OGT-Mediated O-GlcNAcylation Stabilizes c-Myc Activity and Promotes Chemoresistance in Triple-Negative Breast Cancer.

1. Introduction
Triple‐negative breast cancer (TNBC) is a highly heterogeneous subtype of breast cancer with aggressive characteristics, defined by its negativity for the estrogen receptor, progesterone receptor, and human epidermal growth factor receptor 2 [1, 2]. The clinical features of TNBC include invasiveness, metastatic potential, proneness to relapse, and, most importantly, insensitivity to targeted drugs. Although the development of new TNBC‐targeted drugs has made rapid progress, standardized TNBC treatment regimens still heavily rely on chemotherapy [3, 4]. However, intrinsic or acquired resistance to chemotherapy is common, contributing to early treatment failure and poor clinical outcomes [5, 6]. Therefore, exploring the molecular mechanisms underlying drug resistance in TNBC is essential for developing effective targeted interventions.
Recent studies have provided new insights into TNBC progression and treatment strategies, emphasizing the molecular complexity and clinical challenges associated with therapy resistance [7–9]. These advances underscore the urgent need to explore molecular pathways driving TNBC aggressiveness and drug resistance. Among the molecules involved in TNBC progression is the transcription factor c‐Myc, which is overexpressed in TNBC [10]. c‐Myc is involved in several biological processes during cancer progression, including cell cycle regulation, metabolism, and maintenance of stemness. c‐Myc is also closely related to tumor aggressiveness and drug resistance [11–15]. c‐Myc stability and function are regulated not only by phosphorylation but also by O‐linked β‐N‐acetylglucosamine (O‐GlcNAc) modification (O‐GlcNAcylation) [16]. O‐GlcNAcylation is mainly modulated by O‐GlcNAc transferase (OGT), which is known to be closely associated with drug tolerance, cell stemness, and metabolic reprogramming in various malignancies [17, 18]. O‐GlcNAcylation is known to be responsible for chemoresistance by modulating the stability of c‐Myc protein [16]. However, the specific action site and mechanisms involved in TNBC remain unclear and require further exploration.
In this study, we investigated whether OGT‐mediated O‐GlcNAcylation contributed to the stabilization and oncogenic activity of c‐Myc in TNBC. By integrating molecular and functional analyses, we aimed to elucidate the mechanistic basis of c‐Myc regulation by O‐GlcNAcylation and its role in drug resistance. This study provides novel insights into the post‐translational modifications of c‐Myc during chemoresistance in TNBC.

2. Materials and Methods

2.1. Establishment of Drug‐Resistant Cells
To generate the cisplatin (DDP)‐resistant MDA‐MB‐231 cell line, parental MDA‐MB‐231 cells were subjected to a 6‐week regimen of intermittent drug exposure. The cells were treated with DDP (at concentrations gradually increasing from 1 μM to 15 μM) for 24 h, followed by a 48‐h recovery period in drug‐free medium.

2.2. Cell Maintenance and Treatment
The cells were maintained in high‐glucose Dulbecco’s modified Eagle’s medium (HyClone, Cat # SH30243.01) with 10% fetal bovine serum (ExCell Bio, Cat # FSP‐SA‐500) and 1% penicillin–streptomycin (Invitrogen, Cat #15140122), at 37°C in 5% CO2 and humidified air.

2.3. Cell Viability Assay
Cells were seeded at a density of 5 × 103 cells/well in a 96‐well plate and incubated for 24 h. Subsequently, the cells were treated with varying concentrations of DDP for an additional 24 h. Cell viability was detected using the CCK‐8 assay kit (Dojindo, Cat #CK04) and a microplate reader (Bio‐Rad, Cat #1681135) at an absorbance of 450 nm.

2.4. Colony Formation Assay
Cells were seeded in six‐well plates at a density of 600 cells/well. Following attachment, the cells were treated with different drug concentrations and cultured for 10 days. The resulting colonies were washed and fixed prior to staining with 0.1% crystal violet (Beyotime, Cat #C0121) for 20 min. Colony counts were quantified using the ImageJ software.

2.5. Flow Cytometry
After the indicated treatments, cells were rinsed with 1× PBS and harvested. Apoptosis was assessed using an Annexin V‐FITC/propidium iodide (PI) apoptosis detection kit (Vazyme, Cat #A211). Approximately 5 × 105 cells were resuspended in binding buffer and stained with Annexin V/FITC and PI. Apoptotic cells were detected using a FACScan flow cytometer (Becton Dickinson, Cat #34001080), and the resulting data were analyzed using FlowJo software (FlowJo LLC, v.10.8.2).

2.6. Western Blotting
Protein samples were prepared by lysing the cells in RIPA buffer supplemented with 1% protease and phosphatase inhibitor cocktails (Roche, #04693116001; Sigma‐Aldrich, #P0044) for 30 min on ice. Prior to lysis, the cells were rinsed twice with cold 1 × PBS. The resulting lysates were centrifuged, and the protein concentration in the supernatant was quantified using the BCA assay (Thermo Fisher Scientific, #A65453). Equal amounts of protein were electrophoresed on 10% SDS–PAGE gels and transferred to PVDF membranes (Millipore, #GVWP04700) using a Trans‐Blot Turbo apparatus (Bio‐Rad, #1704150). For immunodetection, membranes were first blocked with 5% skim milk in TBST for 1 h at room temperature and then probed with primary antibodies (1:1000) against OGT, total c‐Myc, p‐c‐Myc, O‐GlcNAc, and β‐actin overnight at 4°C. Following a series of washes, the membranes were incubated for 1 h at room temperature with horseradish peroxidase (HRP)–conjugated secondary antibodies. Signal detection was achieved using an enhanced chemiluminescence substrate (Thermo Fisher Scientific, #32209), and images were captured using a ChemiDoc XRS + imaging system (Bio‐Rad, #170–8265). Finally, band intensities were analyzed and normalized to β‐actin using ImageJ software. A comprehensive list of all antibodies used is provided in Table S1.

2.7. Co‐Immunoprecipitation (Co‐IP)
Immunoprecipitation was initiated by cell lysis in IP lysis buffer (Beyotime, Cat #P0013F) containing 1 mM PMSF (Beyotime, Cat #ST506) for 15 min on ice. Subsequently, the lysates were clarified by centrifugation at 13,500 rpm for 15 min at 4°C. The resulting supernatant was incubated overnight at 4°C with gentle rotation in the presence of the indicated primary antibody or a control IgG. To capture the immune complexes, protein A/G agarose beads (Beyotime, Cat #P2012) were added, and incubation continued for a further 2 h at 4°C. Following three washes with the lysis buffer, the immunoprecipitated proteins were eluted from the beads by boiling in SDS loading buffer. The eluted samples were analyzed by SDS–PAGE and western blotting, following standard protocols.

2.8. Cycloheximide (CHX) Chase Assay
The cells were seeded at a density of 1 × 105 cells/well in 6‐well plates and cultured overnight. Experimental groups were pretreated with 10 μM OMSI‐1 for 24 h, while the control group received vehicle only. Subsequently, 100 μg/mL CHX (Sigma‐Aldrich, Cat #C1988), dissolved in DMSO, was added to inhibit protein synthesis. Cells were harvested at 0, 2, 4, 8, and 12 h post‐CHX treatment. At each time point, the cells were lysed, and protein samples were collected via centrifugation, as detailed in the western blotting protocol. The stability of the c‐Myc protein was assessed using primary antibodies against c‐Myc and β‐actin. Protein levels were quantified using the ImageJ software and normalized to the corresponding loading control. The remaining protein at each time point was expressed as a percentage relative to the 0‐h time point.

2.9. Quantitative Polymerase Chain Reaction (qPCR)
Total RNA was isolated using TRIzol reagent (Invitrogen, Cat #15596018), and a NanoDrop spectrophotometer (Thermo Fisher Scientific, Cat # NanoDrop 3300) was used to measure RNA concentration and purity. cDNA synthesis involved reverse transcribing 1 μg of total RNA with the PrimeScript RT Reagent Kit (Takara, Cat #RR037A) according to the kit’s instructions. qPCR amplification was performed on a Real‐Time PCR Detection System (Bio‐Rad, Cat #12011319) using the TB Green Premix Ex Taq II (Takara, Cat #RR820A). The thermal profile included an initial 10‐min denaturation at 95°C and 40 cycles of 95°C for 15 s, 60°C for 30 s, and 72°C for 30 s. All reactions used gene‐specific primers detailed in Table S2. Analysis of relative mRNA expression levels utilized the 2−ΔΔCt method, with β‐actin as the internal control.

2.10. Prediction of O‐GlcNAcylation Sites
Potential O‐GlcNAcylation sites in the c‐Myc protein sequence were predicted using DictyOGlyc 1.1, a neural‐network‐based tool designed to identify serine (Ser) and threonine (Thr) residues likely to undergo O‐GlcNAcylation. We submitted the c‐Myc amino acid sequence to an online interface and selected high‐probability modification sites for further experimental validation.

2.11. Site‐Directed Mutagenesis and Plasmid Construction
The pcDNA3.1 expression vector containing wild‐type (WT) human c‐Myc cDNA was used as a template to create a series of c‐Myc mutants (T58A, S161A, S234A, and S288A) via site‐directed mutagenesis. The Mut Express II Fast Mutagenesis Kit V2 (Vazyme, Cat #C214) was employed, with specific primers designed to introduce each substitution (primers are listed in Table S3). Following transformation into XL1‐Blue competent cells (Agilent Technologies, Cat #200518), positive clones were validated by Sanger sequencing to confirm the desired mutation and the lack of off‐target alterations. Purified plasmid DNA from these constructs, obtained using the Plasmid Midi Kit (QIAGEN, Cat #12143), was subsequently used for cellular transfection and protein expression assays.

2.12. Statistical Analysis
All data were analyzed using GraphPad Prism v.8.0 and are expressed as the mean ± SEM. Student’s t‐test was used to compare two groups, and one‐way analysis of variance was used for multiple group comparisons. Statistical significance was defined as a p‐value < 0.05.

3. Results

3.1. OGT Inhibitor Reversed DDP Resistance of TNBC Cells by Suppressing c‐Myc
To investigate DDP resistance mechanisms in TNBC, we used the established DDP‐resistant cell model (MDA‐MB‐231/R), as detailed in the Methods section. Validation assays confirmed a significantly elevated IC50 for DDP in the resistant cells (13.3 μM) compared with that of the parental line (2.047 μM), resulting in a resistance index (RI) of 6.49 (Figure 1(a)). Subsequent protein analysis revealed concurrent upregulation of OGT, global O‐GlcNAcylation, and c‐Myc in MDA‐MB‐231/R cells (Figure 1(b)). This synchronous increase suggests a potential functional link between O‐GlcNAcylation, c‐Myc stabilization, and acquired chemoresistance. Next, we examined whether inhibition of OGT could reverse DDP resistance. Treatment with the OGT inhibitor, OSMI‐1, significantly enhanced DDP sensitivity in MDA‐MB‐231/R cells, reducing the IC50 from 13.35 μM to 2.45 μM (Figure 1(c)), which emphasizes the possible function of O‐GlcNAcylation. Additionally, MDA‐MB‐231/R cells exhibited impaired colony‐forming ability and a higher rate of apoptosis after OGT inhibition (Figures 1(d) and 1(e)). Furthermore, even when OGT was unaffected, the expression of O‐GlcNAc and c‐Myc in MDA‐MB‐231/R cells was significantly downregulated by the OGT inhibitor, further demonstrating the direct regulatory role of OGT in c‐Myc stabilization (Figure 1(f)). Collectively, these results indicate that the OGT inhibitor reversed DDP resistance in TNBC cells by suppressing c‐Myc expression.
Figure 1OGT inhibitor reverses cisplatin (DDP) resistance of TNBC cells by suppressing c‐Myc. MDA‐MB‐231/R cells were cultured according to the protocol described in the Materials and Methods section. For in vitro studies, MDA‐MB‐231 and MDA‐MB‐231/R cells were treated as follows. (a) Cell viability detection using the CCK‐8 assay and calculated IC50 and resistance index (RI) values. (b) Expression of OGT, O‐GlcNAc, total c‐Myc, and p‐c‐Myc was detected by western blotting. Cells (MDA‐MB‐231/R, unless otherwise indicated) were treated as follows: control group (with culture medium only); DMSO group (with DMSO treatment); and OSMI‐1 group (with 10 μM OSMI‐1 treatment). (c) Cell viability was determined using a CCK‐8 assay, and IC50 and RI values were calculated. Cells were divided into the following treatment groups in subsequent experiments: control group (culture medium only); DDP group (2 μM DDP treated); OSMI‐1 group (10 μM OSMI‐1 treated); and OSMI‐1 + DDP group (10 μM OSMI‐1 + 2 μM DDP treated). (d) Detection of cell proliferation using the colony formation assay. (e) Detection of apoptosis by flow cytometry. (f) Expression of OGT, O‐GlcNAc, total c‐Myc, and p‐c‐Myc was detected by western blotting. Data are presented as mean ± SEM. ns: not significant; ∗∗∗
p < 0.001.

3.2. OGT Interacted With and O‐GlcNAcylated c‐Myc to Enhance Its Stabilization
We evaluated the potential interaction between c‐Myc and OGT using a heterologous overexpression system. Co‐IP assays in HEK293T cells cotransfected with Flag‐c‐Myc and HA‐OGT demonstrated a mutual pull‐down of the two proteins, which supports the potential for OGT and c‐Myc to physically associate with each other when overexpressed (Figure 2(a)). To confirm this interaction at the endogenous level, Co‐IP assays were performed using MDA‐MB‐231/R cells. IP of native OGT resulted in the coprecipitation of endogenous c‐Myc. Additionally, IP of c‐Myc successfully pulled down endogenous OGT, confirming the physiological relevance of this interaction (Figure 2(b)). Given that OGT is the sole enzyme responsible for O‐GlcNAcylation, we evaluated whether c‐Myc was directly modified by O‐GlcNAc. In HEK293T cells transiently expressing FLAG‐c‐Myc, IP with an anti‐FLAG antibody revealed detectable O‐GlcNAcylation, which was significantly suppressed by OGT inhibition (Figure 2(c)). Furthermore, in MDA‐MB‐231/R cells, endogenous c‐Myc exhibited high levels of O‐GlcNAcylation, providing further evidence that this modification was enriched in DDP‐resistant TNBC cells (Figure 2(d)). Since c‐Myc stability is critical for its oncogenic function and chemoresistance, we investigated whether O‐GlcNAcylation affects its protein stability. We performed a CHX chase assay and observed that c‐Myc degradation was significantly accelerated in MDA‐MB‐231/R cells treated with an OGT inhibitor, indicating that O‐GlcNAcylation stabilized c‐Myc in MDA‐MB‐231/R cells (Figure 2(e)). The collective outcomes indicate that OGT interacted with c‐Myc and promoted its O‐GlcNAcylation, thereby enhancing its stabilization under drug‐resistant conditions in TNBC cells.
Figure 2OGT interacts with c‐Myc and O‐GlcNAcylates c‐Myc to enhance its stabilization. (a) HEK293T cells were transfected with HA‐OGT and/or Flag‐c‐Myc for 48 h, after which co‐immunoprecipitation (Co‐IP) was performed using anti‐HA or anti‐Flag antibodies. Western blotting was then performed to detect their interaction. (b) Endogenous interaction between OGT and c‐Myc in MDA‐MB‐231/R cells, confirmed by Co‐IP. (c) HEK293T cells that were transfected with Flag‐c‐Myc were untreated or treated with OSMI‐1. IP with anti‐Flag and WB with anti‐O‐GlcNAc antibodies assessed c‐Myc glycosylation. (d) MDA‐MB‐231/R cells were treated with or without OSMI‐1 and subjected to c‐Myc IP. The cells were then analyzed for OGT and O‐GlcNAcylation. (e) Analysis of c‐Myc protein stability in MDA‐MB‐231/R cells treated with or without OSMI‐1 by CHX chase assay. Cells were harvested at the indicated time points for western blotting. Data are presented as mean ± SEM. ∗∗∗
p < 0.001.

3.3. OGT O‐GlcNAcylated c‐Myc at Thr58 to Promote Its Stability
To identify the specific site on c‐Myc targeted by OGT‐mediated O‐GlcNAcylation, we used DictyOGlyc‐1.1 to predict potential O‐GlcNAcylation sites using a probability threshold of 0.5, as recommended by the developer for high‐confidence predictions. The analysis revealed four high‐probability sites: Thr58, Ser161, Ser234, and Ser288 (Figures 3(a) and 3(b)). To experimentally validate these candidates, endogenous c‐Myc was first knocked out in MDA‐MB‐231/R cells, followed by the reintroduction of either WT or site‐specific mutant forms of c‐Myc (T58A, S161A, S234A, and S288A). Among these mutants, only the T58A mutation significantly reduced both the expression of c‐Myc and its O‐GlcNAcylation, indicating that Thr58 was the predominant O‐GlcNAcylation site (Figure 3(c)). Furthermore, the CHX chase assay revealed that c‐Myc degradation was significantly accelerated in T58A‐mutant cells compared to WT cells, indicating that OGT‐mediated O‐GlcNAcylation at Thr58 enhanced c‐Myc protein stability (Figure 3(d)).
Figure 3OGT O‐GlcNAcylates c‐Myc at Thr58 to promote its stability (a) Possible O‐GlcNAcylation residues predicted by DictyOGlyc 1.1. (b) Targeted O‐GlcNAcylation residues in MDA‐MB‐231/R cells. These cells underwent a c‐Myc knockout and were transfected with c‐Myc wild‐type protein and different mutated c‐Myc proteins, respectively. The cells were treated as follows: control (transfected with c‐Myc WT); T58A (transfected with c‐Myc T58A); S161A (transfected with c‐Myc S161A); S234A (transfected with c‐Myc S234A); and S288A (transfected with c‐Myc S288A). (c) Detection of total c‐Myc and O‐GlcNAcylated c‐Myc expression by western blotting. The cells were treated as follows: control (transfected with c‐Myc WT) and T58A (transfected with c‐Myc T58A). (d) Detection of c‐Myc protein stability by CHX chase assay. Cells were harvested at the indicated time points for western blotting. Data are presented as mean ± SEM. ∗∗∗
p < 0.001.

3.4. OGT Conferred DDP Resistance to TNBC Cells by O‐GlcNAcylating c‐Myc at Thr58
To determine whether OGT‐induced chemoresistance depends on the Thr58 residue of c‐Myc, we established c‐Myc‐knockout MDA‐MB‐231 cells and reconstituted them with WT (MYC‐WT) or Thr58A‐mutant (MYC‐T58A) c‐Myc. OGT was then overexpressed (OGT OE) in each context to evaluate whether its effects on cell viability and apoptosis were Thr58‐dependent. The expression of OGT was greatly increased in cells overexpressing OGT, and these cells were used for subsequent experiments (Figure 4(a)). To dissect the functional relationship between OGT and c‐Myc at Thr58, we generated c‐Myc‐knockout MDA‐MB‐231 cells and reconstituted them with either WT c‐Myc or c‐Myc T58A. Total c‐Myc protein levels were comparable between WT and mutant cells, indicating that the T58A mutation did not affect protein expression (Figure 4(b)). OGT overexpression significantly increased DDP resistance in c‐Myc WT cells, as evidenced by an elevated IC50 value (9.866 μM) compared with that of the control group (2.141 μM) (Figure 4(c)). However, this resistance‐enhancing effect was largely abolished in OGT‐overexpressing cells with the T58A mutation, suggesting that Thr58 was essential for the ability of OGT to promote chemoresistance. Other observations supported this phenomenon. Under DDP treatment, T58A mutant MDA‐MB‐231 cells exhibited lower colony formation ability and higher apoptosis rates than their WT counterparts (Figures 4(d) and 4(e)). These differences were not observed in the absence of drug treatment, indicating that Thr58 plays a drug‐dependent functional role. Consistent with previous findings, c‐Myc stability and O‐GlcNAcylation were significantly decreased when c‐Myc was mutated. However, OGT overexpression did not reverse this effect (Figure 4(f)). These results indicated that Thr58 was essential for the OGT‐mediated regulation of c‐Myc function in DDP resistance in TNBC cells.
Figure 4OGT confers DDP resistance to TNBC cells by O‐GlcNAcylating c‐Myc at Thr58. To determine whether the chemoresistance induced by OGT overexpression depends on the Thr58 residue of c‐Myc, c‐Myc‐knockout MDA‐MB‐231 cells were reconstituted with either wild‐type (MYC‐WT) or Thr58‐mutant (MYC‐T58A) c‐Myc, followed by OGT overexpression (OGT OE). Three experimental groups were established: (i) MYC‐WT, serving as the baseline reconstitution; (ii) MYC‐WT + OGT OE, to examine the effect of OGT overexpression on wild‐type c‐Myc; and (iii) OGT OE + MYC‐T58A, to test whether the Thr58 mutation abolished the OGT‐induced phenotype. (a) Expression levels of OGT detected by qPCR and western blotting. (b) Expression of MYC‐WT and MYC‐T58A in MYC‐knockout MDA‐MB‐231 cells as detected by qPCR and western blotting. Parental cells were used as controls. All subsequent assays were performed on MYC‐KO cells using the three treatment groups described above. (c) Cell viability detection by CCK‐8 assay. The IC50 values are presented. (d) Detection of cell proliferation by colony formation assay. (e) Detection of apoptosis by flow cytometry. (f) Expression levels of OGT, O‐GlcNAc, total c‐Myc, and p‐c‐Myc detected by western blotting. Data are presented as mean ± SEM. ns: not significant; ∗∗∗
p < 0.001.

Consistent with the proposed competitive relationship between O‐GlcNAcylation and phosphorylation at Thr58 [19], OGT overexpression markedly increased global O‐GlcNAc levels while reducing p‐c‐Myc (Thr58) levels, whereas OSMI‐1 treatment produced the opposite effect (Figure S1).

4. Discussion
TNBC lacks specific therapeutic targets, preventing it from benefiting from traditional endocrine or targeted therapeutic strategies. Consequently, chemotherapy remains the mainstream systemic treatment strategy [17, 20]. Although TNBC initially responds to chemotherapy, it frequently develops chemoresistance and exhibits high rates of recurrence and metastasis within a short period [21]. Therefore, addressing chemoresistance is important for TNBC treatment. The molecular mechanisms mediating chemoresistance are complex and often involve multiple signaling pathways and factors [22]. Common resistance mechanisms include abnormal activation of signaling pathways, enhanced DNA damage repair capabilities, and overexpression of drug efflux pumps [23]. These mechanisms are also associated with O‐GlcNAcylation processes [24, 25]. O‐GlcNAcylation is a post‐translational modification that affects various important transcription factors and signaling pathway proteins [26]. As a pivotal transcription factor, c‐Myc contributes to the initiation, advancement, and maintenance of various types of cancers. c‐Myc is overexpressed in TNBC, whereas the PI3K/mTOR/MYC signaling pathway is closely linked to O‐GlcNAcylation in breast cancer [27]. These findings suggested a potential interaction between O‐GlcNAcylation and c‐Myc in TNBC. To investigate the role of c‐Myc and O‐GlcNAcylation in TNBC chemoresistance, we established chemoresistant MDA‐MB‐231 cells using a DDP gradient. We observed the synchronous upregulation of c‐Myc and OGT expression in resistant cells. Concurrently, we noted that inhibition of OGT function in resistant cells, which subsequently downregulated O‐GlcNAcylation levels, significantly repressed cell proliferation and caused apoptosis, accompanied by a decrease in c‐Myc expression and global glycosylation. Our findings suggest that c‐Myc may serve as a substrate for OGT‐mediated O‐GlcNAcylation, indicating that OGT promotes TNBC chemoresistance by enhancing c‐Myc O‐GlcNAcylation.
Current research has not confirmed the precise interaction between c‐Myc and OGT in TNBC cells. Although we observed a synchronous change in global O‐GlcNAcylation and c‐Myc expression levels, given that c‐Myc interacts with multiple signaling pathways, investigating whether the interaction between c‐Myc and OGT is direct or indirect remains crucial. In this study, we performed Co‐IP experiments and successfully verified the direct interaction between OGT and c‐Myc in TNBC background cells by immunoprecipitating intracellular proteins from resistant cells. Next, we further examined the expression and protein stability of c‐Myc after treating resistant cells, confirming the role of OGT‐promoted O‐GlcNAcylation in c‐Myc stability.
c‐Myc is widely recognized for its strong association with chemoresistance in various cancers, and its abnormal expression can promote drug resistance [28]. Our findings shed light on the role of c‐Myc O‐GlcNAcylation in TNBC, complementing the current understanding of TNBC chemoresistance mechanisms and offering a new perspective for studying resistance.
To further explore the potential of c‐Myc as a therapeutic target in TNBC, we investigated the mechanisms influencing c‐Myc stability in TNBC cells. Previous research indicates that c‐Myc stability is highly correlated with its modification sites. Based on the results predicted by DictyOGlyc 1.1, we identified several candidate O‐GlcNAcylation residues in c‐Myc and experimentally evaluated their relevance to protein stability. Among these predicted sites, only the mutation at Thr58 markedly reduced total and O‐GlcNAcylated c‐Myc levels, indicating that Thr58 was the predominant site for OGT‐mediated modification. This finding is biologically meaningful as previous studies have demonstrated that the phosphorylation of key proteins at Ser62 and Thr58 within the N‐terminal regulatory region of c‐Myc dynamically regulates its stability. ERK‐regulated Ser62 phosphorylation stabilizes c‐Myc and promotes cancer progression. Conversely, Thr58 phosphorylation, regulated by GSK3β, promotes c‐Myc degradation and inhibits its pro‐oncogenic activity [29]. Thus, impaired phosphorylation at Thr58 may contribute to the development of cancer. O‐GlcNAcylation often competes with phosphorylation at the same or adjacent residues [30], suggesting that OGT‐catalyzed O‐GlcNAcylation at Thr58 antagonizes Thr58 phosphorylation, thereby stabilizing c‐Myc and enhancing its oncogenic potential in TNBC. By knocking out the WT c‐Myc protein and transfecting c‐Myc mutants into cells, we observed that the loss of Thr58 O‐GlcNAcylation significantly accelerated c‐Myc degradation and reduced cellular chemoresistance. Thus, the identification of Thr58 as a critical O‐GlcNAcylation site provides a mechanistic link between OGT activity and c‐Myc stabilization. These findings, together with the results of the OGT overexpression assays, indicate that the chemoresistance promoted by OGT was dependent on the presence of the Thr58 residue of c‐Myc.
Although our current study showed that O‐GlcNAcylation of c‐Myc, specifically at Thr58, enhances its stability and promotes chemoresistance in TNBC cells, the broader implications of c‐Myc overexpression in drug‐resistant TNBC cells warrant further investigation. Given that c‐Myc functions as a central node integrating multiple oncogenic pathways, including PI3K/AKT, MAPK/ERK, and Wnt/β‐catenin, as well as its well‐documented involvement in modulating DNA repair mechanisms and the expression of drug efflux pumps, future studies should delineate which specific signaling axis or downstream effectors mediate the broader chemoresistance‐promoting effects of elevated O‐GlcNAcylation on c‐Myc, beyond enhancing its stability [27, 31, 32]. Although c‐Myc has previously been implicated in the coordination of these oncogenic networks, the present study specifically focused on defining the upstream post‐translational mechanism by which OGT‐mediated O‐GlcNAcylation stabilizes c‐Myc. Accordingly, the activation status of these pathways was not investigated in this study and will be an important subject for future research.
O‐GlcNAcylation of c‐Myc could potentially enhance the transcription of DNA repair genes, facilitate the repair of chemotherapy‐induced DNA damage, or upregulate efflux pump proteins, thereby increasing drug expulsion from cells. Moreover, O‐GlcNAcylation may modulate c‐Myc transcriptional activity or responsiveness to upstream survival signals. One plausible mechanism is that O‐GlcNAcylation increases c‐Myc activity downstream of the PI3K/AKT pathway, which is often activated in TNBC and is linked to drug resistance [27]. Alternatively, O‐GlcNAcylation may interfere with GSK‐3β‐mediated phosphorylation and degradation of c‐Myc at sites other than Thr58, or modulate its interaction with co‐factors, thereby sustaining its oncogenic activity independently of external inhibitory cues [19]. Consistent with this notion, our data showed that the modulation of OGT in MDA‐MB‐231‐R cells led to reciprocal changes in O‐GlcNAc and p‐c‐Myc (Thr58) levels, with OGT overexpression reducing Thr58 phosphorylation, whereas OSMI‐1 treatment markedly enhanced phosphorylation at this site. Future studies using phosphoproteomics, reporter‐based assays, or pharmacological inhibition of key signaling cascades in OGT‐manipulated models could help delineate these pathway‐specific contributions. Collectively, these findings suggested that OGT‐mediated O‐GlcNAcylation at Thr58 functions as a post‐translational modification that enhances c‐Myc stability and contributes to the development of DDP resistance in TNBC cells.
Despite compelling in vitro evidence, limitations and directions for future research should be acknowledged. Our study lacks in vivo validation, which may have overlooked microenvironment‐related factors that influence drug resistance [33]. For instance, the tumor microenvironment, particularly inflammatory cytokines, such as IL‐6 and TNF‐α, and infiltrating immune cells, can activate multiple signaling pathways, including STAT3 and NF‐κB, thereby contributing to chemotherapy resistance [34–37]. These pathways also enhance the stability and activity of transcription factors associated with drug resistance, such as c‐Myc [38, 39]. Elevated levels of O‐GlcNAcylation in inflammation‐associated malignancies have been shown to promote cell survival and drug tolerance by stabilizing oncogenic proteins [40–42]. Thus, our findings raise the possibility that OGT‐catalyzed O‐GlcNAcylation represents a mechanistic link between inflammation and chemoresistance through the modulation of c‐Myc. To substantiate the clinical relevance of this mechanism further, future studies should establish a DDP‐resistant TNBC xenograft mouse model. By comparing the tumor burden, OGT expression, and c‐Myc O‐GlcNAcylation status in xenografts from treated and control mice, we can verify whether this modification plays a similar role in promoting chemoresistance in vivo.
From a translational standpoint, our results indicate that pharmacological inhibition of OGT may sensitize TNBC cells to DDP by reducing c‐Myc O‐GlcNAcylation. However, because OGT is an essential and ubiquitous enzyme involved in diverse physiological processes, including cell cycle regulation, metabolic homeostasis, and stress responses [43, 44], systemic inhibition may cause off‐target effects or toxicity in normal tissues. OSMI‐1, a small‐molecule OGT inhibitor, has demonstrated preclinical efficacy in suppressing cancer cell proliferation and sensitizing tumor cells to chemotherapy; however, its pharmacokinetic properties, specificity, and safety profile in vivo remain insufficiently characterized. To date, no clinical trials have examined OSMI‐1 activity in TNBC or in any other solid tumor. Additional in vivo studies are required to evaluate the therapeutic potential of OGT inhibition in animal models and to investigate selective or tumor‐targeted strategies to modulate O‐GlcNAcylation. Furthermore, combinatorial approaches that integrate OGT inhibitors with conventional chemotherapy or immune checkpoint blockade could provide synergistic benefits and should be investigated in the context of TNBC.
In summary, our study demonstrates that OGT‐mediated O‐GlcNAcylation of c‐Myc is a crucial factor in promoting TNBC chemoresistance by directly interacting with c‐Myc and enhancing its protein stability, specifically through modification at the Thr58 site. This work not only elucidates a previously unrecognized role for c‐Myc glycosylation in TNBC but also complements the understanding of resistance mechanisms by proposing a direct link between OGT and c‐Myc stability. Our findings highlight the potential of targeting OGT‐mediated c‐Myc glycosylation as a novel therapeutic strategy for overcoming drug resistance in TNBC. Although further in vivo validation and comprehensive studies on the broader impact of c‐Myc O‐GlcNAcylation on diverse resistance pathways are warranted, this study lays a strong foundation for the development of effective interventions against this aggressive and challenging cancer type.

5. Conclusions
This study provides in vitro evidence that TNBC develops chemoresistance by upregulating OGT and c‐Myc expression. This was supported by a higher colony formation rate and lower apoptosis rate in drug‐resistant TNBC cells. OGT was found to O‐GlcNAcylate c‐Myc at Thr58, as supported by protein interaction assays and site‐specific mutagenesis. Mutation of c‐Myc at Thr58 suppresses its stability and function during drug resistance. These results highlight a possible molecular mechanism underlying TNBC.

Funding
This research did not receive any specific grants from funding agencies in the public, commercial, or not‐for‐profit sectors.

Conflicts of Interest
The authors declare no conflicts of interest.

Data Availability Statement
The datasets generated and/or analyzed in the current study are available from the corresponding author upon reasonable request.

Supporting Information
The antibodies used in this study are listed in Supporting Table S1. The primer sequences used for qPCR analysis are provided in Table S2, and the sequences of the mutant constructs employed in the experiments are listed in Table S3.

Supporting information

Supporting Information Additional supporting information can be found online in the Supporting Information section.