Three types of transmembrane protein, IRE1/IRE1, PERK, and ATF6/ATF6 are expressed ubiquitously in vertebrates as transducers of the unfolded protein response (UPR), which maintains the homeostasis of the endoplasmic reticulum. IRE1 is highly conserved from yeast to mammals, and transmits a signal by a unique mechanism, namely splicing of mRNA encoding XBP1, the transcription factor downstream of IRE1 in metazoans. IRE1 contains a ribonuclease domain in its cytoplasmic region which initiates splicing reaction by direct cleavage of XBP1 mRNA at the two stem loop structures. As the UPR is considered to be involved in the development and progression of various diseases, as well as in the survival and growth of tumor cells, UPR inhibitors have been sought. To date, IRE1 inhibitors have been screened using cell-based reporter assays and fluorescent-based in vitro cleavage assays. Here, we used medaka fish to develop an in vivo assay for IRE1 inhibitors. IRE1, IRE1, ATF6 and ATF6 are ubiquitously expressed in medaka. We found that IRE1/ATF6-double knockout is lethal, similarly to IRE1/IRE1-and ATF6/ATF6-double knockout. Therefore, IRE1 inhibitors are expected to confer lethality to ATF6-knockout medaka but not to wild-type medaka. One compound named K114 was obtained from 1,280 compounds using this phenotypic screening. K114 inhibited ER stress-induced splicing of XBP1 mRNA as well as reporter luciferase expression in HCT116 cells derived from human colorectal carcinoma, and inhibited ribonuclease activity of human IRE1 in vitro. Thus, this phenotypic assay can be used as a quick test for the efficacy of IRE1 inhibitors in vivo.
To survive poor nutritional conditions, tumor cells activate the unfolded protein response, which is composed of the IRE1, PERK and ATF6 arms, to maintain the homeostasis of the endoplasmic reticulum, where secretory and transmembrane proteins destined for the secretory pathway gain their correct three dimensional structure. The requirement of the IRE1 and PERK arms for tumor growth in nude mice is established. Here, we investigated the requirement for the ATF6 arm, which consists of ubiquitously expressed ATF6α and ATF6β, by constructing ATF6α-knockout, ATF6β-knockout and ATF6α/β-double knockout in HCT116 cells derived from human colorectal carcinoma. Results showed that these knockout cells grew similarly to wild-type cells in nude mice, contrary to expectations from our analysis of ATF6α-knockout, ATF6β-knockout and ATF6α/β-double knockout mice. We then found that the loss of ATF6α in HCT116 cells resulted in sustained activation of the IRE1 and PERK arms, in marked contrast to mouse embryonic fibroblasts, in which the loss of ATF6α is compensated for by ATF6β. Although IRE1-knockout in HCT116 cells unexpectedly did not affect tumor growth in nude mice, IRE1-knockout HCT116 cells with ATF6α knockdown grew significantly more slowly than wild-type or IRE1-knockout HCT116 cells. These results have unraveled the situation-dependent differential compensation strategies of ATF6α.
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