The effect and related mechanisms of RAC1 GTP on radiotherapy for hepatocellular carcinoma

Introduction

Hepatocellular carcinoma (HCC) represents the third most prevalent cause of cancer-related mortality globally (1). Epidemiological data indicate that the annual incident cases and mortality rates in China account for over 50% of the global burden, constituting a significant public health challenge (2). Current clinical guidelines identify surgical resection and liver transplantation as the primary curative interventions for early-stage HCC (3); however, an epidemiological study indicates that only 30% of patients qualify for these surgical modalities at initial diagnosis (3). Recent advances in radiation oncology, particularly stereotactic body radiotherapy, have demonstrated promising therapeutic outcomes in HCC management, both as monotherapy and in multimodal regimens. Nevertheless, emerging clinical observations have identified a subset of patients exhibiting radioresistant phenotypes, potentially attributable to acquired cellular radioresistance mechanisms (4).

Ras-related C3 botulinum toxin substrate 1 (RAC1), a pivotal regulator within the Rho guanosine triphosphatases (GTPase) family, operates through GTP/ guanosine diphosphate (GDP)-binding state transitions and demonstrates dysregulated expression across multiple malignancies (5,6). A large body of evidence points to RAC1 as being involved in various oncogenic processes including non-small cell lung carcinoma, ovarian cancer, and gastric adenocarcinoma, with its expression levels correlating significantly with histopathological differentiation, tumor-node-metastasis (TNM) staging, metastatic potential, and survival outcomes (7-9). Molecular studies on the pathogenesis of HCC have confirmed RAC1’s involvement in modulating neoplastic proliferation and metastatic dissemination through multiple signaling cascades (10-12).

Emerging radiobiological research has identified RAC1 as a potential mediator of radiotherapy response (13-15). Mechanism-related investigations suggest that RAC1 overexpression facilitates radiation resistance through the induction of G2/M cell cycle arrest and the activation of prosurvival signaling pathways, including the extracellular signal-regulated protein kinase 1 and 2 (ERK1/2) and nuclear factor-κB (NF-κB) (16,17). Notably, Hein et al. reported that upregulation of RAC1 in radioresistant breast cancer models was associated with ERK1/2 and NF-κB pathway activation, which was accompanied by elevated expression of antiapoptotic mediators Mcl-L and Bcl-xL (13). Despite these discoveries, the functional role of RAC1 in HCC radioresistance remains unexplored.

To address this deficiency in research, this study systematically investigated RAC1’s pathophysiological role in HCC radiotherapy through three principal steps: (I) comparative analysis of RAC1 expression patterns in HCC versus those in adjacent nonneoplastic hepatic tissues; (II) evaluation of RAC1’s prognostic significance through survival analysis; and (III) exploration of the mechanism related to the RAC1-mediated radioresistance pathways in HCC models. We present this article in accordance with the TRIPOD and MDAR reporting checklists (available at https://tcr.amegroups.com/article/view/10.21037/tcr-2025-987/rc).

Methods

Cells and clinical specimens

The human hepatic cellular components used in this investigation comprised the human hepatocyte cell line HL-7702 and HCC cell lines HCCLM3, MHCC97-H, HepG2, and Huh7. While HL-7702, HCCLM3, and MHCC97-H were commercially procured (Fuheng Biotechnology, Xi’An, China), HepG2 and Huh7 lines were obtained through academic collaboration with the Ningbo University School of Medicine. Cultivation protocols followed established methodologies: HL-7702 cells were maintained in RPMI-1640 medium (Gibco, Thermo Fisher Scientific, Waltham, MA, USA) supplemented with 10% fetal bovine serum (FBS; Gibco) and 1% penicillin-streptomycin (Thermo Fisher Scientific). HCC cell lines were propagated in Dulbecco’s Modified Eagle Medium (DMEM; Gibco) containing identical serum and antibiotic concentrations. All cultures were incubated under standardized conditions (37 ℃ and a 5% CO₂ humidified atmosphere) with routine subculturing at 80–90% confluence to maintain an exponential growth phase.

A retrospective cohort of 21 paired HCC and adjacent non-tumorous tissues (≤2 cm from tumor margin) was obtained from treatment-naïve patients undergoing curative resection at The Affiliated Lihuili Hospital of Ningbo University (January 2021 to December 2021). Specimens were cryopreserved at −80 ℃ within 30 minutes post-resection in the biorepository at the Ningbo Clinical Pathological Diagnosis Center. The inclusion criteria were as follows: (I) primary HCC diagnosis confirmed by histopathology; (II) tumor-free adjacent tissue verified microscopically; (III) complete clinicopathological documentation; and (IV) absence of preoperative anticancer therapies. Meanwhile, the exclusion criteria were as follows: (I) concurrent malignancies and (II) radiological of histopathological evidence of recurrence or metastasis. The study was conducted in accordance with the Declaration of Helsinki and its subsequent amendments. This study was approved by the Ethics Committee of The Affiliated Lihuili Hospital, Ningbo University (No. KY2022SL322-01). The requirement for individual consent was waived due to the retrospective nature of the analysis.

Reagents and antibodies

The following immunochemical reagents were employed: rabbit monoclonal anti-RAC1 antibody (clone ARC03;cCat #CY-2103; CytoSkeleton, Inc., Denver, CO, USA), phospho-IκBα (Ser32) antibody [Cat #2859S; Cell Signaling Technology (CST), Inc., Danvers, MA, USA], IκBα antibody (Cat #4814S; CST), and rabbit polyclonal Bcl-xL antibody (clone ARC0183; Cat #A19703; ABclonal Technology Co., Ltd., Woburn, MA, USA).

Small-molecule inhibitors

The RAC1 GTPase-specific inhibitor NSC23766 trihydrochloride (CAS 1177865-17-6; lot #SML0952; Sigma-Aldrich/Merck KGaA, Darmstadt, Germany) was reconstituted in sterile ultrapure water to prepare a 100-mM stock solution, with working concentrations titrated to 100 µM in experimental conditions.

Commercial assay kits

The commercial assay kits used in this study include RAC1 Activation Assay Kit (G-LISA BK128; CytoSkeleton, Inc.) and Cell Cycle Analysis Kit (lot #KGA511; KeyGEN Biotech Co., Ltd., Nanjing, China).

Cell culture and experimental design

MHCC97-H cells in a logarithmic growth phase were seeded into six-well plates at a density of 2×10⁵ cells/well and maintained in a humidified incubator until reaching 50–60% confluency as confirmed by phase-contrast microscopy. Experimental cohorts were divided into four groups: (I) untreated control, with cells being cultured in standard growth medium; (II) radiation therapy (RT), with cells being exposed to 15 Gy of X-ray irradiation; (III) pharmacological inhibition (NSC23766), with cells being treated with 100 µM of NSC23766 (RAC1 GTP inhibitor) in serum-free medium; and (IV) combined therapy, with cells being subjected to both NSC23766 treatment and irradiation. For the pharmacological cohorts, NSC23766 was reconstituted in sterile ultrapure water and diluted to 100 µM in prewarmed DMEM. All groups were incubated for 1 hour under standard culture conditions to ensure drug uptake or equilibration.

Radiation protocol

Irradiation was performed using a Primus H linear accelerator (Siemens Healthineers, Erlangen, Germany) calibrated to deliver 6-MV X-rays at a dose rate of 200 cGy/min. Cells were positioned at a source-to-surface distance of 100 cm, with the field size and collimator angles optimized to ensure uniform dose distribution across the plate. Following irradiation, the cells were immediately returned to the incubator for subsequent analyses.

Bioinformatics and survival analysis of RAC1 in HCC

Transcriptomic data and clinical annotations for RAC1 in HCC were retrieved from The Cancer Genome Atlas (TCGA) via the UCSC Xena platform (https://xenabrowser.net/datapages/). RNA sequencing (RNA-seq) data from HCC tissues and adjacent non-tumorous tissues were normalized to transcripts per million (TPM) and log₂-transformed [log₂(1+ TPM)] to mitigate skewness. Differential expression of RAC1 between tumor and normal cohorts was evaluated using a two-tailed Student t-test, with a significance defined as P<0.05. Associations between RAC1 expression and clinicopathological parameters (e.g., TNM stage) were analyzed using Kruskal-Wallis or Wilcoxon rank-sum tests, as appropriate.

For survival analysis, patients with HCC were dichotomized into RAC1-low and RAC1-high subgroups based on an optimal prognostic cutoff as determined by maximally selected rank statistics via the “survminer” R package (The R Foundation for Statistical Computing). Kaplan-Meier curves were generated to compare overall survival (OS), disease-specific survival (DSS), disease-free interval (DFI), and progression-free interval (PFI), with log-rank tests used to assess significance. Univariate and multivariate Cox proportional hazards regression models were implemented to evaluate RAC1 as an independent prognostic factor, with adjustments made for covariates such as age, sex, and tumor stage. Analyses were performed using the “survival” and “ggplot2” packages in R (v.4.1.1). A prognostic nomogram integrating pT stage and RAC1 expression levels was constructed to predict the 1-, 3-, and 5-year OS probabilities. Model performance was validated via calibration plots and quantified using the Harrell concordance index (C-index). All statistical workflows were replicated on the online bioinformatics platform Sangerbox (https://www.home-for-researchers.com/static/index.html#/) to ensure reproducibility.

Immunohistochemical analysis

Antibody staining outcomes were quantified via microscopy under a semiquantitative scoring system that integrated both the proportion of immunopositive cells and staining intensity. Stained sections were evaluated independently by two blinded pathologists to minimize observer bias. Positivity thresholds were defined as follows: samples exhibiting ≤10% antigen-positive cells within the region of interest were classified as negative (score =0); >10% to ≤30% positivity indicated weak expression (score =1); >30% to ≤60% positivity indicated moderate expression (score =2); and >60% positivity indicated strong expression (score =3).

Detection of protein expression levels

Western blotting was employed to quantify the expression levels of target proteins under experimental conditions. MHCC97-H cells were divided into four cohorts: (I) untreated control; (II) a pharmacologically treated group incubated with 100 µM of NSC23766 for 1 hour; (III) a radiation-treated group exposed to 15 Gy of X-ray irradiation via a linear accelerator; and (IV) a combined treatment group. Post-irradiation, cells were maintained in a humidified incubator (37 ℃ in a 5% CO₂ atmosphere) for 2 and 48 hours to evaluate temporal changes in p-IκBα, IκBα, and Bcl-xL expression.

Total protein was extracted using radioimmunoprecipitation assay buffer (RIPA) lysis buffer supplemented with protease and phosphatase inhibitors. Protein concentrations were determined via bicinchoninic acid assay (BCA), and lysates were standardized to equal concentrations prior to separation by 10% sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE). Resolved proteins were electrophoretically transferred to polyvinylidene difluoride membranes at 100 V for 1 hour in transfer buffer. Membranes were blocked for 1 hour at room temperature in 5% nonfat dry milk or 3% bovine serum albumin in Tris-buffered saline with 0.1% Tween-20 (TBST), depending on the primary antibody requirements. Blocked membranes were incubated overnight at 4 ℃ with the primary antibodies diluted in TBST described below. After three 10-minute TBST washes, membranes were probed with species-matched horseradish peroxidase (HRP)-conjugated secondary antibodies for 2 hours at room temperature. Immunoreactive bands were visualized using enhanced chemiluminescence substrate and imaged on an imaging system. Band intensities were quantified using ImageJ software (US National Institutes of Health, Bethesda, MD, USA) normalized to β-tubulin levels.

RAC1 GTP activity assay

MHCC97-H HCC cells were irradiated with a single dose of 15 Gy via a linear accelerator. Cellular proteins were extracted within 30 minutes post-irradiation with ice-cold RIPA lysis buffer to preserve phosphorylation states. RAC1 GTPase activation levels were quantitatively assessed using a G-LISA RAC1 Activation Assay Kit (CytoSkeleton, Inc., Denver, CO, USA) according to the manufacturer’s protocol. Briefly, protein concentrations were determined via BCA. Lysates were mixed with protein detection reagent, and absorbance was measured at 562 nm with a Thermo Fisher Scientific microplate reader (Thermo Fisher Scientific Inc., Waltham, MA USA). Lysate concentrations were normalized to 1.0 mg/mL via dilution buffer. For the G-LISA assay, 50 µL aliquots of blank buffer (negative control), RAC1 GTP-bound positive control, and experimental lysates were loaded in triplicate onto a 96-well affinity plate precoated with a RAC1GTP binding protein. The plate was incubated at 4 ℃ for 30 minutes on a microplate shaker (400 rpm) to facilitate RAC1GTP binding. Post-incubation, wells were aspirated and washed three times with wash buffer. Antigen-presenting buffer was added for 5 minutes to stabilize bound complexes, followed by sequential 1-hour incubations with primary anti-RAC1 antibody and HRP-conjugated secondary antibody at room temperature. After final washes, 50 µL of HRP detection reagent was added, and the reaction was terminated with termination buffer. Absorbance was immediately quantified at 490 nm with enzyme-linked immunosorbent. Raw optical density (OD) values were normalized to total RAC1 levels.

Cell cycle detection

Following irradiation, cells were maintained in a humidified incubator (37 ℃ in a 5% CO₂ atmosphere) for 24 hours. Post-incubation, cell suspensions were harvested from experimental cohorts, and cellular density was quantified via a hemocytometer to standardize concentrations to 1×10⁶ cells/mL. Aliquots of 1-mL single-cell suspensions were subsequently centrifuged and fixed in 4 ℃-prechilled 70% (v/v) ethanol for a duration of 2–24 hours. Following fixation, ethanol was decanted, and cells were resuspended in a propidium iodide (PI)/RNase A staining solution (1:9 ratio; RNase A:PI) to facilitate nuclear DNA content labeling. Stained samples were incubated in darkness at ambient temperature (25 ℃) for 45 minutes to ensure uniform dye penetration. Cell cycle progression was assessed using a BD Accuri C6 flow cytometer. Raw flow cytometry data were analyzed using ModFit LT to quantify G₀/G₁, S, and G₂/M phase distributions.

Statistical analysis

In this study, data analysis and visualization were performed using RStudio (version 4.1.1) for statistical computing, Adobe Photoshop CC 2017 (Adobe, San Jose, CA, USA) for image processing and formatting, GraphPad Prism 7 (Dotmatics, Boston, MA, USA) for graphical representation, and ModFit software (Verity Software House, Topsham, ME, USA) for specialized analytical workflows. Statistical methodologies were selected based on data distribution characteristics and experimental design requirements. Comparative analyses between groups included two-sample independent and paired Student t-tests for parametric data, one-way or multifactorial analysis of variance (ANOVA) for multi-group comparisons, and nonparametric alternatives when assumptions of normality (evaluated via the Shapiro-Wilk test) or homogeneity of variance (assessed by the Levene test) were violated. Survival outcomes were analyzed with Kaplan-Meier curves and log-rank tests, complemented by univariate and multivariate Cox proportional hazards regression models to adjust for potential confounding variables. A two-tailed significance threshold of P<0.05 was applied across all inferential analyses to determine statistical relevance.

Results

Expression of RAC1 in HCC tissues and adjacent non-tumor tissues

RAC1 messenger RNA (mRNA) expression was significantly elevated in HCC tissues as compared to adjacent non-tumor tissues (Figure 1A). This observation was further validated through paired sample analysis, which also demonstrated a statistically significant difference (Figure 1B). Notably, RAC1 mRNA levels were markedly higher in tumor tissues from patients with advanced-stage HCC as compared to those with early-stage HCC (Figure 1C). To further characterize the differential expression of RAC1 at the protein level, immunohistochemical analysis was conducted on postoperative pathological specimens from 21 patients with HCC. The results revealed a significant upregulation of RAC1 protein expression in HCC tissues relative to adjacent non-tumor tissues (Figure 1D-1F).

Figure 1 Expression of RAC1 mRNA and its protein levels. (A,B) Expression of RAC1 mRNA in tumor tissues and adjacent tissues. (C) Expression of RAC1 mRNA in different stages of tumors. (D,E) Immunohistochemical analysis of RAC1 expression in tumor tissues and adjacent tissues. (F) Comparison of differences in RAC1 expression between tumor tissues and adjacent tissues. *, P<0.05; **, P<0.01; ****, P<0.0001. ns, not significant; LIHC, liver hepatocellular carcinoma; RAC1, Ras-related C3 botulinum toxin substrate 1; TPM, transcripts per million.

Association between RAC1 expression and prognosis in patients with HCC

Kaplan-Meier survival analysis was conducted to evaluate the relationship between RAC1 expression levels and clinical outcomes in patients with HCC. The results demonstrated a significant correlation between RAC1 expression and patient survival. Specifically, patients with high RAC1 expression exhibited markedly worse outcomes as compared to those with low RAC1 expression, as evidenced by significant differences in OS, DSS, DFI, and PFI (Figure 2A-2D). Statistical analysis further indicated that patients with low RAC1 expression had a significantly more favorable prognosis than did those with high RAC1 expression.

Figure 2 Relationship between RAC1 expression level and prognosis. (A) OS; (B) DSS; (C) DFI; (D) PFI. DFI, disease-free interval; DSS, disease-specific survival; OS, overall survival; PFI, progression-free interval; RAC1, Ras-related C3 botulinum toxin substrate 1.

Prognostic factor analysis and development of a nomogram prediction model for patients with HCC

Univariate Cox regression analysis revealed that pT stage (P<0.001), pM stage (P=0.02), and RAC1 expression level (P=1e−05) were significantly associated with the survival prognosis of patients with HCC (Figure 3A). To identify independent prognostic factors, multivariate Cox regression analysis was performed, with adjustments made for other clinicopathological variables. The results indicated that pT stage (P<0.001) and RAC1 expression (P=0.002) were independent adverse prognostic factors for OS in patients with HCC (Figure 3B). Furthermore, a nomogram incorporating pT stage and RAC1 expression levels was constructed to predict the 1-, 3-, and 5-year survival rates of patients with HCC (Figure 3C,3D).

Figure 3 Analysis of prognostic factors and establishment of a prognostic prediction model for patients with HCC. (A) Univariate Cox analysis. (B) Multivariate Cox analysis. (C) Column chart. (D) Calibration curve of column chart model. CI, confidence interval; HCC, hepatocellular carcinoma; Mult_Cox, multivariate cox; pM, pathological metastasis; pN, pathological node; Pro, probability; pT, pathological tumor; RAC1, Ras-related C3 botulinum toxin substrate 1; Uni_Cox, univariate cox.

Expression of RAC1 in different HCC cell lines and the effect of radiation on RAC1 activity levels

In this study, we examined the expression levels of RAC1 protein across four distinct HCC cell lines. Western blot analysis revealed that the expression of RAC1 was significantly elevated in the MHCC97-H cell line as compared to the other three HCC cell lines (Figure 4A,4B). This finding suggests that the MHCC97-H cell line may exhibit a higher dependency on RAC1, providing valuable insights for future investigations. Additionally, we examined the effect of radiation on RAC1 activity in MHCC97-H cells. GTPase activity assays demonstrated that the GTP-binding activity of RAC1 was significantly increased in MHCC97-H cells following radiation treatment as compared to a nonirradiated condition (Figure 4C).

Figure 4 The expression of RAC1 in HCC cell lines and the changes in the levels of RAC1 GTP in HCC cells before and after radiation. (A) Protein electrophoresis pattern. (B) Comparison of RAC1 expression levels. (C) Comparison of the changes in the levels of RAC1 GTP. *, P<0.05; **, P<0.01; *** P<0.001. GAPDH, glyceraldehyde-3-phosphate dehydrogenase; GTP, guanosine triphosphate; HCC, hepatocellular carcinoma; IR, ionizing radiation; RAC1, Ras-related C3 botulinum toxin substrate 1.

The role of RAC1 GTPase in regulating the p-IκBα/Bcl-xL pathway in MHCC97-H cells following radiation exposure

In this study, Western blot analysis was employed to assess the impact of radiation on the phosphorylation levels of IκBα in MHCC97-H HCC cells. The results demonstrated that the phosphorylation level of IκBα was significantly upregulated in MHCC97-H cells exposed to 15 Gy of radiation as compared to an untreated condition (P=0.03). Furthermore, the effect of the RAC1 GTPase inhibitor NSC23766 on radiation-induced IκBα phosphorylation was investigated. The findings revealed that the phosphorylation level of IκBα was significantly downregulated in cells treated with a combination of NSC23766 and radiation as compared to a radiation-only treatment (P<0.001) (Figure 5A,5B).

Figure 5 The effect of RAC1 GTP on the p-IκBα/Bcl-xL signaling pathway in MHCC97-H HCC cells after radiation. (A) Protein electrophoresis map. (B) Comparison of p-IκBα expression levels. (C) Comparison of Bcl-xL expression levels. *, P<0.05. GTP, guanosine triphosphate; HCC, hepatocellular carcinoma; IR, ionizing radiation; RAC1, Ras-related C3 botulinum toxin substrate 1.

To further elucidate the role of RAC1 in regulating downstream target gene expression, the protein expression level of Bcl-xL was examined. The results indicated that radiation treatment significantly increased the expression of Bcl-xL in MHCC97-H cells (P=0.01). However, this upregulation was markedly suppressed following treatment with the RAC1 GTPase inhibitor NSC23766 (P=0.008) (Figure 5A,5C), suggesting that RAC1 activation is critically involved in radiation-induced Bcl-xL expression.

The role of RAC1 GTPase in G2/M phase arrest in MHCC97-H HCC cells following radiation exposure

Flow cytometry analysis was used to investigate the impact of radiation on the cell cycle distribution of MHCC97-H HCC cells. The results revealed that within 24 hours of exposure to 15 Gy of radiation, the 4N-DNA content (representing the DNA content of G2/M phase cells) in MHCC97-H cells increased significantly (P<0.001), with the proportion of G2/M phase cells exceeding 70%. This observation indicates that radiation treatment induced pronounced cell cycle arrest at the G2/M phase. Furthermore, in cells treated with a combination of the RAC1 GTPase inhibitor NSC23766 and radiation, the proportion of G2/M phase cells was significantly reduced compared to that in the group treated with radiation alone (P<0.001), suggesting that NSC23766 can mitigate radiation-induced G2/M phase arrest. Notably, treatment with NSC23766 alone, in the absence of radiation, did not significantly alter the 4N-DNA content, indicating that the effect of NSC23766 on the cell cycle may be contingent upon the presence of radiation (Figure 6A-6C). These findings provide critical insights into the potential mechanisms underlying the radiosensitizing effects of NSC23766.

Figure 6 The effect of RAC1 GTP on G2/M phase arrest in MHCC97-H HCC cells after radiation. (A) Cell cycle distribution map. (B) Comparison of different cell cycle proportions. (C) The proportion of different cell cycles. ****, P<0.0001. GTP, guanosine triphosphate; HCC, hepatocellular carcinoma; RAC1, Ras-related C3 botulinum toxin substrate 1.

Discussion

Radiotherapy has gained increasing prominence in the treatment of HCC, demonstrating favorable therapeutic outcomes. However, a subset of patients exhibits suboptimal response, which may be attributed to the development of radiation resistance in hepatocellular cells. This resistance is a critical factor contributing to tumor recurrence (18). Consequently, elucidating the molecular mechanisms underlying the evasion of radiotherapy-induced cell death in HCC has become an urgent research priority. Furthermore, the identification of effective combination therapeutic strategies to enhance the efficacy of radiotherapy holds significant potential for improving overall treatment outcomes in patients with HCC.

As a pivotal member of the Rho GTPase family, RAC1 has a unique molecular mechanism, with a characteristic ability to alternate between the GTP-bound active state and the GDP-bound inactive state. Emerging evidence suggests that RAC1 is aberrantly expressed in various malignancies and plays a crucial regulatory role in multiple cellular processes, including cell proliferation, apoptosis, invasion, metastasis, and angiogenesis, thereby significantly contributing to tumorigenesis and cancer progression (5). In the context of non-small cell lung cancer, Zhou et al. (7) reported significantly elevated RAC1 expression levels in tumor tissues as compared to adjacent non-tumor tissues, with a strong correlation observed between RAC1 expression and both TNM staging and metastatic potential. Similarly, Leng et al. (8) identified a positive association between RAC1 expression levels and advanced disease characteristics in epithelial ovarian cancer, including poor differentiation, elevated serum CA-125 levels, and larger residual tumor volumes. Their prognostic analysis further revealed that patients with high RAC1 expression were more susceptible to early tumor recurrence. In our study, we observed significant upregulation of RAC1 expression in HCC tissues as compared to adjacent non-tumor tissues. Comprehensive analysis of clinicopathological correlations demonstrated that RAC1 expression levels were substantially higher in patients with advanced HCC as compared to those with early-stage disease. Through Kaplan-Meier survival analysis, we established a significant association between elevated RAC1 expression levels and poorer OS, DSS, DFI, and PFI in patients with HCC. To validate these bioinformatics findings, we conducted immunohistochemical analysis of postoperative pathological specimens, which consistently demonstrated higher RAC1 protein expression levels in HCC tissues as compared to adjacent non-tumor tissues. Collectively, these findings suggest that RAC1 may play a critical role in HCC pathogenesis and progression, potentially serving as a valuable biomarker for predicting poor prognosis in patients with HCC. Furthermore, to assess the prognostic value of RAC1 in HCC management, we developed a comprehensive nomogram incorporating pT stage and RAC1 expression levels to the predict 1-, 3-, and 5-year survival rates. This predictive model demonstrated excellent prognostic efficacy and may be valuable tool for more accurate prognosis prediction and may serve as a reference for developing personalized treatment strategies among patients with HCC.

Research has gradually confirmed the pivotal role of RAC1 in modulating radiotherapy efficacy, with proposed mechanisms centering on the upregulation of RAC1 expression and activity, followed by the subsequent activation of downstream antiapoptotic or proapoptotic signaling pathways, which ultimately contribute to the development of radioresistance or radiosensitivity in tumor cells (16). In the context of breast cancer radiotherapy response, Yan et al. demonstrated that the pharmacological inhibition of RAC1 activity or genetic suppression of RAC1 expression could effectively attenuate radiation-induced phosphorylation of mitogen-activated protein MEK1/2 and ERK1/2, thereby significantly enhancing the radiosensitivity of breast cancer cells (17). Furthermore, their research revealed that elevated RAC1 expression correlated with enhanced activation of both ERK1/2 and NF-κB signaling pathways, accompanied by increased expression of downstream antiapoptotic proteins Bcl-xL and Mcl-L (13). Notably, upon radiation stimulation, the levels of activated (GTP-bound) RAC1 increase significantly within cancer cells, accompanied by a pronounced enhancement in its binding to p21-activated kinase1 (PAK1) (19). Subsequently, activated PAK1 can augment IκB kinase (IKK) complex activity, indirectly via the activation of the upstream NF-κB-inducing kinase. The activated IKK complex then specifically phosphorylates the IκBα protein. This phosphorylation event targets IκBα for degradation via the ubiquitin-proteasome pathway, resulting in the liberation of the NF-κB dimer. This liberation facilitates the nuclear translocation of NF-κB, ultimately initiating the transcription of genes encoding anti-apoptotic proteins, including Bcl-xL, Bcl-2, and Mcl-1 (20,21). Consistent with this mechanism, our study observed significant upregulation of RAC1 activity levels in irradiated MHCC97-H HCC cells. Specifically, MHCC97-H cells exposed to 15 Gy of radiation demonstrated increased IκBα phosphorylation and elevated Bcl-xL expression as compared to nonirradiated cells. Notably, concurrent administration of the RAC1 GTPase-specific inhibitor NSC23766 effectively suppressed both radiation-induced IκBα phosphorylation and Bcl-xL expression. These findings suggest that RAC1 GTPase may contribute to the radioresistance mechanism in HCC cells through the activation of the p-IκBα/Bcl-xL signaling pathway. Consequently, the RAC1 GTPase inhibitor NSC23766 emerged as a potential radiosensitizer with promising clinical applications for enhancing radiotherapy response in patients with HCC.

The activation of the G2/M phase cell cycle checkpoint in response to radiation-induced DNA double-strand breaks facilitates cell cycle arrest, thereby providing cells with the temporal opportunity to initiate DNA damage repair mechanisms prior to mitotic entry, ultimately enabling cellular resistance to radiation-induced cell death (22). Gogineni et al. (23) and Anastasov et al. (24) have demonstrated there to be a strong correlation between G2/M phase arrest and radiation resistance in meningioma and breast cancer cells, respectively. Notably, Herst et al. (25) reported enhanced radiosensitivity in glioblastoma through the pharmacological inhibition of G2/M phase arrest via ascorbic acid. Furthermore, in breast cancer models, radiation-induced RAC1 activation has been shown to mediate G2/M phase arrest in MCF-7 cells, thereby enhancing tumor cell viability. Specifically, RAC1 induces oxygen species -dependent DNA damage to activate the ataxia telangiectasia and Rad3-related kinase (ATR)-checkpoint kinase 1 (CHK1) checkpoint pathway. Activated CHK1 phosphorylates cell division cycle 25C (CDC25C) at serine 216, leading to its inactivation and cytoplasmic sequestration. This subsequently inhibits cyclin-dependent kinase 1 (CDK1) activity, ultimately preventing the Cyclin B-CDK1 complex from driving the G2/M transition. CHK1 represents the most direct upstream regulator through which RAC1 triggers G2/M arrest. Importantly, pharmacological inhibition of RAC1 GTPase activity has been demonstrated to attenuate G2/M phase arrest, consequently improving radiosensitivity (17). In our study, we observed significant upregulation of RAC1 activity levels in HCC cells following radiation exposure, concomitant with the induction of G2/M phase arrest. These findings indicate that RAC1 likely functions as a core upstream regulatory component of the G2/M checkpoint in HCC cells. Its upregulated activity may modulate checkpoint regulation via activation of the CHK1-CDC25C pathway, resulting in G2/M arrest, enhanced DNA repair capacity, and ultimately, radiotherapy resistance. Insights into these radioresistance mechanisms may reveal novel therapeutic targets, such as targeting the G2/M checkpoint (e.g., with CHK1 inhibitors) or the RAC1 signaling pathway, to enhance tumor cell radiosensitivity and overcome radioresistance.

However, this study was subject to several limitations that warrant consideration. To begin, analysis of the TCGA cohort revealed that almost all HCC patients included in this dataset had not undergone radiotherapy. This absence of patients treated with radiotherapy constitutes a significant limitation of our study. Although our results demonstrated a significant association between high RAC1 expression and adverse prognosis in HCC patients, the lack of samples from radiotherapy-treated patients within the cohort precludes assessment of any potential association between RAC1 expression levels and tumor response to radiotherapy (radiosensitivity or radioresistance). Future research requires the inclusion of prospective or retrospective cohorts comprising HCC patients who have received curative-intent or adjuvant radiotherapy. This would enable direct analysis of longitudinal changes in RAC1 expression within paired tumor tissue samples collected pre- and post-radiotherapy, investigating their correlation with radiotherapy efficacy, local control rates, and survival outcomes. However, the routine acquisition of such longitudinally paired tissue specimens from HCC patients in clinical practice presents a major challenge. To circumvent this limitation, subsequent research directions will focus on evaluating the correlation between RAC1 expression levels and radiotherapy efficacy through the analysis of RAC1 expression in peripheral blood samples collected pre- and post-radiotherapy in patients with HCC. Additionally, the experimental investigation was confined to a single HCC cell line, potentially limiting the generalizability and broader applicability of the findings. Consequently, future investigations should encompass a more comprehensive panel of HCC cell lines to validate the role of RAC1 in radiation response and establish its consistency across diverse cellular contexts. Moreover, while this study demonstrated that radiation-induced upregulation of RAC1 activity in HCC cells triggers G2/M phase arrest, the exact molecular mechanisms underlying RAC1 GTPase-mediated regulation of cell cycle checkpoints remain to be fully elucidated. Therefore, future studies should aim to delineate the specific molecular pathways through which RAC1 GTPase influences G2/M phase arrest following radiation exposure in HCC cells. This work could provide a robust scientific foundation for developing strategies that can enhance the efficacy of radiotherapy in HCC treatment.

In conclusion, RAC1 has an abnormally high expression in HCC tissues, which is significantly associated with the poor prognosis of patients. Moreover, radiation is able to induce an enhancement of RAC1 activity, an effect that may activate an antiapoptotic signaling pathway downstream of RAC1, and provide a time window for DNA damage repair through the triggering of cell cycle checkpoint arrest in the G2/M phase, which may promote the resistance of HCC cells to radiation.

Conclusions

This study demonstrated that RAC1 is significantly overexpressed in HCC tissues and exhibits a strong correlation with poor clinical outcomes in patients with HCC. In terms of mechanism, radiation-induced aberrant activation of RAC1 appears to contribute to the development of radioresistance through two parallel pathways: (I) upregulation of the p-IκBα/Bcl-xL antiapoptotic signaling cascade and (II) induction of G2/M phase cell cycle arrest. These findings represent the first clarification of the molecular mechanisms underlying RAC1 GTPase-mediated radiation resistance in HCC cells, thereby providing a scientific rationale for the development of novel therapeutic strategies aimed at enhancing the efficacy of radiotherapy in HCC treatment.

Acknowledgments

None.

Footnote

Reporting Checklist: The authors have completed the TRIPOD and MDAR reporting checklists. Available at https://tcr.amegroups.com/article/view/10.21037/tcr-2025-987/rc

Data Sharing Statement: Available at https://tcr.amegroups.com/article/view/10.21037/tcr-2025-987/dss

Peer Review File: Available at https://tcr.amegroups.com/article/view/10.21037/tcr-2025-987/prf

Funding: This work was supported by funding from the Beijing Sci-Tech Innovation Medical Development Foundation (No. KC2023-JX-0288-BM148) and the Wu Jieping Medical Foundation (No. 320.6750.2024-13-68).

Conflicts of Interest: All authors have completed the ICMJE uniform disclosure form (available at https://tcr.amegroups.com/article/view/10.21037/tcr-2025-987/coif). The authors have no conflicts of interest to declare.

Ethical Statement: The authors are accountable for all aspects of the work in ensuring that questions related to the accuracy or integrity of any part of the work are appropriately investigated and resolved. The study was conducted in accordance with the Declaration of Helsinki and its subsequent amendments. This study was approved by The Ethics Committee of The Affiliated Lihuili Hospital, Ningbo University (No. KY2022SL322-01). The requirement for individual consent was waived due to the retrospective nature of the analysis.

Open Access Statement: This is an Open Access article distributed in accordance with the Creative Commons Attribution-NonCommercial-NoDerivs 4.0 International License (CC BY-NC-ND 4.0), which permits the non-commercial replication and distribution of the article with the strict proviso that no changes or edits are made and the original work is properly cited (including links to both the formal publication through the relevant DOI and the license). See: https://creativecommons.org/licenses/by-nc-nd/4.0/.

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(English Language Editor: J. Gray)