Introduction

Renal cancer, specifically clear cell renal cell carcinoma (RCC) (ccRCC), is a solid malignancy of the genitourinary system1, and its incidence is increasing2. ccRCC is characterized by an abundance of clear cytoplasm within tumour cells, distinguishing it from other RCC subtypes3. Although the exact pathogenesis of ccRCC remains unclear, genetic and environmental factors are believed to significantly contribute to its development4. The low overall survival rate in patients with ccRCC is attributed to the limited efficacy of conventional treatments and the development of acquired resistance, which presents a significant challenge in the management of this disease5. Consequently, it is imperative to explore the underlying mechanisms that drive ccRCC progression to devise more efficacious therapeutic approaches.

GA-binding protein transcription factor subunit alpha (GABPA) is a member of the erythroblast transformation-specific transcription factor family; it directly binds to the mutated telomerase reverse transcriptase promoter and plays a pivotal role in activating telomerase6. This activation leads to the upregulation of telomerase expression, which is essential for maintaining telomere length7,8. Extensive research has consistently demonstrated that GABPA exhibits oncogenic properties and significantly contributes to the development and progression of various cancers9,10. Surprisingly, recent studies have shown that GABPA also exhibits remarkable tumour-suppressive properties in specific types of tumours11,12. Therefore, further research on GABPA expression in ccRCC is necessary.

In ccRCC, dysregulated cellular metabolism leads to the excessive production of reactive oxygen species (ROS) during abnormal glucose metabolism and the accumulation of fatty acids13. The dysregulation of metabolic pathways within a cell can result in the excessive accumulation of lipid peroxides, ultimately leading to ferroptosis14, a form of iron-dependent regulated cell death. Ferroptosis is characterized by the disruption of metabolic pathways, causing an abnormal buildup of lipid peroxides15. The close association between iron metabolism and lipid homeostasis requires renal cancer cells to counteract these factors to evade ferroptosis and survive. This process involves the dysregulation of multiple genes through the modulation of ROS levels, thereby enabling renal cancer cells to augment their resistance to lipid peroxidation13. Recently, ferroptosis has been reported to regulate tumour development16,17. Acyl-coenzyme A (CoA) synthetase long chain family member 4 (ACSL4) is an enzyme involved in regulating lipid metabolism and plays a critical role in ferroptosis18. Lipid metabolism is altered during ferroptosis due to the activation of ACSL4, ultimately leading to cellular death. However, the exact mechanisms underlying GABPA-induced ferroptosis in ccRCC remain unclear. Consequently, a comprehensive study on the role of the GABPA-ACSL4 axis in ferroptosis is necessary to gain a more in-depth understanding of the regulation of cell death pathways in ccRCC.

This study revealed a significant association between GABPA deficiency and unfavourable prognosis in patients with ccRCC, as well as the potential impact of GABPA on ferroptosis sensitivity. Furthermore, our investigation revealed a close correlation between GABPA and ACSL4 transcription, thereby enhancing our understanding of the GABPA-induced ACSL4-associated ferroptosis pathway. These findings provide novel insights into the functional importance of GABPA in the intricate progression of ccRCC.

Materials and methods

RCC specimens and cell culture

ccRCC tissues and corresponding normal tissues were obtained from surgical resection specimens at the Department of Urology, Peking University Third Hospital, with the approval of the Ethics Committee of Peking University Third Hospital. Each patient signed an informed consent form. Human ccRCC tissue microarrays (TMAs) were purchased from Shanghai Outdo Biotechnology Co., Ltd. (Shanghai, China). The clinicopathological data of the TMAs are shown in Table S4. ccRCC cell lines (786-O, 769-p, A498, and Caki-1) and HK2 cells were obtained from the American Type Culture Collection (ATCC) and were cultured in Roswell Park Memorial Institute (RPMI) 1640 medium (GIBCO, CA, USA) supplemented with 10% foetal bovine serum (FBS) (GIBCO, CA, USA) in a humidified incubator (5% CO2) at 37 °C.

Establishment of stable cell lines, construction of plasmids, and transfection

A lentiviral vector for the overexpression of GABPA (GABPA-OE) and a negative control lentiviral vector were acquired from GeneChem (Shanghai, China). After infecting ccRCC cells with lentiviruses, the cells were exposed to 4 µg/mL puromycin and maintained in medium containing 2 µg/mL puromycin. si-GABPA was obtained from Sango Biotech (Shanghai, China), and the corresponding sequences are provided in Supplementary Table S1. The luciferase reporter plasmid pGL4.10-ACSL4 promoter was constructed utilizing the ACSL4 promoter region (-2,000 ~ + 200 bp). Lipofectamine RNAiMAX Reagent and Lipofectamine 2000 (Invitrogen, USA) were used to transfect ccRCC cells with siRNAs or plasmids, respectively.

Histology and immunohistochemistry

TMA specimens were baked in an oven at 60 °C for 2 h. Subsequently, the microarray chips were deparaffinized using xylene three times and dehydrated using a graded ethanol series. Then, the microarray chips were heated in a microwave in citrate buffer (pH 6.0) to facilitate antigen recovery. To mitigate the activity of endogenous peroxidase, the microarray chips were treated with 0.3% H2O2 for 15 min at room temperature. After blocking nonspecific binding sites with 10% horse serum for 1 h, the chips were incubated overnight at 4 °C with a primary antibody against GABPA (ab224325, Abcam, USA), followed by incubation with an HRP-conjugated secondary antibody for 1 h. Finally, diaminobenzidine tetrahydrochloride was used as the reaction substrate. Haematoxylin was used to counterstain nuclei, and neutral resin was used to seal the slides. In addition, the TMA was stained with haematoxylin–eosin (HE).

Reverse transcription qPCR

An RNA isolation kit (R0024, Beyotime, China) was used to extract total RNA, which was then quantified following the manufacturer’s instructions. Relative RNA levels were calculated using the 2 − ΔΔCT method, with glyceraldehyde-3-phosphate dehydrogenase (GAPDH) serving as the housekeeping gene. The primer sequences for GABPA and GAPDH are provided in Supplementary Table S2.

Western blotting

Total protein was extracted from tissues or cells using a Total Protein Extraction Kit (E211-02, Vazyme, Nanjing, China). Proteins were transferred to polyvinylidene difluoride (PVDF) membranes (Millipore, USA), which were incubated overnight at 4 °C with primary antibodies against the following proteins: GABPA (ab224325, Abcam, USA), ACSL4 (ab251419, Abcam, USA), and GAPDH (10494-1-AP, Proteintech, China).

Cell proliferation assay

Cell proliferation was measured using CCK-8 and colony formation assays. The CCK-8 assay was performed using ccRCC cells seeded in 96-well plates at a density of 2 × 103 cells per well in RPMI-1640 medium supplemented with 10% FBS. The cell growth rate was assessed using CCK-8 (CK04, Dojindo, Japan) according to the manufacturer’s instructions, and the optical density was measured on a spectrophotometer at 450 nm.

Colony formation was assessed through limiting dilution analysis after the cells were treated. Colonies were fixed using 4% paraformaldehyde, stained with 0.1% crystal violet for 30 min at room temperature, and washed with PBS.

Transwell assays

RCC cells (2 × 105) were cultured in the upper Transwell chamber (pore size, 8 μm; 3422, Corning, USA) in serum-free RPMI-1640 medium (100 µL); the lower chamber contained 20% FBS RPMI-1640 medium (600 µL). Prior to the invasion assay, the upper chamber was coated with 1 mg/ml Matrigel (354480, Corning, USA). For the migration and invasion assays, cells were incubated for 17 h and 30 h, respectively. Following incubation, the cells were fixed with 4% paraformaldehyde and stained with crystal violet. The experiments were repeated independently a minimum of three times.

In vivo studies

The Ethics Committee of the Peking University Third Hospital approved the use of nude mice for experiments, and the National Institutes of Health guidelines were followed for all animal experiments. In summary, for tumour xenograft experiments, approximately 5 × 106 ccRCC cells were subcutaneously injected into the left flank of 3–5-week-old male BALB/c nude mice (n = 5 per group). The tumour size was assessed every 7 days. After 6 weeks, the mice were euthanized humanely, and the tumours were surgically excised.

ChIP

ChIP was conducted using the Pierce Magnetic ChIP Kit (Thermo Scientific, USA) according to the manufacturer’s instructions. Briefly, 786-O cells were fixed with 1% formaldehyde, followed by lysis using MNase and sonication. Immunoprecipitation was then performed using an anti-GABPA antibody (ab224325, Abcam, USA). The obtained DNA was amplified using primers (primer sequences are listed in Supplementary Table S3), quantified through qPCR, and identified through agarose gel electrophoresis after washing and reverse cross-linking.

Luciferase assay

HEK293T cells were transfected with a firefly luciferase reporter gene, pRL-TK (Renilla luciferase plasmid), and either a GABPA overexpression plasmid or an empty vector (pcDNA3.1). A dual-luciferase reporter (Promega, USA) assay was conducted to quantify luciferase activity after 48 h of transfection. Firefly luciferase activity was normalized to Renilla luciferase activity.

Iron, lipid peroxidation, and ROS quantification

Ferrous ion levels in treated cells were assessed using an iron assay kit (ab83366, Abcam, USA), lipid peroxidation levels were assessed using BODIPY-C11 (D3861, Invitrogen, USA) staining, and ROS levels were assessed using an ROS assay kit (R253, Dojindo, Japan).

Statistical analysis

GraphPad Prism (version 8.0) was used for data analysis, and the results are expressed as the mean ± SD. Student’s t test was used to determine the statistical significance of differences between two groups. Pearson’s correlation analysis was utilized to determine the correlation between two variables. Statistical significance was set at p < 0.05. The levels of significance are reported as *p < 0.05, **p < 0.01, ***p < 0.001, and ****p < 0.0001.

Results

GABPA expression is downregulated in ccRCC

To compare the expression of GABPA between ccRCC and normal kidney tissues, we conducted a preliminary assessment of GABPA expression using The Cancer Genome Atlas (TCGA)-Kidney Renal Clear Cell Carcinoma (KIRC) dataset. Compared with corresponding normal tissues, ccRCC tissues showed significantly lower GABPA expression (Fig. 1A). Moreover, an analysis of TCGA data using the Gene Expression Profiling Interactive Analysis website (http://gepia.cancer-pku.cn/) revealed a significant correlation between low GABPA expression and poor survival in patients with ccRCC (Fig. 1B). Additionally, we conducted tissue microarray analysis using samples from a cohort of 60 patients to investigate GABPA levels in ccRCC tissues. Immunohistochemical analyses revealed notably low GABPA expression in ccRCC tissues, as indicated by the relatively low H-scores (Fig. 1C). As shown in Fig. 1D, GABPA expression was lower in ccRCC cells than in human HK2 cells. In addition, the GABPA levels in ccRCC tissues were significantly lower than those in adjacent normal tissues, as demonstrated by western blot analysis (Fig. 1E). In summary, these findings suggest that GABPA plays a substantial role in the progression of ccRCC.

Fig. 1
figure 1

GABPA expression was down-regulated in ccRCC. (A) Analysis of GABPA mRNA expression in TCGA-KIRC; (B) Survival analysis of TCGA data using GEPIA online software; (C) Representative H&E and IHC staining of the TMA of 60 ccRCC patients’ samples to show the histology and the protein level of GABPA in tumors or adjacent tissues; (D) The protein level of GABPA was detected by Western blot analysis in ccRCC cell lines and HK2 cells (n = 3); GAPDH was used as internal standards; ***indicates that compared with HK2 cells group; (E) The detection of the protein level of NAT10 by WB in 12 matched ccRCC tumor (C) and adjacent (N) tissues. The data were presented as mean SD. **P < 0.01, ***P < 0.001, ****P < 0.0001. Abbreviations: KDa, kilodalton; P1-P12, Patient 1-Patient 12.

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GABPA overexpression effectively suppresses tumorigenesis both in Vitro and in vivo

The purpose of this study was to examine the role of GABPA in tumorigenesis. We overexpressed GABPA in ccRCC cells to explore its functions. Lentivirus infection was performed to establish stable ccRCC cell lines overexpressing GABPA. Western blot analysis confirmed the successful overexpression of GABPA in 786-O, 769-P and Caki-1 cells (Fig. 2A and S1A). The results obtained from the Cell Counting Kit-8 (CCK-8) and colony formation assays revealed that compared to the control (puro), the overexpression of GABPA led to a significant decrease in cell proliferation and colony numbers, respectively. This finding indicates that GABPA effectively suppresses the proliferative capacity of ccRCC cells (Fig. 2B, C and S1B). Migration and invasion are crucial indicators of tumour progression. Therefore, we used Transwell migration and invasion assays to examine the motility of ccRCC cells. Remarkably, compared with the control (puro), GABPA overexpression significantly decreased the migratory and invasive potential of 786-O, 769-P and Caki-1 cells (Fig. 2D, E and S1C).

A tumour xenograft mouse model was established to investigate GABPA in vivo. In this model, 786-O cells were injected subcutaneously into male BALB/c nude mice. GABPA overexpression remarkably delayed tumour growth in the tumour xenograft model (Fig. 2F). After 6 weeks, we sacrificed the mice, dissected the transplanted tumours, and analysed their weight changes (Fig. 2G and H). We observed a significant difference in tumour weight between the two experimental groups.

Downregulation of GABPA expression promotes ccRCC aggressiveness in Vitro

Next, we transiently knocked down GABPA in 786-O, 769-P and Caki-1 cells using small interfering RNA (siRNA) to investigate the role of GABPA in ccRCC progression. GABPA knockdown efficiency was confirmed using western blotting (Fig. 3A and S1D). Compared to those in the control group, cell proliferation and colony formation were significantly greater in the GABPA knockdown group, as measured by CCK-8 (Fig. 3B and S1E), and colony formation assays (Fig. 3C), respectively, suggesting that knocking down GABPA effectively promoted cell proliferation. Furthermore, compared with the control, GABPA knockdown significantly enhanced the migration and invasion abilities of 786-O, 769-P and Caki-1 cells, as demonstrated by the Transwell assay results (Fig. 3D, E and S1F).

Fig. 2
figure 2

GABPA overexpression suppresses the proliferation, migration, and invasion of ccRCC. (A) The efficiency of GABPA overexpression was confirmed by Western blot analysis in 786-O and 769-P cells transfected with GABPA overexpression vectors (puro) or GABPA overexpression lentivirus (GABPA OE) (n = 3); Quantitative analysis of WB results by Image J; GAPDH was used as internal standards; (B–C) Cell proliferation assays: CCK-8 and colony formation assays were performed to evaluate the cell proliferation ability in 786-O and 769-P GABPA overexpressing cells; (D-E) Transwell migration and invasion analyses using 786-O and 769-P GABPA overexpressing cells. The migrated and invaded cells were quantified in three randomly chosen fields; Magnification: 100×; (F) 786-O cells were used in nude mice transplantation experiments in vivo (puro group and GABPA OE group). Male nude mice were subcutaneously inoculated with 786-O overexpressing cells in the axilla for 1 week, and tumor size was measured every 7 days (n = 5 per group), and tumor volume growth curves were plotted. (G) Representative images of tumor formation were presented; (H) The resected tumors were weighed and compared using t test; The data were presented as mean SD. **P < 0.01, ***P < 0.001, ****P < 0.0001.

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Fig. 3
figure 3

Interference of GABPA expression promotes the proliferation, migration, and invasion of ccRCC. (A) Protein expression levels of GABPA in 786-O and 769-P cells following GABPA siRNA interference; The interference efficiency was observed by Western blot analysis; Quantitative analysis of these results was conducted using Image J software; GAPDH was used as internal standards; Control: si-GABPA-NC; Experimental groups: si-GABPA-1; si-GABPA-2; si-GABPA-3; (B-C) Inhibition of GABPA can promote the proliferation of ccRCC cells in vitro. CCK-8 and colony formation assays were performed to evaluate the cell proliferation ability in 786-O and 769-P GABPA knockdown cells; The CCK-8 assay required daily detection of cell proliferation activity, and growth curves were drawn after 7 days. The experiment was repeated three times; (D-E) Inhibition of GABPA can promote the migration and invasion of ccRCC cells in vitro. Transwell migration and invasion analyses using 786-O and 769-P GABPA knockdown cells. The migrated and invaded cells were quantified in three randomly chosen fields. These experiments were performed as described in Materials and Methods; Magnification: 100×. The data were presented as mean SD. ***P < 0.001, ****P < 0.0001.

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GABPA overexpression inhibits tumorigenesis in ccRCC by regulating ferroptosis

In our previous study, we found that GABPA may play an important role in inhibiting tumour progression. Other studies have demonstrated a close relationship between ferroptosis and kidney diseases19. We conducted Spearman’s correlation analysis using the KIRC dataset and found a strong correlation between the glycerolipid metabolism score and GABPA expression (R = 0.22, p = 3.45e-07) (Fig. 4A). Previous studies have convincingly demonstrated that the regulation of ferroptosis depends heavily on cellular lipids and lipid metabolism20,21. We hypothesized that ferroptosis is involved in ccRCC tumorigenesis through GABPA. To test this hypothesis, two ferroptosis-specific inhibitors, ferrostatin-1 (Fer-1) and liproxstatin-1 (Lip-1), were used to treat 786-O and 769-P cells stably overexpressing GABPA. Cell viability was assessed using CCK-8 assays (Fig. 4B). We observed that the suppression of cell viability caused by GABPA overexpression was reversed by Fer-1 and Lip-1 treatment. Furthermore, in the context of ferroptosis, reduced mitochondrial size and alterations in mitochondrial structure are significant characteristics of cellular demise15. To assess the ferroptosis-related morphological attributes of ccRCC cells, we used transmission electron microscopy (TEM) (Fig. 4C). TEM revealed that the mitochondria in GABPA-overexpressing (OE) 786-O cells exhibited a spherical shape, diminished dimensions, and disrupted architecture. Additionally, GABPA overexpression increased the intracellular levels of MDA (Fig. 4D). We also measured the levels of lipids and ROS in both the GABPA-OE and puro groups following treatment with RSL-3, which is widely used as a ferroptosis inducer (Fig. 4E–G). There was a noteworthy increase in ROS generation in the GABPA-OE group compared to the puro group, suggesting that GABPA-OE cells are more vulnerable to ferroptosis. Collectively, our findings provide evidence that the upregulation of GABPA expression in ccRCC cells enhances ferroptosis and may exert inhibitory effects on tumorigenesis by regulating ferroptosis.

Fig. 4
figure 4

GABPA overexpression affects ferroptosis sensitivity in ccRCC. (A) Spearman correlation between Glycerolipid metablism score and GABPA expression in KIRC (R = 0.22, p = 3.45e-07); (B) Cell viability was detected by CCK-8 assay. Two ferroptosis-specific inhibitors, ferrostatin-1 (Fer-1) and liproxstatin-1 (Lip-1), were used to treat 786-O and 769-P cells stably overexpressing GABPA. The GABPA overexpression (GABPA-OE) group was more sensitive to ferroptosis-specific inhibitors. (C) The TEM images reveal shrinking mitochondria; In 786-O GABPA-OE group, mitochondria shrink even more significant than puro group; The red arrows indicate this; (D) The relative levels of Malondialdehyde (MDA) comparable sensitivity of ferroptosis; The MDA level was significantly increased in 786-O and 769-P GABPA-OE cells, suggesting a greater sensitivity to ferroptosis; (E) Flow cytometry measurement of relative BODIPY 581/591 C11 fluorescence for lipid ROS; GABPA-OE cell lines are more sensitive to ferroptosis inducer RSL3 (peak shift to the right); (F) The level of lipid ROS showed by Fluorescence microscopy images; GABPA-OE cell lines were sensitive to ferroptosis inducer RSL3, and the green fluorescence was more obvious (scale bar = 100 μm); (G) Relative levels of lipid ROS in ccRCC cells. GABPA-OE cell lines are more sensitive to ferroptosis inducer RSL3; The data were presented as the mean ± SD. *P < 0.05, **P < 0.01, ***P < 0.001, ****P < 0.0001.

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GABPA expression is positively correlated with ACSL4 expression in ccRCC

We utilized a preconstructed GABPA-OE 769-P cell line model and RNA sequencing to investigate the association between the ACSL4 gene and ferroptosis (Fig. 5A–C). The KEGG enrichment analysis indicated that the differentially expressed genes were implicated in crucial lipid metabolism pathways, including “Sphingolipid metabolism”, “Glycerophospholipid metabolism”, and “Glycerolipid metabolism” (Supplementary Figure. S2A). In the GSEA analysis, the results for “GLYCEROLIPID_METABOLISM” and “FATTY_ACID_DEGRADATION” were not statistically significant (Supplementary Figure. S2B). Nonetheless, ACSL4, a gene intimately associated with lipid metabolism and ferroptosis, was identified among the differential genes based on prior literature and was subsequently validated18,22. We further corroborated the correlation between GABPA and ACSL4 utilizing diverse methodologies. In the analysis of the TCGA database, GABPA was strongly positively correlated with ACSL4 expression (p = 0; R = 0.62) (Fig. 5D). Furthermore, in the TCGA database, analysis of the KIRC dataset revealed the downregulation of ACSL4 expression in tumours, with high ACSL4 expression being linked to a favourable prognosis, which aligns with the results pertaining to GABPA (Fig. 5E and F). Hence, these findings offer compelling evidence in support of a substantial positive correlation between GABPA and ACSL4 expression. This correlation strongly suggests that GABPA regulates ACSL4 transcription. Furthermore, the experimental manipulation of GABPA levels through both overexpression and knockdown notably increased and decreased the expression of ACSL4 (Fig. 5G–H), respectively. These findings further reinforce the notion that GABPA and ACSL4 are intricately and positively associated.

Fig. 5
figure 5

GABPA expression is positively correlated with ACSL4 expression in ccRCC. (A-C) The 786-O cell control group and GABPA overexpression group were subjected to transcriptome analysis. Differentially expressed genes were identified and visualized using Venn diagram, volcano plots and heat maps; (D) GABPA and ACSL4 expression is highly correlated with each other in the TCGA ccRCC tumors; (E) The ACSL4 expression in TCGA data using GEPIA online software; (F) Survival analysis of TCGA data using GEPIA online software; (G,H) Western blot analysis of the expression levels of ACSL4 protein in ccRCC with overexpression and knockdown of GABPA (Left). The relative ratio of GABPA/GAPDH or ACSL4/GAPDH was analyzed using the ImageJ software (Right); The data were presented as the mean ± SD. *P < 0.05, ***P < 0.001, ****P < 0.0001.

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GABPA regulates ACSL4 transcription in ccRCC cells by binding to its promoter region

GABPA is widely recognized as a prominent transcription factor, and our previous studies provided evidence for a correlation between GABPA and ACSL4 transcription (Fig. 5D and G-H). Consequently, we hypothesized that GABPA binds to the promoter region of ACSL4, thereby regulating its transcription and translation processes. We used the JASPAR database to predict the GABPA binding motifs within the ACSL4 promoter region (Fig. 6A). Several putative GABPA binding sites were predicted within a region spanning 2,000 and 200 bp upstream and downstream of the transcription start site, respectively, of the human ACSL4 promoter. Four binding sites with high prediction scores were selected for further analyses (Fig. 6B). ChIP and dual-luciferase reporter experiments revealed that binding sites 1, 3 and 4 were direct targets of GABPA (Fig. 6C and D). Furthermore, ChIP–qPCR analysis revealed GABPA enrichment in the ACSL4 promoter (Fig. 6E). These findings suggest that GABPA directly influences the transcription of ACSL4 by interacting with specific sites on its promoter.

Taken together, our results provide evidence that GABPA interacts with the promoter region of ACSL4, resulting in the activation of its transcription, which leads to the accumulation of ROS and mitochondrial degradation, thereby facilitating the onset of ferroptosis.

Fig. 6
figure 6

GABPA directly regulates ACSL4’s transcription. (A) The GABPA motif discovered by JASPAR in the ACSL4 promoter; (B) Four GABPA binding sites in the ACSL4 promoter (P1-P4); (C) ChIP assays were conducted to identify the binding sites in the ACSL4 promoter regions by using GABPA antibody in 786-O cells; (D) Luciferase assays were carried out as described in Materials and Methods. The firefly luciferase activity of the reporter was normalized with the renilla luciferase activity of control reporter, and relative activity was calculated; The luciferase activity revealed that binding sites 1, 3 and 4 (MUT1, MUT3 and MUT4) were direct targets of GABPA; (E) ChIP-qPCR assay revealed GABPA enrichment in the ACSL4 promoter (P1, P3 and P4); The data were presented as mean ± SD. **P < 0.01, ***P < 0.001, ****P < 0.0001.

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Inhibition of ACSL4 restored the effect of GABPA overexpression in ccRCC

Previous findings have indicated a correlation between ACSL4 and susceptibility to ferroptosis, suggesting that ACSL4 plays a significant role in this process18,23. To ascertain the pivotal role of ACSL4 in the suppression of tumorigenesis mediated by GABPA, we employed RNAi to suppress ACSL4 expression in GABPA-OE ccRCC cell lines and monitored tumorigenesis.

First, the efficiency of ACSL4 knockdown in GABPA-OE cells was verified by western blot analysis of GABPA-OE ccRCC cell lysates (Fig. 7A). In GABPA-OE ccRCC cell lines, ACSL4 knockdown significantly reversed the growth inhibition caused by GABPA overexpression, as determined by CCK8 assays (Fig. 7B).

Comparable findings were obtained using a xenograft mouse model, wherein tumor growth was markedly inhibited in mice injected with GABPA-OE 786-O cells. Conversely, the suppression of ACSL4 in GABPA-OE 786-O cells significantly enhanced tumor proliferation rates (Supplementary Figure. S3A-C). Additionally, TEM revealed mitochondrial damage subsequent to GABPA overexpression. However, the concurrent overexpression of GABPA and knockdown of ACSL4 expression mitigated mitochondrial damage (Fig. 7C). Interestingly, we also observed that the sensitivity to ferroptosis induced by GABPA overexpression decreased after ACSL4 knockdown following the addition of a ferroptosis inducer (RSL3) (Fig. 7C). The intracellular MDA levels further supported these findings (Fig. 7D). These results indicate that ACSL4 knockdown can mitigate the GABPA-mediated increase in ferroptosis sensitivity in ccRCC cells. Collectively, these findings substantiate the role of GABPA in modulating ferroptosis through the regulation of ACSL4, thereby influencing tumorigenesis. This mechanism plays a crucial role in the suppression of tumorigenesis in ccRCC (Fig. 7E).

Fig. 7
figure 7

Inhibition of ACSL4 restored the effect of GABPA overexpression. (A) The efficiency of ACSL4 siRNA interference in GABPA-overexpressing (GABPA-OE) 786-O and 769-P cell lines was verified by Western blot (n = 3) (up); GAPDH was used as internal standards; Quantitative analysis of these results was conducted using Image J software (down); (B) CCK-8 assays were performed to evaluate the cell proliferation ability in 786-O and 769-P cells GABPA-overexpressing and/or inhibition of ACSL4; (C) The TEM images reveal shrinking mitochondria; In GABPA-OE 786-O cells, mitochondria exhibit thickened membranes and are highly susceptible to ferroptosis upon treatment with RSL3; But inhibition of ACSL4 could suppress the above results; (D) Relative levels of MDA in 786-O and 769-P cells GABPA-overexpressing and/or inhibition of ACSL4; And sensitivity to ferroptosis (RSL3 treatment); (E) Schematic representation of how dysregulation of ACSL4 through GABPA inhibits tumorigenesis in ccRCC cells by promoting ferroptosis sensitivity. The relative ratio of ACSL4/GAPDH was analysed using the ImageJ software; The data were presented as the mean ± SD. **P < 0.01, ***P < 0.001, ****P < 0.0001.

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Discussion

Metabolic abnormalities are particularly prominent in ccRCC24,25. ccRCC exhibits significant metabolic reprogramming, characterized by the suppression of the tricarboxylic acid cycle and activation of fatty acid synthesis and the pentose phosphate pathway. These metabolic alterations ultimately resulting a metabolic imbalance within ccRCC cells.

ccRCC cells are particularly prone to ferroptosis26,27. Specifically, they exhibit a heightened oxidative stress response, resulting in the generation of substantial quantities of ROS. Concurrently, ccRCC cells activate antioxidant pathways that affect ROS accumulation, thereby establishing metabolic equilibrium. Nevertheless, the disruption of the equilibrium between glucose and lipid metabolism increases ROS production and accumulation, culminating in ferroptosis and subsequently influencing the initiation and progression of tumours28. Metabolic dysregulation has been implicated in the initiation and advancement of cancerous tumours20. Therefore, the modulation of lipid peroxidation and ROS metabolism through a variety of pathways can affect tumorigenesis.

Previous studies have suggested that therapeutics targeting cellular and molecular pathways should be utilized for the management of ccRCC; however, patients have shown limited responses to these therapies29,30. Investigating the pathogenesis of ccRCC and identifying new therapeutic targets present considerable challenges. Consequently, modulating ferroptosis to influence tumour occurrence and progression can serve as an innovative therapeutic approach. Ferroptosis, a recently identified variant of apoptosis-independent programmed cell death, presents a promising avenue for cancer treatment. As shown in Fig. 1, a noteworthy reduction in GABPA expression was observed in ccRCC patients. In addition, we also revealed that GABPA could directly target ACSL4 and induce ferroptosis by activating ACSL4, thereby impeding ccRCC progression (Fig. 6C-E). Additionally, the restoration of GABPA expression effectively suppressed the carcinogenicity of ccRCC cells.

Although the results of our study suggest that GABPA functions as a tumour suppressor gene in renal cancer, its role in different tumour types remains controversial. Sharma et al.9 reported that GABPA regulates androgen receptor (AR) expression and has a profound effect on prostate cancer. Specifically, GABPA interacts with AR to mediate the aggressive phenotype of AR-positive prostate cancer cells. Yuan et al.11 discovered that the transcription factor GABPA is crucial for regulating the expression of DICER1, thereby influencing the production of the Dicer protein and positioning it as a tumour suppressor. Furthermore, Guo et al.12 reported evidence supporting the role of GABPA as a tumour suppressor in bladder cancer; they concluded that GABPA promotes the differentiation of bladder cancer cells by activating forkhead box A1 (FOXA1) and GATA binding protein 3 (GATA3). Therefore, further investigation of the role of GABPA in modulating the biological behaviour of ccRCC cells is necessary. Our study demonstrated that overexpressing GABPA markedly inhibited ccRCC tumorigenesis both in vitro and in vivo. These findings provide valuable insights into the therapeutic potential of targeting GABPA to prevent the initiation and progression of ccRCC.

Recently, there has been a notable surge in research on ferroptosis-based tumour treatments. A wealth of evidence has emerged highlighting the anticancer properties of ferroptosis and demonstrating its ability to sensitize drug-resistant tumour cells to ferroptosis-inducing agents31,32. Hence, it is plausible that effective treatment outcomes can be achieved for specific chemotherapy-resistant tumours. Consequently, ferroptosis could provide a viable treatment option for renal cancer. Our findings established a relationship between GABPA and lipid peroxidation levels and further elucidated the various phenotypic manifestations associated with ferroptosis. In our analysis of transcriptome sequencing results following GABPA overexpression, we identified ACSL4, a gene closely associated with ferroptosis, among the differentially expressed genes. Subsequent ChIP assays and dual-luciferase reporter assays confirmed that GABPA modulates the expression of ACSL4 by recognizing and binding to specific sequences within the promoter region of the ACSL4 gene (Fig. 6C-E). This study provides the first evidence of a direct interaction between GABPA and ACSL4. Furthermore, our findings provide a detailed analysis of the molecular mechanism through which GABPA modulates ferroptosis by regulating ACSL4. Specifically, the downregulation of ACSL4 expression was found to reduce the susceptibility of GABPA-overexpressing cells to ferroptosis, thereby restoring their tumorigenic potential. This study successfully identified and validated the role of ACSL4 in ferroptosis, thereby demonstrating that GABPA impedes the progression of ccRCC by modulating ACSL4 to influence ferroptosis (Fig. 7E).

Nevertheless, it is important to acknowledge the limitations of our study, which include a restricted sample size attributed to time constraints and the necessity for further investigation into the underlying mechanisms. Moreover, additional clinical investigations are needed to validate the influence of GABPA on ferroptosis and the consequential implications for the progression of RCC. Furthermore, there is a need for a comprehensive exploration of the role of GABPA in drug-resistant ccRCC cells.

Conclusions

Our study provides compelling evidence that supports the involvement of GABPA in mediating ACSL4 transcription and translation, resulting in the suppression of the migration and invasion of ccRCC cells through the regulation of ferroptosis. Moreover, we identified a noteworthy correlation between ferroptosis and ccRCC progression in humans, highlighting ferroptosis as a potential promising therapeutic avenue for the treatment of this disease. GABPA activation is a potent mechanism for combating tumour progression and is therefore a novel target for ccRCC treatment.