GCN2-SLC7A11 Axis Regulates Cell Growth and Survival in Retinoblastoma Under Arginine Deprivation

Background

Retinoblastoma (RB) is a common intraocular malignant tumor in children, accounting for 4% of all childhood cancers. Although current treatments such as chemotherapy are effective in some patients, they have drawbacks such as multidrug resistance, renal toxicity, and the induction of secondary cancers. Therefore, there is an urgent need to develop alternative therapeutic strategies with fewer side effects. Arginine deprivation has been shown to be an effective treatment for various solid and non-solid tumors. By inhibiting tumor cell proliferation and inducing cell death, arginine deprivation demonstrates potential anticancer effects. However, the specific mechanisms by which retinoblastoma cells respond to arginine deprivation remain unclear.

This study aims to explore the effects of arginine deprivation on retinoblastoma cells, particularly the mechanisms by which the GCN2 (General Control Nonderepressible 2) and SLC7A11 (Solute Carrier Family 7 Member 11) axis regulates autophagy, cell cycle arrest, and apoptosis. The research team hopes to provide new insights into the treatment of retinoblastoma by uncovering these mechanisms.

Source of the Paper

This paper was co-authored by Dan Wang, Wai Kit Chu, Jason Cheuk Sing Yam, Chi Pui Pang, Yun Chung Leung, Alisa Sau Wun Shum, and Sun-On Chan. The research team is affiliated with the School of Biomedical Sciences, Faculty of Medicine, The Chinese University of Hong Kong; the Department of Ophthalmology and Visual Sciences; the Hong Kong Hub of Pediatric Excellence; the Department of Ophthalmology, Hong Kong Children’s Hospital; the Hong Kong Eye Hospital; and the Department of Applied Biology and Chemical Technology, The Hong Kong Polytechnic University. The paper was published in 2024 in the journal Cancer & Metabolism, titled “GCN2-SLC7A11 Axis Coordinates Autophagy, Cell Cycle, and Apoptosis and Regulates Cell Growth in Retinoblastoma Upon Arginine Deprivation.”

Research Process and Results

1. Arginine Deprivation Inhibits the Growth of Retinoblastoma Cells

The study first evaluated the effects of arginine deprivation on the proliferation of three retinoblastoma cell lines (Y79, WERI-RB-1, and RB-YAM10). Using the CCK-8 assay, the research team found that arginine deprivation significantly inhibited the proliferation of these cells in a concentration-dependent manner. Further high-performance liquid chromatography-mass spectrometry (HPLC-MS) analysis confirmed that arginine levels in the culture medium were significantly reduced after arginine deprivation. Microscopic observations showed that the growth pattern of cells shifted from multicellular clusters to single cells or small clusters, indicating that arginine deprivation significantly affected cell growth.

2. Arginine Deprivation Induces Autophagy, Cell Cycle Arrest, and Apoptosis

The research team further investigated the effects of arginine deprivation on autophagy, cell cycle, and apoptosis. Using Cyto-ID autophagy detection and LC3 immunoblot analysis, the study found that arginine deprivation significantly increased autophagy activity. Cell cycle analysis revealed that arginine deprivation caused cell arrest in the S and G2 phases, with G2 phase arrest becoming more pronounced over time. Additionally, arginine deprivation induced an increase in early apoptotic cells. These results indicate that arginine deprivation inhibits the growth and survival of retinoblastoma cells through multiple mechanisms.

3. Regulation of GCN2 and mTOR Signaling Pathways

The research team found that arginine deprivation activated the GCN2 signaling pathway and inhibited the mTOR (Mechanistic Target of Rapamycin) signaling pathway. Activation of GCN2 led to increased expression of its downstream transcription factor ATF4, while phosphorylation levels of downstream mTOR molecules p70S6K and 4E-BP1 were significantly reduced. These results suggest that arginine deprivation regulates cell metabolism and growth through the GCN2 and mTOR signaling pathways.

4. Role of SLC7A11 in Arginine Deprivation

RNA sequencing analysis revealed that arginine deprivation significantly upregulated the expression of SLC7A11. Further studies confirmed that the expression of SLC7A11 is regulated by GCN2. By knocking down SLC7A11 using shRNA, the research team found that the loss of SLC7A11 rendered retinoblastoma cells partially resistant to arginine deprivation. Additionally, knockdown of SLC7A11 affected the regulation of autophagy and the cell cycle, indicating that SLC7A11 plays a key role in the cellular response to arginine deprivation.

Conclusions and Significance

This study reveals the mechanisms by which the GCN2-SLC7A11 axis regulates the growth and survival of retinoblastoma cells under arginine deprivation. The research found that arginine deprivation inhibits tumor cell growth by activating GCN2 and inhibiting the mTOR signaling pathway, thereby inducing autophagy, cell cycle arrest, and apoptosis. Furthermore, the expression of SLC7A11 is regulated by GCN2 and plays an important role in the cellular response to arginine deprivation. These findings provide new potential targets for the treatment of retinoblastoma and lay the theoretical foundation for developing therapeutic strategies based on arginine deprivation.

Research Highlights

1. Mechanistic Insights: The study systematically elucidates for the first time the mechanisms by which the GCN2-SLC7A11 axis regulates the growth and survival of retinoblastoma cells under arginine deprivation.
2. Multipathway Regulation: The research reveals the complex network by which arginine deprivation regulates cell metabolism and growth through the GCN2 and mTOR signaling pathways.
3. Potential Therapeutic Targets: The discovery of SLC7A11 provides a new potential target for the treatment of retinoblastoma.
4. Innovative Experimental Methods: The study employs advanced experimental techniques such as RNA sequencing, shRNA knockdown, and flow cytometry, providing strong support for in-depth mechanistic analysis.

Additional Valuable Information

The research team also found that arginine deprivation induces changes in the expression of ferroptosis-related genes, further enriching the potential applications of arginine deprivation in cancer therapy. These findings open new directions for future research, particularly in exploring the combined application of arginine deprivation with other anticancer therapies.

This study not only deepens the understanding of the mechanisms of arginine deprivation but also provides new insights and potential targets for the treatment of retinoblastoma, holding significant scientific and practical value.