GCN2-SLC7A11 axis coordinates autophagy, cell cycle and apoptosis and regulates cell growth in retinoblastoma upon arginine deprivation

Background: Arginine deprivation was shown previously to inhibit retinoblastoma cell proliferation and induce cell death in vitro. However, the mechanisms by which retinoblastoma cells respond to arginine deprivation remain to be elucidated. Methods: Human derived retinoblastoma cell lines Y79 and WERI-Rb-1 were exposed to arginine depletion, and the effects on inhibiting cell growth and survival were evaluated. The study investigated potential mechanisms, including autophagy, cell cycle arrest, and apoptosis. Moreover, the roles of GCN2 and mTOR signaling pathways in these processes were examined. Results: We demonstrated that arginine deprivation effectively inhibited the growth of retinoblastoma cells in vitro. This treatment caused an increase in autophagic response. Additionally, we found that prolonged arginine shortage induced G2 cell cycle arrest and was accompanied by an increase in early apoptotic cells. Importantly, arginine depletion also caused an activation of GCN2 and an inhibition of mTOR signaling. We also discovered that the activation of SLC7A11 was regulated by GCN2 upon arginine deprivation. Knockdown of SLC7A11 rendered retinoblastoma cells partially resistant to arginine deprivation. Furthermore, we found that knockdown of GCN2 led to a decrease in autophagic response in WERI-Rb-1 cells and arrested more cells in S phase, which was accompanied by fewer apoptotic cells. Moreover, knockdown of GCN2 caused constant expression of ATF4, phosphorylation of 70S6K, and 4E-BP1 regardless of arginine deprivation. Conclusions: Collectively, our findings suggest that the GCN2-SLC7A11 axis regulates cell growth and survival upon arginine deprivation through coordinating autophagy, cell cycle arrest, and apoptosis in retinoblastoma cells. This work paves the way for the development of a novel treatment for retinoblastoma. mRNA profiles of Y79 cell lines cultured in the presence or absence of arginine for 72 hours

背景：既往研究证实，精氨酸剥夺（arginine deprivation）可在体外（in vitro）抑制视网膜母细胞瘤（retinoblastoma）细胞增殖并诱导细胞死亡。然而，视网膜母细胞瘤细胞对精氨酸剥夺的应答机制仍有待阐明。
方法：将人源视网膜母细胞瘤细胞系Y79与WERI-Rb-1经精氨酸剥夺处理，评估其对细胞生长抑制与细胞存活的影响。本研究探究了潜在的作用机制，包括自噬（autophagy）、细胞周期阻滞（cell cycle arrest）与细胞凋亡（apoptosis）。此外，还考察了GCN2（general control nonderepressible 2）与mTOR信号通路（mTOR signaling pathway）在上述过程中的调控作用。
结果：本研究证实，精氨酸剥夺可有效在体外抑制视网膜母细胞瘤细胞的生长。该处理可增强自噬反应。此外，研究发现，长时间精氨酸缺乏可诱导G2期细胞周期阻滞，并伴随早期凋亡细胞数量增加。值得注意的是，精氨酸剥夺还可激活GCN2并抑制mTOR信号通路。本研究还发现，在精氨酸剥夺条件下，SLC7A11的激活受GCN2调控。敲低SLC7A11可使视网膜母细胞瘤细胞对精氨酸剥夺产生部分抗性。进一步研究发现，敲低GCN2可降低WERI-Rb-1细胞的自噬反应，并使更多细胞阻滞于S期，同时伴随凋亡细胞数量减少。此外，无论是否存在精氨酸剥夺，敲低GCN2均可导致ATF4（activating transcription factor 4）持续表达，以及70S6K（ribosomal protein S6 kinase）与4E-BP1（eukaryotic translation initiation factor 4E binding protein 1）的磷酸化。
结论：综上，本研究结果表明，在精氨酸剥夺条件下，GCN2-SLC7A11轴可通过协调视网膜母细胞瘤细胞的自噬、细胞周期阻滞与细胞凋亡过程，调控细胞生长与存活。本研究为视网膜母细胞瘤的新型治疗手段开发铺平了道路。将Y79细胞系在含精氨酸或不含精氨酸的培养基中培养72小时后，获取其mRNA表达谱。