PO-054 Traditional chinese medicine Ze-Qi-tang formula induces apoptosis and S phase arrest via ROS-dependent JNK and ERK activation in lung cancer

Abstract

IntroductionGuanine nucleotide binding protein like 1 (GNL1) is an evolutionarily conserved high molecular weight GTP binding nucleolar protein belonging to HSR1-MMR1 subfamily of GTPases. These family of nucleolar GTPases are emerging as crucial co-ordinators of signalling cascades and reported to regulate ribosome biogenesis and rRNA processing. GNL1 is a nucleo-cytoplasmic shuttling protein and localises in different cellular compartments in a cell cycle dependent manner. GNL1 regulates cell cycle progression at G2/M phase and found to be upregulated in majority of cancers. However, the functional role of GNL1 during cell proliferation remains unknown.Material and methodsCell lines used HCT116p53+/+, HCT116p53-/-, AGS and HEK293T. Human leukocyte cDNA library as prey and full length GNL1 in pGBKT7 vector as bait was used in Yeast two hybrid screening assay. GNL1 and RPS20 were cloned into pcDNA3 vector as GFP and Flag tag fusion respectively. GNL1 shRNA (TRCN0000189397, Sigma-Aldrich, USA), RPS20 shRNA (TRCN0000117625, Sigma-Aldrich, USA) were used for knockdown of GNL1 and RPS20 expression.Results and discussionsYeast-two hybrid screening identified Ribosomal protein S20 (RPS20), a component of 40S subunit of ribosome, as a novel interacting partner of GNL1. Higher expression of RPS20 is correlated with poor survival of Glioblastoma and Medulloblastoma patients. Our results reveal that GNL1 positively modulates the RPS20 expression and promotes cell proliferation. Interestingly, GNL1 induced cell proliferation was impaired upon RPS20 knockdown suggesting its critical role in GNL1 function. Depletion of endogenous GNL1 and RPS20 slowed down G1 to S phase transition of cell cycle by reducing the level of hyper-phosphorylated Rb. In addition, knockdown of GNL1 and RPS20 reduced the colony forming ability of colon cancer cell line. Furthermore, the positive correlation of GNL1 and RPS20 expression profiles as well as their inverse correlation with patient survival from online databases indicate the prognostic implications of their interplay.ConclusionThe present study demonstrates that A) RPS20 is a novel interacting partner of GNL1. B) GNL1 promotes cell proliferation by modulating RPS20 levels. C) GNL1 and RPS20 helps in G1 to S cell cycle phase transition by hyper-phosphorylating Rb. Collectively, our data provides evidence that the cross talk between GNL1 and RPS20 might play a critical role in tumorigenesis.