lgals9b Stabilizes eef1d Protein and Activates the PI3K/AKT Signaling Pathway to Promote Gastric Cancer Occurrence and Metastasis

Academic Background

Gastric cancer ranks as the fifth most prevalent cancer globally, with over one million new cases diagnosed annually. Due to late detection, it exhibits a high mortality rate and is the fourth leading cause of cancer-related deaths. Despite advancements in treatments such as surgical resection, adjuvant chemotherapy, and immune checkpoint blockade, gastric cancer continues to show limited efficacy and high recurrence rates. These challenges primarily stem from unclear pathogenesis and mechanisms of drug action. Therefore, a comprehensive molecular analysis of gastric cancer’s pathogenesis is essential to identify pivotal targets and explore novel therapeutic strategies.

In recent years, galectins have garnered considerable attention in gastric cancer research, being recognized as promising biomarkers and therapeutic targets. Among them, galectin-9 (lgals9) has received significant attention due to its role in immune regulation within the tumor microenvironment and its potential association with poor prognosis in gastric cancer. However, the function of galectin-9b (lgals9b) in gastric cancer remains relatively unexplored. This study aims to investigate the role of lgals9b in gastric cancer progression, revealing its mechanism of promoting gastric cancer occurrence and metastasis by stabilizing eef1d protein and activating the PI3K/AKT signaling pathway.

Source of the Paper

This paper was co-authored by Huolun Feng, Wei Yao, Yucheng Zhang, Yongfeng Liu, Bin Liu, Ji Zhou, Jiehui Li, Zhuosheng Jiang, Fa Ling, Jianlong Zhou, Deqing Wu, Yong Li, Juan Yang, and Jiabin Zheng. The authors are affiliated with the School of Medicine at South China University of Technology, Guangdong Provincial People’s Hospital (Guangdong Academy of Medical Sciences), Ganzhou Municipal Hospital, Gannan Medical University, and other institutions. The paper was published in 2024 in the journal Oncogene, with the DOI: https://doi.org/10.1038/s41388-024-03247-2.

Research Process and Results

1. lgals9b Enhances the Proliferation, Migration, and Invasion Abilities of Gastric Cancer Cells

To investigate the function of lgals9b in gastric cancer, the research team constructed lgals9b-overexpressing AGS cell lines (AGS-OE) and validated the overexpression through Western blot. Subsequently, the proliferation of AGS-OE and control (Ctrl) cells was assessed using CCK-8 and colony formation assays, revealing that lgals9b significantly promoted cell proliferation. Transwell migration, scratch wound healing, and Transwell invasion assays demonstrated that lgals9b overexpression significantly enhanced the migration and invasion abilities of AGS cells. RNA sequencing (RNA-seq) results showed significant upregulation of cancer progression-related signaling pathways (such as epithelial-mesenchymal transition, angiogenesis, KRAS signaling, etc.) in AGS-OE cells. Similar results were observed in N87 gastric cancer cells.

2. Knockdown of lgals9b Inhibits the Proliferation, Migration, and Invasion Abilities of Gastric Cancer Cells

To further validate the function of lgals9b, the research team constructed lgals9b-knockdown AGS cell lines and confirmed the knockdown efficiency through qRT-PCR and Western blot. CCK-8 and colony formation assays revealed that knockdown of lgals9b significantly inhibited the proliferation of AGS cells. Transwell migration, scratch wound healing, and invasion assays demonstrated that knockdown of lgals9b significantly suppressed the metastatic ability of AGS cells. RNA-seq results indicated that cancer progression-related signaling pathways (such as E2F targets, G2M checkpoint, mTORC1 signaling, etc.) were downregulated following lgals9b knockdown. Similar results were observed in N87 gastric cancer cells.

3. lgals9b Enhances the Growth of AGS Cell Xenografts

To further examine the effect of lgals9b on gastric cancer growth, the research team established xenograft models using AGS cells. The results showed that the tumor growth rate in the AGS-OE group was significantly faster than that in the control group, with larger tumor volumes and masses, while there was no significant difference in body weight between the two groups. Similar results were observed in experiments with AGS-sh3 and AGS-shRNA cells.

4. lgals9b Activates the PI3K/AKT Signaling Pathway by Binding to eef1d

To further elucidate the potential mechanism of lgals9b in gastric cancer, the research team conducted RNA-seq signaling pathway enrichment analysis and immunoprecipitation-mass spectrometry (IP-MS). RNA-seq results showed that several signaling pathways, especially the PI3K/AKT pathway, were enriched in both lgals9b-overexpressing and knockdown groups compared to the control group. Western blot results indicated that the phosphorylation levels of PI3K and AKT were positively correlated with lgals9b protein levels. Through IP-MS screening, the research team identified an interaction between lgals9b and eef1d. Further experiments demonstrated that lgals9b binds to eef1d, preventing its ubiquitination-mediated degradation by the E3 ubiquitin ligase HERC5, thereby stabilizing eef1d protein and activating the PI3K/AKT signaling pathway.

5. eef1d Promotes the Occurrence and Metastasis of Gastric Cancer

The research team constructed eef1d-overexpressing AGS cell lines and assessed the effect of eef1d overexpression on cell proliferation using CCK-8 and colony formation assays. The results showed that eef1d significantly promoted the proliferation of AGS cells. Transwell migration, scratch wound healing, and invasion assays indicated that eef1d overexpression significantly enhanced the metastatic ability of AGS cells. Similar results were observed in eef1d-knockdown cells.

6. lgals9b Antagonizes HERC5-Mediated Ubiquitination Degradation of eef1d

The research team further explored the regulatory role of lgals9b on eef1d protein. The results showed that lgals9b overexpression significantly increased eef1d protein levels, while knockdown of lgals9b significantly decreased eef1d protein levels. Through IP-MS analysis, the research team identified HERC5 as the key E3 ubiquitin ligase responsible for eef1d ubiquitination degradation. Further experiments demonstrated that lgals9b competitively binds to eef1d, reducing its ubiquitination degradation by HERC5, thereby stabilizing eef1d protein and activating the PI3K/AKT signaling pathway.

Conclusion and Significance

This study is the first to reveal that lgals9b regulates eef1d protein levels through competitive binding with HERC5, thereby activating the PI3K/AKT signaling pathway and ultimately promoting the proliferation, migration, and invasion of gastric cancer. This discovery provides new insights into the pathogenesis of gastric cancer and offers a theoretical basis for developing novel therapeutic strategies targeting the lgals9b/eef1d/PI3K/AKT signaling pathway.

Research Highlights

1. Key Finding: lgals9b promotes gastric cancer occurrence and metastasis by stabilizing eef1d protein and activating the PI3K/AKT signaling pathway.
2. Methodological Innovation: The research team employed various high-throughput techniques, including RNA-seq and IP-MS, combined with in vitro and in vivo experiments, to comprehensively elucidate the function and mechanism of lgals9b.
3. Application Value: This study provides a new potential target for molecular targeted therapy in gastric cancer, with significant clinical application prospects.

Other Valuable Information

The experimental design of this study is rigorous, with ample data and reliable results. The research team validated the function of lgals9b in gastric cancer and its underlying mechanisms through multiple experimental approaches, laying a solid foundation for subsequent clinical translation research.