Abbreviations
3‐methyladenine
interferon regulatory factor 4
negative control with empty vector group
overexpression
rapamycin
Introduction
Cervical cancer remains the fourth most prevalent cancer among women globally and is among the top four malignancies affecting women [ 1 ]. For advanced cervical cancer, surgery, radiotherapy, and chemotherapy constitute the primary treatment modalities; nonetheless, due to tumor recurrence and metastasis, the prognosis for these patients remains grim [ 2 ]. Radiotherapy is currently recognized as a pivotal adjuvant treatment following cervical cancer surgery, with nearly 80% of cervical cancer patients requiring radiotherapy at some point during their diagnosis and treatment [ 3 ]. Despite this, due to radiotherapy tolerance, postoperative tumor recurrence and metastasis can still ensue [ 4 , 5 ] Hence, it is imperative to delve deeper into the molecular mechanisms underlying radiotherapy resistance in cervical cancer, pinpoint relevant biological targets, and enhance cervical cancer radiosensitivity.
The mechanisms governing radiotherapy resistance in malignant tumors are not fully elucidated. Potential mechanisms encompass tumor hypoxia, alterations in the tumor microenvironment, DNA damage repair, cancer stem cells, metabolic reprogramming of tumors, the cell cycle, apoptosis, and various other signaling pathways, all of which are intricately linked to the radiosensitivity of malignant tumors [ 6 , 7 ]. Autophagy is a cellular stress response mechanism that transports cytosolic components, senescent or damaged organelles, misfolded proteins with aggregation tendencies, and intracellular pathogens to lysosomes for degradation, thereby producing essential products or energy for biosynthetic processes that ensure cell survival [ 8 ]. Cellular autophagy can be classified into macroautophagy, microautophagy, and chaperone‐mediated autophagy, and comprises four primary stages: (1) induction and activation, (2) autophagosome nucleation, (3) autophagosome elongation and expansion, and (4) autophagosome fusion with lysosomes and subsequent degradation of contents [ 9 , 10 ]. Numerous signaling pathways influence cellular autophagy, with the PI3K/Akt/mTOR pathway being a prominent one [ 11 ]. The PI3K/Akt/mTOR signaling pathway, comprising phosphatidylinositol‐3 kinase (PI3K), its downstream serine/threonine kinase Akt (protein kinase B), and the mammalian target of rapamycin (mTOR), serves as a central regulator of multiple cellular processes including proliferation, apoptosis, metabolic homeostasis, and angiogenesis [ 12 ]. Notably, mTOR functions as a critical negative regulator of autophagy initiation, with its activation suppressing autophagosome formation while its inhibition promotes autophagic flux [ 13 ]. This pathway integrates various extracellular and intracellular signals to coordinate cellular responses to environmental cues, nutrient availability, and therapeutic interventions, making it a crucial determinant of cell fate decisions under both physiological and pathological conditions [ 14 , 15 , 16 ].
The interferon regulatory factor (IRF) family proteins were initially characterized as a group of DNA‐binding proteins inducible by type I interferon (IFN) signals [ 17 , 18 ]. Subsequently, other stimuli, including type II IFN, cytokines, and viral infections, have been shown to induce and activate IRF proteins [ 19 ]. Notably, IRF4, a member of the IRF family, exhibits several unique features compared to other IRF family members. For instance, IRF4 is induced not by type I or II IFN but rather by the activation of T‐cell receptor (TCR) signaling in T cells [ 20 ], B‐cell receptor (BCR), IL‐4, and CD40 signaling in B cells [ 21 , 22 ], and toll‐like receptor (TLR) signaling triggered by lipopolysaccharide (LPS) in macrophages. IRF4 has been implicated in tumor suppression in various cancers, including multiple myeloma [ 23 ], Sjogren Syndrome [ 24 ], colon cancer [ 25 ], and breast cancer [ 26 ], among others. Studies have screened for IRF4 target genes by constructing protein–protein interaction (PPI) networks and mapped them onto initial networks to reconstruct circRNA‐miRNA‐mRNA networks, revealing that hsa_circ_0000301/hsa‐miR‐1228‐3p/IRF4 may play a role in the initiation and progression of cervical cancer [ 27 ].
To date, the mechanisms underlying IRF4‐mediated cell death during radiotherapy remain unexplored. Through transcriptome sequencing of 16 radiotherapy‐resistant and 9 radiotherapy‐sensitive cervical cancer specimens, we identified significant IRF4 downregulation in radioresistant tumors, with low expression correlating with favorable prognosis. Functional studies demonstrated that IRF4 suppresses cervical cancer proliferation while enhancing radiosensitivity in both cellular and animal models. Mechanistically, IRF4 overexpression inhibited DNA replication, colony formation, and cell motility. Following irradiation, IRF4‐expressing cells exhibited markedly increased DNA damage and impaired proliferative capacity. This study provides the first evidence that IRF4 regulates cervical cancer progression and radiosensitivity through autophagy modulation, as IRF4 overexpression robustly activated autophagic flux in cervical cancer cells. Our in vitro and in vivo findings collectively establish that IRF4 upregulation suppresses tumor progression and significantly enhances X‐ray sensitivity.
Materials and Methods
Online Database Analysis
To verify IRF4 expression in cervical cancer, we utilized the Human Protein Atlas database [ 28 ]. The specific steps taken were: accessing the official website at www.proteinatlas.org , inputting “IRF4” into the search box, selecting the “Pathology” option in the “RNA Expression Overview” module, filtering for “Cervical Cancer” samples, and ultimately obtaining survival analysis results and associated graphs.
Sample Source
Between January 2021 and November 2024, this study included 25 cases of primary cervical cancer tissues (tumor diameter > 4 cm) sourced from Fujian Cancer Hospital. These samples comprised 16 radiotherapy‐resistant and 9 radiotherapy‐sensitive samples for transcriptome sequencing. All cases were pathology/cytology‐confirmed and approved by the Ethics Committee of Fujian Cancer Hospital (No. K2024‐222‐01). Patients provided informed consent for the use of their tissues in scientific research experiments.
Immunohistochemistry (IHC)
Immunohistochemical staining adhered to previously established standard methods [ 29 ]. Specifically, 4 μm‐thick paraffin tissue sections were dewaxed at 60°C, followed by xylene dewaxing, gradient ethanol hydration, and high‐pressure thermal repair using pH 6.0 citrate buffer (121°C, 15 min). Sections were then blocked with 3% H₂O₂ and incubated overnight at 4°C with rabbit anti‐human IRF4 monoclonal antibody (Proteintech, Cat. No. 60004‐1‐Ig, diluted 1:400). After incubation with HRP‐labeled goat anti‐rabbit IgG secondary antibody (Beyotime, Cat. No. A0216, diluted 1:500) at room temperature for 1 h, sections underwent DAB staining and hematoxylin counterstaining. Staining results were evaluated using a semi‐quantitative scoring system that considered both staining intensity (0–3 points) and the proportion of positive cells (0–4 points). A final score ≥ 4 was deemed positive expression, with all interpretations independently performed by two senior pathologists using a double‐blind method.
Cell Culture, Adenoviral Infection, and Irradiation
Cells were sourced from the Cell Bank of the Chinese Academy of Sciences. Cells were cultured in RPMI 1640 medium for H8, Siha, and Caski cell lines, and in high‐glucose DMEM medium for C33a and HeLa cell lines, both supplemented with 10% FBS and 100 U/mL penicillin–streptomycin (37°C, 5% CO 2 ). From the available cell lines (H8, Siha, HeLa, C33a, Caski, and Me180) (Use the product from Procell), radiation‐resistant Siha and HeLa cell lines were selected for experimentation. Cells were infected with GeneChem's Ad‐IRF4 and Ad‐NC adenoviruses for 72 h, following the manufacturer's instructions, before being irradiated using a Rad Source Technologies linear accelerator at a dose rate of 1.15 Gy/min.
CCK‐8 Assay
After infection with Ad‐NC and Ad‐IRF4‐OE, Siha or HeLa cells were seeded into 96‐well plates at a density of 3 × 10 3 cells per well, with 8 parallel wells per group. Cell proliferation was assessed using the CCK‐8 assay at 24, 48, and 72 h post‐X‐ray treatment at doses of 2 Gy, 4 Gy, 6 Gy, and 8 Gy. Briefly, 10 μL of CCK‐8 solution and 100 μL of complete medium were added to each well and incubated at 37°C for 2 h. Optical density (OD) was measured at 450 nm using a microplate reader (Bio Rad Laboratories Inc.). The viability index was calculated by dividing the experimental OD value by the control OD value. The experiment was replicated three times.
Colony Formation Assay
Cells were seeded into 6‐well plates at varying densities. Specifically, 1.5 × 10 3 cells per well were seeded for both cell lines at 0 Gy, 3 × 10 3 cells per well for Siha and HeLa at 2 Gy, 6 × 10 3 cells per well for Siha and HeLa at 4 Gy, 8 × 10 3 cells per well for Siha and HeLa at 6 Gy, and 1 × 10 4 cells per well for Siha and HeLa at 8 Gy, with 3 parallel wells per group. After 24 h, Siha and HeLa cells were exposed to X‐rays at 0, 2, 4, 6, and 8 Gy for 20 min. Following incubation at 37°C for 14 days, colonies were fixed with 4% paraformaldehyde at room temperature for 20 min and stained. They were then incubated with Giemsa at room temperature for 30 min. Colonies with at least 50 viable cells were counted under an optical microscope (Olympus), and cloning efficiency was calculated.
5‐Ethynyl‐2′‐Deoxyuridine (EdU) Incorporation Assay
Cell proliferation was further evaluated using the Cell Light EdU DNA Kit (RiboBio, Guangzhou). Briefly, cells were incubated with 50 μM EdU at 37°C for 2 h, fixed with 4% paraformaldehyde at room temperature for 30 min, permeabilized with 0.5% TritonX‐100 for 10 min, and stained with EdU at room temperature for 30 min. Cells were then stained with 5 μg/mL Hoechst 33,342 at room temperature for 30 min and visualized under a fluorescence microscope (Leica Microsystems GmbH). The red‐to‐blue ratio was analyzed using GraphPad software.
Flow Cytometry (FCM) Analysis
FCM analysis was employed to assess apoptosis and cell cycle distribution. For apoptosis analysis, logarithmically growing cells were prepared as a single‐cell suspension and seeded into 6‐well plates at 2 × 10 5 cells/ml, with 3 parallel wells per group. After 48 h of irradiation (0 and 6 Gy), cells were collected and centrifuged at 300 g for 5 min at 4°C. After washing with PBS and discarding the supernatant, cells were centrifuged again at 300 g for 5 min at 4°C. Cells were stained with FITC‐conjugated Annexin V (Shanghai Yisen Biotechnology Co. Ltd.) and PI (Shanghai Yisen Biotechnology Co. Ltd.), and apoptotic cells were counted using the BD FACSCalibur flow cytometer system (CytExpert). GraphPad software was used for statistical analysis.
For cell cycle analysis, cells infected with Ad‐NC and Ad‐IRF4 were collected during logarithmic growth after 48 h of 0 Gy and 4 Gy irradiation and fixed overnight with 70% pre‐cooled ethanol at 4°C. After staining with 10 μg/mL propidium iodide, cells were incubated at 37°C in the dark for 30 min and analyzed using the BD FACSCalibur flow cytometer system (CytExpert).
Indirect Immunofluorescence
Treated cells were placed in 24‐well plates and fixed with 4% paraformaldehyde at a concentration of 50%–60% for 10 min at room temperature after 0 and 6 h of X‐ray irradiation. Cells were then permeabilized with 1% Triton X‐100 at room temperature for 10 min. After blocking with 1% BSA (Beyotime Institute of Biotechnology) at room temperature for 1 h, cells were incubated overnight at 4°C with IRF4 antibody (dilution: 1:400; Cat. No. 60004‐1‐Ig; Proteintech) and then incubated with FITC‐conjugated goat anti‐rabbit secondary antibody (dilution: 1:1000; Cat. No. A0562; Beyotime) at room temperature for 2 h. Cells were stained with DAPI (Sigma Aldrich) and observed under a microscope (Olympus Corporation).
Immunoblotting
Cells from distinct groups were lysed utilizing 1× RIPA lysis buffer (Beyotime, P0013B), and protein concentrations were ascertained using a BCA kit (Beyotime, P0010S). Following centrifugation at 12,000 g for 15 min at 4°C, supernatants were harvested, boiled, and loaded at 20 μg of protein per well. Proteins were separated via 12% or 15% SDS‐PAGE (Beyotime, P0012A) and transferred onto PVDF membranes (Millipore, IPVH00010). Membranes were blocked with 5% BSA for an hour and then incubated overnight at 4°C with primary antibodies targeting IRF4 (1:1000, 60,004‐1‐Ig), LC3 (1:1000, 14,600‐1‐Ig), Beclin‐1 (1:1000, 66,665‐1‐Ig), P62 (1:1000, 66,184‐1‐Ig), p‐AKT (1:5000, 80,455‐1‐RR), AKT (1:1000, ab8805), p‐mTOR (1:10000, 80,596‐1‐RR), mTOR (1:5000, ab109268), and GAPDH (1:5000, 60,004‐1‐Ig). Goat anti‐rabbit or goat anti‐mouse secondary antibodies (Beyotime A0286/A0277, 1:1000) were added and incubated for an hour at room temperature. After ECL development, images were captured using a G:BOX Chemi XRQ, and band intensities were analyzed with ImageJ (Version 1.8.0).
In Vivo Tumor Formation and Irradiation Experiments in Nude Mice
Twenty‐four female BALB/c nude mice (aged 5 weeks, weighing 18–22 g, sourced from Guangdong Pharmaceutical Biology) were housed in an SPF environment (21°C–23°C, 40%–60% humidity, 12‐h light/dark cycle) with ad libitum access to sterile feed and water. Operations adhered to NIH guidelines for laboratory animals [ 30 ]. Mice were randomly divided into five groups ( n = 5): Ad‐IRF4, Ad‐NC, Ad‐IRF4 + IR, Ad‐NC + IR. Siha cells (5 × 106/100 μL PBS) were injected subcutaneously into the left hind flank of each mouse, and tumor volumes were measured every 3 days. Local irradiation of 8 Gy was administered on day 5 post‐injection (when tumors were ≈80 mm 3 ), and tumor growth was monitored every 4 days. Mice were euthanized on day 29, and tumor tissues were collected for storage at −80°C or fixation in 4% paraformaldehyde. This experiment was approved by the Experimental Animal Ethics Committee of Fujian Provincial Hospital. (Serial number: IACUC‐FPH‐SL‐20231207[0275]).
Statistical Analysis
All experimental data were statistically analyzed using GraphPad Prism 6.01 software and presented as mean ± standard deviation. Comparisons between two groups were conducted using unpaired two‐tailed Student's t ‐tests, while multiple group comparisons utilized one‐way ANOVA combined with Tukey's test. Five biological replicates were set up for in vitro experiments, and three biological replicates were set up for each group in vivo. Statistical significance was set at p < 0.05.
Results
IRF4 Is Upregulated in Radiotherapy‐Responsive Cervical Cancer and Radiosensitive Tissues
By comparing transcriptome sequencing data from 16 radiotherapy‐resistant and 9 radiotherapy‐sensitive cervical cancer patients, over 2000 differentially expressed genes were obtained. After cross‐analysis with the TCGA database, 257 genes with prognostic significance were screened. Key genes were precisely identified based on criteria encompassing gene sets related to prognosis in patients before and after 10 radiotherapy sessions, gene sets with expression patterns consistent with prognosis ( p < 0.05), and gene sets with significant differential expression and prognostic value. Ultimately, 14 core genes were identified (Figure 1A ). This study further searched the TCGA database and found IRF4 downregulated in cervical cancer tissues (Figure 1B ). The 5‐year survival rate of patients with high IRF4 expression was significantly higher than that of patients with low IRF4 expression (Figure 1C ). Transcriptome sequencing results from radiotherapy‐sensitive and ‐resistant cervical cancer patient tissues indicated IRF4's potential association with radiosensitivity (Figure 1D ). Q‐PCR revealed higher IRF4 expression in adjacent non‐cancerous tissues compared to cervical cancer tissues (Figure 1E ). Q‐PCR and Western blotting further confirmed IRF4's highest expression in H8 normal cervical squamous epithelial cells, followed by the C33a and HeLa cell lines, and then the Siha cell line (Figure 1F,G ). The expression levels of messenger RNA (mRNA) indicated in Figures 1E,F , and 2A . The protein expression levels indicated in Figures 1G and 2B . Immunohistochemical results showed weaker IRF4 expression in cervical cancer tissues compared to post‐radiotherapy tissues and significantly lower expression in cervical cancer tissues than in adjacent tissues (Figure 1H ).
IRF4 Inhibits Cervical Cancer Cell Growth In Vitro
Two cell lines (HeLa and Siha) were used for in vitro overexpression experiments to establish IRF4‐overexpressing cell models. RT‐qPCR and immunoblotting demonstrated upregulated IRF4 expression (Figure 2A,B ). Immunofluorescence detection revealed IRF4 expression in both the cytoplasm and nucleus, with particularly strong nuclear expression, which significantly increased 8 h post‐irradiation (Figure 2D ). CCK‐8 assays showed IRF4 overexpression inhibiting cervical cancer cell proliferation after 24 h of cell culture (Figure 2C ). Transwell migration and scratch assays found IRF4 overexpression significantly reducing cervical cancer cell migration ability without complete scratch healing within 48 h (Figure 2E,F ). Transwell invasion assays demonstrated IRF4 overexpression inhibiting cervical cancer cell invasion (Figure 2E ). Simultaneously, CCK‐8 monitoring revealed IRF4 upregulation inhibiting the proliferation of Siha and HeLa cells both before and after irradiation (Figure 2G ). EdU staining observed IRF4 reducing the proliferation rate compared to the control group before and after irradiation (Figure 2H ).
IRF4 Enhances Radiosensitivity of Cervical Cancer In Vitro
The effect of IRF4 overexpression on the radiosensitivity of Siha and HeLa cells in vitro was further investigated. Colony formation assays showed IRF4 upregulation significantly reducing the clonogenicity of Siha and HeLa cells both before and after radiotherapy (Figure 3A,B ). Resistance to apoptosis is a critical radiation injury in tumors [ 31 ]. This study explored IRF4's effect on X‐ray‐induced apoptosis using FCM, revealing IRF4 upregulation significantly increasing apoptosis in cervical cancer cells with or without X‐ray exposure (Figure 3C ). The bar chart displays Annexin V + /PI + double‐positive cells and Annexin V + /PI − cells. Meanwhile, cell cycle analysis showed a significant decrease in the S/G2 phase after IRF4 upregulation (Figure 3D ). Under radiation conditions (0–8 Gy), CCK‐8 determined the relative survival rates of Siha and HeLa cells treated with 3‐MA and Rapamycin (RAPA) after exposure to various radiation doses (0, 2, 4, 6, 8 Gy). The experimental results demonstrated that IRF4 overexpression in combination with either 3‐MA or RAPA (rapamycin, autophagy inducer) treatment led to significant inhibition of cell viability in both Siha and HeLa cervical cancer cell lines (Figure 3E,F ).
Overexpression of IRF4 Enhances Radiosensitivity of Cervical Cancer Cells Through Activation of Autophagy
Transcriptome sequencing of IRF4‐overexpressing Siha and HeLa cells, followed by KEGG enrichment analysis, revealed a potential association between IRF4 and the regulation of autophagy and apoptosis in cervical cancer cells (Figure 4A ). This suggests that IRF4 may influence the biological behavior of cervical cancer by modulating autophagy. Transmission electron microscopy (TEM) demonstrated a significant increase in the number of autophagic vacuoles (AVs) in irradiated cells overexpressing IRF4 ( p < 0.001), with autophagy evident across all cell groups (Figure 4B ). To further investigate, indirect immunofluorescence was employed to detect the expression and localization of autophagy‐related proteins LC3, P62, and Beclin‐1, while γ‐H2AX protein expression served as an indirect indicator of successful radiation induction (Figure 4C ). Immunoblotting assays showed that, compared to control cells, the IRF4‐overexpression (IRF4‐OE) group exhibited decreased SQSTM1/p62 protein levels, an increased LC3II/LC3I ratio, and elevated Beclin 1 expression (Figure 4D,E ). Given the crucial role of the PI3K/AKT/mTOR pathway inactivation in controlling tumor cell autophagy, with PI3K, AKT, and mTOR kinases acting as key autophagy regulators, we examined whether IRF4 activates or inhibits PI3K/AKT/mTOR pathway‐mediated autophagy. Immunoblotting revealed that IRF4 overexpression inhibited the expression of PI3K, Akt, mTOR, and their phosphorylated forms (Figure 4E ). Additionally, we explored the relationship between IRF4 expression and autophagy in cervical cancer tissue samples, with immunohistochemical staining showing a positive correlation between LC3B protein expression and IRF4 expression in tumor tissues ( r = 0.867, p < 0.001). Notably, radiation increased LC3B expression. Collectively, these results indicate that IRF4 enhances the radiosensitivity of cervical cancer cells by activating autophagy in vitro.
In Vivo Validation of IRF4‐Mediated Autophagy in Enhancing Radiosensitivity of Cervical Cancer
To assess the impact of IRF4 on cervical cancer growth in vivo, nude mice were subcutaneously inoculated with HeLa cells transfected with either Ad‐NC or Ad‐IRF4‐OE. The results demonstrated that the tumor growth rate was slower in the Ad‐IRF4‐OE group compared to the control Ad‐NC group (Figure 5A ), with a lower final tumor volume in the Ad‐IRF4‐OE group (Figure 5B ). When treated with RAPA, a known autophagy activator, in combination with radiation (IR), nude mice in both the Ad‐NC and Ad‐IRF4‐OE groups exhibited smaller tumor volumes compared to RAPA or IR alone. Notably, when treated simultaneously with RAPA + IR, the tumor volume in the Ad‐IRF4‐OE group was smaller than that in the Ad‐NC group (Figure 5A ), indicating that IRF4 enhances the sensitivity of cervical cancer to radiation and combined inhibitor treatment in vivo. Further investigation of IRF4's regulatory role on autophagy in vivo, based on tumor tissues, revealed reduced SQSTM1/p62 protein levels and increased LC3 and Beclin 1 expression in the Ad‐IRF4‐OE group (Figure 5B ). Additionally, the expression of PI3K, Akt, mTOR, and their phosphorylated forms was significantly lower in the Ad‐IRF4‐OE group compared to the Ad‐NC group (Figure 5C ). When 6G radiation was administered, the expression of PI3K, Akt, mTOR, and their phosphorylated forms was further inhibited in the Ad‐IRF4‐OE group compared to the Ad‐NC group (Figure 5C ). These findings suggest that IRF4 enhances the radiosensitivity of cervical cancer in vivo by regulating PI3K/Akt/mTOR‐mediated autophagy. Mechanism shown in Figure 6 .
Discussion
Currently, radiotherapy for cervical cancer confronts challenges such as radiotherapy resistance, which can lead to recurrence or metastasis in some patients [ 32 ], late toxicity impacting quality of life [ 33 ], uneven distribution of medical resources, obstacles in advancing precise radiotherapy in primary hospitals, inadequate individual optimization, and the absence of reliable biomarkers to guide treatment [ 34 , 35 ]. IRF4, a member of the IRF gene family, is a transcription factor protein with a basic leucine zipper (bZIP) structure [ 36 ]. Research has demonstrated that IRF4 can form heterodimers with IRF8 to bind ISREs, inhibiting the transcription of interferon‐induced genes and exerting transcriptional inhibitory effects in T cells and macrophages [ 37 , 38 ]. In B cells, IRF4 inhibits BCL6 expression by binding to its promoter, thereby promoting the terminal differentiation of B cells [ 39 ]. Notably, the interaction between IRF4 and BCL6 protein also inhibits IRF4's own transactivation capacity, creating a negative feedback regulatory loop [ 40 ]. These findings underscore IRF4's pivotal regulatory role in cell development, immune response, and tumorigenesis [ 41 ]. Building on previous transcriptome sequencing analysis of radiotherapy‐sensitive and resistant cervical cancer tissues, this study identified IRF4 as a potential key regulator of radiotherapy sensitivity in cervical cancer. This study delved into the role of IRF4 in mediating radiotherapy sensitivity in cervical cancer through in vivo and in vitro experiments, confirming that IRF4 impacts the radiosensitivity of cervical cancer by modulating autophagy via the PI3K/AKT/mTOR pathway.
Currently, there is a scarcity of reports on the role of IRF4 expression in tumor radiosensitivity. Although autophagy is one of the most intensively studied mechanisms linking IRF4 to cervical‐cancer proliferation and radiosensitivity, it is by no means the only route through which IRF4 exerts its effects. Studies have indicated that IRF4 could serve as a potential radiotherapy target for primary central nervous system lymphoma (PCNSL) [ 42 ]. Methylation patterns of IRF4 hold promise as indicators of radiochemotherapy response [ 43 ]. This study, for the first time, systematically unveiled the expression characteristics and clinical significance of IRF4 in cervical cancer. Through multi‐omics analysis, it was discovered that IRF4 is significantly underexpressed in cervical cancer tissues and its expression level is closely associated with patient prognosis. Notably, patients with high IRF4 expression exhibit greater sensitivity to radiotherapy, and its expression level can function as an independent molecular marker for predicting the efficacy of radiotherapy, potentially offering a novel strategy for clinical prognosis assessment. Wang et al. [ 25 , 44 ] reported that IRF4 overexpression can facilitate the transdifferentiation of Tregs into macrophage‐like cells, thereby inhibiting the progression of colon cancer. We further validated the role of IRF4 in mediating radiotherapy sensitivity in cervical cancer through in vivo and in vitro radiation experiments. Both in vivo and in vitro experimental findings support that IRF4 overexpression can hinder the progression of cervical cancer cells and augment their radiosensitivity. Therefore, this study provides compelling evidence for exploring the role of IRF4 in radiotherapy sensitivity in cervical cancer.
An in‐depth exploration of the molecular mechanisms underlying tumor radioresistance (IR) will provide crucial theoretical foundations for optimizing radiotherapy regimens and enhancing clinical efficacy. IR triggers cell death by eliciting cellular stress responses, including the generation of DNA damage responses, reactive oxygen species, endoplasmic reticulum stress, and mitochondrial damage [ 45 ]. However, cancer cells may develop radiation resistance by activating damage repair signaling pathways [ 46 , 47 ] or inhibiting death‐related signaling pathways [ 48 ]. Apoptosis and autophagy, as two fundamental forms of programmed cell death, play pivotal roles in regulating cell proliferation and survival maintenance in cervical cancer cells. Autophagy is a highly conserved catabolic process employed to maintain energy homeostasis, whereby damaged organelles and dysfunctional proteins are sequestered by autophagosomes and transported to lysosomes for degradation and recycling [ 49 , 50 ]. Some studies suggest that autophagy can function as a tumor suppressor [ 51 , 52 , 53 , 54 ]. The PI3K/Akt/mTOR pathway plays a vital role in regulating cellular autophagy [ 55 , 56 , 57 ]. This study utilized PCR array analysis and publicly accessible data to investigate the modes of cell death involved in IRF4‐mediated radiosensitivity. KEGG enrichment analysis revealed that IRF4 is closely linked to the regulation of apoptosis and autophagy in cervical cancer cells. We further verified that IRF4 overexpression can induce apoptosis in cervical cancer cells and activate PI3K/Akt/mTOR‐mediated autophagy.
3‐MA is a well‐known autophagy inhibitor widely used to disrupt autophagic flux by inhibiting class III PI3K [ 58 ], and Rapamycin (RAPA) is a recognized autophagy activator [ 59 ]. Studies have reported that the combination of trametinib and rapamycin can synergistically inhibit the MAPK and mTOR signaling networks, thereby more effectively inhibiting IRF4 expression [ 60 ]. This study found through KEGG enrichment analysis that the mTOR signaling pathway in cervical cancer is a downstream target of IRF4. At the cellular level, we observed that IRF4 overexpression combined with 3‐MA/RAPA significantly inhibits the proliferation of Siha and HeLa cells. We further confirmed through nude mouse xenograft models that, compared to RAPA or radiotherapy (IR) alone, RAPA combined with IR treatment markedly inhibits tumor growth; importantly, the tumor inhibition effect is more pronounced in IRF4‐overexpressing nude mice receiving combined treatment. These in vivo experimental results imply that IRF4 overexpression can synergistically enhance the sensitivity of cervical cancer to combined radiotherapy and mTOR inhibitor treatment.
In summary, this study confirms that IRF4 is significantly underexpressed in adjacent tissues of cervical cancer and can serve as a significant indicator for prognosis assessment in cervical cancer patients. IRF4 may act as a novel tumor suppressor gene in cervical cancer, regulating autophagy in cervical cancer cells and augmenting their radiosensitivity by inhibiting the PI3K/AKT/mTOR signaling pathway in vivo and in vitro. Therefore, IRF4 can serve as a potential biomarker for predicting radiosensitivity, and future combined treatment strategies targeting IRF4 (such as IRF4 agonists combined with radiotherapy) may considerably improve the local control rate and overall survival prognosis of patients with advanced cervical cancer by enhancing tumor radiosensitivity.
Author Contributions
Meihui Gao: conceptualization, writing – original draft. Yang Sun: conceptualization, funding acquisition. Zhaolei Cui: writing – review and editing. Huachun Song: data curation. Yanhong Li: resources. Jianfeng Zheng: conceptualization. Wankai Fu: validation.
Funding
Major Scientific Research Program for Young and Middle‐aged Health Professionals of Fujian Province, China (Grant No. 2022ZQNZD008); High‐level Talents Training Project of Fujian Cancer Hospital(Grant number: 2022YNG04); Clinical Research Center for Precision Treatment of Gynecological Malignancies of Fujian Province, China (Grant number: 2022Y2015).
Disclosure
Approval of the Research Protocol by an Institutional Reviewer Board: Approval Number for Review and Consent (K2024‐222‐01). Animal Studies: This experiment was approved by the Experimental Animal Ethics Committee of Fujian Provincial Hospital (Serial number: IACUC‐FPH‐SL‐20231207[0275]).
Consent
All informed consent was obtained from the subject(s) and/or guardian(s).
Conflicts of Interest
The authors declare no conflicts of interest.
Acknowledgments
The authors have nothing to report.
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