Abstract 353: ROR1 inhibits ASK1-mediated pro-apoptotic signaling in lung adenocarcinoma
Authors: Ida Lisa , Yamaguchi Tomoya , Kajino Taisuke , Yanagisawa Kiyoshi , Shimada Yukako , Suzuki Motoshi , Takahashi Takashi 
Published: July 2017

Abstract
We previously reported that the receptor tyrosine kinase-like orphan receptor 1 (ROR1) is transcriptionally activated by TTF-1/NKX2-1 lineage-survival oncogene in lung adenocarcinoma and maintains a favorable balance between pro-survival PI3K-AKT and pro-apoptotic ASK1-p38 signaling. Although in-depth mechanistic insight into how ROR1 sustains EGFR-mediated PI3K-AKT signaling in both kinase-dependent and -independent manners has been obtained, it remains elusive how ROR1 inhibits pro-apoptotic signaling. In the present study, we investigated the underlying mechanism of ROR1-mediated inhibition of the ASK1-p38MAPK signaling pathway. Co-treatment with siASK1 resulted in partial but significant alleviation of siROR1-mediated growth inhibition in the lung adenocarcinoma cell lines PC-9 and NCI-H1975, which readily express both ROR1 and ASK1. Significant decreases in auto-phosphorylation of ASK1 at threonine 845 and p38 phosphorylation at threonine 180 and tyrosine 182 were observed in ROR1-transfected MSTO-211H cells in both steady state and oxidative stress-elicited conditions, suggesting ROR1-mediated negative regulation of the ASK1-p38 axis. ROR1 was shown to interact physically with ASK1 through the C-terminal serine/threonine-rich domain of ROR1. An in vitro kinase assay using cellular ASK1 prepared from ASK1-transfected 293T cells as well as recombinant MBP-tagged kinase-deficient MKK6 revealed that co-incubation of ASK1 with recombinant GST-tagged ROR1 clearly diminished ASK1 auto-phosphorylation and MKK6 phosphorylation. We further examined whether the kinase activity of ROR1 is required for inhibition of ASK1 activity using MSTO-211H cells stably transfected with either a wild-type or a kinase-dead mutant of ROR1. In contrast to wild-type ROR1, kinase-dead ROR1 failed to significantly repress hydrogen peroxide-induced ASK1 phosphorylation as well as consequential p38 phosphorylation and increase in sub-G1 cells. Since we previously found that ROR1 activates SRC, we also examined whether SRC is involved in the ROR1-sustained ASK1 inhibition. Introduction of constitutively active SRC significantly reduced siROR1-induced ASK1activity in PC-9 cells, while co-transfection of SRC and ASK1 resulted in tyrosine phosphorylation of ASK1, which was accompanied with diminished ASK1 phosphorylation at threonine 845. Finally, the interaction between SRC and ASK1 was found to be enhanced by the presence of ROR1 by an in vitro pull-down assay. Taken together, the present findings support our notion that ROR1 sustains lung adenocarcinoma survival, at least in part, through direct physical interaction with both ASK1 and SRC, which consequently results in repression of the pro-apoptotic ASK1-p38 axis in a ROR1 kinase activity-dependent manner.
Citation Format: Lisa Ida, Tomoya Yamaguchi, Taisuke Kajino, Kiyoshi Yanagisawa, Yukako Shimada, Motoshi Suzuki, Takashi Takahashi. ROR1 inhibits ASK1-mediated pro-apoptotic signaling in lung adenocarcinoma [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2017; 2017 Apr 1-5; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2017;77(13 Suppl):Abstract nr 353. doi:10.1158/1538-7445.AM2017-353