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GPX2 inhibition enhances antitumor efficacy of lenvatinib via promoting immunogenic cell death in hepatocellular carcinoma

Journal of Translational Medicine volume 23, Article number: 456 (2025) Cite this article

Abstract

Background

Immunogenic cell death (ICD) is a distinct subtype of regulatory cell death, and represents a potential mechanism to remodel the tumor microenvironment. Lenvatinib is established as the first-line therapy for advanced hepatocellular carcinoma (HCC), but drug resistance limits its efficiency. Our previous research showed that lenvatinib can inhibit GPX2 expression and induce reactive oxygen species (ROS)-related cell apoptosis in HCC. The present study intends to explore whether lenvatinib can induce ICD and clarify its underlying mechanisms in HCC.

Methods

Flow cytometry was utilized to detect the expression levels of CRT and CD markers, measure intracellular ROS levels, and assess cell apoptosis. Western blot analysis was employed to determine changes in protein levels, while qRT-PCR analysis was used to quantify alterations in mRNA levels. Subcutaneous allograft tumor models were established to investigate the mechanism of lenvatinib against HCC. Immunohistochemical (IF) staining were used to detect the ratio of CD8+GZMB+ cells.

Results

Herein, we found that HCC cells treated with lenvatinib or si-GPX2 showed increased ICD markers, such as CRT exposure, ATP secretion, and HMGB1 release. Notably, we demonstrated that lenvatinib promoted dendritic cells (DCs) maturation and CD8+ T cells activation, thus inducing HCC cell apoptosis when co-cultured with peripheral blood mononuclear cells. Additionally, we further demonstrated that lenvatinib or GPX2 inhibition triggers endoplasmic reticulum stress (ERS) in HCC cells, which is mediated by the accumulation of ROS. Our findings indicate that pre-treatment with the antioxidant N-acetylcysteine suppressed lenvatinib-induced expression of CRT on the cell membrane, ATP secretion and HMGB1 release, and inhibited lenvatinib-induced cell apoptosis. Furthermore, we also found that ERS inhibitor ISRIB could reverse lenvatinib-induced upregulation of ICD biomarkers. Moreover, we further identified that downregulation of GPX2 enhanced the efficacy of lenvatinib via triggering ERS-mediated ICD in HCC.

Conclusions

This study uncovered that lenvatinib could be a potent ICD inducer, which could trigger ERS via increasing ROS levels in HCC cells, which present valuable insights into the mechanism of lenvatinib-induced ICD in HCC cells. Collectively, our findings highlight the significant therapeutic potential of the combination of targeting GPX2 and treatment with lenvatinib for HCC.

Background

Hepatocellular carcinoma (HCC) is one of the most common malignancies of the digestive system. Approximately 368,000 people were diagnosed with HCC in 2022, and about 317,000 patients died from the disease in China [1, 2]. Because of lacking specific early symptoms and effective examination methods, most cases are diagnosed at advanced stages. In recent years, the progress of systematic treatment has improved the prognosis of some patients, but the overall 5-year survival rate of HCC is still less than 15% [3, 4]. Therefore, identifying biomarkers for the early diagnosis of HCC and exploring novel molecular targets are crucial to improve the prognosis of patients with HCC.

Lenvatinib has been approved as the first-line therapy for patients with advanced HCC, based on the REFLECT trial, which demonstrated its non-inferiority to sorafenib with respect to efficacy [5]. Recently, combination therapies of multi-targeted kinase inhibitors and immune checkpoint inhibitors (ICIs) have emerged as the premier first-line therapeutic approach for advanced HCC, owing to their superior response rates and survival benefits as evidenced in numerous Phase III clinical trials [6,7,8]. It would be logical to expect that lenvatinib combined with an ICI might exhibit superior performance compared to lenvatinib monotherapy. However, the LEAP 002 trial demonstrated that lenvatinib plus pembrolizumab failed to achieve improved progression-free survival (PFS) and overall survival (OS) [9]. Therefore, further studies to explore the impact of lenvatinib on immunotherapy and to elucidate its underlying mechanisms are urgently important to improve the prognosis for advanced HCC patients.

Immunogenic cell death (ICD) is a specific type of regulatory cell death that can trigger adaptive immune responses directed against antigens of dead cells [10]. This type of cell death was initially observed during treatment with conventional chemotherapy drugs, and it may serve as a trigger for immune responses [11,12,13]. Upon stimulation by ICD inducers, cancer cells produce novel antigenic epitopes and release damage-associated molecular patterns (DAMPs), including exposure of calreticulin (CRT) on the cell membrane (“eat me” signal), ATP secretion (“find me” signal), and HMGB1 release (“present me” signal). Subsequently, these patterns are recognized and bound by dendritic cells (DCs), and then presented to T cells, thereby activating adaptive immune responses [14]. Previous studies have identified that lenvatinib could reduce the infiltration of Treg cells, monocytes, macrophages, while increasing the populations of CD8+ T cells in HCC tumor microenvironment [15]. Besides, it could also exert immunomodulatory effects through suppression of programmed death-L1 (PD-L1) expression and inhibition of the TGF-β signaling pathway [16, 17]. However, the potential of lenvatinib to stimulate the release of novel tumor antigens, subsequently initiating ICD, remains to be further investigated.

In this study, we demonstrated that lenvatinib could induce ICD through inhibiting GPX2 expression in vivo and in vitro. Knockdown of GPX2 could augment endoplasmic reticulum stress (ERS) and further enhance ICD induced by lenvatinib. These results support the potential of combining GPX2 inhibition with lenvatinib or immunotherapy as a promising therapeutic strategy to improve the prognosis of patients with HCC.

Materials and methods

Cell lines and culture

The human HCC cell line Huh7 and the mouse HCC cell line Hepa 1–6 were procured from the Cell Bank of the Chinese Academy of Sciences (Shanghai, China). HCC cells were cultured in RPMI 1640 medium or Dulbecco's Modified Eagle's Medium (DMEM; Gibco), supplemented with 10% fetal bovine serum (FBS; Gibco). The cells were then incubated in a 5% CO2 environment at 37 °C.

Metarials and antibodies

Lenvatinib was purchased from Selleck (Houston, TX, USA). N-acetyl-L-cysteine (NAC, A9165) and ISRIB (SML0843) were obtained from Sigma-Aldrich (St. Louis, MO). Primary antibodies included ATF4 (ET1612-37), CHOP (ET1703-05), PERK (ER64553), calreticulin (CRT, ET1608-60), and β-actin (HA722023), were purchased from HUABIO (Hangzhou, China). Anti-Phospho-PERK antibody (p-PERK, DF7576) was obtained from Affinity Biosciences, while anti-GPX2 (ab140130), anti-Granzyme B antibodies were from Abcam. CD8 monoclonal antibody (66868-1) was obtained from Proteintech.

Lentivirus and siRNA transfection

siRNAs targeting GPX2 and a negative control (si-NC) were designed and synthesized by Sangon Biotech (Shanghai, China). The siRNAs were transfected into HCC cells using RNATransMate (Sangon Biotech) according to the manufacturer’s instructions. The target sequences of GPX2 for human and mouse are 5′-ACATCAAGCGCCTCCTTAA-3′ and 5′-CCACCTTTAGTCTTACCCAAA-3′. To knockdown GPX2 in mouse HCC cells, Hepa 1–6 cells were transfected with sh-GPX2 (LV-GPX2-RNAi) lentivirus, along with a negative control sh-NC lentivirus. All lentiviruses were purchased from GeneChem (Shanghai, China).

ATP release assays

Huh7 and Hepa 1–6 cells were seeded in 6-well plates and transfected with si-GPX2 or treated with lenvatinib for 24 h. ATP levels were measured using the ATP Assay Kit (Beyotime) according to the manufacturer’s instructions. The luminescence (RLU) values of the ATP standards were plotted against their respective concentrations, enabling a direct readout of extracellular ATP levels from the standard curve.

Enzyme-linked immunosorbent assay (ELISA)

Huh7 and Hepa 1–6 cells were seeded in 6-well plates and transfected with si-GPX2 or treated with lenvatinib for 24 h. Subsequently, the supernatants were collected for detection using an ELISA kit. HMGB1 concentrations in the conditioned medium were measured using the E-UNEL-H0065 ELISA kit (Elabscience). Similarly, the levels of interleukin (IL)-2 and interferon (IFN)-γ in the cell culture supernatants were quantified using ELISA kits (SM2000 and MIF00, respectively; R&D Systems) following the manufacturer's instructions.

DCs culture and T cells isolation

Peripheral blood mononuclear cells (PBMCs) were acquired from LEIDEBIO in Guangzhou, China. Following overnight culture to allow cell adhesion, suspended cells were removed. The PBMCs were then cultured in complete medium supplemented with GM-CSF (20 ng/mL) and IL-4 (20 ng/mL) for 5 days to promote differentiation into DCs, which were collected for further experiments. To assess T cell activation, PBMCs were stimulated with CD3 (2 μg/mL) and CD28 (1 μg/mL, BioLegend) for 48 h [18]. Huh7 cells were then treated with lenvatinib for 24 h and co-cultured with the activated PBMCs for another 24 h. Finally, the cells were harvested and labeled with CD3 and CD8 fluorescent antibodies for analysis.

Flow cytometry detection of surface CRT and CD markers

Flow cytometry was employed to precisely detect CRT on the cell membrane, quantify the percentage of dendritic cells (DCs) and CD8+ T cells. The lenvatinib-treated tumor cells were collected and washed twice with PBS, then incubated with Alexa Fluor 488-conjugated CRT (Cell Signaling Technology, USA). Flow cytometry was used to quantify and analyze surface CRT levels. Additionally, antibodies against CD80 (FITC-conjugated, Biolegend), CD86 (PE-conjugated, Biolegend), CD3 (PerCP/cyanine5.5-conjugated, BioLegend) and CD8 (PE/cyanine7-conjugated, BioLegend) were used to stain mature DCs and CD8+ T cells.

Western blotting, qRT-PCR, ROS levels and cell apoptosis detection

The ROS assay kit sourced from Beyotime (China) was utilized to quantify the levels of intracellular reactive oxygen species (ROS). Cell apoptosis was assessed through flow cytometry, employing Annexin V-FITC and propidium iodide (PI) as staining reagents. Western blot and qRT-PCR assays were conducted following previously described. The procedural details for the experiment were carried out in accordance with the methods described in our previous study [19]. The PCR primers are shown in Table S1.

Mouse model and treatments

C57BL/6 mice were purchased from GemPharmatech Co. Ltd and housed in a pathogen-free environment at the laboratory animal center of Sun Yat-Sen University, adhering to the guidelines of the National Institutes of Health of the United States. A total of 5 × 106 Hepa1‐6 cells transfected with sh-NC or sh-GPX2 were resuspended in 200 µL of PBS and subsequently injected into the dorsal region of C57BL/6 mice. The mice were then treated with lenvatinib (20 mg/kg/d) or control solvent. The length and width of subcutaneous tumors were measured with a caliper every three days, and the subcutaneous tumor volume was calculated as (length × width2)/2. The mice were euthanized when the subcutaneous tumor reaches 1.5 cm in length and the tumors were removed for further processing. The Committee on the Ethics of Animal Experiments at Sun Yat-sen University approved the animal experiments (permit number: SYSU-IACUC-2024-002020).

Immunohistochemical (IF) and immunofluorescence (IHC) stainings

The tumor tissue was embedded, sectioned, and deparaffinized in xylene twice for 10 min each. Then, the sections were dehydrated in 70%, 85%, and 100% ethanol for 5 min each. After rehydration, slides were immersed in boiling EDTA-retrieval solution for 15 min to facilitate antigen retrieval. Subsequently, they were permeabilized with 0.4% Triton X-100 for 30 min, and then blocked with goat serum for 1 h at room temperature. Next, the sections were incubated with antibodies (Granzyme B, CD8, CRT, HMGB1, and GPX2) at 4 °C overnight. For IHC staining, samples were washed and incubated with horseradish peroxidase (HRP)-conjugated secondary antibodies, and then detected with diaminobenzidine (DAB). For IF staining, sections were incubated with CoraLite594/488-conjugated secondary antibodies (Elabscience) for 1 h, and then counterstained with DAPI. Finally, fluorescence images were obtained with a fluorescence microscope (IX73, Olympus).

Statistical analyses

The data are presented as the mean ± SD of three independent assays. Statistical analysis was performed using IBM SPSS Statistics 26.0 (IBM Corp., Armonk, NY, USA) with unpaired t-tests, paired t-tests, and one-way and two-way ANOVA. P < 0.05 was considered to indicate a statistically significant difference.

Results

Lenvatinib triggers ROS mediated ICD in HCC cells

HCC cells were treated with lenvatinib to detect the release of DAMPs, including CRT, HMGB1, and ATP. IF assays showed that lenvatinib treatment could promote the cytomembrane translocation of CRT in HCC cells (Fig. S1A and B), which delivered the "eat me" signal to the immune system and stimulated DC maturation. Numerous evidence support the notion that excessive generation of reactive oxygen species (ROS) can induce ICD in tumor cells [20,21,22]. Recently, we found that lenvatinib can elevate ROS levels and induce apoptosis in HCC cells [19]. Herein, we also found that lenvatinib markedly increased the ROS levels in Huh7 and Hepa 1–6 cells (Fig. S1C and D). Then, we pre-treated HCC cells with NAC (4 mM) to neutralize cellular ROS [23], and we found that NAC suppressed the lenvatinib-induced apoptosis in HCC cells (Fig. S1E and F).

Additionally, flow cytometry assay demonstrated that NAC pre-treatment reversed lenvatinib-induced CRT translocation to the cell membrane (Fig. 1A and B). The extracellular release of HMGB1 into the supernatant was quantified with ELISA, and the results identified that HMGB1 levels were much higher in the supernatant of the lenvatinib-treated group compared to the control group, while pre-treated with NAC markedly impaired the secretion of HMGB1 in Huh7 and Hepa 1–6 cells (Fig. 1C and D). Moreover, the ATP levels in the supernatant of the lenvatinib-treated group were significantly elevated compared to the control group, and this increase was attenuated by NAC treatment (Fig. 1E and F). Collectively, these findings suggest that lenvatinib-mediated upregulation of ICD markers is triggered by elevated ROS levels.

Fig. 1
figure 1

The antioxidant N-Acetylcysteine (NAC) could impair the upregulation of ICD markers induced by lenvatinib in HCC cells. A and B Flow cytometry demonstrated the effect of 4 mM NAC pretreatment on CRT translocation to the membranes of HCC cells induced by lenvatinib. C and D The levels of HMGB1 in cell supernatant after different treatments of Huh7 and Hepa1-6 cells were quantified using ELISA assays. E and F The ATP level was analyzed after lenvatinib or NAC treatment in the cell supernatant of Huh7 and Hepa1-6 cells. Each experiment is conducted in triplicate, and the represented data are SD (±) mean for each experiment. *P < 0.05

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Lenvatinib induces ERS in HCC cells

Growing evidence indicates that excessive ROS can lead to endoplasmic reticulum stress (ERS) and subsequently trigger ICD [24]. To evaluate whether lenvatinib could cause ERS in HCC cells, we measured the ERS-related markers in HCC cells by Western blotting. The results showed that lenvatinib increased the expression of p-PERK, ATF4 and CHOP in a dose-dependent manner (Fig. S2A–D). Comparable results were replicated in the mouse HCC cell line Hepa 1–6 (Fig. S2E–H). Subsequently, we employed the ERS inhibitor ISRIB (0.2 μM) in combination with lenvatinib in HCC cells [25]. Notably, ISRIB effectively reversed the up-regulation of p-PERK, ATF4, and CHOP in both Huh7 and Hepa 1–6 cells (Fig. 2A and B and S3A, B). Furthermore, flow cytometry identified that lenvatinib could markedly induce CRT to translocation to the cell membrane, while ISRIB impaired these effects in Huh7 and Hepa 1–6 cells (Fig. 2C and S3C). Additionally, similar trends were observed in ATP and HMGB1 secretion by HCC cells treated with lenvatinib and ISRIB (Fig. 2D-E, and S3D-E). Collectively, these findings suggest that lenvatinib triggers ICD in HCC cells by inducing ERS.

Fig. 2
figure 2

Lenvatinib triggers ERS and further inducing ICD in HCC cells. A Western blotting was used to detect the effect of lenvatinib combined with an ERS inhibitor ISRIB (0.2 μM) on the expression of ATF4, CHOP, and p-PERK in Huh7 cells. B The gray value results of the western blotting experiment in Huh7 cells. C Flow cytometry demonstrated the effect of ISRIB combined with lenvatinib on CRT translocation to the membranes of HCC cells. D The HMGB1 levels in cell supernatant after treatments of ISRIB combined with lenvatinib in Huh7 cells. E The ATP level was analyzed after lenvatinib or ISRIB treatment in the cell supernatant of Huh7 cells. Each experiment is conducted in triplicate, and the represented data are standard deviation (SD) (±) mean for each experiment. *P < 0.05

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Inhibiting GPX2 triggers ERS and promotes ICD in HCC cells

Since the accumulation of ROS elicits oxidative stress responses, ultimately resulting in ERS, and we recently demonstrated that inhibiting GPX2 could increase ROS levels and enhance the antitumor effect of lenvatinib [19]. Therefore, we further explored the correlation of GPX2 expression on ERS activation to induce ICD in HCC cells. The western blotting analysis showed that inhibition of GPX2 increased the protein expression of p-PERK, ATF4, and CHOP, and qRT-PCR results also confirmed that si-GPX2 increased the mRNA expression of ATF4 and CHOP in HCC cells (Fig. 3A–C). We pre-treated HCC cells with NAC prior to GPX2 knockdown. Notably, GPX2 depletion robustly upregulated ER stress markers (p-PERK, ATF4, CHOP) in both Huh7 and Hepa1-6 cells, while NAC pretreatment completely abolished these ER stress responses (Fig. S4A and B). In addition, the IF assay demonstrated that inhibition of GPX2 could promote exposure of CRT on the cell membrane in HCC cells (Fig. 3D). Moreover, ELISA assay also confirmed that downregulation of GPX2 could induce secretion of HMGB1 and ATP in the supernatant of HCC cells (Fig. 3E and F).

Fig. 3
figure 3

Inhibiting GPX2 triggers ERS and promotes ICD in HCC cells. A and B The protein levels of p-PERK, ATF4, and CHOP were quantitatively analyzed by western blotting after inhibiting GPX2 in Huh7 and Hepa1-6 cells. C qRT-PCR analysis determined the mRNA expression levels of PERK, ATF4, and CHOP in Huh7 and Hepa1-6 cells. D Immunofluorescence detected CRT translocation to the membranes of Huh7 and Hepa1-6 cells following GPX2 knockdown. Scale bar: 100 μm. E and F The levels of HMGB1 and ATP in the supernatant of Huh7 and Hepa1-6 cells were analyzed after GPX2 knockdown. Each experiment is conducted in triplicate, and the represented data are SD (±) mean for each experiment. *P < 0.05

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GPX2 downregulation enhances the efficacy of lenvatinib against HCC via activating DCs-mediated T cells killing ability

It is thought that the release of HMGB1 and ATP serves as late apoptotic markers, and is crucial for the presentation of tumor antigens to DCs. Thus, we assessed if downregulation of GPX2 or treatment with lenvatinib triggered the activation of DCs and T cells. Briefly, Huh7 cells were treated with si-GPX2 or lenvatinib for 24 h, followed by co-culture with activated PBMCs. Flow cytometry analysis revealed a significant upregulation of the maturation biomarkers of DCs, CD80 and CD86, in co-cultured HCC cells treated with si-GPX2 or lenvatinib. Notably, the combination of these two treatments yielded a stronger induction of CD80 and CD86 compared to either treatment alone (Fig. 4A and B). Consistently, our results also indicated a significant increase in the percentage of CD3+CD8+ T cells in co-cultures with HCC cells treated with si-GPX2 or lenvatinib (Fig. 4C). Furthermore, the supernatant levels of T cell-derived cytokines IL-2 and IFN-γ were substantially upregulated in co-cultures with the treated tumor cells. Notably, the combination therapy group exhibited the highest levels of CD3+CD8+ T cells, IL-2, and IFN-γ (Fig. 4D and E). Additionally, we conducted flow cytometry to detect apoptosis in HCC cells, with or without co-culture with PBMCs. The results confirmed that inhibiting GPX2 markedly enhanced apoptosis in Huh7 cells. Remarkably, when co-cultured with PBMCs, a much higher level of apoptosis was induced in Huh7 cells (Fig. S5). These findings indicated that si-GPX2 or lenvatinib facilitated the maturation of DCs, thereby inducing the activation of CD8+ T cells.

Fig. 4
figure 4

GPX2 downregulation augments lenvatinib's efficacy against HCC by activating DC-mediated T-cell cytotoxicity. A and B Flow cytometry was utilized to quantify the expression of CD80 and CD86 biomarkers in mature DCs co-cultured with Huh7 cells treated with either si-GPX2 or lenvatinib. C The percent of CD3+CD8+ T cells was detected by flow cytometry following the co-culture of PBMCs with Huh7 cells treated with either si-GPX2 or lenvatinib. D and E ELISA assay detected IFN-γ and IL-2 levels in the cell supernatant of different treatment groups. Each experiment is conducted in triplicate, and the represented data are SD (±) mean for each experiment. *P < 0.05

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Inhibition of GPX2 effectively improves the therapeutic effects of lenvatinib via promoting ERS against HCC in vivo

To explore the correlation of lenvatinib and GPX2 inhibition in regulating ERS in vivo, Hepa1-6 cells transfected with sh-GPX2 or sh-NC were randomly assigned and then injected into the backs of C57BL/6 mice. The analysis of tumor growth and tumor weight demonstrated that treatment with either lenvatinib or sh-GPX2 alone partially reduced tumor burden compared to the control group, whereas the combination of sh-GPX2 and lenvatinib resulted in the most pronounced inhibition of tumor growth (Fig. 5A–C). Subsequently, Western blotting results identified that lenvatinib and sh-GPX2 could markedly decrease the expression of GPX2 in HCC tumors (Fig. 5D and E). Western blotting also revealed that treatment with either lenvatinib or sh-GPX2 alone significantly upregulated p-PERK, ATF4, and CHOP expression, whereas the combined treatment of lenvatinib and sh-GPX2 resulted in a pronounced increase in the levels of these ERS-related markers (Fig. 5F-H).

Fig. 5
figure 5

Inhibition of GPX2 improves the therapeutic effects of lenvatinib against HCC via promoting ERS in vivo. A Images of subcutaneous tumors in C57BL/6 mice treated with lenvatinib after subcutaneous injection of Hepa1-6 cells transfected with sh-NC or sh-GPX2 lentivirus (n = 6 per group). B A quantification of the tumor weight was performed for each group of subcutaneous tumors. C Time-dependent growth curves of subcutaneous tumor volumes were plotted. D Western blotting to detect the effect of lenvatinib or sh-GPX2 on the expression of GPX2, ATF4, CHOP, and p-PERK in subcutaneous tumors. E–H The gray value results of the Western blotting experiment in subcutaneous tumor groups. Each experiment is conducted in triplicate, and the represented data are SD (±) mean for each experiment. *P < 0.05

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GPX2 downregulation enhances the antitumor effect of lenvatinib via inducing ICD in HCC in vivo

We further investigated whether inhibiting GPX2 expression can modulate the immune microenvironment and enhance the antitumor efficacy of lenvatinib in vivo. Consistently, IHC analysis confirmed that either lenvatinib or sh-GPX2 effectively inhibited the expression of GPX2, while concurrently enhancing the expression of HMGB1 in HCC tumors (Fig. 6A–C). Notably, IHC and IF assays revealed that the combination of lenvatinib and sh-GPX2 led to a greater upregulation of HMGB1 and CRT expression compared to either treatment alone (Fig. 6C and D). In addition, our findings identified that both lenvatinib treatment and sh-GPX2 individually enhanced the infiltration of CD8+GZMB+ T cells by IF assay, while the combination of lenvatinib and sh-GPX2 treatment resulted in a remarkable increase in CD8+GZMB+ T cell infiltration compared to individual treatments (Fig. 6E). The combination of targeted therapies with immune checkpoint inhibitors like anti-PD-1 and its ligand PD-L1, has emerged as a promising strategy for HCC. Our extended analysis revealed that both lenvatinib treatment and GPX2 downregulation suppress PD-L1 expression in tumor tissues, with the combination therapy showing additive inhibitory effects (Fig. S6A and B). Collectively, these findings indicate that inhibition of GPX2 not only effectively improves the therapeutic effects of lenvatinib by promoting the induction of ICD in HCC in vivo, but also may enhance PD-L1 blockade efficacy through coordinated molecular regulation.

Fig. 6
figure 6

GPX2 downregulation enhances the lenvatinib’s efficiency against HCC via inducing ICD in vivo. A Representative images of GPX2 and HMGB1 stained by IHC of allograft subcutaneous tumors. Scale bar: 100 μm. B and C Quantitative analysis of GPX2 and HMGB1 expression in subcutaneous tumor tissue by IHC. D Representative images of CRT stained by IF of allograft subcutaneous tumors. Scale bar: 100 μm. E Representative images of CD8+GZMB+ T cell infiltration stained by IF of allograft subcutaneous tumors. Scale bar: 100 μm. Each experiment is conducted in triplicate, and the represented data are SD (±) mean for each experiment. *P < 0.05

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Discussion

Lenvatinib has been approved as the first-line therapeutic option for advanced HCC, and has been shown to significantly prolong overall survival. Nevertheless, drug resistance significantly limits its clinical efficacy [26]. To date, the precise molecular mechanisms of lenvatinib resistance remain poorly understood. Previous studies have revealed that activation of the EGFR-MAPK signaling pathway confers lenvatinib resistance, while combined lenvatinib and EGFR inhibitors (erlotinib or gefitinib) exhibit potent synergistic effects against HCC [27, 28]. Recently, we identified that lenvatinib increases ROS levels and induces apoptosis in HCC cells, and HCC patients with higher expression of GPX2 are more resistant to lenvatinib treatment [19]. In this study, we further confirmed that lenvatinib is a potent ICD inducer, with the ability to elicit antitumor adaptive immune responses. Additionally, we present compelling evidence that lenvatinib induces tumor cell ICD by specifically targeting GPX2. These findings suggest a pivotal role for inhibition of GPX2 in mediating the activation of immune microenvironment that potentiates the efficacy of lenvatinib.

The integration of ROS-generating agents holds promise as a potential strategy for cancer therapy [29]. Excessive ROS accumulation triggers oxidative stress responses, and leads to ERS. Previous studies have shown that sorafenib elevates intracellular ROS levels, and further promotes DNA damage to induce ERS, ultimately triggering apoptosis [30,31,32]. The excessive ERS response represents a novel strategy that harnesses the potential of ROS-mediated ERS augmentation to induce programmed cell death. As a pivotal apoptotic sensor during ERS, CHOP has garnered interest as a potential therapeutic target in cancer [24]. A recent study reveals that higher expression of CHOP is correlated with improved prognosis and survival in HCC patients, and lenvatinib increases CHOP levels via its targeting of LAMTOR5, thereby enhancing apoptotic responses in HCC cells [33]. Consistently, our results demonstrated that lenvatinib dose-dependently elevated ERS markers, including p-PERK, ATF4, and CHOP. Given that suppression of GPX2 leads to enhanced ROS production and subsequently triggers cell apoptosis, we further demonstrated that inhibition of GPX2 significantly potentiates the antitumor efficacy of lenvatinib by eliciting ERS-mediated ICD. These findings reveal that inhibiting GPX2 expression enhances the antitumor efficiency of lenvatinib against HCC through regulating ERS signaling.

It has been demonstrated that chemotherapeutics like oxaliplatin could induce CRT exposure, ATP secretion, and HMGB1 release, consequently triggering ICD and transforming “cold” tumors into more immunogenic “hot” ones [21, 22]. Similarly, other researchers discovered that arsenic trioxide not only elicits ICD, resulting in the recruitment and activation of CD8+ T cells, but also upregulates the expression of PD-L1 on tumor cells, thereby enhancing the therapeutic efficacy of ICIs in HCC [34,35,36]. In the present study, our results confirmed that lenvatinib could promote the release of new tumor antigens that activate the immune microenvironment. An important finding in this study is that lenvatinib potentially prompts ICD in tumor cells, promotes DCs maturation, triggers robust T cells activation, and enhances GZMB and IFN-γ production by CD8+ T cells infiltrating tumors, thereby harnessing T cells immunity for its antitumor efficacy. Previous studies have elucidated the immunomodulatory effects of lenvatinib. On the one hand, lenvatinib exerts extensive influence on immunocytes, notably by diminishing the infiltration of Treg cells, M2 macrophages, and PD-1+ T cells [37], ultimately facilitating the development of long-lasting immune memory. On the other hand, lenvatinib also modulates immune response by inhibiting PD-L1 or TGF-β signaling in tumor cells, highlighting its diverse immunomodulatory capabilities [16]. Although the underlying mechanisms of lenvatinib-induced ICD remain unclear, further investigation is needed to determine whether GPX2 depletion modulates the tumor microenvironment, including T cells, NK cells, and macrophages. Our findings provide pre-clinical evidence that lenvatinib can be used as an ICD inducer, which supports the potential application of combining GPX2 inhibition, lenvatinib, or immunotherapy to improve survival rates among HCC patients.

While our study clarifies the role of GPX2 in HCC progression, several limitations should be noted. First, the possibility of inhibiting GPX2 as a treatment for cancers beyond HCC has not been investigated. Our recent study indicates that GPX2 was overexpressed in multiple gastrointestinal cancers using TCGA data, but its functional importance in other tumor types still needs validation. Second, the generalizability of our results needs confirmation in additional preclinical models that better capture HCC heterogeneity, such as patient-derived xenografts. Moreover, further clinical translation studies are warranted to determine the clinical relevance of these findings and to facilitate the development of personalized treatment strategies [38, 39]. Future research should explore the specific roles of GPX2 across different cancer types and HCC subtypes.

Conclusion

Based on our prior observations that lenvatinib inhibits GPX2 expression and subsequently elevates ROS levels, we have further elucidated that inhibition of GPX2 triggers ERS-mediated ICD and enhances the effectiveness of lenvatinib against HCC via activating DCs-mediated T cells killing ability (Fig. 7). Our findings clearly elucidate the regulatory role of lenvatinib in modulating the HCC immune microenvironment, thereby offering a rationale for the potential exploitation of combined therapeutic strategies involving lenvatinib and GPX2 inhibitors as promising treatments for HCC patients.

Fig. 7
figure 7

Illustration model shows the proposed mechanism of lenvatinib in HCC. Lenvatinib inhibits GPX2 expression and elevates ROS levels, thus promotes ERS and induces ICD in HCC cells, while inhibition of GPX2 triggers ERS-mediated ICD and enhances the effectiveness of lenvatinib against HCC via activating DCs-mediated T cells killing ability

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Data availability

Not applicable.

Abbreviations

HCC:

Hepatocellular carcinoma

ROS:

Reactive oxygen species

ERS:

Endoplasmic reticulum stress

ICD:

Immunogenic cell death

DCs:

Dendritic cells

PBMCs:

Peripheral blood mononuclear cells

NAC:

N-acetylcysteine

ICIs:

Immune checkpoint inhibitors

DAMPs:

Damage-associated molecular patterns

IHC:

Immunohistochemical

ELISA:

Enzyme-linked immunosorbent assay

IF:

Immunofluorescence

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Acknowledgements

Not applicable.

Funding

This project was funded by grants from the Natural Science Foundation of Hunan Province (2025 JJ40100, 2024 JJ6728, and 2025 JJ70052), Health Commission of Hunan Province (Z2023100), National Natural Science Foundation of China (82103221), Hunan Provincial Health High-Level Talent Scientific Research Project.

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Author notes

  1. Yingzheng Tan, Lei Yang and Tao Xu have equally contributed to this work.

Authors and Affiliations

  1. Department of Hepatobiliary and Pancreatic Surgery, Medical Center of Digestive Disease, Zhuzhou Hospital Affiliated to Xiangya School of Medicine, Central South University, Zhuzhou, 412007, Hunan, China

    Tao Xu, Zhijian Zhao, Xun Chen, Caixi Tang & Wenliang Tan

  2. Department of Infectious Diseases, Zhuzhou Hospital Affiliated to Xiangya School of Medicine, Central South University, Zhuzhou, 412007, Hunan, China

    Yingzheng Tan

  3. Department of General Surgery, The Second Affiliated Hospital of Anhui Medical University, Hefei, 230000, Anhui, China

    Lei Yang

  4. Department of Hepatobiliary Surgery, Sun Yat-sen Memorial Hospital, Sun Yat-sen University, Guangzhou, 510120, Guangdong, China

    Qingbin Wang

  5. Department of Pathology, Zhuzhou Hospital Affiliated to Xiangya School of Medicine, Central South University, Zhuzhou, 412007, Hunan, China

    Meiyuan Huang

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Contributions

Caixi Tang and Xun Chen: Conceptualization and Project administration. Lei Yang: Investigation and Writing—Original Draft. Wenliang Tan: Data curation and Funding acquisition. Meiyuan Huang: Methodology and Validation. Zhijian Zhao: Resources and Supervision. Qingbin Wang and Tao Xu: Software and Visualization. Yingzheng Tan: Writing—Review & Editing and Funding acquisition. All authors contributed to the article and approved the submitted version.

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Correspondence to Xun Chen, Caixi Tang or Wenliang Tan.

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Tan, Y., Yang, L., Xu, T. et al. GPX2 inhibition enhances antitumor efficacy of lenvatinib via promoting immunogenic cell death in hepatocellular carcinoma. J Transl Med 23, 456 (2025). https://doiorg.publicaciones.saludcastillayleon.es/10.1186/s12967-025-06468-5

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Journal of Translational Medicine

ISSN: 1479-5876

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