p53-R280S mutation confers lenvatinib resistance in hepatocellular carcinoma via Bcl-2-mediated anti-apoptotic signaling

Abstract

Lenvatinib is approved as a first-line treatment for patients with unresectable hepatocellular carcinoma (HCC), however, its clinical efficacy is frequently limited by the emergence of drug resistance. This study aimed to elucidate the underlying mechanisms by which HCC develops resistance to lenvatinib. Lenvatinib-resistant HCC cell lines were established and subjected to RNA sequencing (RNA-seq) to identify genes associated with drug resistance. HCC cell lines stably overexpressing the p53 R280S mutation were constructed via lentiviral transduction. The effects of p53 R280S on HCC cell proliferation, apoptotic, and lenvatinib sensitivity were evaluated using Cell Counting Kit-8 (CCK-8) assays, flow cytometry, colony formation assays, TUNEL staining, and a mouse subcutaneous xenograft model. The underlying molecular mechanisms were further investigated by Western blotting, immunohistochemical staining (IHC), and quantitative real-time PCR (qRT-PCR). RNA sequencing of lenvatinib-resistant HCC cell lines revealed p53 dysregulation, and sanger sequencing identified the p53 R280S missense mutation, which was shown to enhance lenvatinib resistance in vitro and in vivo. The p53 R280S mutant attenuated lenvatinib-induced apoptosis. Furthermore, mutant p53 upregulated Bcl-2 expression and downregulated BAX expression, thereby suppressing apoptotic signaling. The p53 R280S mutation promotes lenvatinib resistance in HCC by inhibiting apoptosis.

Supplementary Information

The online version contains supplementary material available at https://doi.org/10.1007/s11010-026-05650-7.

Introduction

Hepatocellular carcinoma (HCC) is a major malignancy of the digestive system, with both its incidence and mortality among the highest worldwide [1]. Despite continuous advances in diagnostic and therapeutic strategies, the prognosis of patients with HCC remains poor, with five-year survival rates below 20% [2, 3]. In this context, lenvatinib was approved by the U.S. Food and Drug Administration (FDA) in 2018 as a first-line systemic therapy for patients with unresectable advanced HCC. By targeting multiple tyrosine kinase receptors, including FGFRs, PDGFRα/β, RET, KIT, and VEGFR [4, 5], lenvatinib induces apoptosis, suppresses cellular proliferation and angiogenesis, thereby inhibiting tumor progression [6]. However, the overall response rate to lenvatinib is only 24.1% [7], indicating a major clinical challenge, as many patients exhibit intrinsic resistance or acquire resistance. Consequently, elucidating the mechanisms underlying lenvatinib resistance and identifying effective therapeutic targets are essential for improving clinical outcomes for patients with HCC.

The tumor suppressor protein p53 plays a fundamental role in maintaining cellular homeostasis by regulating multiple biological processes, including cell cycle, apoptosis, cellular differentiation, and DNA damage repair [8, 9]. Despite its essential protective functions, p53 is the most frequently mutated gene among human malignancies with mutations occurring in more than 50% of malignancies. These mutations can be categorized into missense, silent, and nonsense mutations, among which missense mutations account for approximately 75% [10, 11]. Notably, mutant p53 not only loses its canonical tumor-suppressive functions but can also exert dominant-negative effects on wild-type p53. Accumulating evidence indicates that mutant p53 plays a pivotal role in tumor initiation, progression and drug resistance [12, 13].

Recently, numerous p53 mutation subtypes have been extensively characterized. Clinical studies have confirmed that p53 mutations predominantly occur at the highly conserved codons 175, 245, 248, 249, 273, and 282. For example, Kentaro et al. reported that patients harboring p53 mutations rapidly developed resistance to entrectinib after a therapeutic response of brain metastases [14]. The p53 R273H mutant has been shown to enhance cancer cell survival and promote resistance to detachment-induced apoptosis via activation of the PI3K/AKT signaling network and inhibition of Bcl-2-modifying factor (BMF) expression [15]. In addition, Su et al. demonstrated that the hotspot mutant p53 R175H facilitates tumor growth by suppressing ferroptosis through abolishing BACH1-mediated downregulation of SLC7A11. Inactivation of BACH1 or LSD2 effectively inhibits p53 R175H-driven tumor metastasis [16]. However, whether p53 mutations modulate the efficacy of small-molecule drug therapeutics remains largely unexplored. The transcriptional activity of p53 R280S has previously been restored in arsenic trioxide (ATO) relevant studies [17]. Nevertheless, the biological functions and underlying mechanisms of the p53 R280S mutation in tumor progression and drug resistance remain poorly understood.

In the present study, a drug-induced screening strategy was applied to establish a lenvatinib-resistant HCC cell line. RNA sequencing analysis revealed a significant enrichment of the p53 R280S mutation in resistant cells, suggesting its potential involvement in lenvatinib resistance. To date, the potential contribution of the p53 R280S mutation to therapeutic resistance, particularly in response to lenvatinib, has not been fully elucidated. Therefore, this study was designed to systematically investigate the functional role of p53 R280S in lenvatinib resistance in HCC.

Materials and methods

Cell line and cell culture

The human HCC cell lines SNU-449 and Hep3B were obtained from the American Type Culture Collection (ATCC, USA), whereas the SK-Hep-1 and SMMC-7721 cell were provided by the National Collection of Authenticated Cell Cultures (Shanghai, China). SNU-449 cells were routinely cultured in RPMI-1640 medium(Gibco). Hep3B cells were routinely cultured in MEM medium(Gibco). SMMC-7721 and SK-Hep-1 cell were maintained in DMEM medium(Gibco). All culture media were supplemented with 10% fetal bovine serum (FBS), 100 µg/mL streptomycin and 100 U/mL penicillin (Gibco). Cells were maintained at 37℃ in a humidified incubator with 5% CO₂.

Establishment of lenvatinib-resistant HCC cell lines

Lenvatinib-resistant HCC cell lines were generated using a stepwise dose-escalation method, as previously described [18]. Briefly, the half-maximal inhibitory concentration (IC₅₀) values of SNU-449 and SMMC-7721 cells were initially evaluated by the CCK-8 assay (TransGen Biotech Ltd., China). Cells at the exponential growth stage were subsequently treated with incrementally increasing doses of lenvatinib over a period of approximately three months. After prolonged drug exposure, the IC₅₀ values of the surviving cell populations were determined. The successfully generated lenvatinib-resistant cell lines were continuously cultured in medium supplemented with lenvatinib to sustain the drug-resistant phenotype.

Transcriptome sequencing assay

Transcriptome sequencing was performed by Berry Hekang Biotechnology Corporation (Beijing, China). Total RNA was isolated from each sample, and RNA integrity and purity were evaluated by gel electrophoresis. After RNA quality assessment, RNA samples were fragmented and transcribed into generate complementary DNA (cDNA), followed by depletion of ribosomal RNA (rRNA). RNA sequencing libraries were generated using the Illumina TruSeq RNA Sample Prep Kit and subsequently sequenced on the Illumina HiSeq platform with paired-end 150-bp sequencing. Raw sequencing reads were subjected to quality control to remove adaptor sequences, low-quality reads, and reads with high proportions of ambiguous bases. Clean reads were aligned to the human reference genome (GRCh38) using a standard RNA-seq alignment pipeline provided by the sequencing service. Gene expression quantification and normalization were performed using established methods. Differential expression analysis was performed using the DESeq2 package with |log2 fold change|≥1 and adjusted p-value (false discovery rate, FDR) < 0.05 were considered as differentially expressed genes (DEGs).

Construction of stably modified cell lines

Primers targeting the p53 cDNA sequence were designed and synthesized. The primers used for amplification of p53 cDNA were as follows: Forward: 5-GCTCTA GAATGGAGGAGCCGCAGTCAGATCCTAGCGTCGAGCCCCCTCT-3, Reverse: 5-CGGGATCCTCAGTCTGAGTCAGGCCCTTCTGTCTTGAACATGAGTTTTTTAT-3. The amplified p53 PCR product was cloned into PCDH lentiviral vector using T4 ligase. The p53 R280S point mutation was introduced using the Mut Express II Fast Mutagenesis Kit V2 (Vazyme, China) according to the manufacturer’s instructions, and the resulting mutant fragment was subsequently cloned into PCDH vector to generate p53 R280S overexpression construct. The presence of the mutation was verified by Sanger sequencing. Recombinant plasmid was packaged into lentivirus and used to infect Sk-Hep-1 and SNU-449 cells. Stable cell lines overexpressing p53 R280S, as well as control cells transduced with empty vector, were maintained using puromycin. Overexpression efficiency was confirmed by Western blotting.

Cell proliferation assay

The CCK-8 assay was performed to assess the cell viability, as previously described [18]. Cell suspensions were plated in 96-well plates at a density of 2000–3000 cells per well, with each sample plated in triplicate to quintuplicate. Cells were subsequently cultured at 37 °C with 5% CO₂ for 24, 48, 72, and 96 h. Wells containing only culture medium served as blank controls. Ten microliters of CCK-8 reagent were added in each well, and cells were incubated for 3 h at 37℃. Absorbance at 450 nm was measured, and cell viability was determined following the manufacturer’s protocol.

Colony formation assay

Cells were plated into six-well culture plates at a density ranging from 2000 to 3000 cells per well, with three replicate wells per group. The cells were cultured under conditions for 14 days, during which the culture medium was refreshed at three-day intervals. Upon completion of the incubation, the medium was discarded, and cells were washed three times with PBS. Subsequently, the colonies were fixed using 4% paraformaldehyde (1 mL per well) for 15 min at room temperature, followed by staining with 0.1% crystal violet for 15–25 min. Excess stain solution was removed by gentle washing with distilled water. After air-drying, visible colonies were photographed and quantitatively analyzed by ImageJ software.

Cell apoptosis assay

To assess apoptosis, cells were plated into six-well culture plates at a density of 2 × 10⁵ cells per well and exposed to lenvatinib-containing medium for 72–96 h at the indicated concentrations. Following drug treatment, cells were collected and stained with an Annexin V-APC/PI staining kit (KeyGEN BioTECH Corp.,Ltd, Jiangsu, China) in accordance with the manufacturer’s protocol. Annexin V-APC fluorescence (Ex = 633 nm, Em = 660 nm) was detected using the FL4 channel, while PI fluorescence (Ex = 488 nm, Em ≧ 630 nm) was measured through the PE channel, on a BD FACSVerse flow cytometer (BD Biosciences, San Jose, CA, USA).

TUNEL staining was additionally performed using the One Step TUNEL Apoptosis Assay Kit (Beyotime Biotechnology, China). Briefly, cells were fixed in 4% paraformaldehyde (1 mL per well) and permeabilized with 0.3% Triton X-100 (1 mL per well). The TUNEL working solution was prepared by mixing terminal deoxynucleotidyl transferase (TdT) enzyme with fluorescein-labeled dUTP at a 1:9 ratio. After washing with PBS, 50 µL of the TUNEL working solution was mixed in each sample at 37℃ for 60 min under dark conditions. The samples were subsequently washed three times with PBS and counterstained with DAPI. Fluorescence labeling was examined and imaged with a confocal laser scanning microscope.

Dual-luciferase reporter assay

The regulatory effect between p53 R280S mutant and Bcl-2 was examined using pGL3-Basic dual-luciferase reporter system. The promoter region of the BCL2 gene was inserted into a pGL3-Basic plasmid to construct the firefly luciferase reporter construct. Luciferase assays were performed according to the manufacturer’s guidelines. Briefly, HEK-293T cells were seeded into 24-well plates and temporarily transfected with the indicated pGL3-Basic reporter plasmids, the Renilla luciferase control plasmid, and p53 R280S expression plasmid. At 48 h of after transfection, luciferase activities were measured using the TransDetect^® Double-Luciferase Reporter Assay Kit (Code#FR20102, TransGen, Beijing, China) following the manufacturer’s protocol. Firefly reporter luciferase activity was normalized against Renilla luciferase activity to account for transfection efficiency.

Western blot analysis

Total cellular proteins were extracted using RIPA lysis buffer (Beyotime, China) supplemented with protease inhibitor cocktail (Roche Diagnostics, Germany) and phenylmethanesulfonyl fluoride (PMSF, Shanghai Macklin Biochemical Ltd., China). Protein concentrations were quantified using a bicinchoninic acid (BCA) protein assay kit (Beyotime, China) according to the manufacturer’s instructions. Equal amounts of protein were loaded onto sodium dodecyl sulphate-polyacrylamide gels electrophoresis (SDS-PAGE) gels and subsequently transferred to nitrocellulose (NC) membranes. Membranes were blocked with 5% skim milk at room temperature for 1 h to minimize nonspecific binding and then incubated with primary antibodies at 4 °C overnight. Following three washes with TBST, the membranes were incubated with appropriate secondary antibodies for 2–3 h at room temperature. Protein signals were detected by an enhanced chemiluminescence (ECL) reagent (Thermo Fisher Scientific, USA) and imaged with ChemiDoc Imaging System (Bio-Rad, USA). The primary antibodies used in this study are listed as follows: p53 (CST, #2527, 1:1000), Bcl-2 (CST, #3498, 1:1000), GAPDH (CST, #5174, 1:1000), β-actin (Abcam, ab115777, 1:4000), BAX (CST, #2772,1:1000).

Animal experimentation

All animal procedures were conducted in compliance with the protocol approved by the Animal Ethics Committee of Mengchao Hepatobiliary Hospital of Fujian Medical University (IACUCFJMU 2024-Y-0376). All methods were performed in accordance with the relevant guidelines and regulations. Six-week-old male NCG (NOD/Shi-scid/IL-2Rγnul) mice were allocated into two experimental groups. Subcutaneous injections of 8× 10⁶ cells/100 µL of either control or p53 R280S-overexpressing cell suspensions were administered into the right dorsal flank near the hind limb. Tumor volume was calculated using the formula: V = 0.5 × length × width² and tumor size was monitored every three days. When the xenograft tumors reached an approximate volume of 200 mm³, mice were treated by oral lenvatinib at a dose of 4 mg/kg. Mice were humanely euthanized according to ethical guidelines when the tumor volume exceeded 1000 mm³, the longest tumor dimension surpassed 15 mm, or body weight declined by more than 15%.

Statistical analysis

Statistical analyses were performed using GraphPad Prism software (version 9, GraphPad Software, USA). Quantitative data are expressed as mean±standard deviation (SD) from at least three independent biological replicates (n ≥ 3). Differences between two groups were analyzed using an unpaired two-tailed Student’s t-test. Comparisons among multiple groups were performed using one-way analysis of variance (ANOVA). A P-value of < 0.05 was considered statistically significant.

Results

Construction of lenvatinib-resistant cell lines

To explore genes associated with lenvatinib resistance, lenvatinib-resistant HCC lines were generated using a stepwise intermittent drug induction approach (Fig. 1A). SNU-449 and SMMC-7721 cells were originally treated with 2 µM lenvatinib and the concentration was subsequently enhanced every two weeks. Following three months of continuous exposure, the sensitivity of the resulting resistant cell lines was determined using the CCK-8 assay. The half-maximal inhibitory concentration (IC₅₀) of lenvatinib in lenvatinib-resistant SNU-449 (named SNU-449-LR) cells was approximately 11 folds higher than SNU-449 wild-type (named SNU-449-WT) cells, and lenvatinib-resistant SMMC-7721 (named SMMC-7721-LR) cells were 3.6 folds higher than SMMC-7721 wild-type (named SMMC-7721-WT) cells (Fig. 1B and C). Moreover, both SNU-449-LR and SMMC-7721-LR cells exhibited significantly increased survival under lenvatinib treatment for 96 h (p < 0.01 and p < 0.001 respectively, Fig. 1D and E). Consistently, more colonies were formed by SNU-449-LR and SMMC-7721-LR cells than by their wild-type cells under lenvatinib treatment (p < 0.001, Fig. 1F-H). These findings suggest that the established cell models exhibit reduced sensitivity to lenvatinib.

Fig. 1.

Screening of lenvatinib-resistant HCC cell lines and effects on cell proliferation. A Flowchart of the establishment process for lenvatinib-resistant (LR) HCC cell lines. B, C Half-Maximal Inhibitory Concentrations (IC₅₀) of Wild-Type (WT) and LR HCC cells. D, E Cell viability assessed by CCK-8 assays in SNU-449 and SMMC-7721 WT and LR cells following lenvatinib treatment, n = 5. F–H Colony formation assays of SNU-449 and SMMC-7721 WT and LR cells under lenvatinib exposure, n = 3. Data are presented as mean ± SD. Statistical analysis was performed using an unpaired two-tailed Student’s t-test. **p < 0.01, ***p < 0.001

Discovery and identification of the p53 R280S mutation

Next, RNA-seq was performed to evaluate the candidate genes potentially driving lenvatinib resistance in HCC cells. A total of 4440 and 6620 differently expressed genes (DEGs) were found in SNU-449-LR and SMMC-7721-LR cells, respectively, compared with their wild-type cells (Fig. 2A and C). KEGG pathway analysis showed that these DEGs were primarily involved in metabolic pathways and cancer related signaling pathways (Fig. 2B and D). Notably, the p53 signaling pathway was among the most significantly enriched pathways in both cell lines. To further examine this finding, the protein expression of p53 was examined by Western blot. As shown in Fig. 2E, p53 levels were higher in resistant cells compared with wild-type cells. It is well established that p53 mutations occur in the context of drug resistance [19, 20]. To investigate potential genetic alterations, Sanger sequencing was performed to analyze p53 gene. The result showed that a point mutation (R280S) of the p53 gene was identified in resistant cells (Fig. 2F).

Fig. 2.

Transcriptomic analysis and identification of the p53 R280S mutation. A, B Volcano plots and KEGG pathway enrichment analyses of differentially expressed genes derived from RNA sequencing of SMMC-7721 WT and LR cells, C, D Corresponding analyses of SNU-449 WT and LR cells. E Western blot analysis of p53, GAPDH protein levels in both SNU-449 WT and LR cells, as well as SMMC-7721 WT and LR cells. F Validation of the p53 mutation site was achieved through sanger sequencing analysis

p53 R280S mutation increases the lenvatinib resistance of HCC cells by inhibiting apoptosis

Stable overexpression of the p53 R280S mutation in SK-Hep-1 and SNU-449 cells was confirmed by Western blotting as illustrated in Fig. 3A. To evaluate its effect on lenvatinib sensitivity, colony formation and CCK-8 assays were performed. Cells were continuously incubated with lenvatinib (SK-Hep-1: 12.27 µM, SNU-449: 19.23 µM). Colony formation assay showed that p53 R280S-overexpressing exhibited a greater number of clonal clusters in SK-Hep-1 and SNU-449 cells(Figure 3B and C, p < 0.001). Consistently, CCK-8 assay reflected a similar result in cell viability (Fig. 3D, p < 0.001). Apoptosis was further assessed by flow cytometry and TUNEL assays. The results showed that apoptosis was significantly suppressed by p53 R280S overexpression in the presence of lenvatinib (Fig. 3E, F, G and H, p < 0.01). To further address the concern that the observed phenotypes might be influenced by overexpression-based systems, we performed additional validation experiments in p53-deficient HCC cells and experiments comparing WT p53 and p53 R280S in the original cell model. As shown in Fig S3 and Fig S4C-F, p53 R280S consistently reduced apoptosis and enhanced and promoted cell survival under lenvatinib treatment.

Fig. 3.

p53 R280S promotes resistance to lenvatinib. A Western blot analysis confirming successful overexpression of p53 R280S in SNU-449 and SK-Hep-1 cells. The colony-forming ability, n = 3 (B, C) and CCK 8 assay, n = 5 (D) evaluating the effects of p53 R280S overexpression on cell proliferation following lenvatinib treatment. E, G Flow cytometry analysis assessing apoptosis in p53 R280S overexpression compared with control group under lenvatinib exposure, n = 3. F, H Representative TUNEL staining illustrating cell apoptosis in SK-Hep-1 and SNU-449 cells expressing p53 R280S, n = 3. Typical tumor growth curve (K) for xenograft tumors, n = 5 (I), tumor weight (J) and body weight (L) of mice with or without lenvatinib treatment. M Representative H&E staining and IHC images showing p53 expression in xenograft tumors. N TUNEL staining of tumor tissues from p53 R280S-overexpressing and control xenografts to assess apoptosis in vivo. Data are presented as mean ± SD. Statistical analysis was performed using an unpaired two-tailed Student’s t-test. *p < 0.05, **p < 0.01, ***p  < 0.001, ****p < 0.0001

Similarly, the effect of p53 R280S overexpression was further evaluated in vivo. NCG mice bearing approximately 180 mm³ HCC tumors were treated with lenvatinib for two weeks (at 4 mg/kg daily, five days a week). As shown in Fig. 3I, J and K, tumor weights and tumor volumes exhibited a marked increase in the p53 R280S overexpression group compared with the control group (p < 0.05), whereas no difference in body weight was observed (Fig. 3L). TUNEL analysis further confirmed that overexpression of p53 R280S markedly reduced apoptosis in tumor tissues under lenvatinib treatment (Fig. 3N). Afterwards, p53 expression and morphology of HCC cells were confirmed by pathological analysis (Fig. 3M). Collectively, these findings suggest that p53 R280S overexpression is associated with promoted lenvatinib resistance, primarily through decreased apoptosis.

BAX and Bcl-2 were involved in mediating the effect of p53 R280S on apoptosis in HCC cells

To explain the molecular mechanisms through which p53 R280S regulates apoptosis, RNA-seq was performed. GO enrichment of genes exhibiting differential expression showed that apoptosis as one of top regulated process were identified consistent with our previous results (Fig. 4A, B). Among these DEGs, several BCL2 family genes, including BCL2L11 and BCL2L13, as well as other apoptosis-related genes were identified to be downregulated in cells with p53 R280S overexpression. qRT-PCR analysis of BCL2L11 subsequently confirmed the RNA-seq results (Fig. 4C and D). Given that Bcl-2 and BAX are well-known downstream factors of p53 involved in cellular apoptosis [21], we next examined the expression levels of these two proteins. The results indicated that Bcl-2 were upregulated by p53 R280S overexpression in the presence of lenvatinib (12.27 and 19.23 µM) in SK-Hep-1 and SNU-449 cells, while BAX were downregulated by that (Fig. 5A). Meanwhile, the expressions of Bcl-2 and BAX were also investigated in tumor tissues of the mice. Consistent trends were observed in tumor tissues, as indicated by IHC staining (Fig. 5C). Furthermore, a luciferase reporter assay was applied to investigate whether p53 R280S regulates Bcl-2 expression at the transcriptional level. The results demonstrated that p53 R280S was associated with increased Bcl-2 promoter activity (Fig. 5B).

Fig. 4.

Transcriptomic analysis of p53 R280S-regulated genes potentially involved in lenvatinib resistance. EnrichGo enrichment dot plots between p53 R280S-overexpressing and control groups in SK-Hep-1 cells (A) and SNU-449 cells (B). RT-PCR was performed to quantify BCL2L11 expression in SK-Hep-1 cells C and SNU-449 cells D overexpressing p53 R280S and control cells. Data are presented as mean ± SD (n = 3). Statistical analysis was performed using an unpaired two-tailed Student’s t-test. **p < 0.01, ***p < 0.001

Fig. 5.

p53 R280S promotes Bcl-2 transcriptional activation and contributes to lenvatinib resistance in HCC cells. A Western blot analysis of p53, Bcl-2, BAX, GAPDH protein expression in SNU-449 and Sk-Hep-1 cells overexpressing p53 R280S and control vectors. B Dual-luciferase reporter assay demonstrating that p53 R280S significantly enhances the transcriptional activity of the promoter. C Representative IHC images of Bcl-2 and BAX expression in the subcutaneous xenograft tumors. D, E Cell viability of SK-Hep-1 and SNU-449 cells treated with lenvatinib in the presence or absence of the BCL2 inhibitor Venetoclax. F–I Flow cytometric analysis of apoptosis in SK-Hep-1 and SNU-449 cells following treatment with lenvatinib and Venetoclax. Data are presented as mean ± SD (n = 5); *p < 0.05, **p < 0.01, ****p  < 0.0001

Functional rescue experiments using the selective Bcl-2 inhibitor venetoclax were performed to further investigate whether Bcl-2 contributes to p53 R280S-mediated resistance to lenvatinib. The IC50 value of v enetoclax was first determined in HCC cells, and a sublethal concentration was selected for subsequent combination studies (Fig S4A-B). As shown in Fig. 5D-I, p53 R280S-expressing cells exhibited significantly higher viability and reduced apoptosis following lenvatinib treatment compared with control group cells. Importantly, co-treatment with venetoclax partially decreased cell viability and increased apoptosis in p53 R280S-expressing cells.These findings suggest that Bcl-2-associated anti-apoptotic signaling may contribute to p53 R280S-mediated lenvatinib resistance.

Taken together, these results suggest that p53 R280S overexpression is associated with altered expression of apoptosis-related genes, including increased Bcl-2 and decreased BAX, which may contribute to reduced sensitivity to lenvatinib.

Discussion

As a first-line therapeutic agent, lenvatinib has been widely used for the treatment of advanced HCC, particularly in patients without prior systemic therapy who are ineligible for surgical resection, demonstrating significant antitumor efficacy [22, 23]. Nonetheless, the emergence of lenvatinib resistance adequately weakens its clinical efficacy and poses a major obstacle to improving patient survival outcomes [24]. Increasing evidence suggests that lenvatinib resistance is a multifaceted and dynamic process involving target gene mutations, an immunosuppressive tumor microenvironment, cancer stem cell (CSC) properties and tumor cell plasticity-including epithelial-mesenchymal transition (EMT), vascular remodeling, hypoxia, aberrant activation of signaling pathways and variations in pharmacokinetics [25]. p53 mutations are one of the most prevalent genetic alterations in HCC, occurring in approximately 12% to 48% in advanced-stage cases [26]. Such mutations often result in dysfunctional p53 protein, impairing apoptotic signaling, cell cycle arrest and DNA damage response, thereby promoting the therapeutic resistance in tumor cells [27]. For instance, mutant p53 disrupts the miR-34a-LRPPRC-MDR1 axis that normally sensitizes cells to chemotherapy [28].

In addition to the previously reported mechanisms, mutant p53 exerts gain-of-function (GOF) activities such as PI3K/AKT and MAPK that drive therapeutic resistance [13]. Lenvatinib primarily targets angiogenesis-related receptors such as VEGFR and FGFR. however, mutant p53 can bypass these receptor tyrosine kinases and sustain downstream signaling, thereby limiting drug efficacy. Moreover, mutant p53 promotes CSC, EMT and tumor microenvironment remodeling, all of which contribute to drug resistance [9, 12, 13]. These effects collectively provide a mechanistic basis for the reduced sensitivity to lenvatinib observed in p53-mutant HCC cells. In the present study, we report for the first time the identification of a p53 R280S mutation associated with lenvatinib resistance in HCC. Both in vitro and in vivo experiments demonstrated that the p53 R280S mutation decreases the cellular sensitivity to lenvatinib.

Dysregulation of cell proliferation and apoptosis is widely recognized as fundamental hallmarks of cancer. A substantial portion of the antitumor activity exerted by targeted drugs such as nilotinib, imatinib, vemurafenib, and trastuzumab is mediated by the induction of apoptosis in cancer cells [29–31]. Consequently, disruption of apoptotic activation mechanisms often results in increased resistance of tumor cells to these targeted agents. In our study, cells overexpressing p53 R280S exhibited significantly reduced apoptosis compared with control cells following lenvatinib treatment, indicating that p53 R280S promotes lenvatinib resistance by inhibiting apoptosis responses. Previous studies have shown that wild-type p53 (WTp53) suppresses Hsp90α secretion, whereas mutant p53 (mutp53) facilitates its vesicular transport and exosomal release. Rab coupling protein (RCP) suppression led to a reduction in eHsp90α secretion, thereby limiting tumor malignancy. Reintroduction of recombinant Hsp90α significantly restored tumor migration and invasiveness impaired by RCP deficiency [32]. Although the role of p53 mutations in targeted therapy resistance has not been fully elucidated, and the R280S variant is considered relatively rare, our findings demonstrate that under sustained drug pressure, this mutation can significantly enhance resistance in HCC cells. The results fill a critical gap in the field and provide novel mechanistic insights into lenvatinib resistance.

Wild-type p53 primarily functions as a transcription factor that restricts tumor cell proliferation and survival by activating multiple downstream genes. Beyond its transcription activity, p53 directly interacts with members of the Bcl-2 family to promote mitochondrial apoptosis [33]. To explore the role of p53 R280S in regulating lenvatinib resistance, transcriptome sequencing was performed on cells overexpressing p53 R280S and their corresponding controls. The analysis revealed that BCL2L11, a Bcl-2 family protein with established pro-apoptotic activity, was significantly downregulated in the p53 R280S-overexpression group. BCL2L11 is a well-established pro-apoptotic protein that facilitates programmed cell death and regulates multiple cellular processes [34], and prior research has shown that BCL2L11 modulates the sensitivity of lung cancer to osimertinib treatment [35]. In agreement with these observations, our results showed that Bcl-2 expression was markedly upregulated in p53 R280S-overexpressing cells following lenvatinib treatment. A well-characterized regulatory interplay exists between BAX and Bcl-2. The anti-apoptotic protein Bcl-2 resides within the mitochondrial membrane, where it sequesters and neutralizes BAX, thereby preventing the initiation of apoptosis [36]. When the balance between BAX and Bcl-2 is disrupted, cancer cells are promoted to proliferate and evade apoptosis. Our results further demonstrated that overexpression of p53 R280S significantly suppressed BAX expression, supporting a role for the mutant protein in attenuating apoptotic signaling. This discovery highlights the potential of targeted P53 mutation inhibitors as a tactic to overcome lenvatinib resistance.

This study has several inherent limitations. Our research identified a novel mutation site that contributes to the development of lenvatinib resistance. To strengthen the biological relevance of our findings, we additionally validated the effects of p53 R280S in p53-deficient HCC cells. The consistent phenotypic changes observed across different cellular contexts support the robustness of the mutation-associated effects. Nevertheless, future studies employing endogenous knock-in models will be required to further define the physiological role of p53 R280S in lenvatinib resistance. Furthermore, our transcriptomic data indicate a potential association between the p53 mutation and metabolic pathways; further experimental validation are required to elucidate this metabolic observation. Although substantial progress has been made in translating p53-targeted therapies from preclinical models to clinical settings [37, 38] the diversity and complexity of p53 mutations make it difficult for a single inhibitor to cover all mutation types. Nevertheless, our study offers novel insights of the molecular mechanisms contributing to therapeutic resistance in HCC and highlights potential avenues for improving treatment responses in patients with lenvatinib-resistant disease.

In summary, our findings suggest that p53 R280S contributes to lenvatinib resistance in HCC by suppressing apoptosis and modulating Bcl-2 family signaling. These results provide new insights into the role of mutant p53 in targeted therapy resistance and may offer potential therapeutic strategies for overcoming lenvatinib resistance.

Supplementary Information

Below is the link to the electronic supplementary material.

Author contributions

Niangmei Cheng: Investigation, writing–original draft, data curation, funding acquisition. Kexin Liu: Data curation, validation. Xin Qi and Leijie Cai: Data curation, methodology. Yang Zhou and Fei Wang: Resources, validation, Bixing Zhao: Funding acquisition, conceptualization, writing–review & editing. Yingchao Wang: Formal analysis, supervision, funding acquisition, conceptualization, project administration.

Funding

This work was supported by the National Natural Science Foundation of China. (32470612), Joint Funds for the innovation of science and Technology, Fujian province (2023Y9268), Natural Science Foundation of Fujian Province (2024J011217, 2023J011467), Medical Innovation Project of the Fujian Provincial Health Commission Science and Technology Program (2023CXA050, 2025CXA053).

Data availability

No datasets were generated or analysed during the current study.

Declarations

Competing interests

The authors declare no competing interests.

Ethical approval

All animal procedures were conducted in compliance with the protocols approved by the Animal Ethics Committee of Institutional Animal Center (IACUCFJMU 2024-Y-0376).