Ferroptosis is induced by lenvatinib through fibroblast growth factor receptor‐4 inhibition in hepatocellular carcinoma

Abstract The tyrosine kinase inhibitor lenvatinib is used to treat advanced hepatocellular carcinoma (HCC). Ferroptosis is a type of cell death characterized by the iron‐dependent accumulation of lethal lipid reactive oxygen species (ROS). Nuclear factor erythroid‐derived 2‐like 2 (Nrf2) protects HCC cells against ferroptosis. However, the mechanism of lenvatinib‐induced cytotoxicity and the relationships between lenvatinib resistance and Nrf2 are unclear. Thus, we investigated the relationship between lenvatinib and ferroptosis and clarified the involvement of Nrf2 in lenvatinib‐induced cytotoxicity. Cell viability, lipid ROS levels, and protein expression were measured using Hep3B and HuH7 cells treated with lenvatinib or erastin. We examined these variables after silencing fibroblast growth factor receptor‐4 (FGFR4) or Nrf2 and overexpressing‐Nrf2. We immunohistochemically evaluated FGFR4 expression in recurrent lesions after resection and clarified the relationship between FGFR4 expression and lenvatinib efficacy. Lenvatinib suppressed system Xc − (xCT) and glutathione peroxidase 4 (GPX4) expression. Inhibition of the cystine import activity of xCT and GPX4 resulted in the accumulation of lipid ROS. Silencing‐FGFR4 suppressed xCT and GPX4 expression and increased lipid ROS levels. Nrf2‐silenced HCC cells displayed sensitivity to lenvatinib and high lipid ROS levels. In contrast, Nrf2‐overexpressing HCC cells displayed resistance to lenvatinib and low lipid ROS levels. The efficacy of lenvatinib was significantly lower in recurrent HCC lesions with low‐FGFR4 expression than in those with high‐FGFR4 expression. Patients with FGFR4‐positive HCC displayed significantly longer progression‐free survival than those with FGFR4‐negative HCC. Lenvatinib induced ferroptosis by inhibiting FGFR4. Nrf2 is involved in the sensitivity of HCC to lenvatinib.


| INTRODUC TI ON
Hepatocellular carcinoma (HCC) is a common cancer worldwide. 1 Hepatic resection has been established as a safe and effective treatment for patients with HCC. However, the number of patients who develop recurrence remains high. 2,3 With the evolution of science and technology, systemic therapies that improve the prognosis of patients with HCC have been developed. 4 Lenvatinib is an oral multikinase inhibitor that targets vascular endothelial growth factor (VEGF) receptors 1-3, fibroblast growth factor receptors 1-4 (FGFR1-4), platelet-derived growth factor receptor, rearranged during transfection, and KIT. Lenvatinib is approved for the treatment of radioiodine-refractory differentiated thyroid cancer, advanced renal cell carcinoma, and HCC. [5][6][7] Ferroptosis is morphologically, biochemically, and genetically distinct from apoptosis, various forms of necrosis, and autophagy. This process is characterized by the iron-dependent accumulation of lethal lipid reactive oxygen species (ROS). 8 Previously, the induction of apoptosis was considered the main mechanism of cancer cell death for conventional treatments. However, studies have reported that inducing ferroptosis can significantly improve the efficacy of killing cancer cells, indicating that ferroptosis is another important process for cancer treatment. 9,10 Previous studies illustrated that sorafenib, a multikinase inhibitor, induced ferroptosis. 11 However, whether lenvatinib induces cell death via ferroptosis remains unknown.
Nuclear factor erythroid 2-related factor 2 (Nrf2) and its repressor protein Kelch-like ECH-associated protein 1 (Keap1) are key transcription factors for processing intracellular ROS. 12 Keap1 suppresses Nrf2 at low levels via ubiquitination and degradation. However, under oxidative conditions, Nrf2 is phosphorylated, after which it translocates to the nucleus. 13 Consequently, Nrf2 binds to the conserved anti-oxidant response element at the promoter regions of a battery of anti-oxidative and cellular defense targets, such as NADPH quinone oxidoreductase 1 (NQO1), hemoxidase-1, and ferritin heavy polypeptide 1, and then elicits robust anti-toxification responses. 14 Nrf2 has been described as a crucial stress response mediator in solid tumors. 15 We previously reported that NQO1 activation was related to metastasis and poor prognosis by enabling HCC cells to overcome anoikis during anchorage-independent culture. 16 Recent studies clearly demonstrated the central role of Nrf2 in protecting HCC cells against ferroptosis via the p62-Keap1-Nrf2 pathway, which upregulates multiple genes involved in iron and ROS metabolism. 11 It is unclear whether cell death induced by lenvatinib is associated with ferroptosis.
In this study, we investigated the relationship between lenvatinib and ferroptosis and clarified the involvement of Nrf2 in lenvatinibinduced cytotoxicity.

| Lipid peroxidation analysis
Lipid ROS analysis was performed using a Lipid Peroxidation (MDA) Assay Kit, which measures the lipid peroxidation marker malondialdehyde (MDA). Cells (2 × 10 6 /well) were seeded into a 10-cm plate and incubated at 37°C for 24 h in 10% CO 2 . Cells were homogenized in lysis solution on ice using a homogenizer and centrifuged at 13,000 g for 10 min. The supernatant was collected, mixed with TBA reagent, incubated at 95℃ for 60 min, and subjected to an optical density measurement at 532 nm on a microplate reader (BioTek).

| Caspase-3/7 Fluorescence Assay
Caspases-3/7 analysis was performed using a Caspasse-3/7 Fluorescence Assay Kit. Cells (1 × 10 4 /well) were seeded into a 96well plate and incubated at 37°C for 24 h in 10% CO 2 . After treatment with lenvatinib, at 37°C for 24 h, the plates were centrifuged in a plate centrifuge at 800 g for five minutes. Finally, the cells were incubated with a caspase-3/7 substrate solution at 37°C for 90 m.
Caspase-3/7 activity was determined based on the fluorescence intensity in cells using a microplate reader (BioTek).
Immunohistochemical data for FGFR4 staining and P-Nrf2 was evaluated by two experienced researchers (N.I. and S.I.) who were blinded to the clinical status of the patients. The final assessments were achieved by consensus. Cancer cells with membranous staining for FGFR4 were considered positive staining. 17

| Assessment of cell viability
The cells were further treated with lenvatinib, erastin, or ML385 for 12-72 h. Cell viability was evaluated using a CellTiter-Glo luminescent cell viability assay kit (cat. G7570, Promega), which determined cellular viability using ATP levels.

| Clinical and laboratory evaluation
The following test values were collected from medical records within 1 month before surgery.

| Statistical analysis
Statistical differences were determined using the Mann-Whitney test. P < 0.05 was considered statistically significant. All analyses were performed using JMP 15.0.0 software (SAS Institute).

| Increased susceptibility to lenvatinib after silencing endogenous nuclear factor erythroidderived 2-like 2 in hepatocellular carcinoma cells
To investigate whether silencing of Nrf2 contributes to the sensitivity to lenvatinib-induced ferroptosis, we silenced Nrf2 protein expression using the RNA interference technique. NQO-1 is a downstream enzyme in the Nrf2 signaling pathway. Nrf2, p-Nrf2, and NQO-1 protein expression was significantly reduced after Nrf2 silencing (Figure 2A). We investigated lipid ROS levels following Nrf2 silencing in HuH7 and Hep3B cells treated with lenvatinib and ferrostatin-1. Lipid ROS levels were higher in Nrf2-silenced cells treated with lenvatinib than in lenvatinib-exposed cells trans-  Figure 2D). The viability of cells treated with lenvatinib was measured using the CellTiter-Glo assay. Nrf2-overexpressing cells treated with lenvatinib displayed greater survival than control cells ( Figure 2D).

| Fibroblast growth factor receptor-4 and nuclear factor erythroid-derived 2-like 2 expression are related to lenvatinib therapeutic efficacy in patients
Next, we examined whether FGFR4 protein expression is related to the efficacy of lenvatinib. Patient characteristics are shown in Tables 1 and 2. Table 1 shows clinical data at the time of surgery, and Table 2 shows clinical data at the time of treatment of lenvatinib.
As presented in Figure S7A, B, the cancer cells exhibited positive FGFR4 staining. Figure 3A illustrates the relationship between had significantly longer progression-free survival than those with negative FGFR4 expression (log-rank P = 0.0052, Figure 3B). We investigated protein expression of P-Nrf2 associated with efficiency of lenvatinib. As presented in Figure S8A, B, the cancer cells exhibited positive P-Nrf2 staining. Figure S8C illustrates the relationship between P-Nrf2 expression and lenvatinib resistance in 31 patients. had significantly shorter progression-free survival than those with negative P-Nrf2 expression (log-rank P = 0.0124, Figure S8D).

| DISCUSS ION
In the present study, we analyzed the cytotoxicity of lenvatinib in HCC cells. We demonstrated that lenvatinib suppressed xCT expression and induced lipid ROS accumulation through FGFR4 inhibition. The accumulated lipid ROS induced ferroptosis in HCC cells.
Additionally, activated Nrf2 suppressed ferroptosis induced by lenvatinib ( Figure 4). We demonstrated that the inhibition of activated Nrf2 could sensitize HCC cells to lenvatinib. To the best of our knowledge, this is the first study to demonstrate the interaction between lenvatinib and ferroptosis and its regulation by Nrf2.
Ferroptosis is a regulated cell death pathway with unique morphological, biochemical, and genetic hallmarks, and it is associated with xCT and GPX4 signaling. all of which are indispensable for GSH synthesis. 32,33 In this study, lenvatinib inhibited xCT expression and lipid ROS accumulation, leading to ferroptosis. However, the activation of Nrf2 by lipid ROS accumulation suppressed ferroptosis. Therefore, Nrf2 inhibitors, such as ML385, encouraged that lenvatinib-induced ferroptosis could be efficiently cause.
In this study, we demonstrated that FGFR4 expression in cancer cells is related to the therapeutic efficacy of lenvatinib in patients with HCC. Yamauchi et al. reported that the tumor FGFR4 level was an independent predictor of the response to lenvatinib. 17 The sample size was relatively small in the two studies, and thus, further research is necessary.
Lenvatinib also suppresses vascular endothelial growth factor receptor in vivo, leading to tumor ischemia. 34

TA B L E 2 Association between FGFR4
expression and patient clinicopathological factors at the time for lenvatinib treatment to ferroptosis-mediated tissue injury in the intestine, 35 brain, 36 cardiomyocytes, 37 and kidneys. 38 Considering the lack of angiogenesis in vitro, it is possible that lenvatinib more strongly induces ferroptosis in vivo, and therefore, it will be interesting to evaluate the contribution of lenvatinib to ferroptosis in vivo in future studies.
In conclusion, we first revealed that lenvatinib induces ferroptosis by suppressing xCT expression in HCC cell lines. Lenvatinib-induced ferroptosis was regulated by Nrf2. Therefore, functional characterization of Nrf2 in ferroptosis may offer insights into the treatment of HCC.

ACK N OWLED G M ENTS
We thank Ms Saori Tsurumaru, Ms Asuka Nakamura, Ms Yuko Kubota, and Ms Miki Nakashima for their technical support. We thank Joe Barber Jr, PhD, from Edanz Group (https://en-autho r-servi ces.edanz.com/ac) for editing a draft of this manuscript.

CO N FLI C T O F I NTE R E S T
The authors have no conflicts of interest to declare.