Research Background and Problem Introduction

Protein O-GlcNAc glycosylation is an important post-translational modification that plays a critical role in cellular metabolic plasticity. Aberrant O-GlcNAc glycosylation levels have been observed in various cancers, including endometrial cancer (EC), a common malignancy in women. However, clinical characterization and interrogation of the molecular mechanisms underlying dysregulated O-GlcNAc glycosylation in EC remain incomplete.

The incidence of endometrial cancer has increased by 50% over the past two decades, becoming one of the most common cancers within the female reproductive system in developed countries. In China, as of 2022, there were approximately 77,700 newly diagnosed cases and 13,500 estimated deaths from endometrial cancer. Obesity and conditions associated with metabolic syndrome, such as diabetes, are risk factors for this disease. Endometrial cancer can be classified into Type I or Type II tumors based on clinical and endocrine features, or categorized as endometrioid carcinoma, serous carcinoma, carcinosarcoma, or clear-cell carcinoma according to histopathological characteristics.

In recent years, integrating proteomics analysis into genomic classification has accelerated the identification of clinically actionable molecular targets in endometrial cancer. However, post-translational modifications (PTMs) that contribute to the functional complexity of the proteome have not been fully characterized and need to be incorporated into the current classification system. O-GlcNAc glycosylation, an important PTM sensitive to cellular metabolism and stress, has been linked to the molecular etiology of endometrial cancer.

Paper Source and Author Information

This study was conducted by researchers from multiple institutions, including Xiangya Hospital, Central South University, School of Life Sciences, Tsinghua University School of Pharmaceutical Sciences, among others. The paper was published in the journal Nature Communications, DOI: 10.1038/s41467-025-56633-z.

Research Process and Methods

Study Subjects and Sample Processing

Researchers first performed immunohistochemistry (IHC) analysis on a tissue microarray containing 23 non-tumor and 31 tumor endometrial samples. They then expanded their analysis to a cohort of 219 patients with endometrial cancer, all of whom had undergone hysterectomy. By staining these samples with IHC, they assessed the O-GlcNAc glycosylation levels and their correlation with clinical parameters.

Experimental Design and Data Analysis

Tissue Microarray Analysis

Researchers used anti-O-GlcNAc monoclonal antibody RL2 to detect O-GlcNAc glycosylation levels in the tissue microarray. Results showed that O-GlcNAc glycosylation and OGT expression were significantly higher in tumor tissues compared to controls, while OGA expression showed no significant difference.

Cohort Analysis

Patients were divided into high O-GlcNAc glycosylation (High-RL2) and low O-GlcNAc glycosylation (Low-RL2) groups based on IHC scores. Statistical analysis indicated that O-GlcNAc glycosylation levels were significantly associated with tumor histologic grade, FIGO stage, and distant metastasis. Kaplan-Meier survival analysis revealed that patients in the high O-GlcNAc glycosylation group had significantly shorter progression-free survival (PFS) and overall survival (OS) compared to those in the low O-GlcNAc glycosylation group.

TCGA Dataset Validation

To validate these findings, researchers further analyzed the TCGA UCEC dataset. They calculated a virtual O-GlcNAc index and correlated it with clinical parameters. Results showed that the O-GlcNAc index was significantly associated with histologic grade, FIGO stage, and patient age. Patients in the high O-GlcNAc glycosylation group exhibited significantly shorter PFI and OS compared to those in the low O-GlcNAc glycosylation group.

In Vitro Experiments and Mechanistic Investigation

Generation of Endometrial Organoids and Functional Impact

Researchers generated organoids from normal and cancerous endometrium to study the impact of altered O-GlcNAc glycosylation levels on their functions. Results showed that inhibiting OGA increased O-GlcNAc glycosylation levels, promoting proliferation and stem cell properties in normal endometrial organoids; whereas inhibiting OGT decreased O-GlcNAc glycosylation levels, limiting the growth of cancerous endometrial organoids.

Screening and Validation of F-box Only Protein 31 (FBXO31)

Using a genome-wide CRISPR-Cas9 knockout screen, researchers identified FBXO31 as a key regulator of O-GlcNAc glycosylation homeostasis. FBXO31, as a substrate recognition component in the SCF ubiquitin ligase complex, directly binds and ubiquitinates OGT, thereby controlling its degradation. The loss of FBXO31 stabilized OGT, leading to elevated intracellular O-GlcNAc glycosylation levels, which promoted endometrial malignancy.

Mouse Model Experiments

Researchers validated the anti-tumor activity of the OGT inhibitor OSMI-1 using xenograft mouse models. OSMI-1 treatment reduced tumor volume and extended the survival of mice. Additionally, FBXO31 deficiency promoted tumor formation but enhanced tumor sensitivity to OSMI-1.

Key Findings and Conclusions

Correlation Between O-GlcNAc Glycosylation Levels and Clinical Characteristics

The study demonstrated that O-GlcNAc glycosylation levels in endometrial cancer tissues were significantly associated with histologic grade, FIGO stage, and patient prognosis. Higher O-GlcNAc glycosylation levels correlated with more advanced histologic grades, higher FIGO stages, and poorer survival rates.

Mechanism of FBXO31 Regulating O-GlcNAc Glycosylation Homeostasis

FBXO31 regulates O-GlcNAc glycosylation homeostasis by ubiquitinating OGT to control its protein stability. Loss of FBXO31 leads to increased OGT stability and O-GlcNAc glycosylation levels, promoting endometrial cancer progression. FBXO31 expression is significantly downregulated in endometrial cancer, and low expression correlates with poor prognosis.

Therapeutic Potential of Inhibiting O-GlcNAc Glycosylation

Inhibiting OGT to decrease O-GlcNAc glycosylation levels effectively suppressed the proliferation and survival of endometrial cancer cells both in vitro and in vivo. This suggests that targeting O-GlcNAc glycosylation homeostasis may be a promising strategy for treating high-grade endometrial cancer.

Highlights and Significance

Key Discoveries

1. Correlation Between O-GlcNAc Glycosylation Levels and Endometrial Cancer Characteristics: Higher O-GlcNAc glycosylation levels correlate with more advanced histologic grades, higher FIGO stages, and poorer survival rates.
2. FBXO31 Regulates O-GlcNAc Glycosylation Homeostasis: FBXO31 maintains O-GlcNAc glycosylation homeostasis by ubiquitinating OGT. Loss of FBXO31 increases OGT stability and O-GlcNAc glycosylation levels, promoting endometrial cancer progression.
3. Therapeutic Potential of Inhibiting O-GlcNAc Glycosylation: Inhibiting OGT to lower O-GlcNAc glycosylation levels effectively suppresses the proliferation and survival of endometrial cancer cells in vitro and in vivo.

Scientific Value and Application Prospects

This study reveals the crucial role of O-GlcNAc glycosylation in endometrial cancer and identifies FBXO31 as a key regulator of O-GlcNAc glycosylation homeostasis. These findings provide new insights into the molecular mechanisms of endometrial cancer and lay the foundation for developing novel therapies targeting O-GlcNAc glycosylation homeostasis. Particularly for high-grade endometrial cancer patients, targeting O-GlcNAc glycosylation homeostasis may become a promising therapeutic strategy.

Methodological Innovations

1. Genome-Wide CRISPR-Cas9 Screening: Researchers identified key regulators of O-GlcNAc glycosylation homeostasis, such as FBXO31, through genome-wide CRISPR-Cas9 screening, demonstrating the powerful application potential of gene editing technology in cancer research.
2. Endometrial Organoid Models: Researchers generated organoids from normal and cancerous endometrium to study the impact of altered O-GlcNAc glycosylation levels on their functions, providing a reliable model system for in vitro studies.
3. Xenograft Mouse Models: Researchers validated the anti-tumor activity of the OGT inhibitor OSMI-1 using xenograft mouse models, showcasing a complete research chain from in vitro to in vivo.

This study not only elucidates the important role of O-GlcNAc glycosylation in endometrial cancer but also provides significant clues for developing novel therapies, offering important scientific value and application prospects.