Glycogen synthase 1 promotes breast cancer progression by promoting IκBα ubiquitination and degradation independent of its canonical enzyme function

Abstract

Breast cancer remains a global health challenge, with triple-negative breast cancer (TNBC) posing a particular therapeutic difficulty. Glycogen synthase 1 (GYS1), a key glycogen metabolic enzyme, is upregulated in various cancers under hypoxic stress and is associated with therapy resistance, but its functional role in BRCAs remains incompletely defined. Here, we identify a non-canonical, glycogen-independent mechanism by which GYS1 promotes breast cancer progression. We demonstrate that Gys1 knockout suppresses proliferation and migration of breast cancer cells in vitro and inhibits tumor growth in vivo. Crucially, neither ablation of other glycogen synthesis enzymes nor pharmacological inhibition of GYS1 recapitulates these effects, uncoupling the oncogenic role of GYS1 from its metabolic function. Mechanistically, GYS1 activates the canonical NF-κB pathway by promoting the ubiquitination and degradation of IκBα. GYS1 stabilizes the interaction between IκBα and its E3 ubiquitin ligase BTRC, facilitating IκBα proteasomal degradation and enabling nuclear translocation of NF-κB. Our findings establish GYS1 as a novel scaffold protein and an upstream regulator of NF-κB signaling independent of its enzymatic function, nominating it as a promising therapeutic target for breast cancer particularly for TNBC subtype that lacks effective treatment.

Supplementary Information

The online version contains supplementary material available at 10.1186/s12964-026-02728-z.

Introduction

Breast cancer (BRCA) persists as a formidable global health challenge, maintaining its status as the most frequently diagnosed malignancy among women and ranking among the top three most common cancers worldwide [1, 2]. Over recent decades, concerted efforts in screening and therapeutic advancements have led to a consistent decline in breast cancer mortality rates in several high-income regions, including North America and the European Union. This encouraging trend is largely attributable to the widespread implementation of early detection programs and the development of increasingly effective systemic therapies [3]. Despite this progress, a stark global disparity remains, with breast cancer continuing to be the leading cause of cancer-related mortality among women in many less developed countries, where access to modern diagnostics and treatments is often limited. BRCA could be categorized into three groups; BRCA expressing hormone receptor (estrogen receptor (ER +) or progesterone receptor (PR +), BRCA expressing human epidermal receptor 2 (HER2 +) and triple-negative breast cancer (TNBC) (ER −, PR − and HER2 −) [4, 5]. Nevertheless, for a substantial proportion of patients, conventional chemotherapy and hormone therapy remain the cornerstone of first-line treatment, highlighting the ongoing reliance on non-specific cytotoxic agents. As a result, TNBC is still considered the most challenging type to define and treat due to the limited targets identified [6], which further underscores the significance of identifying new molecular targets in this battle.

Glycogen synthase 1 (Gys1), in contrast to the liver-enriched Gys2 isoform, is ubiquitously expressed across a range of metabolic and structural tissues, including skeletal muscle, adipose tissue, and the kidney [7]. As the rate-limiting enzyme in the final step of glycogen biosynthesis, GYS1 catalyzes the transfer of glucose monomers from UDP-glucose to the growing glycogen chain, extending it via α−1,4-glycosidic linkages. Beyond its classical role as a cytosolic glucose reservoir, glycogen metabolism is increasingly recognized as a critical regulator in diverse physiological processes. These include pivotal roles in adipose tissue differentiation and thermogenesis [8], as well as in fueling the macrophage-mediated acute immune response [9]. In the context of tumor biology, a growing body of evidence points to the dysregulation of glycogen metabolism as an important marker of cancers. Studies have demonstrated that GYS1 expression is rapidly induced under hypoxic conditions, a common feature of the tumor microenvironment [10, 11]. Furthermore, a positive correlation between GYS1 expression and intracellular glycogen accumulation has been observed in diverse cancer cell lines, including those derived from glioblastoma, breast, and colon carcinomas [12], suggesting a potential adaptive mechanism for cancer cell survival. Notwithstanding these observations, the specific functional impact of GYS1 on breast cancer pathogenesis remains incompletely characterized and constitutes an active area of inquiry. Existing literature indicates that glycogen accumulation in breast cancer cells can be induced by radiation exposure and is associated with the development of radiation resistance [13], positioning glycogen as a potential mediator of treatment failure. More recently, Gys1 knockdown was shown to impair the proliferation of triple-negative breast cancer cells and to sensitize them to agents targeting mitochondrial protein homeostasis [14]. While these findings underscore the oncogenic relevance of GYS1, a critical gap in knowledge persists: the existing evidence does not conclusively determine whether the observed effects of GYS1 on breast cancer progression are mediated solely through its canonical function in glycogen synthesis, or whether it exerts additional, non-canonical functions independent of glycogen storage. Elucidating this precise mechanistic link is paramount for validating GYS1 as a therapeutic target.

Our study initially demonstrated that Gys1 promotes tumor proliferation and migration in both mouse and human TNBC cells. This oncogenic role was further supported by in vivo xenograft models, where Gys1 knockout significantly suppressed tumor growth. Interestingly, neither genetic ablation of other glycogen synthesis enzymes nor pharmacological inhibition of GYS1-induced glycogen reduction recapitulated the tumor-suppressive phenotype, suggesting that the pro-tumorigenic function of GYS1 is independent of its role in glycogen metabolism. Subsequently, we identified a novel molecular link between GYS1 and a major oncogenic signaling hub. Strikingly, we found that Gys1 deletion potently suppresses the activation of Nuclear Factor-kappa B (NF-κB), a transcription factor complex widely recognized as a master regulator of immune responses [15], cellular development and differentiation [16], and, most notably, a critical driver of tumor cell survival, proliferation, and inhibition of apoptosis [17, 18]. The canonical NF-κB pathway, which represents the primary activation mechanism in response to pro-inflammatory cytokines and cellular stress, is tightly controlled by the inhibitory protein IκBα. Pathway activation hinges upon the phosphorylation, ubiquitination, and subsequent proteasomal degradation of IκBα [19], a process mediated by the IKK complex (comprising IKKα, IKKβ, and NEMO). The degradation of IκBα unshackles the p65/p50 heterodimer, allowing its nuclear translocation to initiate the transcription of target genes [20]. In line with this mechanism, Gys1 knockout increased IκBα protein levels without altering its mRNA expression. Further mechanistic investigation revealed that GYS1 facilitates the ubiquitination and degradation of IκBα by enhancing its interaction with the E3 ubiquitin ligase β-Transducin Repeat Containing Protein (BTRC), thereby activating NF-κB signaling to drive tumor progression.

In summary, our findings uncover a non-canonical, glycogen-independent role for GYS1 as a critical regulator of the NF-κB pathway through post-translational control of IκBα stability, positioning it as a potential therapeutic target in breast cancer.

Results

Gys1 deficiency inhibits the proliferation and migration of breast cancer in vitro

To establish the clinical relevance of GYS1 in breast cancer, we first investigated the correlation between GYS1 expression levels and patient survival outcomes. Interrogation of the GEPIA database (http://gepia.cancer-pku.cn/) revealed a significant inverse relationship: breast cancer patients with higher GYS1 levels experienced substantially lower overall survival rates compared to those with low GYS1 expression (Fig. 1A). This is consistent with the findings from a previous study using the METABRIC database, which specifically reported that high GYS1 expression is associated with worse overall survival, particularly in TNBC patients [14]. Therefore, these compelling clinical association positions GYS1 as a potential oncogene with prognostic significance in breast cancer pathogenesis. We next analyzed Gys1 mRNA expression across different breast cancer subtypes. GYS1 expression was significantly elevated in the ER-HER2 + and TNBC subtypes compared to the ER + HER2 + group (Fig. 1B). To functionally validate this observation, we employed CRISPR/Cas9-mediated genome editing to generate a stable Gys1 knockout in the mouse 4T1 breast cancer cell line. The successful and efficient ablation of GYS1 protein was confirmed by Western blot analysis (Fig. 1C). As the primary enzyme catalyzing glycogen synthesis, Gys1 knockout resulted in a marked and significant reduction in intracellular glycogen levels (Fig. 1D), thereby verifying the metabolic consequence of its loss.

Fig. 1.

Gys1 knockout suppresses proliferation and migration in both mouse and human breast cancer cells. A Overall survival curve of breast cancer (BRCA) patients with low versus high GYS1 expression levels, obtained from the GEPIA database. B Tukey boxplots of GYS1 mRNA levels in primary breast tumors stratified by clinical subtype from the METABRIC dataset (n = 108, 1398, 139, 258). C Western blot analysis validating the knockout efficiency of GYS1 in murine 4T1 cells. D Intracellular glycogen levels in sgCtrl and sgGys1 4T1 cells (n = 6 for each group). E Cell proliferation measured by CCK-8 assay in 4T1 cells following GYS1 knockout (n = 18 for each group). F (Left) Representative images from wound healing assays showing the migratory capacity of sgCtrl and sgGys1 4T1 cells. (Right) Quantification of migration distance (n = 12 for each group). Scale bar: 200 µm. G Representative images from Transwell migration assays assessing the migratory capacity of sgCtrl and sgGys1 4T1 cells. Scale bar: 100 µm. H Cell proliferation measured by CCK-8 assay in MDA-MB-231 cells following Gys1 knockdown (n = 18 for each group). I (Left) Representative images from wound healing assays showing the migratory capacity of shCtrl and shGys1 MDA-MB-231 cells. (Right) Quantification of migration distance (n = 12 for each group). Scale bar: 200 µm. J Representative images from Transwell migration assays assessing the migratory capacity of shCtrl and shGys1 MDA-MB-231 cells. Scale bar: 100 µm. All statistical data are shown as mean ± SD and analyzed by Student's t test (D,F, I), one-way ANOVA (B) and two-way ANOVA (E, H). *P < 0.05, ***P < 0.001, ns for not significant

We next assessed the functional impact of Gys1 deficiency on core oncogenic phenotypes. CCK-8 cell proliferation assays demonstrated that Gys1 knockout markedly suppressed the growth kinetics of 4T1 cells (Figure E). Moreover, wound healing and Transwell migration assays revealed that Gys1 knockout significantly impaired the migratory capacity of 4T1 cells (Fig. 1 F and G). To ascertain whether the oncogenic role of Gys1 is conserved across species and relevant in human breast cancer, we extended our findings to the human TNBC cell line MDA-MB-231. Using shRNA-mediated knockdown, we achieved a significant reduction in GYS1 protein level (Figure S1A). Consistent with the results in the 4T1 model, Gys1 downregulation in MDA-MB-231 cells led to a significant decrease in glycogen content (Figure S1B). Crucially, this metabolic perturbation was again associated with a significant inhibition of both cellular proliferation (Fig. 1H) and migration (Fig. IJ and 1J).

Therefore, this complementary set of experiments, conducted in both murine and human breast cancer cell models, consistently demonstrates that GYS1 is not merely a metabolic enzyme but plays a critical and evolutionarily conserved role in regulating glycogen metabolism, cellular proliferation, and migratory capacity in vitro. These findings solidify GYS1 as a key regulator of aggressive tumor cell behaviors in breast cancer.

Gys1 ablation impairs tumor growth and metastasis in vivo

To further elucidate the functional role of GYS1 in breast cancer progression, we established a syngeneic tumor model by subcutaneously inoculating BALB/c female mice with control (sgCtrl) or Gys1-knockout (sgGys1) 4T1 cells. Tumors derived from sgGys1 cells displayed markedly slower growth kinetics over time and reached significantly smaller final volumes compared to those in the sgCtrl group (Fig. 2A–C). Immunohistochemical (IHC) analysis of tumor sections confirmed highly efficient knockout of GYS1 in the sgGys1 group in vivo, validating our genetic targeting approach (Fig. 2D). In line with the enzymatic function of GYS1 in glycogen biosynthesis, periodic acid–Schiff (PAS) staining demonstrated a substantial depletion of intracellular glycogen stores within sgGys1 tumors, confirming a loss of glycogen synthesis resulting from Gys1 ablation (Fig. 2E). Moreover, IHC staining for Ki-67, a well-established marker of cell proliferation, indicated a pronounced reduction in the number of proliferating tumor cells upon Gys1 depletion, underscoring the importance of GYS1 in sustaining tumor growth (Fig. 2F).

Fig. 2.

Gys1 knockout suppresses tumor growth and lung metastasis in a syngeneic mouse model. A Representative photographs of mice bearing subcutaneous tumors derived from sgCtrl or sgGys1 4T1 cells, taken prior to dissection. B Excised subcutaneous tumors from sgCtrl and sgGys1 groups. C Tumor growth curves showing volume over time in sgCtrl and sgGys1 groups (n = 5 for each group). D Representative IHC staining for GYS1 in tumor sections. Scale bar: 20 µm. E Representative PAS staining for glycogen content in tumor sections. Scale bar: 100 µm. F Representative IHC staining for Ki-67 in tumor sections. Scale bar: 100 µm. G (Left) Representative macroscopic images of lungs from mice 21 days after intravenous injection with sgCtrl or sgGys1 4T1 cells. (Right) Quantification of metastatic nodules in the lung tissues (n = 4 for each group). H Representative H&E staining of lung sections, showing metastatic foci. Scale bar: 100 µm. I Representative IHC staining for E-cadherin in lung sections. Scale bar: 100 µm. All statistical data are shown as mean ± SD and analyzed by two-way ANOVA (C) and Student's t test (G). *P < 0.05, **P < 0.01, ***P < 0.001

We next evaluated the impact of GYS1 on metastatic dissemination. Mice were intravenously injected with either sgCtrl or sgGys1 4T1 cells to assess their potential to form lung metastases. The sgCtrl group developed numerous and extensive metastatic nodules throughout the lung parenchyma. In stark contrast, the sgGys1 group showed a significant reduction in pulmonary metastasis (Fig. 2G). This dramatic impairment in metastatic colonization was further corroborated by histological examination of lung sections using hematoxylin and eosin (H&E) staining (Fig. 2H). Additionally, IHC analysis of E-cadherin expression in lung tissues revealed a notable upregulation of this key epithelial adhesion marker in the sgGys1 group (Fig. 2I). This shift suggests that Gys1 loss may promote the maintenance of a more epithelial phenotype, potentially reducing the epithelial-to-mesenchymal transition (EMT) and thereby weakening the invasive and metastatic capabilities of the cancer cells.

Collectively, these in vivo findings provide compelling evidence that genetic knockout of Gys1 effectively curbs primary tumor growth, impairs cellular proliferation, and severely restricts distant metastasis in a syngeneic mouse model of breast cancer, further highlighting GYS1 as a critical metabolic regulator of tumor progression and aggressiveness.

The tumor inhibitory effect of Gys1 knockout is independent of glycogen biogenesis

Glycogen biogenesis is initiated by glycogenin-1 (GYG1), which catalyzes its own glycosylation to generate a short, covalently attached oligosaccharide primer. This primer serves as the essential substrate for glycogen synthase (GYS), which progressively elongates the glucose chain through the formation of α−1,4-glycosidic linkages. The final maturation of the glycogen particle is achieved by the branching enzyme (GBE1), which introduces α−1,6-glycosidic linkages at regular intervals, thereby giving rise to the characteristic, highly branched macromolecular structure of glycogen (Fig. 3A). To systematically dissect whether the tumor-suppressive effects observed upon Gys1 ablation are mechanistically linked to the depletion of intracellular glycogen, we extended our genetic targeting approach to two other critical enzymes in the glycogen synthesis pathway, GYG1 and GBE1, in 4T1 cells. Genetic knockout of Gyg1 or Gbe1 effectively reduced intracellular glycogen to an extent comparable to Gys1 knockout (Fig. 3B and C). Strikingly, however, and in direct contrast to the consequences of Gys1 depletion, the loss of Gyg1 or Gbe1 failed to recapitulate the inhibitory effects on cellular proliferation and migration. Comprehensive functional assays, including CCK-8 for cell viability, wound healing for two-dimensional migration, and Transwell assays for cell invasion and migration, collectively demonstrated that the growth and motile capacities of Gyg1- and Gbe1-deficient cells remained largely unaltered (Fig. 3D-F).

Fig. 3.

Glycogen levels are not causally linked to proliferation and migration of breast cancer cells. A Schematic diagram of the metabolic pathway from extracellular glucose uptake to glycogen synthesis, highlighting key enzymes. B Western blot analysis validating the knockout efficiency of GBE1 and GYG1 proteins in 4T1 cells. C Intracellular glycogen levels in cells following knockout of Gbe1 or Gyg1 in 4T1 cells (n = 6 for each group). D Cell proliferation measured by CCK-8 assay in cells after knockout of Gbe1 or Gyg1 (n = 18 for each group). E (Left) Representative images from wound healing assays showing the migratory capacity of cells after knockout of Gbe1 or Gyg1. (Right) Quantification of migration distance (n = 12for each group). Scale bar: 200 µm. F Representative images from Transwell migration assays assessing the migratory capacity of cells after knockout of Gbe1 or Gyg1. Scale bar: 100 µm. G Intracellular glycogen levels measured after a 12-h fasting period, followed by treatment with 10 µM MZ-101 and a 6-h refeeding phase (n = 6 for each group). H Cell proliferation measured by CCK-8 assay following MZ-101 treatment (n = 18 for each group). I (Left) Representative images from wound healing assays showing the migratory capacity of cells after MZ-101 treatment. (Right) Quantification of migration distance (n = 12 for each group). Scale bar: 200 µm. J Representative images from Transwell migration assays assessing the migratory capacity of cells after MZ-101 treatment. Scale bar: 100 µm. All experiments in this figure were performed using the 4T1 cell line. All statistical data are shown as mean ± SD and analyzed by one-way ANOVA (C, E), Student's t test (G, I) and two-way ANOVA (D, H). ***P < 0.001

To further corroborate this finding using a complementary pharmacological strategy, we treated parental 4T1 cells with MZ-101, a potent and highly selective small-molecule inhibitor of GYS1 [21]. While MZ-101 treatment successfully mirrored the glycogen-lowering effect seen in the genetic models (Fig. 3G), it did not phenocopy the anti-proliferative and anti-migratory outcomes observed in the Gys1-knockout cells (Fig. 3H-J). This critical discrepancy between genetic ablation and enzymatic inhibition of GYS1 further underscores the complexity of its functional role.

Collectively, these results indicate that the suppression of breast cancer progression upon Gys1 loss is independent of its canonical role in glycogen synthesis and the resulting reduction in cellular glycogen level, suggesting a non-canonical, kinase-independent function for GYS1 in driving oncogenic processes.

GYS1 regulates IκBα protein level and NF-κB activation

The canonical NF-κB signaling pathway is a well-established driver of tumorigenesis, promoting cell survival, proliferation, and inflammation [22–24]. In our investigation of the molecular mechanisms underlying the tumor-suppressive phenotype of Gys1 knockout, we discovered a profound inhibition of this pathway. Specifically, we observed a substantial decrease in the levels of phosphorylated p65 (Ser536), a key marker of NF-κB activation, in both Gys1-knockout 4T1 cells and the corresponding tumor tissues derived from them (Fig. 4A and B), as well as in Gys1-knockdown MDA-MB-231 human breast cancer cells (Fig. 4C). In contrast, treatment with the GYS1 inhibitor MZ-101 showed no significant impact on phosphorylated p65 levels (Figure S2). Furthermore, reintroduction of Flag-tagged Gys1 into Gys1-deficient 4T1 and MDA-MB-231 cells reversed the observed decrease in phosphorylated p65 protein levels (Fig. 4D and E). These findings prompted us to hypothesize that the nuclear translocation and activation of the NF-κB transcription factor complex is compromised upon Gys1 loss.

Fig. 4.

Gys1 knockout increases IκBα protein expression and suppresses p65 phosphorylation and nuclear translocation. A (Left) Western blot analysis of IκBα and phosphorylated p65 (p-p65) protein levels in sgCtrl and sgGys1 4T1 cells. (Right) Quantification of relative protein expression levels (n = 3 for each group). B (Left) Western blot analysis of IκBα and p-p65 protein levels in MDA-MB-231 cells expressing shCtrl or shGys1. (Right) Quantification of relative protein expression levels (n = 3 for each group). C (Left) Western blot analysis of IκBα and p-p65 protein levels in subcutaneous tumors from sgCtrl and sgGys1 groups. (Right) Quantification of relative protein expression levels in tumor tissues (n = 3 for each group). D (Left) Western blot analysis of IκBα and p-p65 protein levels in 4T1 cells expressing sgCtrl, sgGys1, or sgGys1 followed by 3 × Flag-Gys1 rescue. (Right) Quantification of relative protein expression levels (n = 3 for each group). E (Left) Western blot analysis of IκBα and p-p65 protein levels in MDA-MB-231 cells expressing shCtrl, shGys1 or shGys1 followed by 3 × Flag-Gys1 rescue. (Right) Quantification of relative protein expression levels (n = 3 for each group). F Immunofluorescence staining showing the subcellular localization of p65 in sgCtrl and sgGys1 4T1 cells. Scale bar: 10 µm. G Western blot analysis of p65 protein levels in cytoplasmic and nuclear fractions of sgCtrl and sgGys1 4T1 cells following cellular fractionation. H mRNA expression levels of Nfkbia (encoding IκBα) in sgCtrl and sgGys1 4T1 cells, as determined by quantitative PCR. All statistical data are shown as mean ± SD and analyzed by Student's t test (A-C, H) and one-way ANOVA (D, E). *P < 0.05, **P < 0.01, ***P < 0.001, ns for not significant

To directly test this hypothesis, we examined the subcellular localization of the p65 subunit. Immunofluorescence staining revealed a marked difference: while sgCtrl-expressing cells exhibited a greater intensity of p65 within the nuclei, sgGys1-expressing cells showed predominantly cytoplasmic p65 localization (Fig. 4F). This impaired nuclear translocation was further quantified by cellular fractionation followed by Western blot analysis. The results confirmed a significant retention of p65 in the cytoplasmic fraction and a concomitant reduction in the nuclear fraction of sgGys1-expressing cells compared to sgCtrl-expressing cells (Fig. 4G).

Mechanistically, we sought to identify the upstream event responsible for suppression of NFkB pathway by Gys1 depletion. We found that genetic ablation of Gys1 led to a pronounced upregulation of IκBα protein expression (Fig. 4A-C), which was fully rescued by the re-expression of Gys1 (Fig. 4D and E). IκBα is the primary inhibitory protein that binds to and sequesters NF-κB complexes in the cytoplasm, thereby preventing their nuclear import. Intriguingly, this increase in IκBα protein was not associated with a corresponding elevation in its mRNA transcript levels, which remained unchanged between sgCtrl and sgGys1 cells (Fig. 4H). This critical discrepancy strongly suggests that the accumulation of IκBα protein and the subsequent inhibition of NF-κB signaling upon Gys1 loss are governed by a post-transcriptional regulatory mechanism, rather than enhanced gene expression.

GYS1 regulates the ubiquitination degradation of IκBα by influencing the interaction between BTRC and IκB

To elucidate the molecular mechanism underlying IκBα accumulation upon Gys1 knockout, we hypothesized that GYS1 may regulate the proteasomal degradation pathway of IκBα. To test this hypothesis, we first assessed IκBα protein stability using the protein synthesis inhibitor cycloheximide (CHX). Our time-course experiments revealed a significantly prolonged half-life of IκBα protein in sgGys1 cells compared to control cells (Fig. 5A), indicating impaired degradation in the absence of Gys1. This stabilization effect was completely abolished upon co-treatment with the proteasome inhibitor MG132, which equalized IκBα levels between the two groups (Fig. 5B). These pharmacological interventions strongly suggested that GYS1 facilitates the ubiquitin-mediated proteasomal degradation of IκBα. Consistent with this, co-immunoprecipitation assays using an IκBα antibody demonstrated markedly reduced ubiquitination of IκBα in Gys1-deficient 4T1 and MDA-MB-231 cells, a defect that was rescued upon re-expression of 3 × Flag-GYS1 (Fig. 5C and D).

Fig. 5.

GYS1 serves as a molecular scaffold facilitating the IκBα-BTRC interaction to promote ubiquitin-mediated degradation of IκBα. A Western blot analysis of IκBα protein degradation kinetics in sgCtrl and sgGys1 4T1 cells treated with 50 μg/mL cycloheximide (CHX) for the indicated time periods (n = 3 for each group). B Western blot analysis of IκBα protein levels in sgCtrl and sgGys1 4T1 cells co-treated with 50 μg/mL CHX and 20 μM MG132 for the indicated durations (n = 3 for each group). C, D 4T1 or MDA-MB-231 cells expressing sgCtrl, sgGys1, or sgGys1 followed by 3 × Flag-Gys1 were treated with 20 μM MG132 for 6 h prior to immunoprecipitation with an anti-IκBα antibody. Western blot was then performed to detect ubiquitin levels. E, F Endogenous co-immunoprecipitation (co-IP) of GYS1 and IκBα in 4T1 cells. Cell lysates were immunoprecipitated with antibodies against GYS1 or IκBα, followed by immunoblotting with the indicated antibodies. G, H Co-IP assay assessing the interaction between GYS1 and IκBα. HEK293T cells were co-transfected with Myc-tagged IκBα and 3 × Flag-tagged GYS1. I Co-IP assay validating the interaction between GYS1 and BTRC. HEK293T cells were co-transfected with HA-tagged BTRC and 3 × Flag-tagged GYS1. J Schematic diagram of the GYS1 protein domain architecture. K Three-dimensional structural model from molecular docking (generated using AlphaFold 3) illustrating the respective binding sites of IκBα and BTRC on GYS1. L, M Co-IP assays validating the binding of GYS1's Rossmann fold 1 (R1) and Rossmann fold 2 (R2) to IκBα and BTRC, respectively. HEK293T cells were co-transfected with 3 × Flag-tagged R1 or R2, along with Myc-tagged IκBα or HA-tagged BTRC. N Co-IP assay examining the effect of increasing GYS1 expression on the IκBα-BTRC interaction. HEK293T cells were co-transfected with fixed amounts of Myc-tagged IκBα and HA-tagged BTRC, along with increasing doses of 3 × Flag-tagged GYS1. All statistical data are shown as mean ± SD and analyzed by two-way ANOVA. ***P < 0.001

We next investigated whether GYS1 interacts directly with IκBα to facilitate its degradation. Co-immunoprecipitation assays in 4T1 cells using antibodies against GYS1 and IκBα confirmed the interaction of endogenous GYS1 with endogenous IκBα (Fig. 5E and F). This interaction was further validated in HEK293T cells through reciprocal co-immunoprecipitation experiments following the co-expression of IκBα and GYS1 (Fig. 5G and H). Since the E3 ubiquitin ligase BTRC is well-established as the primary mediator of IκBα ubiquitination [25–27], we examined whether GYS1 also associates with BTRC. Remarkably, co-immunoprecipitation assays demonstrated that GYS1 directly binds BTRC (Fig. 5I), suggesting that GYS1 may function as a scaffold bridging the substrate and its ligase.

Structurally, GYS1 comprises two distinct Rossmann-like folds with characteristic N-terminal and C-terminal domains (Fig. 5J). To map the precise interaction interfaces, we performed molecular docking simulations using AlphaFold 3. These predictions revealed that IκBα preferentially binds to Rossmann fold 1 of GYS1, while BTRC predominantly interacts with Rossmann fold 2 (Fig. 5K). We experimentally validated these structural predictions in HEK293T cells by overexpressing individual Rossmann folds with BTRC, which confirmed the specific domain requirements for these interactions (Fig. 5L and M). Based on these comprehensive findings, we propose a novel molecular model wherein GYS1 functions as a critical scaffolding platform, simultaneously engaging IκBα and BTRC through its two distinct Rossmann folds, thereby spatially facilitating the ubiquitination of IκBα by its cognate E3 ligase. Supporting this model, increasing expression of GYS1 enhanced the interaction between IκBα and BTRC in a dose-dependent manner in a co-immunoprecipitation assay (Fig. 5N), demonstrating that GYS1 actively promotes the formation of the ubiquitination complex.

In summary, these results establish that beyond its canonical role in glycogen metabolism, GYS1 functions as an essential molecular platform that promotes the ubiquitination and degradation of IκBα by bridging its physical association with the E3 ubiquitin ligase BTRC, thereby providing a mechanistic link between glycogen synthesis and NF-κB signaling pathway regulation.

Overexpression of the full-length GYS1 but not the individual Rossmann domain promotes breast cancer proliferation and migration

Based on our previous structural and biochemical findings that identified distinct binding interfaces for IκBα and BTRC on separate Rossmann folds of GYS1, we hypothesized that only the full-length GYS1 protein, functioning as an integral molecular scaffold, would be capable of promoting IκBα ubiquitination and degradation, thereby driving tumor progression. Isolated structural domains, we reasoned, would lack the necessary spatial architecture to coordinate this protein complex. To test this hypothesis, we established stable 4T1 cell lines overexpressing either full-length GYS1, the Rossmann fold 1 domain (R1, responsible for IκBα binding), or the Rossmann fold 2 domain (R2, responsible for BTRC interaction). Western blot analysis confirmed comparable overexpression levels of the respective constructs (Fig. 6A). We then systematically assessed the functional consequences of these expressions. A battery of functional assays revealed a striking functional dichotomy: while overexpression of full-length GYS1 significantly enhanced cellular proliferation (as measured by CCK-8 assay) and potently stimulated migration (in both wound healing and Transwell assays), the expression of either the R1 or R2 domain alone produced no discernible effect on these oncogenic phenotypes (Fig. 6B-D).

Fig. 6.

Only full-length GYS1 promotes oncogenic phenotype and reduces IkBa in 4T1 cells. A Western blot analysis confirming the stable overexpression of Rossmann fold 1 (R1), Rossmann fold 2 (R2), and full-length GYS1 in 4T1 cells. B Cell proliferation measured by CCK-8 assay in 4T1 cells overexpressing vector control, R1, R2, or full-length GYS1 (n = 18 for each group). C (Left) Representative images from wound healing assays showing the migratory capacity of the indicated cell lines. (Right) Quantification of migration distance (n = 12 for each group). Scale bar: 200 µm. D Representative images from Transwell migration assays assessing the migratory capacity of the indicated cell lines. Scale bar: 100 µm. E Western blot analysis of IκBα protein degradation kinetics in the indicated cell lines treated with 50 µg/mL CHX for the specified time periods (n = 3 for each group). F Western blot analysis of IκBα protein levels in the indicated cell lines co-treated with 50 µg/mL CHX and 20 µM MG132 for the indicated durations (n = 3 for each group). G (Left) Western blot analysis of IκBα and p-p65 protein levels in 4T1 cells overexpressing vector control, R1, R2, or full-length GYS1. (Right) Quantification of relative protein expression levels (n = 2 for each group). All experiments in this figure were performed using the 4T1 cell line. All statistical data are shown as mean ± SD and analyzed by two-way ANOVA (B, E, F) and one-way ANOVA (C, G). ***P < 0.001, ns for not significant

We next interrogated the mechanistic basis for this observed functional requirement for the full-length protein. Using cycloheximide chase experiments, we found that only the full-length GYS1 markedly shortened the half-life of IκBα protein, accelerating its turnover (Fig. 6E). This effect was specifically dependent on proteasomal activity, as it was completely abolished by co-treatment with the proteasome inhibitor MG132 (Fig. 6F). Consistent with the enhanced degradation of its inhibitory protein, only the full-length GYS1 exhibited a significant reduction in total IκBα levels and a concomitant increase in the phosphorylation of p65 (Fig. 6G).

Collectively, these data validate our central hypothesis that the tumor-promoting function of GYS1 is intrinsically tied to its structural integrity. We demonstrate that the full-length GYS1 protein acts as an indispensable molecular platform, spatially facilitating the ubiquitination and degradation of IκBα to unleash NF-κB-driven oncogenic programs. This non-canonical, scaffolding function, which is entirely distinct from its role in glycogen anabolism, nominates GYS1 as a compelling and mechanistically defined therapeutic target for the intervention of breast cancer progression.

Discussion

As the rate-limiting enzyme in glycogen biosynthesis, GYS1-mediated glycogen metabolism is well-established as a pivotal regulator of cellular energy homeostasis. Despite this fundamental role, the specific relationship between GYS1 and tumor development remains relatively understudied. A growing body of evidence has begun to establish a connection between GYS1 and BRCA. I It was initially identified as a potential oncogene in TNBC through a multi-omics analysis of disulfidptosis regulators. Subsequent work further demonstrated that targeting GYS1 triggers a novel form of regulated cell death, disulfidptosis, by inducing aberrant F-actin contraction in TNBC models [28]. Additional evidence indicates that GYS1 targeting exposes a critical metabolic vulnerability in TNBC, where its knockdown not only suppresses breast cancer cell proliferation but also synergistically enhances sensitivity to perturbations in mitochondrial protein homeostasis [14]. These seminal studies collectively emphasize the role of GYS1 in the context of glycogen synthesis, leading to the prevailing hypothesis that glycogen metabolism and resultant glycogen levels are the primary regulators of its tumor-promoting effects. Consequently, such findings have prompted calls for the development of GYS1 inhibitors as potential therapeutic strategies, focusing on its metabolic function.

In our study, we first validated the robust tumor-promoting function of GYS1 using a comprehensive approach involving both in vitro human and mouse TNBC cell lines and in vivo syngeneic models. Surprisingly, we discovered for the first time that this oncogenic activity is largely independent of intracellular glycogen levels and overall glycogen metabolism (Fig. 3). A critical piece of evidence supporting this paradigm shift is that neither pharmacological inhibition of GYS1’s enzymatic activity nor genetic interference with other key glycogen metabolic enzymes (GYG1, GBE1) could replicate the potent anti-tumor phenotypes observed with Gys1 knockout. These findings not only guided our subsequent exploration of the non-canonical mechanisms by which GYS1 exerts its functions but also carry significant therapeutic implications, suggesting that simply developing catalytic inhibitors of GYS1 to modulate glycogen levels may not be a viable therapeutic approach for breast cancer.

Although the functional profile of GYS1 in cancer remains closely tied to glycogen metabolism, a recent study in clear cell renal cell carcinoma (ccRCC) also suggested a potential non-metabolic role. That work implicated a GYS1-RPS27A interaction in promoting p65 phosphorylation and nuclear translocation, thereby facilitating tumor progression [10]. To systematically investigate how GYS1 regulates BRCA independently of glycogen, we explored multiple avenues—including the possibility that GYS1 itself could localize to the nucleus and exert direct transcriptional regulation, analogous to p65. However, in both normoxic and hypoxic breast cancer cells, GYS1 exhibited exclusively cytoplasmic localization with no detectable nuclear presence (data not shown).

Fortunately, our investigation took a pivotal turn when we observed that Gys1 knockout in both breast cancer cells and tumor tissues similarly led to suppressed p65 phosphorylation and a consequent inhibition of the pro-tumorigenic NF-κB signaling pathway. Notably, we also detected a significant accumulation in the protein level of IκBα, the key inhibitory protein of the canonical NF-κB pathway. The IκB family encompasses multiple members, including p100, p105, IκBα, IκBβ, IκBε, IκBζ, BCL-3, and IκBNS, which function as central negative regulators of the NF-κB pathway [29]. These proteins share a common mechanism of action: through their C-terminal ankyrin repeats, they bind to and mask the nuclear localization signal (NLS) of NF-κB dimers, thereby sequestering the transcription factor in an inactive state within the cytoplasm. Their signal-responsive degradation is governed by specific motifs, primarily within the N-terminal regions, which harbor critical phosphorylation and ubiquitination sites [30]. Among the classical IκB proteins constitutively present in the cytoplasm—IκBα, IκBβ, and IκBε—IκBα stands out for its role in mediating the most rapid and transient activation of NF-κB in response to a wide array of stimuli [31, 32]. Further investigation revealed that GYS1 promotes the proteasome-mediated ubiquitination and degradation of IκBα. Mechanistically, we established that GYS1 does not function as a typical signaling molecule but acts as a critical molecular scaffold that facilitates the physical interaction between IκBα and its cognate E3 ubiquitin ligase BTRC. This scaffolding function, mediated by distinct structural domains within GYS1, enhances the binding of IκBα to BTRC and thereby accelerates IκBα degradation. This finding unveils a previously unappreciated, non-metabolic function of GYS1 in promoting NF-κB activation through the direct regulation of IκBα protein stability, providing a novel mechanistic insight into its role in breast cancer progression (Figure S3).

Our findings have profound implications for targeting GYS1 in breast cancer therapy. The discovery of its glycogen-independent, scaffolding function necessitates a fundamental re-evaluation of therapeutic strategies. Simply inhibiting the enzymatic activity of GYS1, as attempted with MZ-101, is unlikely to yield significant clinical efficacy, as evidenced by our data showing a disconnect between glycogen reduction and anti-tumor effect. Instead, the future of GYS1-targeted therapy lies in the development of novel agents, such as proteolysis-targeting chimeras (PROTACs) or protein–protein interaction (PPI) inhibitors, specifically designed to disrupt its scaffold function within the IκBα-BTRC complex. Such an approach would aim to dismantle the GYS1-mediated signaling hub that drives NF-κB activation, thereby suppressing tumor growth and metastasis without directly affecting glycogen synthesis. This strategy could be particularly impactful in TNBC, a subtype with limited therapeutic options and known NF-κB pathway hyperactivation. However, a significant challenge remains: achieving high specificity to avoid disrupting the essential metabolic functions of GYS1 in normal tissues, which could lead to on-target toxicities. Future efforts must therefore focus on precise structural mapping of the interaction interfaces to enable the rational design of disruptive molecules that spare the enzyme's catalytic site.

Our mechanistic data establish GYS1 as a key regulator of p65 activation. Future studies validating the correlation between GYS1 expression and p65 phosphorylation or nuclear localization in clinical tumor samples will be crucial. Assessing their co-expression could reveal a combined biomarker with enhanced prognostic power, potentially identifying patients who might benefit from therapies targeting this pathway.

In summary, we identify a non-canonical role for GYS1 as a molecular scaffold that activates NF-κB signaling by facilitating IκBα ubiquitination, independently of glycogen metabolism. This paradigm-shifting finding redefines GYS1's oncogenic mechanism and nominates its scaffolding function as a compelling therapeutic target in breast cancer.

Methods

Animals

Mice were housed in a specific-pathogen-free (SPF) facility with 12-h:12-h light: dark cycles and given free access to food and water. All mice were maintained under specific-pathogen-free (SPF) conditions with a 12-h light/dark cycle and provided with ad libitum access to food and water. For the xenograft tumor model, 4–6-week-old female BALB/c nude mice were randomly allocated into experimental groups. Subsequently, 4T1 cells transduced with either sgCtrl or sgGys1 were resuspended in PBS and subcutaneously injected into the right flank of each mouse. Tumor volumes were measured every three days and calculated using the formula: 0.5 × a² × b, where a and b represent the shortest and longest diameters, respectively. For the lung metastasis model, the same genetically modified 4T1 cells were intravenously injected into the tail vein of mice. After 21 days, all mice were euthanized, and lung tissues were harvested for macroscopic and histological analysis of metastatic nodules. All animal experimental protocols were approved by Institutional Animal Care and Use Committee Institutional Animal Care and Use Committee of the Shanghai Institute of Nutrition and Health, Chinese Academy of Sciences (CAS) with an approval number SINH-2024-CY-1.

Cell culture

MDA-MB-231 and HEK293T cells were cultured in DMEM (Gibco), while 4T1 cells were maintained in 1640 medium (Procell). All culture media were supplemented with 10% fetal bovine serum (Gibco), 100U/mL penicillin, and 100 μg/mL streptomycin (Procell).

Reagents and antibodies

Antibodies used in this study were purchased as follows: anti-mouse-GYS1 (sc-81173), anti-β-actin (sc-47778), anti-Ub (sc-8017) and anti-H3 (sc-8654) were purchased from Santa Cruz (Dallas, TX, USA), anti-α-tubulin (T6199) and anti-Flag (F3165) were from Sigma-Aldrich (St. Louis, MO), anti-p65 (8242S), anti-p-p65 (ser536) (3033S) and anti-IκBα (9242S) were purchased from Cell Signaling Technology (Boston, MA), anti-GBE1 (A6599), anti-GYG1 (A10218), anti-GAPDH (A19056), anti-Myc (AE070), anti-HA (AE008) and anti-human-GYS1 (A2519) were obtained from Abclonal (Boston, MA). Polyethylenimine (PEI) were purchased from Sigma-Aldrich. The TRIzol reagent was from Invitrogen (Carlsbad, CA, USA). The RIPA buffer was from Yeasen (Shanghai, China). The nuclear and cytoplasmic protein extraction kit (C510001) was from Sangon Biotech (Shanghai, China).

Plasmids and lentivirus

Gene knockout plasmid was constructed using the lentiCRISPRv2 vector system following the CRISPR/Cas9 methodology, with a target-specific sgRNA sequence inserted into the plasmid backbone. For Gys1 knockdown in MDA-MB-231 cells, lentiviral particles were produced using the pLKO.1-puro vector encoding short hairpin RNA (shRNA) targeting Gys1 transcripts. The specific sgRNA and shRNA sequences are provided in Table S1. The plasmids pMD2. G and psPAX2 were used for lentivirus packaging.

Cell proliferation assay

Cells were seeded at a density of 2000–3000 cells per well in a 96-well plate. Cell proliferation was detected by using the Cell Count Kit-8 (MA0218, MeilunBio) according to the manufacturer’s instructions. At indicated time point, CCK8 solution was added to each well and incubated for 1 h. Absorbance was detected at a wavelength of 450 nm.

Wound healing assay

Cells were plated in 12-well plates and when it reached 90% confluence, sterile pipette tips were used to scratch the wound uniformly. Cell motility was assessed by measuring the distance of the wound at 0 h and 24 h.

Transwell migration assay

Cell migration was measured using Transwell inserts (Corning Life Sciences, Tewksbury, MA). Logarithmic growth cells were taken and counted after routine digestion. 1 × 10⁵ cells were re-suspended in 200ul serum-free medium and added to the upper chamber of transwell chamber, and 550ul complete medium was added to the lower chamber. Attention was paid to avoid bubbles during the process. After cultured cells for 24 h, the 24-well plate was removed, the medium in the upper chamber was poured, the cells that did not migrate in the upper chamber were wiped with a PBS wet cotton swab, fixed and stained in 1% crystal purple methanol solution for 10 min, gently rinsed with running water and dried, the transferred cells were observed under a microscope, counted and photographed.

Co-immunoprecipitation

HEK293T cells were transiently transfected with the indicated plasmids using the PEI method. For co-immunoprecipitation assays, cells were washed three times with ice-cold phosphate-buffered saline (PBS) and lysed in IP lysis buffer (20 mM Tris–HCl, pH 7.5, 137 mM NaCl, 5 mM EDTA, 1% NP-40, 10% glycerol, 50 mM NaF, 1 mM Na₃VO₄, and protease inhibitor cocktail) for 30 min at 4 °C with gentle agitation. The lysates were clarified by centrifugation at 12,000 × g for 30 min at 4 °C. Approximately 10% of the supernatant was reserved as input for immunoblotting analysis. The remaining lysate was incubated overnight at 4 °C with primary antibodies. Protein A/G Plus Agarose beads (Santa Cruz) were then added and incubation continued for an additional 2 h at 4 °C. The beads were collected and washed five times with ice-cold lysis buffer. Bound proteins were eluted and subjected to western blot analysis.

Western blotting

Tissue and cell samples were lysed in RIPA buffer supplemented with fresh protease inhibitors (MCE) and phosphatase inhibitors (Sigma-Aldrich). Lysates were centrifuged at 13,200 rcf (g) for 10 min at 4 °C, and the supernatant was collected. Total protein concentration was determined using a BCA Protein Assay kit (Beyotime) following the manufacturer’s instructions. Proteins were separated on 10% SDS‑PAGE gels and subsequently transferred to PVDF membranes (Fisher Scientific). The membranes were incubated with primary antibodies overnight at 4 °C, followed by incubation with horseradish peroxidase (HRP)-conjugated secondary antibodies. Blots were visualized using a Tanon-5200 imaging system. Primary and secondary antibodies were used at dilutions of 1:1000 and 1:5000, respectively. Band intensities were quantified with ImageJ software. For quantitative analysis, band intensities were normalized to internal loading controls. Apparent variations in absolute band intensities between different figures may result from differences in exposure times for visualization and do not affect within-experiment statistical comparisons.

Real-time quantitative PCR analysis

Total RNA was isolated using TRIzol reagent. Complementary DNA (cDNA) was synthesized from equal amounts of total RNA using the FastQuant RT Kit (Tiangen) according to the manufacturer's instructions. Quantitative PCR was performed on an ABI QuantStudio 6 system using the SYBR Green PCR master mix (TOYOBO) and gene-specific primers (provided in Table S1). The reaction conditions were as follows: initial denaturation at 95 °C for 2 min, followed by 40 cycles of 95 °C for 15 s and 60 °C for 1 min. All samples were run in technical duplicates. The relative expression levels of the target genes were calculated using the 2^(-ΔΔCt) method and normalized to the endogenous reference gene β-actin.

Measurement of glycogen content

The kit to detect glycogen content (D799398) was from Sangon Biotech (Shanghai, China). The measurement of glycogen content was referred to the manufacturer’s instructions, and the glycogen levels were normalized by cell number.

Haematoxylin and eosin (H&E) staining, Periodic acid-schiff (PAS) staining and immunohistochemistry

Tissues were isolated from the mice, fixed overnight with 4% paraformaldehyde (PFA) for paraffin‐embedding. For H&E or PAS staining, the tissue blocks were cut into at a thickness of 5 μm and stained with H&E or PAS. For immunohistochemistry (IHC), the slides were processed by blocking with hydrogen peroxide, antigen retrieval, blocking with serum and then incubation with anti‐GYS1 and anti-Ki-67 antibodies. The secondary antibodies were applied after washing, followed by coloration with diaminobenzidine (DAB). The mounted slides were imaged using an Olympus BX51 microscope.

Immunofluorescence staining

Cells were seeded on glass coverslips in 12-well plates and cultured under standard conditions until they reached 60–70% confluence. After designated treatments, cells were fixed with 4% paraformaldehyde in PBS for 15 min at room temperature, followed by permeabilization with 0.1% Triton X-100 for 10 min. To block nonspecific binding, samples were incubated in blocking buffer (5% bovine serum albumin in PBS) for 1 h at room temperature. Following this, the sections were incubated overnight at 4 °C with primary antibodies, followed by incubation with corresponding secondary antibodies at room temperature for 1 h. Nuclei staining was achieved by treating the sections with Hoechst 33,342 (Molecular Probes) in PBS for a duration of 8 min. Fluorescence images were captured using ZEISS (LSM880). The dilution ratios of primary antibodies and secondary antibodies used were respectively set as 1:200 and 1:500.

In silico molecular docking

The protein structure of human GYS1, human IκBα and human BTRC were predicted via Swiss-Model: target FASTA sequences were submitted for automatic modeling using high-homology, high-resolution PDB templates [33, 34]. Protein–protein binding was performed with ClusPro online server (https://cluspro.org/), the top ranked energy cluster in balanced energy mode was illustrated using PyMOL.

Statistical analysis

All data are shown as mean ± SD. Comparisons of two groups were performed by a two-tailed Student’s t test. One-way ANOVA was used to assess comparisons across three or more experimental groups. The effects of two independent variables were assessed by two-way ANOVA. All statistics were performed using the GraphPad Prism software and values of P < 0.05 were considered statistically significant.

Authors’ contributions

Yan Chen and Shixuan Zhuo designed the experiments. Shixuan Zhuo and Zinan Wang performed normal experiments and analyzed data. Zinan Wang helped with the molecular docking part. Yu Zhang provided technical assistance. Shixuan Zhuo, Zinan Wang and Yan Chen wrote the manuscript and prepared the figures. All authors read and approved the manuscript.

Funding

This study was funded by National Natural Science Foundation of China (32230047 to YC), Chronic Non-Communicable Diseases-National Science and Technology Major Project of China (2404ZD0531300 to YC), the National Key Research and Development Program of China (2023YFA1801100 to YC), and the Research Funds of Hangzhou Institute for Advanced Study, UCAS (to YC).

Data availability

No datasets were generated or analysed during the current study.

Declarations

Competing interests

The authors declare no competing interests.