAP1/NRF2 signaling pathway serves as a key regulator of cellular defense against oxidative stress and is frequently dysregulated in cancer, promoting tumor cell survival and therapeutic resistance (11). Given this context, we sought to explore the potential interaction between ASPN and the KEAP1/NRF2 pathway in GC. Following ASPN knockdown in MKN45 and MKN28 GC cell lines, a significant decrease in NRF2 protein expression was observed, whereas KEAP1 levels remained unchanged, as confirmed by Western blot analysis (Figure 4A). These results suggest that ASPN may influence the stability or turnover of NRF2 protein. To further investigate the underlying mechanism, we employed cycloheximide (CHX), a potent inhibitor of protein synthesis, to evaluate the half-life of NRF2 in ASPN-silenced cells. Our findings revealed a significantly accelerated degradation of NRF2 in ASPN-depleted cells compared to control cells (Figure 4B), indicating that ASPN may protect NRF2 from proteolytic degradation. Furthermore, we assessed NRF2 ubiquitination levels following ASPN silencing and observed a marked increase in ubiquitinated NRF2, suggesting enhanced targeting of NRF2 for proteasomal degradation (Figure 4C). Collectively, these findings support a model in which ASPN competes with NRF2 for binding to KEAP1. By interfering with KEAP1-mediated ubiquitination and subsequent degradation of NRF2, ASPN contributes to NRF2 stabilization and potentially enhances its transcriptional activity. This mechanism represents a novel mechanism by which ASPN promotes oncogenic phenotypes in GC through modulation of the KEAP1/NRF2 signaling pathway.

Figure 4 ASPN activates the KEAP1/NRF2 signaling pathway in GC cells. (A) Protein expression levels of NRF2 and KEAP1 were analyzed in MKN45 and MKN28 cells transfected with shASPN#1 and shASPN#2 or a non-targeting negative control (sh-NC). (B) MKN45 cells were transfected with sh-NC, shASPN#1, or shASPN#2. Following transfection, cells were treated with CHX for 0, 2, 4, and 8 hours. Western blot analysis was conducted to assess the expression levels of NRF2 and ASPN. (C) An immunoprecipitation assay was carried out to evaluate the ubiquitination status of NRF2 in MKN45 and MKN28 cells transfected with shASPN#2 and treated with CHX. ASPN, asporin; CHX, cycloheximide; GC, gastric cancer.

Mechanistic role of ASPN in KEAP1/NRF2 interaction

Given the central role of KEAP1 in mediating NRF2 degradation, we hypothesized that ASPN modulates NRF2 stability through its interaction with KEAP1. To validate this hypothesis, we silenced KEAP1 expression in both MKN45 and MKN28 cell lines and subsequently knocked down ASPN. Western blot analysis demonstrated that in the absence of KEAP1, ASPN silencing had no significant effect on NRF2 protein levels (Figure 5A). These results strongly suggest that ASPN-mediated regulation of NRF2 is dependent on the presence of KEAP1. To examine the subcellular localization of ASPN and KEAP1, we performed immunofluorescence staining in both cell lines. The results revealed that both proteins predominantly localize to the cytoplasm and exhibit substantial colocalization (Figure 5B), supporting the possibility of a direct physical interaction between ASPN and KEAP1. To further confirm this interaction, Co-IP experiments were carried out. Immunoprecipitation of ASPN led to the successful co-precipitation of KEAP1 from cell lysates of both MKN45 and MKN28 cells (Figure 5C), providing direct evidence of an endogenous interaction between ASPN and KEAP1. Building upon these findings, we investigated the impact of ASPN knockdown on the interaction between KEAP1 and NRF2. Co-IP assays revealed that ASPN silencing enhanced the binding affinity between KEAP1 and NRF2 (Figure 5D), indicating that in the absence of ASPN, KEAP1 more effectively associates with NRF2, thereby promoting its ubiquitination and subsequent degradation. Collectively, these findings provide compelling evidence for a novel regulatory mechanism in which ASPN competes with NRF2 for binding to KEAP1. By sequestering KEAP1, ASPN prevents its interaction with NRF2, thereby stabilizing NRF2 and potentially enhancing its transcriptional activity. This stabilization may confer survival advantages to GC cells and contribute to oncogenic progression. This newly identified mechanism not only expands our understanding of ASPN’s functional role in cancer but also highlights potential therapeutic strategies targeting the KEAP1/NRF2 signaling axis in GC.

Figure 5 ASPN competes with NRF2 for binding to KEAP1 in GC cells. (A) Protein expression levels of ASPN, KEAP1, and NRF2 were analyzed by Western blot in MKN45 and MKN28 cells expressing the indicated shRNAs. (B) Immunofluorescence staining was performed to visualize the localization of ASPN, KEAP1, and DAPI in MKN45 and MKN28 cells. (C) Cell lysates were subjected to immunoprecipitation using an anti-KEAP1 antibody or an isotype-matched IgG control. Endogenous ASPN binding was detected in MKN45 and MKN28 cells by immunoprecipitation assay. (D) An immunoprecipitation assay was conducted to evaluate the binding affinity of NRF2 or ASPN to KEAP1 in MKN45 and MKN28 cells transfected with shASPN#2 and treated with CHX. Scale bar =50 µm. ASPN, asporin; CHX, cycloheximide; DAPI, 4',6-diamidino-2-phenylindole; GC, gastric cancer.

NRF2 overexpression counteracts the effects of ASPN knockdown

To further elucidate the functional relationship between ASPN and NRF2 in GC, we performed a series of functional assays in MKN45 and MKN28 cell lines. Cells were transfected with either a non-targeting control shRNA (sh-NC), an shRNA targeting ASPN (sh-ASPN#2), or a combination of sh-ASPN#2 and an NRF2 overexpression vector. Western blot analysis confirmed the effective knockdown of ASPN and overexpression of NRF2 (Figure 6A). Subsequently, cell proliferation was assessed using colony formation and EdU DNA incorporation assays. The results showed that ASPN depletion significantly inhibited cell proliferation in both cell lines (Figure 6B,6C). However, co-expression of NRF2 partially restored the proliferative capacity, suggesting that NRF2 can partially compensate for the growth-suppressive effects of ASPN knockdown. In addition, we evaluated the invasive potential of the transfected cells using Transwell invasion assays. Consistent with our observations in proliferation assays, ASPN knockdown led to a significant reduction in cell invasiveness. However, this inhibitory effect was partially reversed by NRF2 overexpression (Figure 7), indicating that NRF2 may promote invasion and counteract the effects of ASPN depletion. To validate these in vitro findings, in vivo tumor xenograft experiments were conducted. The results demonstrated that ASPN knockdown significantly suppressed tumor growth, as reflected by reduced tumor volumes and weights (Figure 8A-8C). Notably, concurrent overexpression of NRF2 partially rescued tumor growth, further supporting the notion that NRF2 can antagonize the tumor-suppressive effects of ASPN depletion in vivo. Collectively, these findings reveal a compensatory mechanism in which NRF2 partially offsets the inhibitory effects of ASPN knockdown on both cell proliferation and invasion in GC. This functional interplay between ASPN and NRF2 may have significant implications for the development of targeted therapeutic strategies in GC.

Figure 6 NRF2 overexpression abrogates the inhibitory effects of ASPN knockdown on GC cell proliferation. MKN45 and MKN28 cells were transfected with sh-NC, sh-ASPN#2, or sh-ASPN#2 combined with an NRF2 overexpression vector. (A) Protein expression levels of ASPN, NRF2, and GAPDH were assessed by Western blot analysis. (B,C) Cell proliferation was evaluated using colony formation assay (B) and EdU incorporation assay (C). *, P<0.05. ASPN, asporin; DAPI, 4',6-diamidino-2-phenylindole; EdU, 5-ethynyl-2'-deoxyuridine; GC, gastric cancer.

Figure 7 NRF2 overexpression abrogates the inhibitory effect of ASPN knockdown on GC cell invasion. MKN45 and MKN28 cells were transfected with sh-NC, sh-ASPN#2, or sh-ASPN#2 in combination with an NRF2 overexpression vector. Cell invasion was evaluated using Transwell assays. Crystal violet staining. Scale bar: 100 µm. *, P<0.05. ASPN, asporin; GC, gastric cancer; NC, negative control.

Figure 8 ASPN knockdown suppresses xenograft tumor formation in vivo. Representative images of xenograft tumors (A), tumor volume measurements (B), and tumor weight data (C) are presented for the indicated experimental groups. *, P<0.05. ASPN, asporin; NC, negative control.

Discussion

In this study, we systematically investigated the molecular mechanisms and biological significance of ASPN in GC. Significant overexpression of ASPN was observed in GC tissues, a finding consistent with reports in other malignancies such as pancreatic, colorectal, prostate, bladder, and certain subtypes of breast cancer, suggesting its involvement in oncogenic processes (12-17). However, the functional role of ASPN varies across different cancer types (18).

We examined the contribution of cancer-associated fibroblasts (CAFs) to ASPN secretion. CAFs are known to support cancer cell invasion and metastasis, and our findings indicate that ASPN secreted by CAFs further enhances these malignant properties (19,20). While the overexpression of ASPN in cancer cells and its function as an oncoprotein have been well documented, the underlying molecular mechanisms remain incompletely understood (21). Previous studies have demonstrated that ASPN can modulate key signaling pathways, including TGF-β, EGFR, and CD44. In lung adenocarcinoma and esophageal squamous cell carcinoma, somatic alterations in key genes driving the Nrf2 pathway are frequently observed, mainly including inactivating mutations in the KEAP1 gene or activating mutations in the NRF2 gene itself. These mutations disrupt the normal regulation of Nrf2, leading to its continuous accumulation in the cell nucleus (22). Our results reveal a novel interaction between ASPN and the KEAP1/NRF2 signaling pathway, which plays a critical role in cellular survival under oxidative stress conditions (23). The KEAP1/NRF2 pathway is frequently exploited by cancer cells to enhance survival and develop resistance to therapeutic interventions (24). Under homeostatic conditions, NRF2 is primarily regulated at the post-translational level by KEAP1, which functions as an adaptor molecule to anchor Nrf2 in the cytoplasm and mediate its ubiquitination and degradation, thereby maintaining Nrf2 at a low activity level (25,26). Under oxidative stress, KEAP1 undergoes cysteine thiol modifications, leading to NRF2 stabilization and nuclear translocation (27,28). We demonstrated that ASPN disrupts the interaction between KEAP1 and NRF2, thereby inhibiting NRF2 ubiquitination and proteasomal degradation. Functional rescue experiments revealed that NRF2 overexpression partially reversed the inhibitory effects of ASPN knockdown on cell proliferation and invasion, confirming a significant functional interplay between these two proteins. Collectively, our findings indicate that ASPN is consistently overexpressed in GC tissues and plays a pivotal role in tumor progression through its modulation of the KEAP1/NRF2 pathway. Specifically, ASPN competes with NRF2 for binding to KEAP1, resulting in enhanced NRF2 stabilization and accumulation. This interaction promotes the aggressive behavior of GC cells in both in vitro and in vivo models, highlighting ASPN as a potential therapeutic target in GC. Helicobacter pylori and other environmental risk factors can activate the Nrf2 pathway by inducing chronic inflammation and oxidative stress (29). Meanwhile, ASPN has been confirmed to stabilize and enhance the activity of Nrf2. Additionally, CAFs are an important source of ASPN secretion, and risk factors such as inflammation and aging can promote the activation of CAFs (15). This indicates that risk factors may increase the secretion of ASPN by activating CAFs, thereby reshaping the ASPN-rich tumor microenvironment and further strengthening the Nrf2-dependent pro-cancer mechanism, ultimately promoting the progression of GC. Taken together, ASPN is likely to serve as a functional hub that closely links key environmental risk factors with the Nrf2-dependent pro-cancer mechanism.

Conclusions

In summary, our study demonstrates that ASPN is significantly upregulated in GC tissues and promotes tumor progression through its interaction with the KEAP1-NRF2 signaling pathway. Specifically, ASPN competes with NRF2 for binding to KEAP1, thereby preventing KEAP1-mediated ubiquitination and degradation of NRF2. This leads to NRF2 stabilization, which contributes to the development and aggressive behavior of GC in both in vitro and in vivo models. This interaction reveals a key regulatory mechanism in cancer biology, wherein ASPN modulates the KEAP1-NRF2 pathway to support tumor cell survival and progression. By enhancing NRF2 stability, ASPN promotes cancer cell proliferation and resistance to stress conditions. Targeting ASPN therefore represents a promising therapeutic strategy to counteract the cytoprotective effects of NRF2 in cancer cells and enhance their susceptibility to treatment. These findings highlight the potential of ASPN as a novel therapeutic target for improving clinical outcomes in GC.

Acknowledgments

None.

Footnote

Reporting Checklist: The authors have completed the ARRIVE and MDAR reporting checklists. Available at https://tcr.amegroups.com/article/view/10.21037/tcr-2025-aw-2442/rc

Data Sharing Statement: Available at https://tcr.amegroups.com/article/view/10.21037/tcr-2025-aw-2442/dss

Peer Review File: Available at https://tcr.amegroups.com/article/view/10.21037/tcr-2025-aw-2442/prf

Funding: This research was supported by a grant from the Yancheng City Science and Technology Project (No. YK2024030 to J.Y.).

Conflicts of Interest: All authors have completed the ICMJE uniform disclosure form (available at https://tcr.amegroups.com/article/view/10.21037/tcr-2025-aw-2442/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. The study was conducted in accordance with the Declaration of Helsinki and its subsequent amendments. The study was approved by the Ethics Committee of Nantong First People’s Hospital (No. 2023KT039) and informed consent was taken from all the patients. All animal experiments were performed under a project license (No. P20260305-023) granted by the Institutional Animal Care and Use Committee of Nantong First People’s Hospital, in compliance with Nantong University national or institutional guidelines for the care and use of animals.

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/.

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