ASCT2 suppresses proliferation and epithelial-mesenchymal transition of renal cell carcinoma cells via NLRP3 inflammasome-induced pyroptosis

Introduction

Renal cell carcinoma (RCC) is a frequently diagnosed urological cancer. Its morbidity and mortality rate are on the rise. Based on the worldwide oncology data from 2020, China reported 555,000 newly diagnosed RCC cases, ranking third of all malignant tumors (1). The number of male and female patients was 319,000 and 236,000, respectively, with higher proportion of males than that of females (2). The mortality rate was 12.0/100,000, ranking fifth (3). The number of deaths from RCC was 165,000 for males and 121,000 for females, and the mortality rate was 14.8/100,000 and 9.4/100,000, respectively (4).

Although surgery is a prevalent therapeutic approach for localized RCC, 20–30% of patients still experience disease recurrence postoperatively. Currently, advancements in immunotherapy, radiotherapy, and molecularly targeted agents have transformed treatment paradigms, and advances in treatment have enhanced the clinical outcomes of advanced RCC, achieving a 75% 5-year survival probability among diagnosed individuals (5). Nevertheless, despite pharmacological interventions, the long-term survival of RCC patients remains suboptimal due to local recurrence and metastatic progression (6). Consequently, identifying prognostic factors is essential for risk classification, therapeutic decision-making, and improving long-term outcomes (7). The common prognostic indicators include tumor, nodes, metastasis (TNM) staging, tumor grade, and histopathological classification (8). Nevertheless, these conventional prognostic markers exhibit certain limitations, including suboptimal predictive accuracy, elevated testing costs, technical complexity in detection, and prolonged result reporting. Therefore, it is necessary to identify additional therapeutic targets and biomarkers. The identification of molecular targets such as alanine serine cysteine transporter 2 (ASCT2) and potential prognostic biomarkers should provide new avenues for treatment to improve responses in RCC. Although considerable progress has been made regarding the treatment of RCC, there is still considerable room for improvement in addressing the issues of recurrence and metastatic disease. In this study, we propose that ASCT2 is a central mediator in the pathogenesis of RCC through its regulation of the NOD-like receptor thermal protein domain associated protein 3 (NLRP3) inflammasome, presenting a new potential therapeutic target.

Pyroptosis represents a programmed cellular demise pathway that is discovered in recent years, which mainly occurs in macrophages (9,10). Its morphological characteristics, occurrence and regulation mechanism are different from necrosis, apoptosis and other types of cell death modes, and it is an important innate immunity of the body (11). In recent years, research investigations have demonstrated that cell proptosis exhibits a strong correlation with cancer (12). NLRP3 inflammasome is one of the most intensively studied inflammasomes, which mainly mediates cell charring and inflammatory reaction through activating caspase-1 (13,14).

ASCT2 is a Na⁺ dependent glutamine transporter and belongs to the ASC family of neutral amino acid transporters system (15). Many studies have shown that the expression of ASCT2 in a variety of tumor, such as breast cancer, cervical cancer, liver cancer, lung cancer, melanoma, RCC, as well as prostate carcinoma, has increased significantly (16,17). Knockdown of ASCT2 markedly suppressed tumor cell proliferation, suggesting that ASCT2 facilitates excessive glutamine uptake, providing an optimal microenvironment for tumor cell survival (18). Therefore, the lead compounds screened with ASCT2 as the target are likely to become promising new targeted therapeutic drugs (19). This study elucidates ASCT2’s function in the pathogenesis of RCC and its underlying molecular mechanisms. We present this article in accordance with the MDAR reporting checklist (available at https://tau.amegroups.com/article/view/10.21037/tau-2025-431/rc).

Methods

Clinical research model

Serum specimens of individuals diagnosed with RCC were collected from the Gaoxin Branch of the First Affiliated Hospital of Nanchang University and preserved at −80 ℃. Inclusion criteria: (I) patients with RCC confirmed by histopathology; (II) patients have complete clinicopathological data and follow-up data were available; (III) patients have not received preoperative radiotherapy, chemotherapy or targeted therapy; (IV) patients aged between 18 and 80 years; except RCC, there were no history of other malignant tumors for the patients in the past 5 years. The study was conducted in accordance with the Declaration of Helsinki and its subsequent amendments. The study was approved by the Ethics Committee at Gaoxin Branch of the First Affiliated Hospital of Nanchang University (No. 2024-7). All enrolled cancer patients signed voluntary participant agreement documents prior to study enrollment.

Cell culture

Human RCC cell lines (such as 769-P, 786-O, ACHN and Caki-1) and human embryonic kidney cell line AAV-293 were routinely cultured in RPMI-1640 medium containing 10% fetal bovine serum (FBS) and 1% penicillin streptomycin at 37 ℃ and 5% CO₂. The experiment was divided into the following groups: (I) blank control group: transfection of empty vector (vector) or negative control siRNA (si-NC); (II) ASCT2 overexpression group (ASCT2): transfected with ASCT2 overexpression plasmid; (III) ASCT2 knockdown group (ASCT2 siRNA, si-ASCT2): transfected with ASCT2 siRNA; (IV) ASCT2-OE + NLRP3 inhibitor group (ASCT2 + NLRP3i): ASCT2 overexpression was treated with NLRP3 inhibitor; (V) ASCT2 knockdown + NLRP3 agonist group (si-ASCT2 + NLRP3), the NLRP3 agonist was added on the basis of ASCT2 knockdown. Lipofectamine 3000 reagent was used for all transfections, and subsequent detection was performed 48 hours after transfection.

Cell Counting Kit-8 (CCK-8)

Cells in logarithmic growth phase were inoculated in 96-well plates with about 5×10³ cells per well, cultured overnight and adhered to the wall, and then transfected or treated with drugs as required. After treatment, 10 µL of CCK-8 reagent was added to each well and incubated in a 37 ℃ incubator for 2 hours without light. Finally, the absorbance value of each well was detected at the wavelength of 450 nm using a microplate reader to evaluate the cell proliferation activity.

5-ethynyl-2'-deoxyuridine (EdU)

Cells in logarithmic growth phase were inoculated into 96-well plates and incubated with EdU reagent with a final concentration of 10 µM for 2 hours after corresponding treatment; discard the culture medium, use 4% paraformaldehyde to fix for 15 minutes after phosphate-buffered saline (PBS) cleaning, add 0.5% Triton X-100 to penetrate the membrane for 10 minutes, then add click reaction solution to incubate in dark for 30 minutes according to the instructions of the kit, and finally add Hoechst 33342 to stain the nucleus for 10 minutes. Observe and count EdU-positive cells under the fluorescence microscope.

Transwell

Cells were resuspended in serum-free medium, 200 µL cell suspension (about 5×10⁴ cells) was added to Transwell upper chamber, 500 µL medium containing 10% FBS was added to the lower chamber, and incubated in 37 ℃ incubator for 24 hours. Take out the chamber, discard the liquid in the upper chamber, use 4% paraformaldehyde to fix for 30 minutes after PBS cleaning, dye with 0.1% crystal violet for 20 minutes, gently wipe off the non-migrated cells in the upper chamber with a cotton swab, and randomly select the visual field under the microscope to count the number of migrated cells on the back of the ventricular membrane.

JC-1

Cells in logarithmic growth phase were inoculated into 6-well plates. After corresponding treatment, the culture medium was discarded, the cells were washed with PBS, 1 mL JC-1 working solution was added to each well, and incubated at 37 ℃ in dark for 20 minutes. Discard the JC-1 dye solution, wash the cells with JC-1 buffer twice, and add the cells to culture for observation under the fluorescence microscope.

Calcein-AM/CoCl₂

Cells in the logarithmic growth phase were inoculated into 96-well blackboards. After corresponding treatment, PBS working solution containing 2 µM calcein-AM and 1 mL CoCl₂ was added to each well and incubated at 37 ℃ in the dark for 30 minutes. The dye solution was discarded and the cells were washed with PBS for three times. The green fluorescence intensity was detected immediately with a fluorescent microplate reader (excitation wavelength 490 nm, emission wavelength 515 nm).

Lactate dehydrogenase (LDH) release

Cells in logarithmic growth phase were inoculated into 96-well plates. After corresponding treatment, 100 µL of supernatant from each well was absorbed and transferred to the new 96-well plates. The substrate reaction solution was added according to the instructions of LDH detection kit, and the reaction was kept away from light for 30 minutes. Finally, the absorbance value was detected at 490 nm wavelength using an enzyme reader.

Enzyme-linked immunosorbent assay (ELISA) for IL-1α

Cell culture supernatants after different treatments were collected. After the impurities were removed by centrifugation at 4 ℃, the operation was carried out according to the instructions of the commercial human IL-1α ELISA Kit: add the standard and sample into the enzyme labeled plate coated with antibody, wash the plate after incubation at room temperature for 2 hours, add biotinylated detection antibody for incubation for 1 hour, wash the plate again, and then add streptomycin and biotin-horseradish peroxidase (HRP) conjugate for incubation for 30 minutes. After thorough washing, add 3,3',5,5'-tetramethylbenzidine (TMB) substrate for color development, and finally add termination solution to terminate the reaction. Immediately use the enzyme labeled instrument to measure the absorbance value of each hole at 450 nm wavelength, and calculate the concentration of IL-1α in the sample according to the standard curve.

Sequencing

Based on RNA-sequencing (RNA-seq) technology, the transcriptome sequencing of RCC cells transfected with ASCT2 overexpression vector or empty vector was analyzed. The specific steps are as follows: firstly, the total RNA of cells was extracted, and after passing the quality inspection, eukaryotic mRNA was enriched with magnetic beads with oligo(dT). Then, the mRNA was broken into short fragments in the fragmentation buffer, and the broken mRNA was used as a template to synthesize the first complementary DNA (cDNA) strand with six-base random primers, and then the second cDNA strand was synthesized. After purification, terminal repair, adding a tail and connecting the sequencing connector, the fragment size was selected and polymerase chain reaction (PCR) amplification was carried out to construct the final cDNA library. After the library passed the quality inspection, double terminal (pe150) sequencing was performed on the Illumina novaseq 6000 platform. The original data were analyzed for quality control, comparison, gene quantification and differential expression to screen the downstream target genes regulated by ASCT2.

Real-time PCR

Total RNA extraction was performed employing the TRIzol reagent (Takara, Dalian, China), followed by using PrimeScript RT Kit for reverse transcription (Takara). Gene expression quantification was performed on the Applied Biosystems 7500 Fast platform with Takara’s PrimeScript kit per standardized protocols, executed employing the Applied Biosystems 7500 Fast qPCR SystemTakara PrimeScript RT reagent Kit, following standardized procedures.

Immunofluorescence

The cell samples were immobilized using 4% paraformaldehyde solution. The samples underwent membrane permeabilization using 0.5% X-100 in PBS containing 0.1% 15-minute treatment with Triton X-100 minutes at ambient thermal conditions, then incubated with 5% bovine serum albumin (BSA) for a blocking solution (30 min, 37 ℃). The cell samples underwent 16-hour cold-room exposure to primary antibodies at 4 ℃: The samples were subjected to detection antibody incubation using Cy3-labelled goat anti-rabbit or DyLight 488-conjugated caprine anti-murine IgG (2 h, 37 ℃). Nuclear staining was performed using DAPI and visualized using an Olympus IX71 fluorescence microscope (Tokyo, Japan).

Western blot

Protein separation and detection were conducted via immunoblotting following established protocols (20). Blots underwent antibody incubation with ASCT2, NLRP3, GSDMD and 4 ℃ overnight incubation with β-actin primary antibody incubation preceding HRP-conjugated secondary antibody exposure. Following a 15-minute Tris Buffered Saline with Tween-20 (TBST) wash, HRP-labelled species-specific enzyme-linked secondary immunoglobulins (sc-2004/sc-2005, 1:5,000 dilution, Santa Cruz Biotechnology, Dallas, TX, USA, USA) were applied for 1 hour within a 37 ℃ environment. Protein luminescent signals were captured using enhanced chemiluminescence (ECL) technology (ECL, Beyotime, Shanghai, China) with band densitometry analyzed via Image Lab v3.0 Bio-Rad software (Hercules, CA, USA).

Statistical analysis

Data are presented as mean ± standard deviation (SD). Student’s t-test has been applied for comparison between two groups, and one-way analysis of variance (ANOVA) has been applied for comparison among multiple groups. Kaplan-Meier method was used to analyse the survival of patients, and log-rank test was used to compare the differences of survival curves between groups. P<0.05 has been considered significant. Sample sizes were determined based on prior studies, power analysis with α=0.05 and power =0.80. Every experiment has been repeated for at least 3 times.

Results

ASCT2 levels in patients with advanced RCC

First, we assessed the levels of ASCT2 expression in patients with RCC by PCR and western blot (WB) methods. We found that ASCT2 was decreased on mRNA (Figure 1A) and protein (Figure 1B) levels in RCC tissue compared with the normal adjacent tissue. Furthermore, in RCC samples with high expression of ASCT2, better overall and disease-free survival (DFS) have been observed (Figure 1B,1C). Finally, ASCT2 mRNA expression in RCC lines was lower than that of human embryonic kidney cells (AAV-293) (Figure 1D), especially Caki-1 cells, so Caki-1 cells were used for the following analysis.

Figure 1 ASCT2 was down-regulated in RCC. Comparison of ASCT2 mRNA (A) and protein (B) expressions in RCC tissue and the adjacent normal tissue in patients with RCC (A,B). (C) OS and DFS for RCC patients with high and low ASCT2 expression. (D) ASCT2 mRNA expression in primary embryonic human kidney cells (AAV-293 cell) and different RCC cell lines. **, P<0.01 compared with normal group or AAV-293 cells. ASCT2, alanine serine cysteine transporter 2; DFS, disease-free survival; OS, overall survival; RCC, renal cell carcinoma.

The ASCT2 gene suppressed the proliferation of RCC cells

Next, ASCT2 gene was over-expressed in the in vitro cell culture system through the ASCT2 over-expression vector (Figure 2A). Furthermore, results of CCK-8 (Figure 2B), Transwell (Figure 2C) and EdU (Figure 2D) assays suggested that ASCT2 over-expression decreased proliferation and motility rate, and inhibited migration of RCC cells. Then, the ASCT2 gene was down-regulated in the cell-based experimental system through the si-ASCT2. si-ASCT2-3 has shown the best inhibitory effects, thus it was used for the following experiments (Figure 2E). We found that ASCT2 gene inhibition increased cell proliferation and motility, meanwhile increased migration of RCC cells (Figure 2F-2H).

Figure 2 The ASCT2 gene suppressed the proliferation and migration of RCC cells. ASCT2 over-expression vector increased ASCT2 mRNA expression (A), cell growth (B), migration rate (crystal violet staining; magnification, 200×) (C), and number of EdU-positive cells (immunofluorescence; magnification, 200×) (D) in RCC cells; ASCT2 siRNA decreased ASCT2 mRNA expression (E), increased cell growth (F), migration rate (crystal violet staining; magnification, 200×) (G), and number of EdU-positive cells (immunofluorescence; magnification, 200×) (H) in RCC cells. **, P<0.01 compared with vector or si-NC group. ASCT2, alanine serine cysteine transporter 2; EdU, 5-ethynyl-2'-deoxyuridine; NC, negative control; OD, optical density; RCC, renal cell carcinoma; siRNA, small interfering RNA.

ASCT2 gene increased pyroptosis of RCC cells

Then, we examined whether ASCT2 could affect pyroptosis of RCC cells. Following ASCT2 up-regulation, JC-1 fluorescence was diminished, along with the calcein-AM/CoCl₂ fluorescence intensities, and RCC cell viability; on the other hand, LDH enzymatic activity, propidium iodide (PI) concentrations, and IL-1α level in RCC cells were increased (Figure3A-3F), these results suggested ASCT2 over-expression increased pyroptosis of RCC cells. Furthermore, ASCT2 gene inhibition increased JC-1 monomer formation and calcein-AM/CoCl₂ fluorescence intensity, enhanced RCC cell proliferation, while reducing LDH release, PI uptake, and IL-1α level in RCC cells (Figure 3A-3F). These results suggested ASCT2 inhibition decreased pyroptosis of RCC cells. Moreover, ASCT2 over-expression promoted GSDMD expression, while si-ASCT2 down-regulated GSDMD protein levels in RCC cells (Figure 3G,3H).

Figure 3 ASCT2 increased pyroptosis of RCC cells. JC-1 disaggregation (A), calcien-AM/CoCl₂ levels (B), cell viability (C), LDH release (D), PI levels (E), IL-1α level (F) and GSDMD protein expressions (G,H) in RCC cells with different treatments. **, P<0.01 compared with vector or si-NC group. ASCT2, alanine serine cysteine transporter 2; LDH, lactate dehydrogenase; NC, negative control; PI, propidium iodide; RCC, renal cell carcinoma.

NLRP3 was a target of ASCT2 in RCC

Next, we explored the underlying mechanism of ASCT2 in the epithelial cells of RCC via microarray profiling. RNA-seq was performed on RCC cells transfected with either ASCT2 over-expression vector or the empty vector. NLRP3 is one of the most significantly up-regulated genes (Figure 4A). Moreover, RCC cells were transfected with ASCT2 over-expression vector or si-ASCT2, and the expression of NLRP3 was examined. The results suggested that ASCT2 up-regulation increased NLRP3 mRNA expression, and ASCT2 gene inhibition suppressed NLRP3 mRNA expression in RCC cells (Figure 4B). Furthermore, confocal laser scanning microscopy results revealed that ASCT2 overexpression elevated ASCT2 and NLRP3 expression in RCC cells (Figure 5A). Moreover, results of WB analysis suggested that ASCT2 up-regulation increased ASCT2 and NLRP3 protein expressions in RCC cells (Figure 5B). On the other hand, ASCT2 inhibition suppressed ASCT2 and NLRP3 protein expressions in RCC cells (Figure 5C).

Figure 4 NLRP3 was a target of ASCT2 in RCC. Results of RNA-seq (A) and NLRP3 mRNA expression in RCC cells with different treatments (B). **, P<0.01 compared with the WT group. ASCT2, alanine serine cysteine transporter 2; NC, negative control; NLRP3, NOD-like receptor thermal protein domain associated protein 3; RCC, renal cell carcinoma; RNA-seq, RNA-sequencing; ROS, reactive oxygen species; WT, wild type.

Figure 5 ASCT2-induced NLRP3 expression in RCC cells. Results of ASCT2/NLRP3 staining by scanning confocal microscopy (immunofluorescence; magnification, 200×) (A), ASCT2/NLRP3 protein expression in RCC cells by ASCT2 up-regulation (B) or down-regulation (C). **, P<0.01 compared with vector or si-NC group. ASCT2, alanine serine cysteine transporter 2; NC, negative control; NLRP3, NOD-like receptor thermal protein domain associated protein 3; RCC, renal cell carcinoma.

ASCT2 regulates the progression of RCC via regulating NLRP3

Lastly, whether ASCT2 regulated the progression of RCC via regulating NLRP3 were examined. As shown in Figure 6A,6B, NLRP3 inhibitor counteracted the impact of ASCT2 on NLRP3 inflammasome/GSDMD pore-forming protein complex expressions and increased viability of RCC cells (Figure 6A,6B). Moreover, NLRP3 inhibitor also counteracted the impact of ASCT2 on migration and proliferation of RCC cells (Figure 6C,6D). On the other hand, NLRP3 agonist counteracted the influences of si-ASCT2, increased the NLRP3 inflammasome/GSDMD pore-forming complex expressions and decreased viability of RCC cells (Figure 6E,6F). NLRP3 agonist also attenuated the impacts of si-ASCT2 on migratory capacity and proliferation of RCC cells (Figure 6G,6H).

Figure 6 Up-regulation of NLRP3 abrogates the effects of ASCT2 in RCC cells. NLRP3/GSDMD protein expressions (A), cell viability (B), migration rate (crystal violet staining; magnification, 200×) (C), and EdU-positive cells (magnification, 200×) (D) in RCC cells by ASCT2 up-regulation or ASCT2 + NLRP3i; NLRP3/GSDMD protein expressions (E), cell viability (F), migration rate (crystal violet staining; magnification, 200×) (G), and EdU-positive cells (magnification, 200×) (H) in vitro model by ASCT2 knockdown or si-ASCT2 + NLRP3. **, P<0.01 compared with Vector or si-NC group; ^##, P<0.01 compared with ASCT2 or si-ASCT2 group. ASCT2, alanine serine cysteine transporter 2; EdU, 5-ethynyl-2'-deoxyuridine; NC, negative control; NLRP3, NOD-like receptor thermal protein domain associated protein 3; NLRP3i, NLRP3 inhibitor; OD, optical density; RCC, renal cell carcinoma.

Discussion

The purpose of this study was to investigate the expression characteristics and functional role of ASCT2 in RCC and its molecular mechanism of regulating pyroptosis through NLRP3 inflammasome. Our results systematically reveal the potential role of ASCT2 as a tumor suppressor gene in RCC for the first time, which affect the proliferation, migration and apoptosis of tumor cells by regulating the NLRP3/GSDMD pathway.

First, we found in clinical tissue samples that the mRNA and protein expression levels of ASCT2 in RCC tissues were significantly lower than those in adjacent normal tissues, suggesting that ASCT2 may have tumor suppressive effect in RCC. Further survival analysis showed that the overall survival (OS) and DFS of patients with high expression of ASCT2 were significantly better than those with low expression, indicating that the expression level of ASCT2 was closely related to the prognosis of RCC patients. This finding is different from previous reports of ASCT2 as an oncogene in breast cancer, prostate cancer and other tumors (21,22), suggesting that ASCT2 may have tissue-specific functions in different tumor types. We also verified the low expression of ASCT2 in a variety of RCC cell lines, which provided a basis for further functional experiments.

In the functional experiment, overexpression of ASCT2 significantly inhibited the proliferation and migration of RCC cells, while knockdown of ASCT2 showed the opposite effect. These results indicate that ASCT2 can inhibit the malignant phenotype of RCC cells in an in vitro model, further supporting its role as a tumor suppressor gene. It is worth noting that the effect of ASCT2 on cell behavior may be related to its regulation of amino acid metabolism, especially glutamine transport (23,24). However, this study does not directly involve the mechanism at the metabolic level, and the metabolic reprogramming effect of ASCT2 in RCC can be further explored in the future.

We further explored whether ASCT2 affects RCC progression by regulating cell pyroptosis. The results showed that overexpression of ASCT2 could significantly induce changes in pyroptosis related indicators, including JC-1 polymer dissociation, calcein-AM/CoCl2 fluorescence intensity decrease, LDH release increase, PI uptake increase and IL-1α level increase. At the same time, GSDMD protein expression was also up-regulated with ASCT2 overexpression. GSDMD is an executive protein of pyroptosis, and its activation is a key marker of pyroptosis (25). These results clearly indicate that ASCT2 can promote the pyroptosis of RCC cells. On the contrary, knockdown of ASCT2 inhibited the cell pyroptosis, which further confirmed the positive role of ASCT2 in the regulation of RCC cell pyroptosis.

In order to clarify the upstream mechanism of ASCT2 regulating pyroptosis, we found that NLRP3 was one of the most significantly up-regulated genes after ASCT2 overexpression through RNA-seq screening. Subsequently, the positive correlation between ASCT2 and NLRP3 at mRNA and protein levels was verified by qPCR and Western blot. NLRP3 inflammasome is a known core component of pyroptosis, and its activation can promote caspase-1-mediated GSDMD cleavage and pyroptosis (26,27). Our results are the first to link ASCT2 with NLRP3 inflammasomes, suggesting that ASCT2 may promote pyroptosis by positively regulating NLRP3 expression.

In order to further verify whether ASCT2 mediates its biological effects through NLRP3, we conducted the rescue experiments. The results showed that NLRP3 inhibitor could reverse the inhibitory effect of ASCT2 overexpression on NLRP3/GSDMD protein expression, cell viability, migration and proliferation; NLRP3 agonist can reverse the promotion effect of si-ASCT2 on the above indicators. These results suggest that ASCT2 can inhibit the proliferation and migration of RCC cells by regulating the expression of NLRP3 and then affecting the GSDMD mediated pyroptosis process. This mechanism is consistent with the results reported by Tan et al. (28) that NLRP3 inhibits tumor progression through pyroptosis in RCC, and also emphasizes the complex role of inflammatory bodies in the tumor microenvironment.

There are some limitations in this study. First of all, although we indirectly proved the functional connection of ASCT2-NLRP3 axis through recovery experiment, we still lacked evidence of direct molecular interaction (such as co-immunoprecipitation, GST pull-down, etc.). In the future, we should further explore whether ASCT2 affects NLRP3 expression through transcriptional regulation or signal pathway activation. Secondly, all experiments were carried out in vitro, lacking in vivo animal model or preclinical research validation. Subsequent studies should establish xenograft tumor models and analyze the correlation between ASCT2 expression and pyroptosis death markers.

Taken together, the ASCT2 gene suppressed the proliferation and epithelial cells of RCC through the activation of pyroptosis in vitro by NLRP3 inflammasome. Our findings demonstrate a potential use of ASCT2 levels as an indicator of RCC. Moreover, the inhibition of ASCT2 offers a promising clinical intervention approach for managing RCC.

Conclusions

In summary, we show that ASCT2 is a key regulator of RCC progression via modulating NLRP3-mediated pyroptosis. Our results suggest that the targeting of ASCT2 could represent a novel therapeutic approach for RCC, especially for patients who have recurrent or metastatic disease.

Acknowledgments

None.

Footnote

Reporting Checklist: The authors have completed the MDAR reporting checklist. Available at https://tau.amegroups.com/article/view/10.21037/tau-2025-431/rc

Data Sharing Statement: Available at https://tau.amegroups.com/article/view/10.21037/tau-2025-431/dss

Peer Review File: Available at https://tau.amegroups.com/article/view/10.21037/tau-2025-431/prf

Funding: This work was supported by the Professional and Technical Backbone Project of Gaoxin Branch of the First Affiliated Hospital of Nanchang University (No. GXGG20247202).

Conflicts of Interest: All authors have completed the ICMJE uniform disclosure form (available at https://tau.amegroups.com/article/view/10.21037/tau-2025-431/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 at Gaoxin Branch of the First Affiliated Hospital of Nanchang University (No. 2024-7). All enrolled cancer patients signed voluntary participant agreement documents prior to study enrollment.

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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