Integrated multi‐omics analysis of post‐translational modifications in head and neck squamous cell carcinoma: elucidating x‐ray and carbon ion irradiation effects

Abstract

Background and purpose

Head and neck squamous cell carcinoma (HNSCC) exhibits heterogeneous responses to radiotherapy; however, the underlying multi‐omics regulatory networks remain poorly characterized. This study elucidated the distinct post‐translational modification (PTM) landscapes induced by X‐ray and carbon‐ion irradiation.

Methods

Using integrated proteomics, phosphoproteomics, and acetylomics, we systematically profiled PTMs in HSC‐3 cells at 30 min post‐irradiation with X‐ray (8 Gy) or carbon ions (4 GyE). Computational analyses included functional annotation, kinase activity prediction, and PTM crosstalk mapping.

Results

Carbon ions induced 21% more phosphosites (2,781 vs. 2,301) and 61% more acetyl sites (3,324 vs. 2,070) than X‐ray, demonstrating broader PTM reprogramming. Notably, carbon ion‐induced histone hypoacetylation (H2AZ1K8/K5, H3C1K28/K24, and H4C1K92) and hyperphosphorylation (H1‐10S2 and H3C1T119) potentially alter chromatin accessibility. Functional enrichment analyses revealed the activation of the non‐homologous end‐joining (NHEJ) pathway and coordinated upregulation of antigen‐presenting machinery (such as CD274, TAP2, HLA‐A, and PSMB8) through PTM remodeling. Distinct PTM signatures in RNA splicing factors implicate alternative splicing in radiation response. Kinase activity profiling revealed ATM hyperactivation and CDK suppression in the carbon ion group.

Conclusions

Carbon ion irradiation drives distinct PTM‐mediated immunomodulatory and DNA damage‐response networks, providing a mechanistic rationale for combining carbon ion radiotherapy with immunotherapy. Future studies should validate the PTM‐associated targets to improve HNSCC treatment.

This study underscores the critical role of PTMs in mediating cellular responses to X‐ray and Carbon ion irradiation radiotherapy in HNSCC. The integration of multi‐omics data provides insights into the molecular mechanisms underlying radiation‐induced changes, offering valuable clues for improving therapeutic outcomes and advancing precision medicine in head and neck cancer.

1. INTRODUCTION

Head and neck squamous cell carcinoma (HNSCC) is the sixth most common cancer worldwide, accounting for over 90% of all head and neck malignancies. ¹ Its anatomical complexity and frequent late diagnosis often necessitate intensive treatment with intensity‐modulated radiotherapy (IMRT). Although IMRT improves local control, treatment‑related toxicities compromise patients’ quality of life owing to the close anatomical proximity of the tumor and planning target volume (PTV) to critical organs. Carbon ion radiotherapy (CIRT), with its precise dose delivery and high relative biological effectiveness (RBE), shows promise for improving outcomes in HNSCC, particularly in recurrent nasopharyngeal carcinoma and sinonasal malignancies. ² , ³ , ⁴ , ⁵ However, the molecular and biological regulatory mechanisms underlying carbon ion radiation within tumor cells remain largely unexplored.

Post‐translational modifications (PTMs) critically regulate cellular signaling, metabolic regulation, and stress responses, with phosphorylation and acetylation being the two most abundant forms. These modifications not only regulate protein functional activity but also influence cellular responses to radiotherapy. ⁶ , ⁷ , ⁸ Previous studies have extensively investigated the effects of X‐ray radiotherapy on tumor protein phosphorylation and acetylation profiles. However, coordinated PTM alterations and their mechanistic interplay induced by X‐ray radiotherapy and CIRT in HNSCC remain unclear.

Previous studies demonstrated that HNSCC cells irradiated with 8 Gy X‐ray or 4 GyE heavy ions in vitro exhibit comparable tumor‐killing efficacy. ⁹ To verify the equivalent cytotoxicity of this dose regimen in HSC‐3 cells, we examined apoptosis‐related markers in the treated cell groups using western blotting. Our results showed that HSC‐3 cells subjected to the two irradiation modalities displayed comparable levels of apoptosis (Figure S6A). To capture the earliest deterministic events of modified signaling post‐irradiation and avoid interference from subsequent feedback loops or bypass signaling, we selected the 30‐minute post‐irradiation time point. Accordingly, we characterized the dynamic changes in the proteome, phosphoproteome, and acetylome of HSC‐3 HNSCC cells 30 min after exposure to X‐rays (8 Gy) or carbon ions (4 GyE). Integrated multi‐omics analysis characterized the PTM characteristics, crosstalk, kinase activity, and functional impact, revealing distinct radiobiological mechanisms. These findings provide important insights for the optimization of therapeutic efficacy.

2. MATERIALS AND METHODS

2.1. Cell lines and culture conditions

The human HNSCC cell line HSC‐3 was obtained from Daping Hospital, Army Medical University, Chongqing, China, and was cultured in Dulbecco's modified Eagle's medium (DMEM) supplemented with 10% heat‐inactivated fetal calf serum (Gibco, USA) and 1% penicillin/streptomycin (Gibco, USA). Cells were maintained at 37°C in a humidified atmosphere containing 5% CO₂.

2.2. X‐ray irradiation

Cells in the log‐growth phase were plated in T‐75 flasks (Corning, NY, USA) and irradiated with an X‐ray beam (Varian Unique; dose rate: 400 MU/min, SSD: 100 cm) at room temperature. X‐ray irradiation was performed with a single exposure to a physical dose of 8 Gy.

2.3. Carbon ion irradiation

For carbon ion irradiation, cells in the log‐growth phase were plated in T‐75 flasks (Corning, NY, USA) and irradiated at room temperature with a carbon ion beam (energy, 334.09 MeV/u) delivered by the Heavy Ion Research Facility at Zhejiang Cancer Hospital. We calibrated the carbon ion beam spread‐out Bragg peak (SOBP) to target the adherent cell surfaces. Carbon ion irradiation was performed using a single exposure to a physical dose of 4 GyE.

2.4. Proteomics, phosphoproteomics, and acetylomics profiling

We subjected the HNSCC cell line HSC‐3 to proteomic, phosphoproteomic, and acetylomic profiling, each performed in three biological replicates, followed by comprehensive bioinformatics analyses.

2.5. LC‐MS/MS analysis

The tryptic peptides were dissolved in solvent A, and directly loaded onto a homemade reversed‐phase analytical column (25 cm length, 100 µm i.d.). The mobile phase consisted of solvents A (0.1% formic acid and 2% acetonitrile in water) and B (0.1% formic acid in acetonitrile). Peptides were separated using the following gradient: 0–14 min, 6%–24% B; 14–16 min, 24%–35% B; 16–18 min, 35%–80% B; 18–20 min, 80% B, all at a constant flow rate of 500 nL/min on a NanoElute UHPLC system (Bruker Daltonics). The peptides were subjected to a capillary source, followed by the timsTOF Pro 2 mass spectrometry. The applied electrospray voltage was 1.75 kV. The precursors and fragments were analyzed at the TOF detector. The timsTOF Pro was operated in data‐independent parallel accumulation serial fragmentation (dia‐PASEF) mode. The full MS scan was set at 300–1500 m/z (MS/MS scan range) and 20 PASEF (MS/MS mode)‐MS/MS scans were acquired per cycle.

DIA data were processed using Spectronaut (v.18) software. Tandem mass spectra were searched against the Homo_sapiens_9606_SP_20231220. fasta (20429 entries) concatenated with the reverse decoy database, and trypsin/P was specified as a cleavage enzyme, allowing up to two missed cleavages. The Cys carbamidomethyl group was specified as a fixed modification. N‐terminal acetylation and Met oxidation were specified as variable modifications. The false discovery rate (FDR) of the proteins, peptides, and peptide‐spectrum matches (PSMs) was adjusted to < 1%. The PTM localization probability threshold was set at > 0.75.

2.6. Bioinformatics analysis

Detailed methodologies for these approaches are provided in the Supplementary Methods.

3. RESULTS

We performed a multi‐omics analysis of HSC‐3 HNSCC cells subjected to X‐ray irradiation (8 Gy), carbon ion irradiation (4 GyE), or sham irradiation (control), with samples harvested at 30 min. Proteomic, phosphoproteomic, and acetylomic analyses were conducted using liquid chromatography‐tandem mass spectrometry for peptide identification and quantification (Figure 1A). We identified 7,698 proteins with 22,912 phosphorylation sites and 6,583 acetylation sites (Figure S1A and Table S1‐S3). Violin plots, PCA, and Pearson correlations (0.92–0.99) confirmed dataset stability and reproducibility (Figure S1B–D). Among the identified proteins, 4,750 were phosphorylated and 2,647 were acetylated, with 1,631 shared (Figure 1B–C). We quantified 19,183 phosphorylation and 6,301 acetylation sites corresponding to 3,953 and 2,519 proteins, respectively (Figure 1D). Most modified proteins were localized to the nucleus (Figure 1E). X‐ray irradiation altered the expression of 109 proteins (59 up‐regulated and 50 down‐regulated), whereas carbon ions modulated a more extensive set of 219 proteins (195 up‐regulated and 24 down‐regulated). Carbon ions modified more phosphorylation (2,781 vs. 2,301 sites) and acetylation (3,324 vs. 2,070 sites) sites than X‐rays (Figure 1F), likely due to their higher relative biological effectiveness (RBE) and linear energy transfer (LET), inducing denser DNA damage and complex cellular responses.

FIGURE 1.

Comprehensive Analysis of Proteomic, Phosphoproteomic, and Acetylomic Changes in HNSCC Following X‐ray and carbon ion irradiation Radiotherapy:(A) Sample Submission Workflow; (B) Distribution Analysis of Identified Proteins and Modification Sites in Phosphoproteomics and Acetylomics;(C) Bubble Plot of Protein Identification Quantity and Significance in Phosphoproteomics and Acetylomics; (D) Quantitative Analysis of Identified Proteins and Modification Sites in Phosphoproteomics and Acetylomics;(E) Subcellular Localization Distribution of phospho/acetyl‐proteins (Stacked Bar Chart); (F) Statistical Overview of Differential Proteins and Modification Sites Among Control, X‐ray, and carbon ion irradiation Groups.

Histones are nuclear proteins that form chromatin and regulate gene expression and DNA stability via PTMs such as acetylation and phosphorylation. As demonstrated in previous studies, post‐translational modifications dynamically modulate chromatin accessibility, thereby influencing a range of fundamental cellular processes, including development and disease. Reduced histone acetylation has been mechanistically linked to radiotherapy efficacy, potentially by repressing the expression of proapoptotic or immunomodulatory genes, thereby increasing radioresistance. X‐ray (8 Gy) and carbon ion (4 GyE) irradiation reduced the acetylation of the histones H2AZ1 (K8, K5, K14, and K12), H2BC12 (K6, K16, K13, and K12), H3C1 (K57, K37, K28, K24, K19, K15, K123, and K10), and H4C1 (K92 and K60), with carbon ions showing stronger effects (Figure 2A‐B). H2AZ1 affects tumor progression, ¹⁰ , ¹¹ and H2B/H3C1 is related to immune infiltration patterns in malignancies such as glioma. ¹² , ¹³ Phosphorylation increased more prominently in H1‐10 (S2), H2BC12 (S65), H3C1 (T119), and H4C1 (T31) after irradiation with carbon ions (Figure 2B). ATM‐driven H3S10 phosphorylation aids in DNA repair; however, aberrant phosphorylation may disrupt the G2/M checkpoint or activate pro‐survival pathways. Our findings demonstrate that carbon‐ion irradiation exhibits a stronger regulatory capacity in epigenetic modifications than X‐ray irradiation, with both histone acetylation inhibition and phosphorylation activation being more pronounced. This may be attributed to the LET of carbon ions, which induces extensive DNA damage, suggesting the potential advantage of carbon ions in inducing cell death or modulating antitumor immunity.

FIGURE 2.

Profiling the Epigenetic Landscape of Histone Modifications in Response to X‐ray and carbon ion irradiation: (A) PTMs of canonical histones of the control, X‐ray, and carbon ion irradiation groups; (B) Heatmap depicting the significance of histone modification sites.

Multi‐omics analysis of HSC‐3 HNSCC cells revealed that acetylation and phosphorylation levels significantly increased after irradiation with X‐rays (8 Gy) or carbon ions (4 GyE), surpassing changes in protein abundance (Figure 1F). Differential analysis of the PTM sites showed that carbon ion irradiation specifically potentiated the phosphorylation of MCM3 at S681, suggesting enhanced tumor cell cycle arrest and significantly reduced acetylation of PHF20L1 at K28, which is potentially linked to improved anticancer efficacy (Figure 3A). ¹⁴ , ¹⁵ Comparative analysis indicated complex PTM crosstalk (Figure 3B and S2A). KEGG and GO enrichment analyses of irradiation‐modified proteins revealed phosphorylation enrichment in the non‐homologous end‐joining (NHEJ) pathway. Carbon ion irradiation induces a substantially stronger activation signature within the NHEJ pathway than X‐rays, a phenomenon potentially associated with immunostimulatory effects. Carbon ion irradiation enriched hyperacetylated proteins in estrogen signaling, focal adhesion, and metabolic pathways and hyperphosphorylated proteins in glucagon and calcium signaling pathways (Figure S2D).

FIGURE 3.

Multi‐functional analysis of PTMs: (A) Volcano plots depict the most significantly altered sites of two PTMs across control, X‐ray, and carbon ion irradiation groups; (B) Venn diagrams illustrate proteins uniquely or commonly altered in both PTM datasets; (C) Clustering analysis summarizes phosphorylation patterns, with the top two enriched terms from GOMF, GOCC, KEGG, GOBP, and Protein Domain enrichment analyses displayed adjacent to corresponding clusters; (D) Clustering analysis summarizes acetylation patterns, with the top two enriched terms from GOMF, GOCC, KEGG, GOBP, and Protein Domain enrichment analyses displayed adjacent to corresponding clusters.

Unsupervised clustering analysis of phosphorylation site abundance across conditions (non‐irradiated, X‐ray, and carbon ion) categorized 22,912 identified sites into six distinct clusters based on their response patterns (Figure S3A). We systematically evaluated the clustering performance across a gradient range of cluster numbers from k = 2 to k = 8 and selected k = 6 based on the membership distribution of the left‐side clustering curves and the consistency of their trends. Functional annotation of these clusters revealed their distinct biological roles (Figure S3B‐F). Proteins in Cluster 2 exhibited significantly increased phosphorylation after X‐ray irradiation, with further enhancement following carbon ion irradiation. Conversely, phosphorylation in cluster 5 decreased after X‐ray irradiation and showed a greater reduction under carbon‐ion irradiation. Enrichment analysis revealed that proteins in Cluster 2 were enriched in processes such as DNA binding and chromatin organization, and were highly represented in the “Antigen processing and presentation” pathway, with domain enrichment in RasGEF and Helicase domains. Proteins in Cluster 5 were enriched in pathways related to brain development and ribosome function, with domain enrichment in the PTEN C2 and Cofilin/tropomyosin domains. The most enriched terms for each cluster are displayed in a clustering graph (Figure 3C). We analyzed the acetylation modifications using clustering methods and identified 6,583 acetylation sites grouped into six clusters (Figure S4A). Cluster 3 exhibited reduced acetylation after X‐ray irradiation but significantly increased acetylation after carbon ion irradiation, whereas cluster 4 showed marked reductions under both conditions. GO enrichment analysis revealed that cluster 3 proteins were enriched in immune response pathways, including leukocyte‐mediated immunity and antigen processing. YTHDF2, a differentially expressed gene in cluster 3, is associated with histone modifications (H3K4me3 and H3K27ac) in its promoter, leading to its upregulation in hepatocellular carcinoma and poor prognosis. ¹⁶ KEGG analysis highlighted enrichment in “antigen processing and presentation” pathways. These results suggest that carbon‐ion irradiation induces a stronger immune response in HNSCC cells than X‐ray irradiation. Finally, to facilitate the presentation of all analysis results, we have displayed the top two most significantly enriched terms from the GOMF, GOCC, KEGG, GOBP, and Protein Domain enrichment analyses on the right side of the corresponding cluster expression pattern clustering graph (Figure 3D).

PTMs can occur at one or more sites on a single protein and diverse PTM types exhibit intricate regulatory crosstalk involving synergistic, competitive, or antagonistic interactions. A previous study has revealed that AF9 regulates transcription and DNA repair under genotoxic stress. CaMKII‐mediated HDAC5 phosphorylation promotes PCAF‐dependent AF9 K339 acetylation, causing monomerization and reduced transcription. K339 acetylation fosters DNA‐PKc interaction, induces S395 phosphorylation, and further decreases AF9‐SEC binding to downregulate transcription and aid in repair. Post‐repair HDAC5 re‐entry restores AF9 deacetylation, resuming transcription. ¹⁷ Correlation analyses were performed across the identified PTM sites to systematically investigate the crosstalk networks induced by radiotherapy. The results of the significant multi‐PTM crosstalk are presented in a results table (Tables S4 and S5). A scatter plot (Figures 4A and 4B) shows the intramolecular and intermolecular crosstalk, with each point representing a crosstalk relationship pair. From the significantly correlated modification‐site pairs, we identified the top 10 core modification sites with the highest number of crosstalk relationships. A circular plot (Figure 4C‐F) illustrates these core sites and the top 10 most significant crosstalk partners. The intramolecular and intermolecular crosstalk are depicted in Figures 4C‐D and 4E‐F, respectively.

FIGURE 4.

Crosstalk Between Different Modifications:(A) Scatter plot of intra‐protein crosstalk with multi‐modifications; (B) Scatter plot of inter‐protein crosstalk with multi‐modifications;(C) Circle plot showing positive correlations of intra‐protein crosstalk with multi‐modifications;(D) Circle plot showing negative correlations of intra‐protein crosstalk with multi‐modifications;(E) Circle plot showing positive correlations of inter‐protein crosstalk with multi‐modifications;(F) Circle plot showing negative correlations of inter‐protein crosstalk with multi‐modifications;(G) Acetylation and phosphorylation modification sites of RAD50, XRCC4, XRCC5, and XRCC6 proteins.

Currently, there is limited research investigating whether crosstalk between PTMs induced by radiotherapy exhibits synergistic or antagonistic effects on DNA repair processes. To address this knowledge gap, we identified significantly differentially modified proteins in the classical homologous recombination repair (HR) and NHEJ pathways. We selected representative proteins with high levels of differential modifications, including RAD50, XRCC4, XRCC5 (Ku80), and XRCC6 (Ku70), and visualized their phosphorylation and acetylation modification sites. Ku70 acetylation releases Bax and c‐FLIP from this complex, resulting in Bax activation, c‐FLIP degradation, and apoptosis initiation. ¹⁸ Mechanistically, Ku80 poly(ADP‐ribosyl)ation (PARylation) and phosphorylation decreased, resulting in the dissociation of the PARP1‐Ku80 complex. After dissociation, Ku80 occupancy at DSBs and the resulting Ku80‐primed NHEJ activity increase. ¹⁹ While crosstalk among PTMs of DNA repair‐related proteins has been documented, this crosstalk has not been systematically characterized following irradiation. Our study provides a reference for potential modification sites and lays the foundation for future investigations in this field.

Phosphorylating kinases catalyze the transfer of phosphate groups from high‐energy donor molecules such as ATP to substrate molecules. This process, known as phosphorylation, typically aims to “activate” or “energize” a substrate, enabling it to perform specific functions. Protein kinases play pivotal roles in cellular signaling and complex biological processes. By integrating phosphoproteomic profiles with kinase‐substrate prediction algorithms, we identified 87,098 regulatory relationships involving 439 protein kinases and 8,138 phosphorylation sites across 2,366 proteins. These findings are detailed in Table S6, offering a comprehensive overview of kinase‐substrate interactions.

We selected the kinases with activity scores and generated heatmaps for the top 25 kinases with the highest and lowest activity scores among the sample groups or subgroups (Figure 5A). ATM (Ataxia‐Telangiectasia Mutated) is a critical phosphatidylinositol‐3‐kinase‐related protein kinase that plays a central role in the DNA damage response and cell cycle regulation. Our data demonstrated that ATM kinase activity exhibited a moderate increase following X‐ray irradiation and a marked increase after carbon ion irradiation. These findings indicate that carbon‐ion irradiation induces more severe DNA damage than X‐ray irradiation, prompting tumor cells to activate a more robust DNA repair response. Similarly, we observed a significant reduction in the phosphorylation activity of the cell cycle‐related proteins CDKL1, CDK7, CDK9, CDK2, CDK5, and CDK6 in cells treated with carbon ion irradiation. This further demonstrates that carbon ion irradiation induces severe DNA damage, leading to the inhibition of cell cycle regulatory mechanisms. We further used western blotting to validate the phosphorylation levels of the ATM substrate γ‐H2AX and the CDK substrate CDC25C in non‐irradiated HSC‐3 cells and in cells exposed to X‐rays (8 Gy) or carbon ions (4 GyE). These results were largely consistent with those of our sequencing analysis (Figure S6B). These kinase activity cluster heatmaps revealed shared kinase activity patterns in similar phenotypes or unique patterns in distinct phenotypes, aiding in the identification of potential connections between kinase activity and phenotypic traits. This approach provides a reference for the selection of kinases of interest for further experimental studies.

FIGURE 5.

Phosphoproteomic Profiling and Regulatory Mechanisms of Kinases in HNSCC Following X‐ray and carbon ion irradiation Radiotherapy:(A) Heatmap of phosphokinase activity scores, with columns representing sample names and rows representing phosphokinases;(B) Bar plot of phosphokinase activity scores for each sample, with the x‐axis showing activity scores and the y‐axis showing the top 10 phosphokinases ranked by activation or inhibition states. Red represents activation, and blue represents inhibition.

We generated bar charts for the top 10 kinases in significantly activated or inhibited states (Figure 5B). Additionally, we extracted the top 20 active or inhibited kinases from each comparison group to generate multi‐omics heatmaps (Figure S5A‐C). These analyses provide a comprehensive framework for kinase‐related biological research and a valuable reference for subsequent experimental studies.

Comparative analysis of X‐ray and carbon ion irradiation in HNSCC cells highlighted distinct molecular responses. Both modalities induced significant perturbations in DNA repair and cell cycle pathways, as demonstrated by changes in protein abundance, acetylation, and phosphorylation (Figure 6). These findings underscore the divergent cellular mechanisms activated by X‐ray and carbon ion therapies, potentially influencing therapeutic outcomes and warranting further investigation to optimize treatment approaches.

FIGURE 6.

Overall expression profiles of representative genes and proteins involved in DNA repair, cell cycle, immune response, and alternative splicing pathways, including multiple modifications.

Carbon ion irradiation induces robust activation of immune response pathways in HNSCC. Notably, CD274 (PD‐L1) exhibited significant changes in protein levels and phosphorylation, with Thr‐285 phosphorylation enhancing PD‐L1 stability. ²⁰ Additionally, TAP2, HLA‐A, and PSMB8 showed elevated protein levels in response to carbon ion irradiation, reflecting enhanced antigen presentation. TAP2 facilitates peptide transport to the endoplasmic reticulum for MHC class I loading, whereas PSMB8, a component of the immunoproteasome, plays a role in antigenic peptide processing. These findings highlight the potential synergy between carbon ion therapy and immunotherapy, thereby providing computational predictions to underpin the optimization of radioimmunotherapy strategies in clinical practice.

In addition, our study revealed that both irradiation methods induced strong translational and post‐translational modifications of splicing‐related proteins, suggesting potential impacts on alternative splicing. Based on the proteomic data, we observed that splicing factors HNRNPC and HNRNPA2B1 exhibited lower protein expression levels in the carbon‐ion irradiation group. Notably, both molecules play oncogenic roles in HNSCC. Furthermore, our findings revealed that carbon ion irradiation markedly enhanced the acetylation levels of HNRNPC and phosphorylation of HNRNPA2B1. However, mechanisms underlying these PTMs remain understood. This study provides a foundation for future investigations into the regulatory mechanisms underlying these modifications. These observations not only highlight the complexity of cellular responses to radiation, but also provide a foundation for future studies aimed at elucidating the role of alternative splicing in therapeutic resistance and radiosensitivity.

4. DISCUSSION

In the present study, comprehensive PTM profiling was performed on HNSCC HSC‐3 cells 30 min after X‐ray or carbon ion irradiation. Our results demonstrated that carbon ion irradiation elicited far more extensive alterations in protein phosphorylation and acetylation than X‐ray irradiation.

A high LET of carbon ions generates denser ionization tracks and stronger oxidative stress than low‐LET X‐rays, ²¹ which robustly activates stress‐responsive signaling cascades (e.g., ATM/ATR and Sirtuin pathways), and thus induces more pervasive PTM reprogramming. ²² This unique PTM landscape, driven by high‐LET carbon ions, further correlates with potent antitumor activity in HNSCC, underscoring the translational potential of leveraging PTM‐regulated signaling networks to optimize carbon‐ion radiotherapy strategies.

The selection of the 30‐min post‐irradiation time point was not intended to capture fully established phenotypic changes, but rather to characterize the earliest signal‐determining events following irradiation, thereby avoiding interference from subsequent feedback loops or collateral signaling pathways. These early PTM events can be regarded as triggers for cell fate decisions, and their functional directionality (e.g., pro‐survival or pro‐death) is fundamentally established during the immediate post‐irradiation phase.

Specifically, carbon ion irradiation induced more pronounced histone PTM changes, including reduced acetylation and increased phosphorylation, than X‐ray irradiation. These modifications, which are influenced by the high LET of carbon ions, may enhance the effectiveness of radiotherapy by promoting cell death and modulating antitumor immunity. We also analyzed phosphokinase activity and regulatory mechanisms in HSC‐3 cells following X‐ray and carbon ion irradiation, identified kinase‐phosphorylation site regulatory networks, and provided a comprehensive framework for understanding kinase‐mediated signaling and therapeutic responses. Our findings demonstrate that different radiotherapy modalities exert a marked impact on key molecular components associated with the immune response, DNA repair, and alternative splicing, as evidenced by differential alterations in protein levels and PTMs. The sequencing data obtained in this study provide a valuable foundation for further investigations of the underlying molecular mechanisms.

The interplay between phosphorylation and acetylation in multi‐PTM networks highlights the cooperative regulation of protein function, underscoring the importance of PTM crosstalk in radiotherapy resistance. Irradiation significantly alters histone acetylation and phosphorylation, implicating transcriptional reprogramming and stress responses. Pathway enrichment analysis revealed that irradiation‐induced protein modifications were closely linked to DNA repair, cell cycle regulation, and apoptosis.

Carbon ion irradiation upregulates TAP2, HLA‐A, and PSMB8, which are critical for MHC class I antigen presentation. ²³ , ²⁴ TAP2 facilitates the transport of antigenic peptides into the endoplasmic reticulum, whereas HLA‐A presents these peptides to CD8⁺ T cells, thereby enhancing tumor immunogenicity. Elevated PSMB8 levels further support immunogenic cell death. ²⁵ , ²⁶ , ²⁷ These findings suggest that carbon ion therapy may synergize with immune checkpoint inhibitors, such as anti‐PD‐1/PD‐L1 therapies, to amplify antitumor immune responses in HNSCC. ²⁷ Clinical trials are warranted to evaluate the efficacy of combining carbon‐ion irradiation with immunotherapy to improve the outcomes in patients with advanced or radioresistant HNSCC. Notably, the upregulation of MHC class I‐related molecules can serve as a predictive biomarker for patient selection in such combinatorial trials, ensuring that patients most likely to benefit from synergistic treatments are prioritized.

These findings provide a framework for understanding the molecular basis of radiotherapy response and resistance in HNSCC. Dynamic changes in phosphorylation and acetylation, coupled with histone PTM, offer potential biomarkers for early detection of treatment failure and personalized treatment strategies. Clinically, these PTM‐based biomarkers can be integrated into liquid biopsy platforms to enable noninvasive, real‐time monitoring of treatment response, allowing for timely adjustment of radiotherapy regimens in patients with HNSCC. ²⁸ , ²⁹ Furthermore, the identification of radiation‐induced changes in kinase activity and signaling pathways provides a foundation for developing novel therapeutic targets, particularly in the context of combining radiotherapy with immunotherapy. For instance, the kinases driving pro‐survival PTM reprogramming identified in this study could be targeted with small‐molecule inhibitors to sensitize radioresistant tumors to carbon ion irradiation, a strategy that merits preclinical validation prior to clinical testing.

However, one limitation is the single time point (30 min post‐irradiation), which may miss late‐phase PTM dynamics. Therefore, further longitudinal studies are required. Furthermore, our study exclusively employed a single HNSCC cell line (HSC‐3). HNSCC is a highly heterogeneous disease. The PTM dynamics and immunomodulatory profiles observed in this study may be specific to the genotype of the HSC‐3 cells. Therefore, validation using a panel of HNSCC cell lines with distinct genetic backgrounds or in vivo models is required before our conclusions can be generalized.

CONFLICT OF INTEREST STATEMENT

The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper.

Supporting information

Supporting Information: pro670097‐sup‐0001‐TableS1.xls

Supporting Information: pro670097‐sup‐0002‐TableS2.xls

Supporting Information: pro670097‐sup‐0003‐TableS3.xls

Supporting Information: pro670097‐sup‐0004‐TableS4.xlsx

Supporting Information: pro670097‐sup‐0005‐TableS5.xlsx

Supporting Information: pro670097‐sup‐0006‐TableS6.xlsx

Supporting Information: pro670097‐sup‐0007‐FigureS1‐S6.docx

Supporting Information: pro670097‐sup‐0008‐SuppMat.docx