Integrated ATAC-Seq and RNA-Seq Analyses Identify the Motif CGTTTCCGGT as an Arginine Deficiency-Responsive DNA Element in Cancer Cells

TL;DR

This study finds that arginine deprivation therapy affects cancer cell growth by altering chromatin structure and suppressing a gene called C11orf54.

Contribution

The study identifies a novel DNA motif, CGTTTCCGGT, as a responsive element to arginine deficiency in cancer cells.

Findings

ADT significantly inhibits colorectal cancer cell proliferation and migration.

The motif CGTTTCCGGT is linked to chromatin remodeling and gene regulation under arginine deficiency.

C11orf54 expression is reduced with ADT, suggesting it as a potential therapeutic target.

Abstract

This study investigates the novel epigenetic mechanism underlying resistance to arginine deprivation therapy (ADT) in tumors, with implications for therapeutic targeting. In brief: 1. ADT potently suppresses CRC proliferation and migration while exerting only a limited pro-apoptotic effect. 2. Integrative ATAC-seq and RNA-seq analyses identify the motif CGTTTCCGGT as an arginine deficiency-responsive DNA element in cancer cells and reveal that the transcriptional inhibition of C11orf54 with ADT is associated with ETV5. 3. ATAC-seq demonstrates reduced chromatin accessibility at the C11orf54 locus in ADT-treated cells and functional validation via luciferase reporter assays confirms suppressed C11orf54 promoter activity with ADT.

Background/Objectives: Cancer is predicted to become the leading cause of premature mortality worldwide within this century. Among the hallmarks of cancer, metabolic reprogramming has received growing attention, and arginine deprivation therapy (ADT) represents a potential treatment strategy for tumors exhibiting arginine auxotrophy. Colorectal cancer cells frequently suppress the expression of argininosuccinate synthetase 1 (ASS1), rendering them dependent on extracellular arginine. However, how CRC cells adapt to and resist ADT remains largely unknown. Methods: We combined ATAC-seq and RNA-seq analyses with multiple functional assays—including CCK-8 viability, apoptosis detection, wound-healing, and transwell migration tests—to investigate the molecular basis of ADT response in cancer cells. Results: ADT markedly inhibited cancer cell proliferation (p < 0.001) and motility (p < 0.05) across three cell lines. Integrative multi-omics analyses revealed substantial chromatin remodeling and transcriptional reprogramming under ADT, with differentially expressed genes enriched in autophagy and cell-growth-related pathways. Among these, the motif CGTTTCCGGT was identified as an arginine deficiency-responsive DNA element in cancer cells, and C11orf54 showed pronounced downregulation accompanied by reduced chromatin accessibility at its genomic locus. Conclusions: These findings suggest that ADT restricts cancer cell proliferation and migration through chromatin remodeling mediated by the motif CGTTTCCGGT and the downregulation of C11orf54, identifying C11orf54 as a potential target for enhancing the efficacy of arginine deprivation therapy in cancer cells.

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1. Introduction

Cancer has surpassed infectious and metabolic diseases to become one of the predominant causes of morbidity and mortality worldwide [1,2]. Current global health statistics indicate that cancer is already the leading cause of death in more than 50 countries and is projected to dominate worldwide mortality profiles by the end of this century [2,3]. Consequently, understanding its biological mechanisms and identifying effective therapeutic strategies remain urgent priorities.

Among the defining characteristics of malignant cells is the reprogramming of their metabolic network [4,5,6]. In particular, the altered utilization of amino acids confers both a growth advantage and potential therapeutic vulnerabilities [7,8,9,10]. Arginine deprivation therapy (ADT) has attracted considerable attention as a metabolic intervention, especially for tumors exhibiting arginine auxotrophy [11,12,13]. More than 70% of human cancers—including colorectal, pancreatic, and hepatocellular carcinomas—display reduced expression of argininosuccinate synthetase 1 (ASS1), which limits endogenous arginine synthesis and renders these cells dependent on extracellular arginine sources [14,15,16,17].

In colorectal cancer (CRC), the activity of both ornithine transcarbamylase and ASS1 is diminished, further constraining the urea cycle and reinforcing arginine dependence [18]. Consequently, CRC cells rely heavily on exogenous arginine to sustain proliferation, making them particularly susceptible to arginine depletion [19].

Combination therapies incorporating ADT with standard treatments—such as radiotherapy, temozolomide, cyclin-dependent kinase inhibitors, or autophagy modulators—have demonstrated enhanced efficacy in preclinical models [20,21]. Nevertheless, therapeutic resistance remains a major challenge. While ASS1-deficient tumor cell lines are highly responsive to arginine-degrading enzymes in vitro, clinical responses are often inconsistent [16]. Such discrepancies may arise from tumor heterogeneity, compensatory metabolic pathways, reactivation of ASS1, or interactions with the tumor microenvironment, including macrophage infiltration [22].

To further clarify the molecular mechanisms underlying ADT resistance, we combined ATAC-seq and RNA-seq to correlate chromatin remodeling with transcriptional output, an approach that can capture both epigenetic and transcriptomic changes associated with resistance. Our results identify the motif CGTTTCCGGT as an arginine deficiency-responsive DNA element in cancer cells, and C11orf54 as a key gene whose downregulation is associated with decreased chromatin accessibility and potential resistance to arginine deprivation. These findings provide new insight into the adaptive responses of CRC cells and suggest that C11orf54 may represent a promising therapeutic target to overcome resistance to arginine starvation.

2. Materials and Methods

RNA Isolation and mRNA Quantification. Total RNA was isolated from cultured cells using TRIzol reagent (Invitrogen, Carlsbad, CA, USA) according to the manufacturer’s instructions. RNA concentration and purity were assessed spectrophotometrically. For mRNA analysis, 1 μg of total RNA was reverse-transcribed into complementary DNA (cDNA) using the PrimeScript RT Master Mix (Takara, Kusatsu, Japan) with random hexamer primers. Quantitative real-time PCR (qRT-PCR) was performed on a Roche LightCycler^®^ 480 II system (Roche, Basel, Switzerland) using SYBR Green Master Mix (Yeasen, Shanghai, China). Relative gene expression levels were calculated using the 2^−ΔΔCt^ method, with appropriate internal controls included for normalization.

Primers targeting C11orf54 and Primers for GAPDH were designed using the Primer-BLAST tool provided by NCBI. All oligonucleotides were synthesized commercially by Genewiz Biotech (Genewiz, Suzhou, China).

Western Blotting. Cells were lysed on ice for 30 min in RIPA buffer (Beyotime, Shanghai, China) supplemented with 1 mM PMSF (phenylmethanesulfonyl fluoride) to inhibit protease activity. Protein concentrations were quantified using a BCA Protein Assay Kit (Thermo Fisher Scientific, Waltham, MA, USA). Equal amounts of protein (40 μg) were separated by 12% SDS-PAGE and subsequently transferred onto PVDF membranes (Millipore, Burlington, MA, USA). Membranes were blocked with 5% non-fat milk in TBST (Tris-buffered saline containing 0.1% Tween-20) for 1 h at room temperature, then incubated overnight at 4 °C with primary antibodies: rabbit anti-C11orf54 (Proteintech, Wuhan, China, Cat. No. #23251-1-AP, 1:1000) and rabbit anti-GAPDH (Huabio, Hangzhou, China, Cat. No. ET1601-4, 1:5000). Following washes in TBST, membranes were incubated with HRP-conjugated goat anti-rabbit IgG (Beyotime, Cat. No. A0208, 1:5000) for 1 h at room temperature. Protein signals were visualized using ECL substrate (Yeasen) and detected with an ImageQuant LAS 4000 system (GE Healthcare, Chicago, IL, USA). Band intensities were quantified by densitometry using ImageJ software (v1.53, National Institutes of Health, MD, USA) and normalized to GAPDH.

Cell Culture and Cell Scratch Assay. Human tumor cell lines HepG2, Huh7, and SW1116 (ATCC, Manassas, VA, USA) were cultured in DMEM (Gibco, Grand Island, NY, USA) supplemented with 10% fetal bovine serum (FBS, Gemini Bio, West Sacramento, CA, USA), 100 U/mL penicillin, and 100 μg/mL streptomycin (Invitrogen) at 37 °C in a humidified atmosphere containing 5% CO_2_. For arginine deprivation experiments, cells were switched to arginine-free DMEM supplemented with 10% FBS, 100 U/mL penicillin, and 100 μg/mL streptomycin. Control cells were maintained in the same arginine-free DMEM base medium with L-Arginine hydrochloride added back to 84 mg/L (0.4 mM).

For the scratch (wound-healing) assay, cells were seeded into 6-well plates at a density of 5 × 10^5^ cells per well and incubated overnight to achieve a confluent monolayer. Three parallel linear scratches were generated per well using a sterile 10 μL pipette tip. Detached cells were removed by washing twice with PBS, and fresh medium with or without arginine was added. Wound closure was documented at 0 and 24 h using a microscope at 10× magnification.

Cell Proliferation Assay (CCK-8). Cells were seeded into 96-well plates at a density of 1 × 10^3^ cells per well and allowed to adhere overnight. Subsequently, cells were cultured under control or arginine-deprived conditions. At designated time points (days 1–4), 10 μL of CCK-8 reagent (Beyotime) was added to each well, followed by incubation at 37 °C for 1 h. The absorbance at 450 nm was recorded using a microplate reader (BioTek Synergy H1) to assess cell viability.

Transwell Migration Assay. For cell migration analysis, 3 × 10^4^ cells suspended in serum-free medium were seeded into the upper chamber of Transwell inserts (8 μm pore size, Corning). For the control group, arginine-free DMEM supplemented with L-Arginine hydrochloride (84 mg/L, 0.4 mM) was used in both chambers; for the AF group, arginine-free DMEM was used in both chambers. The lower chamber contained 10% FBS as a chemoattractant in both conditions. After 36 h of incubation, non-migrated cells on the upper surface of the membrane were removed using a cotton swab. Migrated cells on the lower membrane surface were fixed with 4% paraformaldehyde, stained with Coomassie Brilliant Blue, and quantified under a light microscope.

Flow Cytometry for Apoptosis. Apoptotic cell death was assessed using the Annexin V-FITC/PI Apoptosis Detection Kit (Beyotime) according to the manufacturer’s instructions. Cells were harvested, washed with PBS, and stained with Annexin V-FITC and propidium iodide (PI). Samples were analyzed on CytoFLEX S (Beckman Coulter, Brea, CA, USA) to quantify the proportions of viable, early apoptotic, late apoptotic, and necrotic cells.

Plasmid Construction. The pGL3 luciferase reporter vector and β-Gal control plasmid were obtained from our laboratory [23]. To generate the C11orf54 promoter-driven luciferase reporter plasmid (pGL3-C11orf54-promoter), the human C11orf54 promoter region (−938bp to −100 bp) was amplified from genomic DNA by PCR using the primers. The amplified fragment was cloned into pGL3-vector using the Seamless Cloning Kit (Beyotime). The resulting construct was verified by Sanger sequencing (Genewiz, Suzhou, China) to confirm correct insertion and orientation.

Luciferase Reporter Assay. Cells were seeded in 24-well plates at 5 × 10^4^ cells per well and cultured overnight to achieve 60–70% confluence. Cells were co-transfected with 100 ng of luciferase reporter plasmid (pGL3-C11orf54-promoter or control) and 10 ng of β-Gal plasmid for normalization using Lipofectamine™ 3000 (Thermo Fisher) following the manufacturer’s protocol. Twenty-four hours post-transfection, cells were cultured under control or arginine-deprived conditions for an additional 24 h. Cells were then lysed in 1× Passive Lysis Buffer (Promega, Madison, WI, USA) on ice for 15 min. Lysates were collected by centrifugation (12,000× g, 5 min, 4 °C), and luciferase activity was measured using the GloMax^®^-Multi Detection System (Promega). Results were expressed as fold change relative to the control group.

ATAC-seq Library Preparation and Sequencing. For ATAC-seq profiling, SW1116 cells maintained under control or AF condition were collected in three independent biological replicates (n = 3). A total of 40,000 nuclei per replicate were subjected to Tn5 transposase-mediated tagmentation (Illumina, San Diego, CA, USA). The resulting DNA fragments were purified with the MinElute PCR Purification Kit (Qiagen, Hilden, Germany), amplified using 1× NEBNext High-Fidelity PCR Master Mix (New England Biolabs, Ipswich, MA, USA), and sequenced on the Illumina NovaSeq 6000 with paired-end 150 bp reads.

ATAC-seq Data Processing. Raw reads were quality-trimmed using Trimmomatic (v0.39), and clean reads were aligned to the human reference genome (hg38) using BWA (v0.7.17). Accessible chromatin regions were identified by MACS2 (v2.2.7.1) peak calling. Peak annotation was performed using bedtools (v2.30.0). To compare accessibility between conditions, peaks across all samples were merged and quantified by read count per region. DESeq2 (v1.36.0) was applied to detect differentially accessible regions (|log_2_FC| > 1, p < 0.05). For motif analysis, sequences within ±200 bp of each differential peak were extracted, and HOMER (v4.11, findMotifsGenome.pl) was used for de novo motif discovery. The resulting motifs were cross-referenced against the HOMER and JASPAR databases to assign candidate transcription factors.

RNA-seq Library Preparation and Sequencing. Transcriptomic profiling was conducted on SW1116 cells under control and AF condition using three biological replicates per group (n = 3). Total RNA was isolated, converted into sequencing libraries, and subjected to paired-end sequencing on the Illumina platform.

RNA-seq Data Processing. Raw reads were processed using fastp (v0.23.2) with default parameters to remove adapter sequences and low-quality reads. Clean reads were aligned to the human reference genome (hg38) using HISAT2 (v2.2.1), and gene-level expression was quantified using HTSeq (v2.0.2). Differential expression analysis was performed with DESeq2, applying Benjamini–Hochberg correction for multiple testing. Genes meeting |log_2_FC| > 1 and FDR < 0.05 were considered significantly differentially expressed. Functional annotation was performed through GO enrichment (Fisher’s exact test, p < 0.01) and KEGG pathway analysis (Fisher’s exact test, p < 0.05).

Statistical Analysis. All experiments were conducted in three independent replicates (n = 3) unless stated otherwise. Image quantification for Western blotting, cell density, wound-healing assays, transwell migration assays, and flow cytometry data was performed using ImageJ software (v1.53, National Institutes of Health, MD, USA). Data are expressed as mean ± standard deviation (SD). Statistical analyses were performed using GraphPad Prism (v9.0, GraphPad Software, San Diego, CA, USA). Normality of data distribution was assessed by the Shapiro–Wilk test, and homogeneity of variance was confirmed by Levene’s test. Comparisons between two groups were made using Student’s t-test, while comparisons among multiple groups were conducted using one-way ANOVA followed by Tukey’s post hoc test. A p-value < 0.05 was considered statistically significant.

3. Results

3.1. Arginine Deprivation Impairs Tumor Cell Proliferation and Migration

We first evaluated the effect of arginine deprivation on tumor cell growth and motility in Huh7, SW1116, and HepG2 cells cultured in either complete medium (control) or arginine-free medium (AF) to model their dependence on extracellular arginine sources. In the control condition, cells grew densely, indicating robust proliferative and survival capacity. In contrast, the AF group showed significantly reduced relative cell density across all three cell lines (*** p < 0.001; Figure 1a).

To assess cell migration, we performed a scratch wound-healing assay. At the initial time point (0 h), clear and consistent scratches were present in all groups. After 24 h, control cells exhibited substantial wound closure, particularly in Huh7 and HepG2 cultures. Conversely, arginine-deprived cells showed minimal wound closure, indicating impaired migratory capacity (* p < 0.05 or ** p < 0.01; Figure 1b).

We next examined migratory behavior using a transwell migration assay. Under control conditions, large numbers of Coomassie Brilliant Blue-stained cells were observed on the lower surface of the membrane, indicating strong migratory capacity. However, under arginine-deprived conditions, the number of migrated cells was markedly reduced in SW1116 and HepG2 lines, with Huh7 cells also showing a noticeable decline in migration (all ** p < 0.01 or *** p < 0.001; Figure 1c). Collectively, these data confirm that arginine deprivation reduces tumor cell migratory capacity.

Cell proliferation was assessed in Huh7, SW1116, and HepG2 cells using the CCK-8 assay under control and AF condition. In all three cell lines, cells maintained in the control condition showed a progressive, time-dependent increase in optical density (OD_450_), consistent with active proliferation. By contrast, cells cultured in the AF condition exhibited a markedly attenuated rise in OD_450_ values across all time points, reflecting significant growth inhibition. By day 4, AF treatment reduced cell proliferation by 84% in Huh7, 68% in SW1116, and 51% in HepG2 compared to control (**** p < 0.0001; Figure 1d).

Apoptotic responses were further evaluated using Annexin V-FITC/propidium iodide (PI) staining. In the control condition, most cells localized to the viable population (Q4), with only a small fraction displaying apoptotic signatures (Q2 + Q3). Upon arginine deprivation, the total apoptotic fraction (Q2 + Q3) was approximately 3.5% versus 5.4% in Huh7, 7.7% versus 17.2% in SW1116, and 7.9% versus 8.7% in HepG2 (control versus AF, respectively); however, these differences were not statistically significant (ns; Figure 1e). The proportion of necrotic cells (Q1) remained consistently low across all groups (0.4–2.1%). These findings suggest that arginine starvation primarily limits cell proliferation, rather than inducing significant apoptosis.

Taken together, the results summarized in Figure 1 indicate that arginine deprivation substantially impairs both the proliferative and migratory capacities of tumor cells, and has little impact on cell apoptosis.

3.2. ATAC-Seq and RNA-Seq Identify Motif CGTTTCCGGT as an Arginine Deficiency-Responsive DNA Element

To investigate the molecular basis of adaptive responses to arginine deprivation, SW1116 cells were cultured in either the control condition or the AF condition and analyzed using ATAC-seq and RNA-seq. ATAC-seq profiling revealed substantial alterations in chromatin accessibility between the two groups. Compared with the control condition, cells cultured under AF conditions exhibited a global reduction in chromatin accessibility, with an average of 239 peaks per replicate in the control group versus 160 peaks in the AF group (Figure 2a). Examination of regions surrounding transcription start sites (TSSs) showed divergent accessibility patterns between the two groups (Figure 2b). The functional annotation of differentially accessible regions (DARs) indicated altered distributions across promoters (±1 kb), intronic, and intergenic regions, suggesting locus-specific chromatin remodeling (Figure 2c,d).

Heatmap visualization further highlighted pronounced differences in chromatin accessibility landscapes, revealing distinct regulatory features in AF-treated cells relative to controls (Figure 2e). A volcano plot identified 15 DARs between groups—10 exhibiting decreased accessibility and 5 increased accessibility under arginine deprivation (Figure 2f). Motif enrichment analysis showed upregulation of ZNF16- and GFX-binding motifs and downregulation of ETV5 motifs (Figure 2g). Gene Ontology (GO) enrichment analysis of the AF group revealed significant associations with biological processes such as protein localization, autophagy, and cell growth, linking changes in chromatin structure to potential functional consequences (Figure 2h).

RNA-seq analysis revealed that the AF group exhibited a transcriptional profile distinct from the control group, as shown by hierarchical clustering (Figure 3a). A volcano plot revealed 2955 DEGs (|log_2_FC| > 1, FDR < 0.05), including 1440 upregulated and 1514 downregulated genes under arginine deprivation (Figure 3b). GO enrichment analysis of these DEGs indicated that multiple cellular processes and molecular functions were altered, further supporting the presence of global transcriptional reprogramming (Figure 3c).

Integration of ATAC-seq and RNA-seq data identified loci with concordant changes in chromatin accessibility and transcriptional output (Figure 3d). Among the co-regulated loci, C11orf54 emerged as a candidate gene exhibiting concordant reductions in both chromatin accessibility and transcript abundance. Visualization of the C11orf54 locus showed decreased ATAC-seq signal intensity in AF-treated cells, indicating restricted chromatin accessibility across this genomic region (Figure 3e). Motif analysis identified ETV5-binding sequences (CGTTTCCGGT) within the C11orf54 locus (Figure 3f). Further validation using the JASPAR database confirmed the presence of multiple high-scoring ETV5 motifs (Figure 3g).

In summary, integrative ATAC-seq and RNA-seq analyses demonstrate that the motif CGTTTCCGGT serves as an arginine deficiency-responsive DNA element in cancer cells, and arginine deprivation leads to reduced chromatin accessibility and transcriptional suppression of C11orf54. These results indicate that C11orf54 downregulation may contribute to adaptive resistance under arginine deprivation.

3.3. C11orf54 Is Downregulated Under Arginine-Deprived Conditions

Across all three cell lines, AF condition led to a marked reduction in C11orf54 mRNA levels compared with the control group (* p < 0.05; Figure 4a). Consistently, Western blot analysis confirmed a notable decrease in C11orf54 protein expression under AF conditions. To investigate the transcriptional regulation of C11orf54, a luciferase reporter plasmid containing its promoter region (−938 to −100 bp) was constructed (Figure 4b). Luciferase assays revealed significantly reduced promoter activity in the AF group relative to the control group (* p < 0.05, ** p < 0.01, *** p < 0.001; Figure 4c).

Collectively, these results support the conclusion that arginine deprivation suppresses C11orf54 expression in part by inhibiting its promoter activity at the transcriptional level. To further investigate the functional role of C11orf54, we performed overexpression experiments in HepG2, Huh7, and SW1116 cells. CCK-8 assays revealed that C11orf54 overexpression significantly promoted cell proliferation under control conditions compared to empty vector controls. Moreover, C11orf54 overexpression partially rescued the proliferation inhibition induced by arginine deprivation (Supplementary Figure S1).

4. Discussion

This study demonstrates that arginine deprivation therapy (ADT) suppresses tumor cell proliferation and migration, with only a modest impact on apoptosis. Through integrative ATAC-seq and RNA-seq analyses, we identify the motif CGTTTCCGGT as an arginine deficiency-responsive DNA element in cancer cells and characterize the transcriptional downregulation of C11orf54 as a potential adaptive response to prolonged arginine deprivation. Arginine deprivation reduced chromatin accessibility at the C11orf54 promoter and decreased its transcriptional activity, as confirmed by luciferase reporter assays. These findings suggest that C11orf54 downregulation may be related to the development of ADT resistance and may highlight C11orf54 as a potential molecular target to enhance responses to arginine deprivation-based therapies.

ADT, a metabolic intervention targeting tumors with high arginine auxotrophy, has emerged as a promising strategy to inhibit cancer progression [12,24]. While prior studies have highlighted the efficacy of ADT in suppressing tumor growth by inducing apoptosis or cell cycle arrest [11,12,19], our work uncovers a subtler transcriptional adaptive response. Here, we demonstrate that prolonged arginine deprivation primarily suppresses tumor cell proliferation and migration, with limited induction of apoptosis—a finding consistent with reports that some cancers exhibit metabolic plasticity, prioritizing survival over death even under nutrient stress [25]. This observation highlights the need to dissect adaptive mechanisms beyond apoptosis to optimize ADT efficacy.

Integrative analysis of ATAC-seq and RNA-seq data was key to identifying the CGTTTCCGGT motif as an arginine deficiency-responsive DNA element corresponding to the established ETV5-binding profile. This motif, enriched in regions associated with reduced chromatin accessibility under arginine deprivation, suggests a transcriptional regulatory mechanism involved in the adaptive response. Previous studies have linked nutrient deprivation to dynamic chromatin remodeling; for example, glutamine deprivation alters the transcriptional induction of ATF4 to drive amino acid homeostasis genes [26]. Our findings point to a comparable process in arginine metabolism, though acting through a distinct mechanism involving ETV5-associated chromatin accessibility changes at the C11orf54 locus.

The downregulation of C11orf54 emerged as an adaptive event. This gene, whose role in metabolic stress responses has not been previously explored, exhibited reduced promoter accessibility and transcriptional activity following arginine deprivation, as validated by luciferase assays. C11orf54 functions as a highly conserved ester hydrolase across diverse species. Its knockdown suppresses cell proliferation and potentiates cisplatin-induced DNA damage and apoptosis [27,28,29]. Given its transcriptional suppression, C11orf54 may normally promote processes that sustain proliferation. We hypothesize that its downregulation may represent a resource-sparing program that redirects cellular activities to survive arginine scarcity. Consistent with this, our functional experiments showed that C11orf54 overexpression partially rescued proliferation inhibition under arginine deprivation, suggesting that its downregulation contributes to ADT-induced growth restraint. While these findings were consistent across hepatocellular carcinoma and colorectal cancer models, the generalizability of the ETV5–CGTTTCCGGT–C11orf54 axis to all ASS1-deficient cancers remains to be validated. Given the metabolic and epigenetic heterogeneity among different tumor types, future studies employing a broader panel of ASS1-deficient cancer models will be necessary to confirm the universality of this mechanism in ADT resistance.

From a translational perspective, the ETV5/C11orf54 axis may present a specific therapeutic vulnerability. The observation that ADT primarily induces proliferative arrest rather than apoptosis suggests that the transcriptional suppression of C11orf54 serves as a survival mechanism. Consequently, disrupting this adaptive response—either by preventing C11orf54 downregulation or by combining ADT with agents that exploit C11orf54-associated DNA-repair deficiencies—could shift the cellular response from cytostatic dormancy to active cytotoxic death. Such strategies may effectively lower the apoptotic threshold and overcome ADT resistance in ASS1-deficient cancers.

Notably, similar adaptive gene repression has been observed in other metabolic therapies: for instance, asparagine synthetase (ASNS) is upregulated to resist asparaginase treatment, but its silencing enhances sensitivity by limiting de novo asparagine synthesis [30]. In summary, our study provides insights into the transcriptional adaptive responses to ADT by linking metabolic stress to chromatin remodeling and C11orf54 downregulation. Targeting this axis may offer new strategies to enhance the durability of arginine deprivation-based therapies.

5. Conclusions

These findings indicate that ADT restricts cancer cell proliferation and migration with modest apoptosis, accompanied by reduced chromatin accessibility and transcriptional downregulation of C11orf54 mediated by the CGTTTCCGGT motif. Our data suggest that the C11orf54–motif axis contributes to adaptive responses under arginine deprivation and may represent a potential target for enhancing ADT efficacy in ASS1-deficient cancers.