Surufatinib achieves radiosensitivity in cholangiocarcinoma by suppressing GINS4 expression to induce Ca2+/cAMP signaling activation

Abstract

BACKGROUND

The tyrosine kinase inhibitor surufatinib demonstrates antitumor activity in biliary tract cancer (BTC). Previous evidence suggests that it exhibits a potential radiosensitizing effect when combined with radiotherapy (RT).

AIM

To assess surufatinib’s potential to enhance radiosensitivity and to explore the associated mechanisms.

METHODS

BTC cell lines NOZ and TFK-1 were utilized. Cell proliferation inhibition, radiosensitizing effects, and induction of DNA damage and apoptosis by surufatinib were detected using colony formation, immunofluorescence, and flow cytometry analyses. A cholangiocarcinoma tumor-bearing mouse model was established to validate the in vivo effects. High-throughput sequencing, Gene Set Enrichment Analysis (GSEA), genetic interventions and western blotting were performed to identify key genes and signaling pathways associated with surufatinib treatment and radiosensitization.

RESULTS

Surufatinib significantly inhibited proliferation and was associated with enhanced radiosensitivity in BTC cell lines. Combined with RT, surufatinib increased radiation-induced DNA damage and apoptosis. In vivo, surufatinib markedly inhibited angiogenesis and was associated with a radiosensitizing effect. Transcriptome sequencing identified GINS4 as a differentially expressed gene, with its expression correlating with poor prognosis. Knockdown of GINS4 inhibited tumor proliferation and enhanced the therapeutic efficacy of surufatinib combined with RT. Conversely, GINS4 overexpression promoted tumor proliferation, which was suppressed by surufatinib combined with RT. GSEA analyses suggested that GINS4 is closely associated with the Ca²⁺/cAMP signaling pathway. Surufatinib combined with RT was associated with suppressed GINS4 expression and concomitant activation of Ca²⁺/cAMP signaling in association with increased apoptosis.

CONCLUSION

Our study suggests that surufatinib is associated with radiosensitization in cholangiocarcinoma, potentially through inhibition of GINS4 expression and activation of the Ca²⁺/cAMP signaling pathway, leading to increased radiation-induced cellular damage and apoptosis. Further mechanistic studies are needed to establish causality.

Key Words: Biliary tract cancer; Surufatinib; Radiosensitivity; GINS4; Ca²⁺/cAMP signaling pathway

Core Tip: Surufatinib has been approved for the treatment of advanced cholangiocarcinoma. Our clinical trial demonstrates that surufatinib exerts prominent radiosensitizing effects. Its synergistic anti-tumor activity with radiotherapy is mainly achieved by suppressing the expression of GINS4, a critical proliferation-related gene, while simultaneously triggering the activation of Ca²⁺/cAMP signal cascades. Such molecular changes amplify radiation-triggered DNA injury, aggravate irreversible cellular damage, and ultimately facilitate robust apoptotic death of tumor cells upon radiation exposure. Our study provides more therapeutic options for clinical treatment.

INTRODUCTION

Biliary tract cancer (BTC) is a highly aggressive, and prognostically poor cancer. Over recent decades, its incidence has been steadily increasing[1]. Radiotherapy (RT) induces DNA damage and trigger cell necrosis and apoptosis[2,3]. It could reduce tumor volume, downstage disease, increase surgical resectability, and lower local recurrence rates in BTC[4]. Consequently, RT has become an important treatment modality for BTC[4,5].

BTC exhibit a degree of intrinsic radioresistance[6]. BTC surrounded by critical organs such as the liver, stomach, and duodenum, presents significant challenges for increasing radiation doses[4,6]. In recent years, emerging RT techniques such as stereotactic body RT (SBRT) have been applied. However, current research, predominantly comprising retrospective or small-sample studies, indicates that while escalating the radiation dose to the tumor region is critical for improving efficacy, it remains difficult to avoid damage to surrounding tissues[4]. Beyond boosting target dose, another strategy to improve RT efficacy is to use radiosensitizers. Nevertheless, prospective ABC-07 study showed that adding SBRT to a conventional gemcitabine-based chemotherapy regimen did not confer survival benefits for BTC patients[7]. Existing radiosensitization strategies for BTC, including gemcitabine-based regimens, have shown limited clinical benefit. Key challenges include off-target toxicity, lack of tumor specificity, and incomplete understanding of the molecular determinants of radioresistance in BTC. In particular, the roles of DNA replication-associated genes and calcium signaling pathways in modulating radiosensitivity remain largely unexplored. These findings underscore the necessity of exploring novel radiosensitizers in BTC.

Surufatinib is a small-molecule tyrosine kinase inhibitor that targets vascular endothelial growth factor receptors 1, 2, 3, fibroblast growth factor receptor 1, and colony-stimulating factor 1 receptor. It thereby inhibits tumor angiogenesis, proliferation, and invasion. It gained approval in 2022 for the second-line treatment of advanced BTC[8]. In our preliminary research, we observed that surufatinib combined with hypo-fractionated RT significantly improved survival in BTC patients who had failed standard therapies, suggesting its potential to enhance radiosensitivity in BTC[9]. However, the underlying mechanism remains unclear.

Factors such as mutations of tumor-related genes, dysregulation of DNA damage repair pathways, inactivation of apoptosis-related proteins, and dysregulation of calcium ion signaling can significantly impact tumor radiosensitivity[3,9,10]. For BTC, the intrinsic key factors driving proliferation remain incompletely elucidated. Surufatinib has shown good efficacy in inhibiting BTC cell proliferation in clinical practice and exhibits a radiosensitizing effect when combined with RT[9]. Yet, the mechanisms require further confirmation. Specifically, it is unclear whether surufatinib acts through a specific gene target to modulate radiosensitivity, and whether Ca²⁺/cAMP signaling is functionally involved. Therefore, this study aims to investigate the radiosensitizing effect of surufatinib on BTC cells and to explore whether GINS4 and the Ca²⁺/cAMP pathway are associated with this effect, thereby providing a solid theoretical foundation for subsequent clinical applications.

MATERIALS AND METHODS

Cell culture

The human gallbladder cancer cell line NOZ and the cholangiocarcinoma cell line TFK-1 were purchased from Genechem Co., Ltd. (Shanghai, China). All cells were cultured in RPMI-1640 medium with 10% fetal bovine serum at 37 °C in a humidified atmosphere containing 5% CO₂.

Cell proliferation assessment

For Cell Counting Kit-8 (CCK8) assays, 5 × 10³ cells per well were seeded in 96-well plates. After co-cultured with varying concentrations of surufatinib for 24 hours or 48 hours, cell proliferation of NOZ and TFK-1 cells was assessed using CCK-8, following the product protocol. Absorbance was measured at 450 nm. The half-maximal inhibitory concentration (IC₅₀) and 20% inhibitory concentration (IC₂₀) were determined. IC₂₀ concentration of surufatinib for 24 hours was used in following experiments.

Colony formation was assessed in 6-well plates. Cell densities ranging from 100 cells/well to 5000 cells/well and treated as with/without surufatinib, thus control group and surufatinib group. Subsequently, cells were irradiated with 0 Gy, 2 Gy, 4 Gy, 6 Gy, or 8 Gy using an X-RAD 320 X-ray irradiator (Precision X-Ray, CT, United States) at a dose rate of 1.2 Gy/minute. When visible colonies had formed, the culture was terminated. Colonies were fixed with 4% paraformaldehyde fixation and Giemsa staining. Colony formation efficiency was calculated. The sensitization enhancement ratio (SER) was calculated as the ratio of the mean lethal dose (D₀) of control cells to the D₀ of surufatinib-treated cells. This experiment was performed in triplicate.

Flow cytometry for apoptosis detection

After 24 hours incubation with or without surufatinib (IC₂₀ concentration added 24 hours prior to irradiation), cells were treated with RT (0 Gy, 2 Gy, 6 Gy). Cells were harvested 24 hours post-irradiation for analysis. Apoptosis Detection Kit (Cat# AB_2869082, BD Biosciences, NJ, United States) according to the manufacturer’s instructions as in previous studies[2,3]. Apoptosis was analyzed immediately using a flow cytometer, and data were processed using FlowJo software.

Immunofluorescence detection of γ-H2AX expression

Cells were divided into four groups: Control group, surufatinib group, radiation group (6 Gy), and combination group. Surufatinib was added 24 hours prior to irradiation. Twenty-four hours post-irradiation, cells were fixed with 4% formaldehyde and permeabilized. Cells were incubated overnight at 4 °C with primary antibody against γ-H2AX (ab11174, 1:800, Abcam, United Kingdom), followed by DyLight 549-conjugated secondary antibody for 1 hour at room temperature. Nuclei were stained with DAPI. γ-H2AX foci were counted blindly by two independent investigators in 80-100 tumor cells per group. The average number of foci per cell was calculated for quantitative analysis of γ-H2AX expression.

Establishment of xenograft tumor model in nude mice

A total of 0.1 mL of the single-cell suspension (2 × 10⁷ cells/mL) was injected subcutaneously into the posterior limbs of BALB/c nude mice (female, 4-6 weeks old). When tumor diameters reached approximately 10 mm, the mice were randomly divided into 4 groups: Control (vehicle only), surufatinib alone, RT alone (6 Gy), and combination (surufatinib + 6 Gy). Surufatinib was administered by oral gavage at a dose of 50 mg/kg once daily for 14 consecutive days. For the combination group, surufatinib was started 7 days before irradiation and continued until the end of the experiment. Irradiation (6 Gy) was delivered as a single fraction on day 8 after the start of drug administration. Four mice were assigned to each group initially; one mouse in the combination group died due to anesthesia, resulting in 3 mice for that group. Tumor length (L, longest diameter) and width (W, shortest diameter) were measured every other day. Tumor volume was calculated according to the formula: Volume (mm³) = (L × W²)/2. Tumor growth (volume and time) was recorded. All tumor samples were collected at the endpoint when one group reached a tumor volume of approximately 2 cm³. The animal study protocols employed in this research received approval after review by the Animal Research Ethics Committee of Shanghai Gene Chem (No. GSZE0346553), ensuring strict compliance with the applicable animal welfare guidelines.

Immunohistochemical analysis

Immunohistochemical analysis was performed as previously described[3]. Primary antibodies against Ki-67 and CD31 (Abcam, ab16669, 1:150, United Kingdom) were applied and incubated overnight at 4 °C. Subsequently, sections were incubated with a biotinylated secondary antibody for 20 minutes at room temperature. Color development was performed using the prepared solution (Dako kit, K5007, CA, United States). Nuclei were counterstained with hematoxylin. All slides were coded and scored blindly by two independent pathologists. For each section, five random high-power fields (× 400) were quantified.

Quantitative reverse-transcription polymerase chain reaction analysis

Total RNA was extracted from cells using TRIzol reagent. cDNA was synthesized from RNA using a reverse transcription kit (Thermo Fisher Scientific, MA, United States). Quantitative polymerase chain reaction amplification and analysis were performed using an Applied Biosystems StepOne or StepOnePlus Real-Time PCR System. All experiments were performed in triplicate.

RNA sequencing analysis

RNA sequencing (RNA-seq) was performed by Novogene (Beijing, China). The procedure is described online (https://cn.novogene.com). Sequence analysis was conducted using the Illumina data analysis pipeline. The corresponding RNA-seq data (accession number GSE269494) have been deposited in the Gene Expression Omnibus (GEO) database (https://www.ncbi.nlm.nih.gov/geo).

Datasets and data preprocessing

RNA array dataset GSE10072 was collected from the NCBI/GEO database (https://www.ncbi.nlm.nih.gov/gds/), along with RNA-seq data from the cholangiocarcinoma study in The Cancer Genome Atlas (TCGA-CHOL) database (https://cancergenome.nih.gov/). The expression of differentially expressed genes between normal and tumor tissues was analyzed, as well as their impact on the survival prognosis of BTC. All data were extracted and annotated using R software (version 4.4.2).

Gene Set Enrichment Analysis enrichment analysis

Key signaling pathways were screened through Gene Set Enrichment Analysis (GSEA) and Kyoto Encyclopedia of Genes and Genomes (KEGG) gene set enrichment analysis. KEGG enrichment analysis was carried out using the “clusterProfiler” package in R. GSEA was performed using gene sets from the Molecular Signatures Database. Pathways with adjusted P values < 0.05 were deemed significant.

Cell transfection

The pLenti-GINS4 (OE-GINS4) construct was generated using standard molecular biology techniques and sequenced to ensure its validity. Additionally, GINS4-specific siRNA (si-GINS4) and a negative control siRNA (si-NC) were designed and synthesized. The target sequences used were as follows: Control si-NC: ACGUGACACGUUCGGAGAATT; si-GINS4: AACUGCCCGAAAGAGGUCCTT. Transfection was performed using Lipofectamine 3000 (Thermo Fisher Scientific, MA, United States) according to the manufacturer’s protocol. Cells were used for experiments 48 hours after transfection.

Western blot analysis

Protein extracts were separated by sodium-dodecyl sulfate gel electrophoresis and transferred to polyvinylidene fluoride membranes. Membranes were incubated overnight at 4 °C with the following primary antibodies: PKA (ABclonal, A0798, 1:1000, Wuhan, Hubei Province, China), p-PKA (ABclonal, AP0557, 1:1000, Wuhan, Hubei Province, China), CREB (ABclonal, A1189, 1:1000, Wuhan, Hubei Province, China), p-CREB (ABclonal, AP0019, 1:1000, Wuhan, Hubei Province, China), GINS4 (SLD5) (Abcam, ab101346, 1:2000, United Kingdom), and GAPDH (Proteintech, 60004-1-Ig, 1:2000, Wuhan, Hubei Province, China). The secondary antibodies included HRP-conjugated Affinipure goat anti-rabbit IgG (H + L) (1:7000, SA00001-2, Proteintech, Wuhan, Hubei Province, China). The optical density of target bands was quantified using the AlphaEaseFC software analysis system and normalized to GAPDH. Quantifications were derived from three independent experiments.

Calcium ion detection

Intracellular calcium ion concentrations were measured using the Fluo-4 Calcium Assay Kit (S1061S, Beyotime, China). Cells were stained with Fluo-4 AM (500 ×) in assay buffer for 30 minutes at 37 °C in the dark. Fluorescence was detected using flow cytometry (Ex/Em = 490/525 nm).

cAMP concentration measurement

The concentration of cAMP in samples was measured using the Human cAMP ELISA Kit (E-EL-0056; Elabscience, China) according to the manufacturer’s instructions. Standards and samples were added to the microplate wells, followed by biotinylated detection antibody. After incubation and washing, HRP-conjugated avidin was added. The substrate solution was added and the reaction was terminated with stop solution. Absorbance was measured at 450 nm, and cAMP concentrations were determined from a standard curve.

Statistical analysis

Data are presented as means ± SEMs. Statistical analyses were performed using GraphPad Prism 6. Differentially expressed genes were identified using the DESeq2 R package, with a threshold of |log₂fold change| (log₂FC) > 1 and an adjusted P value < 0.05. Kaplan-Meier survival analysis was performed using the survival R package. Comparisons between two groups were made using Student’s t-test. For multiple comparisons among groups with normally distributed data, one-way analysis of variance (ANOVA) with the Bonferroni correction was applied. Homogeneity of variances was assessed between groups. Exact P values for key comparisons are provided in the figures and figure legends. P < 0.05 was considered statistically significant.

RESULTS

Surufatinib is associated with enhanced radiosensitivity in cholangiocarcinoma cells and increased DNA damage

The CCK-8 assay demonstrated that surufatinib inhibited the proliferation of NOZ and TFK-1 cells in a dose-dependent manner (Figure 1A). The IC₂₀ concentration after 24 hours of drug treatment was selected for subsequent experiments (IC₂₀: NOZ 4.03 μg/mL; TFK-1 4.33 μg/mL). Subsequently, the radiosensitizing effect of surufatinib was investigated using the colony formation assay. Cell survival curves were fitted using the multi-target single-hit model (Figure 1B), and key parameters were calculated: Mean lethal dose (D₀), quasi-threshold dose (Dq), extrapolation number (N), surviving fraction at 2 Gy (SF₂), and SER (Table 1). Compared to radiation alone, surufatinib treatment significantly decreased the D₀, N, and SF₂ values in NOZ and TFK-1 cells, indicating an association with radiosensitization. Specifically, the SF₂ value decreased from 72.47% to 46.37% in NOZ cells and from 74.16% to 41.36% in TFK-1 cells.

Figure 1 Surufatinib is associated with enhanced radiosensitivity of NOZ and TFK-1 and increased DNA damage. A: Cell viability assays were performed by Cell Counting Kit-8. Concentrations are shown in μg/mL. The anti-proliferative effect of surufatinib on NOZ and TFK-1 cells; B: Representative colony formation staining images and survival curves of NOZ and TFK-1 with different treatments. The survival curves were plotted using the multi-target single-hit model; C: Surufatinib increased the fluorescence foci of γ-H2AX in tumor cells after radiotherapy. Representative fluorescence foci images and quantitative detection of γ-H2AX foci in NOZ and TFK-1 cells following different treatments; scoring was performed blindly by two investigators; D and E: Apoptosis assessment by flow cytometry in NOZ and TFK-1 cells following different treatments. Apoptotic rates were calculated as the percentages of annexin-V FITC-positive cells. All columns indicate mean and error bars indicate SEM from three independent experiments. ^aP < 0.05, ^bP < 0.01, ^cP < 0.001. SF: Surufatinib; RT: Radiotherapy.

Table 1 The relative parameters of cell survival curves after irradiation.

Parameter/cell lines	NOZ		TFK-1
	Control	Surufatinib	Control	Surufatinib
D0	2.055	1.926	1.772	1.573
Dq	2.054	0.683	2.202	0.760
N	2.717	1.426	3.464	1.621
SF2	72.47%	46.37%	74.16%	41.36%
SER	/	3.01	/	2.90

D₀: Lethal dose; Dq: Quasi-threshold dose; N: Extrapolation number; SF₂: Surviving fraction at 2 Gy; SER: Sensitization enhancement ratio (calculated as D₀ control/D₀ surufatinib).

Surufatinib, combined with radiation significantly increased the number of γ-H2AX foci in NOZ and TFK-1 cells. Moreover, under 6 Gy, the induction of γ-H2AX expression was significantly greater compared to 2 Gy (6 Gy vs 2 Gy: NOZ 36.83 ± 0.86 vs 7.80 ± 0.65; TFK-1 21.70 ± 0.71 vs 14.08 ± 0.45; P < 0.05) (Figure 1C). Concurrently, apoptosis analysis using flow cytometry showed that surufatinib significantly enhanced radiation-induced apoptosis in both NOZ and TFK-1 cells (Figure 1D and E). The combination with 6 Gy demonstrated a significantly more potent pro-apoptotic effect compared to combination with 2 Gy (6 Gy vs 2 Gy: NOZ 24.71% ± 1.87% vs 18.01% ± 0.94%; TFK-1 32.81% ± 0.34% vs 26.70% ± 1.14%; P < 0.05) (Figure 1E). These results demonstrate that surufatinib is associated with enhanced radiosensitivity of cholangiocarcinoma cells, which correlates with increased radiation-induced DNA damage and apoptosis. Notably, this effect was more pronounced when combined with a hypofractionated radiation dose.

Surufatinib potentiates RT in vivo by inhibiting tumor angiogenesis and proliferation

To investigate the effects of surufatinib on tumor proliferation and RT response in vivo, we established a xenograft tumor model in nude mice. When tumor diameter reached 10 mm, mice were randomly divided into 4 groups: Control, RT (6 Gy/fraction), surufatinib, and combination group. Tumor size was measured every 2 days to generate growth curves (Figure 2A), and all mice were sacrificed when one group reached a tumor volume of approximately 2 cm³.

Figure 2 Surufatinib is associated with radiation sensitization in a nude mouse tumor transplantation model by inhibiting tumor proliferation and angiogenesis. A: Tumor growth curves of different treatment groups. Data are mean values from 4 mice in control, surufatinib, and radiotherapy groups, and 3 mice in the combination group (one mouse died from anesthesia); B: Representative tumor images of different treatment groups (control, surufatinib, radiation, and combination); C: Histogram of tumor weight of each mouse in the 4 groups; D: Representative immunohistochemistry staining image for Ki-67 in the 4 groups and the histogram for Ki-67 positive proportions. All immunohistochemical scoring was performed blindly; E: Representative immunohistochemistry staining image for CD31 in the 4 groups and the histogram for number of tumor vessels. ^bP < 0.01, ^cP < 0.001, ^dP < 0.0001. Ctrl: Control; SF: Surufatinib. RT: Radiotherapy alone.

Differences in tumor volume curves among treatment groups became apparent at day 11 post-treatment. Final tumor volumes were: Control group 2.97 ± 0.55 cm³, surufatinib group 0.98 ± 0.62 cm³, RT group 2.28 ± 0.46 cm³, and combination therapy group 0.70 ± 0.38 cm³. The combination therapy showed significantly enhanced tumor suppression compared to RT (Figure 2A and B). Tumor weight measurements revealed that the combination group had significantly reduced tumor weight compared to RT alone (0.42 ± 0.26 g vs 0.68 ± 0.39 g, P < 0.05) (Figure 2C).

Ki-67 expression showed that the combination therapy significantly inhibited tumor proliferative activity compared to either treatment alone (combination vs RT vs surufatinib: 28.5% ± 1.4% vs 39.2% ± 3.4% vs 51.9% ± 1.2%, P < 0.01 for combination vs RT) (Figure 2D). CD31 staining revealed that microvessel density counts were 26.33 ± 0.88, 19.00 ± 0.57, 9.66 ± 0.33, and 9.33 ± 0.88 in the control, RT, drug therapy, and combination groups, respectively (Figure 2E). Compared to control and RT groups, surufatinib significantly inhibited tumor angiogenesis (P < 0.01). These results demonstrate that surufatinib can effectively inhibit tumor proliferation and angiogenesis in vivo, thereby enhancing the RT response.

GINS4 is involved in the radiosensitizing effect of surufatinib in cholangiocarcinoma

To further explore potential mechanisms associated with surufatinib-induced radiosensitization, we performed transcriptome sequencing on cholangiocarcinoma cells after different treatments. Compared to the combination group, we identified 23 differentially expressed genes (P.adjust < 0.05, log₂FC > 0.5) from the intersection of upregulated genes in control (Ctrl), surufatinib (SF), and 6 Gy groups. Further intersection with TCGA database genes related to poor prognosis in BTC yielded 5 candidate genes (Figure 3A-C).

Figure 3 GINS4 is involved in the radiosensitizing effect of surufatinib on cholangiocarcinoma. A: A Venn diagram from RNA sequencing (RNA-seq) data identifies 23 genes associated with surufatinib (SF) and 6 Gy irradiation; B: Another Venn diagram highlights five genes differentially expressed in SF-induced radiosensitization; C: Heatmap analysis shows expression patterns of these genes across various treatment groups; D: RNA-seq analysis reveals significant downregulation of GINS4 following SF + 6 Gy treatment compared to radiotherapy or SF alone; E: Western blot confirms the changes in GINS4 expression across different groups; F and G: Analysis of GSE10072 and The Cancer Genome Atlas RNA-seq data indicates elevated GINS4 expression in tumors; H: High GINS4 expression correlates with poor prognosis; I: Western blot assesses GINS4 protein levels in NOZ and TFK-1 cells across four groups: Si-NC, si-NC + SF + 6 Gy, si-GINS4, si-GINS4 + SF + 6 Gy. Control (Ctrl) in panels J-L represents untreated control cells; si-NC served as the transfection control (data not shown separately due to similarity to Ctrl); J: Cell proliferation of NOZ and TFK-1 cells measured using the Cell Counting Kit-8 assay; K and L: Apoptosis rates determined by flow cytometry; M: Clonogenic survival rates evaluated using the clonogenic assay. All data are presented as mean ± SEM from three independent experiments. ^aP < 0.05, ^bP < 0.01, ^cP < 0.001, ^dP < 0.0001. NS: Not significant; Ctrl: Control; SF: Surufatinib; RT: Radiotherapy; TCGA: The Cancer Genome Atlas.

High-throughput sequencing revealed that GINS4 expression was closely associated with surufatinib treatment and radiosensitization. Compared to the control group, radiation or surufatinib alone only mildly suppressed GINS4 expression, while the combination treatment significantly inhibited GINS4 expression, showing marked differences compared to the other three groups (P < 0.01). Western blot analysis confirmed these sequencing results (Figure 3D and E). Integrated analysis of GEO dataset GSE107943 and TCGA-CHOL data showed that GINS4 expression was significantly higher in cholangiocarcinoma tissues than in normal tissues (P < 0.01), and prognostic analysis indicated that high GINS4 expression was associated with poor prognosis in BTC (P = 0.027) (Figure 3F-H). These findings suggest that GINS4 expression correlates with malignant progression of BTC and may play an important role in surufatinib-associated radiosensitization.

To validate GINS4 function, we performed siRNA knockdown of GINS4 expression combined with corresponding treatments. The SF + 6 Gy combination inhibited GINS4 expression, with this effect being more pronounced in the GINS4 knockdown group (Figure 3I). GINS4 knockdown alone was associated with significantly inhibited proliferation of NOZ and TFK-1 cells compared to controls (NOZ: 0.61 ± 0.02 vs 0.81 ± 0.03; TFK-1: 0.59 ± 0.02 vs 0.84 ± 0.03; P < 0.01), and SF + 6 Gy treatment further reduced proliferation in knockdown cells (P < 0.01) (Figure 3J). GINS4 knockdown also significantly increased apoptosis rates in both cell lines (NOZ: 21.93% ± 0.27% vs 9.15% ± 0.17%; TFK-1: 26.99% ± 0.15% vs 6.22% ± 0.14%; P < 0.001), which was further enhanced by SF + 6 Gy treatment (P < 0.001) (Figure 3K and L). Additionally, GINS4 knockdown reduced colony formation ability in both cell lines, with SF + 6 Gy treatment causing further suppression of proliferative capacity (Figure 3M).

GINS4 overexpression (Figure 4A) significantly increased tumor proliferative activity compared to controls (NOZ: 1.25 ± 0.05 vs 0.86 ± 0.04; TFK-1: 1.18 ± 0.06 vs 0.82 ± 0.04, P < 0.01), but SF + 6 Gy treatment still effectively reduced GINS4 expression and inhibited tumor proliferation (Figure 4A and B). GINS4 overexpression also suppressed apoptosis (NOZ: 4.64% ± 0.30% vs 7.48% ± 0.16%; TFK-1: 4.65% ± 0.17% vs 8.45% ± 0.09%, P < 0.001) and enhanced colony formation rate (P < 0.01). However, SF + 6 Gy treatment still significantly increased apoptosis in GINS4-overexpressing cells after irradiation (P < 0.001) and markedly inhibited colony formation ability in both cell lines post-radiation (P < 0.001) (Figure 4C-E). These results suggest that GINS4 expression is correlated with surufatinib treatment effects and that inhibition of GINS4 may contribute to reduced tumor proliferation.

Figure 4 Overexpression of GINS4 promotes tumor proliferation and inhibits apoptosis, while surufatinib suppresses GINS4 expression and is associated with radiosensitization. A: The pLenti-GINS4 (OE-GINS4) construct was generated using standard molecular biology techniques. Western blot analysis was performed to detect GINS4 expression levels among different treatment groups in NOZ and TFK-1 cells; control = empty vector control; B: Under conditions of GINS4 overexpression, cell viability was assessed across various treatment groups; C and D: Flow cytometry was employed to evaluate apoptosis in NOZ and TFK-1 cells in OE-GINS4 compared to control cells, with or without SF + 6 Gy treatment. Quantitative analysis of apoptosis rates among different treatment groups is presented in D; E: Clonogenic assays were conducted to assess the proliferative capacity of OE-GINS4 and control cells, with or without SF + 6 Gy treatment. Representative images of colony formation and quantitative analysis of colony counts for different treatment groups in NOZ and TFK-1 cells are shown in E, respectively. All data are presented as mean ± SEM from three independent experiments. ^bP < 0.01, ^cP < 0.001. SF: Surufatinib.

GINS4 expression is correlated with Ca²⁺/cAMP signaling in surufatinib-treated cells

KEGG and GSEA enrichment analyses suggested that GINS4 may function through Ca²⁺ and cAMP signaling pathways (Figure 5A). GSEA revealed that high GINS4 expression was negatively correlated with activation of Ca²⁺ influx channels, intracellular Ca²⁺ transport, and regulation of intracellular Ca²⁺ concentration (Figure 5B and C). Using the Fluo-4 calcium assay, we measured intracellular Ca²⁺ levels across treatment groups. Surufatinib significantly increased post-radiation Ca²⁺ concentrations (Ctrl vs SF + 6 Gy, NOZ: 7.55 ± 0.24 vs 36.22 ± 0.61; TFK-1: 8.78 ± 0.56 vs 35.15 ± 0.65; P < 0.001). Compared to controls, GINS4 overexpression reduced intracellular Ca²⁺ levels (Ctrl vs OE-GINS4, NOZ: 7.55 ± 0.24 vs 4.62 ± 0.20; TFK-1: 8.78 ± 0.56 vs 3.36 ± 0.15; P < 0.001). However, SF + 6 Gy treatment was associated with restored Ca²⁺ levels in GINS4-overexpressing cells (NOZ: 9.66 ± 0.23 vs 4.62 ± 0.20; TFK-1: 11.56 ± 0.67 vs 3.36 ± 0.15; P < 0.001) (Figure 5D and E). Quantitative cAMP enzyme-linked immunosorbent assay and western blot analysis of cAMP signaling proteins (Figure 5F and G) demonstrated that SF + 6 Gy significantly elevated cAMP levels (Ctrl vs SF + 6 Gy, NOZ: 4.12 ± 0.36 vs 7.31 ± 0.40; TFK-1: 4.34 ± 0.20 vs 7.84 ± 0.46; P < 0.001) and phosphorylation of PKA and CREB. GINS4 overexpression suppressed cAMP expression (Ctrl vs OE-GINS4, NOZ: 4.12 ± 0.36 vs 3.07 ± 0.18; TFK-1: 4.34 ± 0.20 vs 2.44 ± 0.35; P < 0.05) and downstream protein phosphorylation. Notably, SF + 6 Gy treatment was associated with reversal of this suppression, increasing cAMP and phosphor-PKA/CREB levels in GINS4-overexpressing cells (NOZ: 4.92 ± 0.37 vs 3.07 ± 0.18; TFK-1: 5.46 ± 0.38 vs 2.44 ± 0.35; P < 0.01) (Figure 5F and G). These results indicate that high GINS4 expression is associated with reduced Ca²⁺/cAMP signaling, whereas surufatinib treatment is associated with restoration of Ca²⁺/cAMP pathway activation. Causality remains to be established by functional inhibition experiments.

Figure 5 GINS4 expression is correlated with Ca²⁺/cAMP signaling in surufatinib-treated cells. A: Kyoto Encyclopedia of Genes and Genomes pathway enrichment analysis of GINS4 reveals significant pathways; B and C: Gene Set Enrichment Analysis highlights the impact of GINS4 on calcium signaling pathways; D and E: The Fluo-4 calcium ion detection kit was employed to measure changes in intracellular calcium ion concentration across different intervention groups, and quantitative analysis was presented in a histogram; F: Enzyme-linked immunosorbent assay was utilized to quantitatively analyze cAMP levels in different treatment groups for both OE-GINS4 and control (Ctrl) samples; G: Western blot analysis was performed to assess the expression of cAMP downstream-related proteins in different treatment groups for both OE-GINS4 and Ctrl samples. All data are presented as mean ± SEM from three independent experiments. ^aP < 0.05, ^bP < 0.01, ^cP < 0.001. Ctrl: Control; GINS4 overexpression: OE-GINS4; SF: Surufatinib.

DISCUSSION

Despite anatomical differences among intrahepatic cholangiocarcinoma, gallbladder cancer, and extrahepatic cholangiocarcinoma, these malignancies share robust proliferative and invasive capacities, often exhibiting intrinsic radioresistance[4,6]. BTC displayed post-irradiation SF₂ values exceeding 50%, indicating marked radioresistance[6]. Given the inherent radioresistance of these tumors and the limited radiation tolerance of surrounding normal anatomical structures, the therapeutic value of RT in BTC remains controversial. Conventional radiosensitizers demonstrate limited efficacy and significant treatment-related toxicities[4,6,7]. Our study demonstrates that surufatinib, effectively enhances radiosensitivity in BTC. Mechanistically, surufatinib is associated with enhanced radiosensitivity in BTC. The observed effects correlate with suppression of GINS4 expression, activation of Ca²⁺/cAMP signaling, and increased radiation-induced DNA damage and apoptosis.

Surufatinib exhibits dual anti-proliferative and tumor microenvironment-modulating effects. Prior studies have established its single-agent efficacy in advanced cholangiocarcinoma. Our work reveals that surufatinib is associated with enhanced post-irradiation apoptosis and DNA damage. Under surufatinib treatment, SF₂ values decreased from 72.47% to 46.37% in NOZ cells and from 74.16% to 41.36% in TFK-1 cells, indicating substantially improved radiosensitivity. In vivo studies corroborated these findings, showing combined therapy suppressed tumor proliferation and angiogenesis to a greater extent than either treatment alone. Our results show that surufatinib combined with RT induced apoptosis that was greater than the sum of individual treatments, suggesting a more-than-additive effect; however, formal combination index (CI) analysis is warranted to confirm synergy. This suggests that the mechanism may involve specific pathway activation beyond conventional targets.

Further investigation revealed that GINS4 exhibited significant differential expression across treatment groups. While radiation or surufatinib alone only modestly reduced GINS4 expression, the combination treatment profoundly suppressed GINS4 levels, suggesting its potential involvement in radiosensitization[11]. The GINS complex, first identified by Boskovic et al[12], is involved in DNA replication initiation and elongation in eukaryotes. As the core structural component, GINS4 facilitates replisome assembly and fork progression, playing essential roles in cell cycle regulation and genomic stability maintenance[12]. Clinical evidence shows GINS4 overexpression across multiple malignancies, with elevated expression correlating with poor survival[13,14]. Our TCGA analysis confirmed GINS4 upregulation in biliary tract malignancies, corroborating its prognostic significance. Functional studies demonstrated that GINS4 knockdown was associated with increased apoptosis and suppressed proliferation. Conversely, GINS4 overexpression promoted tumor proliferation, yet surufatinib combined with RT effectively counteracted this radioresistance-associated phenotype. These findings suggest that GINS4 may be a candidate regulator of cholangiocarcinoma proliferation and a potential target associated with surufatinib’s radiosensitizing effect.

KEGG and GSEA analyses linked GINS4 to Ca²⁺/cAMP signaling. High GINS4 expression inversely correlated with Ca²⁺ influx channel activity, intracellular Ca²⁺ transport, and concentration regulation, suggesting a possible association with calcium homeostasis disruption. The Ca²⁺ signaling system maintains cellular homeostasis through coordinated action of membrane channels, pumps, and organelle transporters[15,16]. Notably, aberrant activation of calcium efflux channels promotes tumor aggressiveness, while excessive influx or pump activity causes cytosolic Ca²⁺ overload, disrupting organelle integrity and activating apoptotic pathways[15-17]. Ca²⁺ elevation inhibits pancreatic cancer proliferation via mitochondrial structural collapse and AMP-activated protein kinase (AMPK) pathway activation[18]. Our study showed that surufatinib plus radiation significantly increased intracellular Ca²⁺ levels and activated pro-apoptotic AMPK signaling. GINS4 overexpression was associated with reduced Ca²⁺ concentrations and downstream signaling, while surufatinib treatment was associated with counteracting this effect on Ca²⁺/cAMP pathway activity.

Several limitations should be acknowledged. First, the sample size in the in vivo study (initially 4 mice per group, with one mouse lost in the combination group) is small, which limits statistical power and generalizability; future studies with larger cohorts are needed. Second, only two cell lines were used; additional BTC cell lines or patient-derived xenograft models would strengthen the conclusions. Third, although we observed associations between GINS4 and Ca²⁺/cAMP signaling, direct molecular interactions (e.g., protein binding or transcriptional regulation) were not demonstrated. Fourth, functional rescue experiments using PKA inhibitors (e.g., H89) or adenylyl cyclase inhibitors are required to establish causality for the cAMP pathway; these experiments are planned for future investigations. Fifth, we did not calculate the CI for drug-radiation interactions, so the term “more-than-additive” should be interpreted descriptively pending formal CI analysis. Sixth, survival data and systemic toxicity were not assessed in the animal model. Seventh, key apoptotic markers such as cleaved caspase-3, cleaved PARP, Bax, and Bcl-2 were not examined; future studies should include these to characterize the apoptotic mechanism more fully. Despite these limitations, our findings provide a basis for further mechanistic and translational studies.

CONCLUSION

Our study provides evidence that surufatinib is associated with enhanced radiosensitivity in cholangiocarcinoma, and that this effect correlates with suppression of GINS4 expression and activation of Ca²⁺/cAMP signaling. These findings suggest a potential mechanism for surufatinib-based radiosensitization, warranting further mechanistic and translational investigation.