Integrated Proteomics and Metabolomics Analysis Provides Insights into Metformin Down-regulating GLUL and SP1 to Inhibit Esophageal Squamous Cell Carcinoma Migration and Invasion
Huayu Luo, Tao Shen, Jing Zhang, Yang Cheng, Zhongyu Zhang, Yujie He, Hongzheng RenIntroduction:
Esophageal Cancer (EC) is a prevalent gastrointestinal malignancy. Despite significant advances in diagnostic and therapeutic approaches, the prognosis of Esophageal Squamous Cell Carcinoma (ESCC) remains poor. The development of new therapeutic agents for ESCC would greatly benefit patients and their families. Metformin has been found to be useful in other tumours, but its role and mechanism in ESCC have not been investigated.
Methods:
The effect of metformin on inhibiting invasive metastasis in ESCC cells was explored by in vitro cytology experiments. 4D-DIA proteomics and untargeted LC-MS metabolomics provided critical technical support for this study. The roles and mechanisms of differential proteins, metabolites, and key metabolic pathways in ESCC migration and invasion were further explored by identifying differentially expressed proteins and metabolites in metformin-treated ESCC cells using proteomics and metabolomics. RT-qPCR and Western blot were used to confirm metformin's inhibitory effects on key proteins in metabolic pathways associated with ESCC cell migration and invasion. Using the ESCC tissue microarray (TMA) to detect the expression levels of GLUL and SP1.
Results:
This study demonstrated that metformin significantly reduced ESCC cells' viability, proliferation, migration, and invasion, while enhancing apoptosis. Integrated multi-omics pathway analysis revealed the activation of several metabolic pathways following metformin treatment in ESCC cells. In vitro experiments confirmed that metformin significantly downregulated the key proteins GLUL and SP1, as identified in the differential proteomics analysis. Immunohistochemistry (IHC) showed that GLUL and SP1 expression were significantly lower in ESCC tumor tissues from patients with a history of preoperative metformin administration than in those without.
Discussion:
Enhanced glutamine metabolism in tumors promotes cancer progression. Downregulation of GLUL reduces glutamine production from the alanine, aspartate, and glutamate metabolic pathways, thereby inhibiting tumor progression. SP1 is an oncogenic factor in esophageal cancer and an unfavorable prognostic factor. Metformin downregulates both GLUL and SP1, affecting the alanine, aspartate, and glutamate metabolic pathways, as well as the choline metabolic pathway in cancer.
Conclusion:
In summary, metformin may inhibit the migration and invasion of ESCC cells through downregulation of GLUL and SP1, which play important roles in the mechanism of metformin's anti-tumor effects. These findings provide new insights into the therapeutic potential of metformin in the treatment of ESCC.