Abstract A18: Geranylgeranoic acid (GGA) induced autophagic cell death through induction of unfolded protein response (UPR) and suppression of mTOR signaling pathway

Abstract

Abstract Background and Aim: Geranylgeranoic acid (GGA) is a natural polyprenoic acid that is expected to prevent a second primary hepatoma. In human hepatoma-derived HuH-7 cells, GGA induces cell death as well as the initial phase of autophagy, but blocks the maturation process of autolysosomes, which may be linked to cell death. However we have not yet revealed the molecular mechanism of GGA-induced initial autophagy. In this meeting, we present a possible mechanism of GGA-induced initial autophagy, which may involve mTOR signaling pathway and unfolded protein response (UPR). Methods: To analyze the cellular process of autophagy, we observed multicolor fluorescent puncta by live-cell imaging in HuH-7/mRFP-GFP-LC3 cells after GGA (10 μM) treatment. We also examined cellular protein levels of phospho-mTOR (Ser2448 or Ser2481), mTOR, Raptor, Rictor, phospho-AKT, Beclin1, LC3β and FoxO1 by western blotting. The cellular levels of XBP1 mRNA splicing, CHOP and PDIA4 mRNAs were measured by RT-qPCR to estimate UPR. Results: In HuH-7 cells, phospho-mTOR (Ser2448) protein expression level was high under normal culture condition. GGA treatment further upregulated phospho-mTOR (Ser2448) and induced a transient surge of phospho-mTOR (Ser2481) at 30 min. However from 1 h to 8 h after GGA treatment, phosphorylation of mTOR was suppressed down to sub-basal levels. The similar up-and-down movements were observed also in the cellular levels of mTOR, Raptor and Rictor, and at 4 h these proteins became undetectable. However phospho-AKT levels did not show significant change after GGA treatment. Rapid and dramatic induction of XBP1 splicing was observed after GGA treatment. In other words, after 15 min of GGA treatment, the upregulation of XBP1s mRNA (spliced form) was observed and the upregulation continued until 4 h. In contrast, XBP1u mRNA (unspliced form) was downregulated in a time dependent manner until 4 h. However at 24 h, these changes were reversed and XBP1u became dominant. CHOP mRNA was upregulated after 1 h GGA treatment and the upregulation continued to 4 h. The cellular level of PDIA4 mRNA was not changed by GGA. The cellular protein levels of Beclin1 and FoxO1 were upregulated by GGA treatment. The accumulations of LC3β-II and autophagosome were also induced with GGA. GGA-induced accumulation of LC3β-II was partially attenuated by co-treatment with 4μ8C, a specific inhibitor of IRE1 ribonuclease activity. Discussion: In the literature, it has been repeatedly reported that signaling pathway of mTOR is activated at a basal level in HuH-7 cells. Therefore, we were interested what would happen on mTOR signaling when GGA induced initial autophagy in HuH-7 cells. As a result, signaling pathway of mTOR, either mTORC1 or mTORC2, was unexpectedly further upregulated by GGA treatment, but this upregulation at 30 min was transient. After 1-h treatment, GGA induced rapid unfolded protein response (UPR), which may trigger suppression of mTOR signaling. Hence, we speculate that UPR-mediated suppression of mTOR signaling may provide permissive effects on GGA-induced initial autophagy. Citation Format: Chieko Iwao, Yoshihiro Shidoji. Geranylgeranoic acid (GGA) induced autophagic cell death through induction of unfolded protein response (UPR) and suppression of mTOR signaling pathway. [abstract]. In: Proceedings of the AACR Special Conference: Targeting the PI3K-mTOR Network in Cancer; Sep 14-17, 2014; Philadelphia, PA. Philadelphia (PA): AACR; Mol Cancer Ther 2015;14(7 Suppl):Abstract nr A18.