Targeting IDH1 mutation-driven Nrf2 signaling to suppress malignant behavior in fibrosarcoma cells

Abstract

Isocitrate dehydrogenase 1 (IDH1) mutations are prevalent in various cancers and have significant implications for tumor biology. It is known that cancer cells with IDH1 mutations, particularly R132C or R132H, exhibit decreased production of nicotinamide adenine dinucleotide phosphate and thus impaired glutathione synthesis. This study investigated the roles of IDH1 mutations in the regulation of nuclear factor erythroid-2-related factor 2 (Nrf2)-mediated signaling pathways in fibrosarcoma HT1080 cells harboring the IDH1 R132C mutation. Knockdown of IDH1 using siRNA in HT1080 cells inhibited Nrf2 stabilization and reduced the expression of antioxidant genes, thereby providing favorable conditions for cancer progression. In addition, inhibition of IDH1 decreased reactive oxygen species (ROS) production and impaired cell migration, highlighting its role in promoting malignant behavior such as colony-forming ability. Small molecule inhibitors targeting the IDH1 R132 mutation suppressed cell migration and colony formation in HT1080 cells. Moreover, we observed that IDH and Nrf2 contribute to immune evasion by modulating the expression of programmed death-ligand 1 (PD-L1) in HT1080 cells. Altogether, our findings provide valuable insights for the development of therapeutic approaches for IDH1-mutant cancers. We suggest targeting the IDH1-Nrf2 axis as a strategy to regulate the immune response and inhibit cell migration in fibrosarcoma.

Supplementary Information

The online version contains supplementary material available at 10.1007/s43188-025-00284-1.

Introduction

Various genetic mutations and metabolites involved in metabolic signaling have been revealed to directly contribute to cancer development. Thus, anticancer drugs targeting cancer cell metabolic pathways have attracted significant attention [1, 2].

Isocitrate dehydrogenase 1 (IDH1) is a component of the citric acid cycle and plays a role in generating nicotinamide adenine dinucleotide phosphate (NADPH) [3]. The NADPH generated by IDH1 reduces glutathione, thereby protecting cells from the toxicity of reactive oxygen species (ROS), and acts as an essential cofactor in various biosynthetic pathways [4]. The arginine residue at position 132 of IDH1 is critical for isocitrate binding, and IDH1 mutations in which this residue undergoes substitution by cysteine or histidine (R132C and R132H) impair its ability to bind [5]. Such IDH1 mutants bind to NADPH and produce tumor-associated D-2-hydroxyglutarate through the reduction of α-ketoglutarate, thereby contributing to cancer progression [5]. IDH1 gene mutations have been widely studied in glioma or leukemia patients [6–11] and are rarely detected in solid tumors, such as cholangiocarcinoma and chondrosarcoma [10, 12–14]. The high prevalence of IDH mutations in chondrosarcoma highlights their potential as therapeutic targets, particularly in dedifferentiated chondrosarcoma, which can progress into more aggressive forms such as osteosarcoma or fibrosarcoma [15, 16]. Mao et al. demonstrated that IDH-mutant fibrosarcoma cells secrete oncogenic factors that promote angiogenesis, including vascular endothelial tube formation, potentially facilitating metastasis [17]. The R132C mutation is a genetic alteration in the IDH1 gene and has been identified in fibrosarcoma and various other cancers [18, 19]. Cancer cells harboring IDH1 mutations show decreased NADPH production, which impairs glutathione synthesis and promotes ROS accumulation [20, 21]. Moreover, it was estimated that D-2-hydroxyglutarate production in IDH1 R132 mutant cells utilizes NADPH at a rate comparable to that of de novo lipogenesis, which is typically regarded as the most significant NADPH-consuming process [18].

As a result of metabolic and signaling aberrations, cancer cells exhibit elevated ROS levels [22]. Accumulating evidence suggests that ROS exert a dual effect, either promoting or inhibiting cancer progression [23]. Moderately elevated ROS levels have been observed in various types of cancer cells, highlighting the role of ROS signaling in tumor progression and metastasis [24]. Sustained ROS production is linked to cancer progression, as it promotes DNA mutations and activates pro-oncogenic signaling pathways [25]. It has been reported that IDH1 mutations reprogram ROS homeostasis in cancer cells, leading to a dependency on antioxidant gene expression for ROS scavenging that is mediated by nuclear factor erythroid-2-related factor 2 (Nrf2) [26]. Based on these reports, this study investigated whether inhibiting the activity of mutant IDH1 in fibrosarcoma cells could modulate Nrf2-mediated PD-L1 expression and suppresses cell migration.

Materials and methods

Reagents

Roswell Park Memorial Institute (RPMI) 1640 medium was purchased from Welgene (South Korea). TRIzol^®, 2′,7′-dichlorofluorescein diacetate (DCF-DA), and bicinchoninic acid (BCA) protein assay reagents were obtained from Thermo Fisher Scientific (Waltham, MA, USA). The following primary antibodies were used: anti-lamin B1 (Cell Signaling Technology, Danvers, MA, USA), anti-IDH1, anti-Nrf2 (both from Abcam, Cambridge, UK), anti-NAD(P)H quinone oxidoreductase (NQO1) and anti-β-actin (both from Santa Cruz Biotechnology, Dallas, TX, USA), anti-glutamate-cysteine ligase catalytic subunit (GCLC) (Abnova, Taiwan), anti-tubulin (AbClon, South Korea). The IDH1 inhibitors, AGI-5198 and IDH305 were purchased from Sigma-Aldrich (St. Louis, MO, USA) and Cayman Chemical Company (Ann Arbor, MI, USA), respectively.

Cell culture

Human fibrosarcoma HT1080 cells were obtained from the Korean Cell Line Bank (KCLB, Seoul, Korea). The cells were maintained in RPMI 1640 medium supplemented with 10% fetal bovine serum (Gibco) and 1% antibiotic-antimycotic mixture (Welgene) in an incubator at 37 °C in a humidified atmosphere containing 5% CO₂.

Western blot analysis

For total protein extraction, cells were lysed in radio-immunoprecipitation assay (RIPA) lysis buffer (20 mM Tris-HCl, pH 7.4, 150 mM NaCl, 1 mM Na₂EDTA, 1 mM EGTA, 1% Triton, 2.5 mM sodium pyrophosphate, 1 mM β-glycerophosphate, 1 mM Na₃VO₄, 1 μg/mL leupeptin and protease inhibitors) on ice for 30 min and then centrifuged at 14,000g for 30 min. Cytoplasmic and nuclear protein fractions were extracted using the NE-PER™ Nuclear and Cytoplasmic Extraction Reagents (Thermo Fisher Scientific). Protein concentrations of the supernatant were measured using bicinchoninic acid reagents. Proteins were separated by electrophoresis on 8–15% sodium dodecyl sulfate-polyacrylamide gel (SDS-PAGE) and transferred onto a polyvinylidene fluoride membrane (Gelman Laboratory, Ann Arbor, MI, USA). The membranes were blocked with 5% non-fat dry milk in Tris-buffered saline containing 0.1% Tween-20 (TBST) for 1 h at room temperature. After blocking, the membranes were incubated overnight at 4 °C with the indicated primary antibodies. The blots were rinsed three times with TBST buffer for 10 min each. Following washing, the blots were incubated with 1:5000 dilution of horseradish peroxidase-conjugated secondary antibody in TBST containing 3% non-fat dry milk for 2 h at room temperature. Finally, the blots were washed three more times in TBST buffer, and the transferred proteins were detected using enhanced chemiluminescence (ECL) detection reagents (Thermo Fisher Scientific).

Reverse transcription-polymerase chain reaction (RT-PCR)

Total RNA was isolated using TRIzol™ reagent (Invitrogen) according to the manufacturer’s protocol. Reverse transcription of total RNA was performed using a cDNA synthesis RT kit (SolGent, Seoul, South Korea) according to the manufacturer’s instructions. One μl of cDNA was amplified in sequential reactions using Slog™ 2 × Taq PCR Smart mix 1 (SolGent) for conventional PCR. Amplification products were resolved by 2% agarose gel electrophoresis, stained with SYBR green and visualized using a Davinch Gel imaging system (Davinch-K, Korea). Real-time PCR was then performed using Real-Time PCR Kit with EvaGreen™ (SolGent) with an initial denaturation at 95 °C for 10 min, followed by 40 cycles of 95 °C for 15 s, 55 °C for 30 s and 72 °C for 30 s, with a final extension at 72 °C for 7 min. The primers and their sequences are listed in Table S1.

Transient transfection with siRNA

HT1080 cells were plated at a confluence of 60% in dish and grown in complete growth medium. Transfection of IDH1 or Nrf2 siRNA was performed using the Lipofectamine RNAiMAX reagent (Invitrogen) according to the manufacturer’s instructions. The sequences of the siRNA constructs are provided in Table S1. The siRNA oligonucleotides targeting IDH1 and Nrf2 were purchased from Bioneer (Seoul, Korea).

Analysis of intracellular reactive oxygen species accumulation

Intracellular ROS accumulation was measured using the redox-sensitive dye DCF-DA (Invitrogen). The cells were washed once with phosphate-buffer saline and loaded with 10 μM DCF-DA for 30 min at 37 °C to detect ROS-mediated oxidation of the fluorescent compound DCF using a fluorescence microscope (Nikon Ti Eclipse, Japan).

Wound migration assay

The migration of HT1080 cells was measured using Culture-Inserts (Ibidi). Cells were seeded into each well of the Culture-Inserts, and after incubation, the culture-inserts were removed, and 500 μm cell-free gaps were created. The dishes containing these cells were then treated with a medium containing siRNA or an IDH1 inhibitor and incubated at 37 °C. Phase-contrast images of the closed gaps were captured at the indicated incubation times using an inverted microscope (Nikon Ti Eclipse).

Clonogenic assay

HT1080 cells were seeded in 60 mm dishes at a density of 1 × 10² cells and treated with AGI-5198 or IDH305. The cells were cultured for 10 days, with the culture medium replaced with fresh medium every 2 days. The colonies were fixed, stained with crystal violet solution, and washed extensively to remove excess dye, and then imaged and counted using an inverted microscope (Nikon Ti Eclipse).

Statistical analysis

Data are presented as the mean ± standard deviations (SD) from at least three independent experiments. Statistical analysis between groups was performed using Student’s t-test.

Results

IDH1 modulates Nrf2-mediated signaling in HT1080 cells harboring the IDH1 mutation

The fibrosarcoma HT1080 cell line is known to carry the heterozygous IDH1 R132C mutation, which results in the substitution of arginine with cysteine [27]. It is well-known for its high metastatic potential and is widely used as a classical model for investigating tumor cell metastasis [28]. To investigate the relationship between mutant IDH1 and the regulation of Nrf2-mediated signaling in fibrosarcoma, we suppressed IDH1 expression in HT1080 cells. Genetic inhibition of IDH1 using siRNA abolished the nuclear translocation of Nrf2 (Fig. 1a). The protein expression levels of NQO1 and GCLC were also attenuated by IDH1 knockdown (Fig. 1b). Under the same experimental conditions, RT-qPCR analysis showed that the silencing IDH1 decreased NQO1 and GCLC transcript levels (Fig. 1c). By contrast, silencing Nrf2 had no effect on IDH1 expression in HT1080 cells but decreased NQO1 mRNA expression (Fig. 1d). These findings indicate that IDH1 contributes to the expression of Nrf2-mediated antioxidant enzymes, which play a role in regulating cellular resistance in fibrosarcoma cells.

Fig. 1.

The effects of IDH1 activity on Nrf2-mediated signaling in HT1080 cells with the IDH1 mutation. a HT1080 cells were transiently transfected with scrambled or IDH1 siRNA for 48 h and then fractionated into nuclear and cytosolic fractions. In each fraction, IDH1 and Nrf2 expression was measured. For immunoblotting, lamin B1 and α-tubulin were used as nuclear and cytosolic markers, respectively. Levels of the indicated antioxidant marker proteins (b) and mRNA (c) were analyzed using western blotting and real-time PCR, respectively. d HT1080 cells were transiently transfected with scrambled or Nrf2 siRNA for 48 h, and the protein and mRNA levels of IDH1, Nrf2, and NQO1 were examined. For the loading control of total protein, the antibody against actin was used

IDH1 knockdown suppresses ROS production and cell migration in HT1080 cells

ROS-induced cellular stress is recognized as a factor that promotes tumor cell migration [29, 30]. Park et al. demonstrated that hydrogen peroxide-mediated ROS generation promotes the migration and invasion of HT1080 cells, which is associated with Rac1 activation and increased phosphorylation of focal adhesion kinase [31]. Conversely, reducing ROS levels was shown to inhibit the migration and metastatic potential of HT1080 cells [31]. We investigated how IDH1 inhibition affects ROS generation and cell migration. As shown in Fig. 2a, IDH1 inhibition in HT1080 cells led to a significant reduction in ROS generation. In addition, knockdown of IDH1 effectively decreased the migratory potential of cells without affecting cell proliferation (Fig. 2b). In fibrosarcoma cells with moderately elevated ROS levels, diminished ROS generation appears to suppress cell migration.

Fig. 2.

The effects of IDH1 knockdown on ROS production and cell migration in HT1080 cells. a Intracellular ROS levels were compared using DCF-DA staining in HT1080 cells transfected with scrambled or IDH1 siRNA. b Cell migration was assessed at the indicated time points using a wound-healing assay after transfection with scrambled or IDH1 siRNA in HT1080 cells

Inhibitors targeting the IDH1 R132 mutation inhibit cell migration and colony formation in HT1080 cells

We investigated whether inhibitors targeting the IDH1 R132 mutation regulate cell migration and colony-forming ability, as observed after IDH1 knockdown. AGI-5198 and IDH305 are potent and selective inhibitors targeting the IDH1 R132 mutation [32, 33]. We observed a concentration-dependent inhibition of cell migration in AGI-5198 and IDH305-treated cells compared to untreated cells under serum-free conditions (Fig. 3a). It is well established that slug and vimentin enhance cancer cell proliferation and migratory ability [34], and vimentin is necessary for slug-induced migration [35]. We observed that AGI-5198 and IDH305 reduced vimentin and slug mRNA expression (Fig. 3b) and decreased colony-forming capacity of HT1080 cells (Fig. 3c). These results indicate that the IDH1 mutation may contribute to the migratory and malignant properties of fibrosarcoma.

Fig. 3.

The effects of R132 mutation inhibitors on cell migration and colony formation in fibrosarcoma HT1080 cells. a Cell migration was evaluated using a wound-healing assay in HT1080 cells treated with AGI-5198 (10 and 20 μM) or IDH305 (10 and 20 μM) for the indicated times. b Vimentin and slug mRNA levels were analyzed by RT-PCR in cells treated with inhibitors targeting IDH1 mutation. c The clonogenic efficiency of HT1080 cells treated with AGI-5198 or IDH305 was assessed as described in the “Materials and methods” section. After 10 days of incubation, the cells were stained with crystal violet for visualization under a microscope. The graph represents the numbers of colonies that were larger than 500 μm. The values are indicated as mean ± SD (n = 3)

Blocking IDH1 suppresses the expression of programmed death-ligand 1

Programmed death-ligand 1 (PD-L1) expression in cancer cells is widely recognized as a protective mechanism against immune system surveillance [36]. We confirmed that inhibiting IDH1 expression using IDH1 siRNA decreased PD-L1 mRNA and protein expression (Fig. 4a). In addition, inhibitors targeting the IDH1 mutation similarly decreased PD-L1 expression (Fig. 4b). In the tumor microenvironment, PD-L1 expression is influenced by various factors, such as inflammatory signals and the oncogenic pathway. It is well established that several transcription factors, including MYC, NF-κB, STAT1/3, HIF-1α, and Nrf2, play key roles in regulating PD-L1 expression [37, 38]. Specifically, PD-L1 is known to be a direct transcriptional target of Nrf2 [39]. In line with this report, Nrf2 knockdown in HT1080 cells led to a decrease in PD-L1 mRNA and protein expression (Fig. 4c). These results suggest that PD-L1 may play a critical role in immune evasion mediated by IDH1 and Nrf2 in fibrosarcoma.

Fig. 4.

Suppression of PD-L1 expression by IDH1 inhibition. In HT1080 cells transfected with scrambled, IDH1 (a), or Nrf2 (c) siRNAs for 48 h, mRNA and protein levels of PD-L1 were assessed using real-time PCR and immunoblot analysis. b PD-L1 mRNA and protein expression levels were also measured in HT1080 cells treated with the IDH1-targeted inhibitors, AGI-5198 and IDH305

Discussion

IDH1 mutations are prevalent genetic alterations in human cancers [6–10, 12–14], however, the development of selective therapeutics inhibitors targeting IDH1 mutations remains unresolved. The neomorphic activity of IDH1 mutations profoundly affects cellular biology, causing metabolic reprogramming, genome-wide DNA hypermethylation, and disrupted redox homeostasis. IDH1 mutations lead to the loss of wild-type IDH1 functions, resulting in decreased NADPH and GSH levels, along with increased ROS levels [40–42]. According to several studies, overexpression of wild-type IDH1 has been observed in various cancers, such as non-small cell lung carcinoma [43–45], melanoma [11] and pancreatic adenocarcinoma [46, 47]. Wild-type IDH1, along with mutant IDH1, is gaining attention as a therapeutic target, emphasizing the increasing necessity for further research. In this study, we revealed that IDH1 affects redox homeostasis, which became the main focus of our investigation.

As previously discussed, ROS function as a double-edged sword in cancer cells. While excessive ROS levels trigger cancer cell death, moderate ROS levels support cell survival and promote tumor progression [23–25]. Our findings demonstrate that IDH1 inhibition reduces ROS generation, thereby suppressing Nrf2 activation, a critical regulator of cellular defense mechanisms. This effect is likely attributed to reduced ROS production, which is essential for maintaining cell survival. Consistent with our findings, salidroside has been reported to inhibit tumor cell metastasis by reducing excess intracellular ROS, thereby downregulating the ROS-PKC signaling pathway [48]. Similarly, dieckol suppresses cell migration by downregulating FAK signaling through scavenging intracellular ROS, suggesting that reducing ROS levels can inhibit migration in HT1080 cells [31].

IDH gene mutations have been detected in various solid tumors, including glioma, cholangiocarcinoma, and chondrosarcoma. Targeting mutated IDH enzymes has emerged as a promising therapeutic strategy for solid tumors, with ongoing advancements in current clinical trials [5, 49, 50]. IDH inhibitors are clinically useful both as standalone treatments and in combination with drugs targeting various pathways. In particular, although inhibiting mutant IDH1 has been suggested to regulate Nrf2 signaling, a combination therapy using mutant IDH1 inhibitors and targeting active Nrf2 regulation may be more effective.

The redox status in tumors may be influenced by the emergence and inhibition of IDH1 mutations. Glioma cells harboring IDH1 mutations display elevated levels of ROS stress compared to their wild-type IDH1 counterparts [51]. IDH1 mutations disturb the NADP⁺/NADPH balance in cancer cells, leading to an increased reliance on glutathione metabolism. In this context, Nrf2 is essential for enhancing glutathione synthesis and mitigating ROS levels, indicating a dependency on Nrf2-regulated signaling pathways [52]. Notably, Nrf2-associated signaling pathways are highly activated to sustain redox homeostasis and support cell survival in IDH1-mutant cancer cells [26, 51]. IDH1 activates Akt, disrupting the Nrf2 degradation pathway and leading to Nrf2 stabilization. As a transcription factor, Nrf2 promotes the expression of ROS-scavenging genes, thereby providing protection against ferroptosis [53]. In line with these findings, our study demonstrated that Nrf2 is activated in fibrosarcoma cells carrying mutant IDH1, and that inhibiting IDH1 attenuates the activity of Nrf2-associated downstream signaling pathways.

Constitutive activation of Nrf2 has been observed in various malignancies, including breast, liver, lung, pancreatic, ovarian, and endometrial cancers [14, 54–60]. Nrf2 plays a pivotal role in supporting cancer cell survival by promoting tumor progression, enhancing therapy resistance, and facilitating immune evasion [39, 54, 57, 61–64]. Nrf2 overexpression has been associated with resistance to PD-L1 blockade therapy, indicating a potential connection between Nrf2 and the PD-L1/PD-1 axis [64]. In melanoma, Nrf2 has been identified as an upstream regulator of PD-L1 expression. Nrf2 inhibition significantly decreases PD-L1 levels while enhancing T-cell infiltration into tumor sites [39, 64]. The present study indicates that IDH1 mutations may facilitate immune evasion in fibrosarcoma cells by upregulating PD-L1 through an Nrf2-dependent mechanism, as illustrated in Fig. 5. Such a regulatory mechanism can also be applied to other types of cancer with IDH1 mutations. In agreement with our study, IDH1 mutations reprogram ROS homeostasis in glioma, creating a dependency on the Nrf2 antioxidant pathway for effective ROS scavenging [26]. In glioma, Nrf2 regulation has also been shown to be associated with immune evasion. IDH1 mutations affect the immune microenvironment by decreasing the number of immune cells, such as T cells, B cells, and macrophages [65, 66]. We anticipate that future studies will require further expansion and validation across various cell lines and animal models.

Fig. 5.

A proposed mechanism for the role of mutant IDH1 in ROS regulation and Nrf2-mediated signaling. Both wild-type and mutant IDH1 influence intracellular NADPH production, ROS levels, and Nrf2 signaling, indicating that these pathways can be modulated according to IDH1 status. a Wild-type IDH1 catalyzes the conversion of isocitrate to α-ketoglutarate, generating NADPH, which contributes to the suppression of ROS and reduction of oxidative stress. b Mutant IDH1 abnormally converts isocitrate into 2-hydroxyglutarate, depleting NADPH and increasing NADP⁺ levels, ultimately leading to reduced NADPH availability and elevated ROS levels. The ROS induced by the IDH1 mutation activate Nrf2, a key transcription factor that regulates the expression of antioxidant proteins and PD-L1, thereby enhancing resistance to oxidative stress and promoting immune evasion in cancer cells. In other words, mutant IDH1 may promote the survival and immune evasion of cancer cells through Nrf2-mediated pathways. In mutant IDH1-expressing cancer cells, inhibiting IDH1 reduces ROS production, which, in turn, suppresses Nrf2 activity and leads to the downregulation of antioxidant enzymes and PD-L1 expression

Previously, research targeting IDH1 mutations primarily focused on glioma and leukemia. Ivosidenib and olutasidenib, the first identified IDH1 inhibitors, have demonstrated clinical efficacy in patients with mutant IDH1 acute myeloid leukemia [67, 68]. Recently, studies have increasingly emphasized their roles in solid tumors, including cholangiocarcinoma and chondrosarcoma [27, 69]. Notably, with 50–80% of chondrosarcomas harboring IDH gene mutations, researchers are focused on identifying inhibitors that target these mutations [70]. In a Phase I trial, ivosidenib demonstrated favorable tolerability, resulting in significant 2-HG reduction and a progression-free survival of 5.6 months in patients with advanced chondrosarcoma [71]. The clinical application of IDH1 mutation-targeting inhibitors shows great promise, underscoring the need for further research on their mechanisms of action and the development of inhibitors. Moreover, depletion of mutant IDH1 has been shown to suppress the tumorigenic potential in chondrosarcoma cell lines harboring IDH1 mutations by decreasing the expression of integrins α5 and β5 [27]. This aligns with our findings, which demonstrate that suppressing Nrf2 expression decreases cell migration. Reports also suggest that chemokines, such as C–C motif chemokine ligand 2 secreted in IDH1-mutant cholangiocarcinoma, contribute to tumor growth and immune suppression [69]. C–C motif chemokine ligand 2 stimulates M2-like macrophages, which in turn release immunosuppressive substances [69]. Additionally, based on our findings, it is evident that Nrf2 may play a critical role in this context, thereby warranting further research. Moreover, our observation that IDH1 regulation suppresses PD-L1 expression appears to be closely related to these findings. Overall, we have highlighted the significance of developing inhibitors for cancers harboring the IDH1 R132 mutation. The findings of this study should be further validated in additional fibrosarcoma cell lines, other cancer types, and more in vivo experiments.

Supplementary Information

Below is the link to the electronic supplementary material.

Funding

This work was supported by Kyonggi University’s Graduate Research Assistantship 2025 and the Basic Science Research Program through the National Research Foundation of Korea (NRF) funded by the Ministry of Science and ICT (NRF-2020R1A2C1103139 to D.-H.K. and IRIS-RS-2024-00451538 to K.-S.C).

Data availability

The datasets generated during and/or analyzed during the current study are available from the corresponding author on reasonable request.

Declarations

Conflict of interest

The authors declare that they have no conflict of interest. Kyung-Soo Chun was the Associate Editor of Toxicological Research while the manuscript of this article was submitted and peer-reviewed. Editorial Board Member status has no bearing on editorial consideration.