Abstract B150: Toyocamycin inhibits ER stress-induced XBP1 mRNA splicing through IRE1α activation

Abstract

Abstract Poorly vascularized solid-tumor cells encounter a range of cytotoxic conditions such as hypoxia, nutrient deprivation and pH changes. These cytotoxic conditions in solid tumor are considered to cause ER stress. There is now abundant evidence to suggest that ER stress response is activated in various tumors to protect tumor cells from cytotoxic condition-induced apoptosis. XBP1 is considered to be a critical transcription factor of this process and required for tumor survival. Therefore, small molecule inhibitors of XBP1 activation would provide a new type of therapeutic strategy for cancer treatment. In this study, we screened small molecule inhibitors of ER stress-induced XBP1 activation from culture broth of microorganisms and we identified toyocamycin (H, Nishimura et al.: J. Antibiot., 9, 60 (1956)). Thus, we investigated the mechanism by which toyocamycin inhibited ER stress-induced XBP1 activation. XBP1 is activated by IRE1α-mediated unconventional splicing of XBP1 mRNA, which resulted in creating a translational frame shift to produce an active XBP1 transcription factor. In HeLa cells, toyocamycin inhibited the transcriptional activity of XBP1, since toyocamycin suppressed tunicamycin or 2-deoxy-glucose, a well known ER stressor, -induced GRP78, EDEM, and ERdj4 expression that are known to be transcriptionaly regulated by active XBP1. Furthermore, toyocamycin inhibited ER stress-induced endogenous XBP1 mRNA splicing evaluated by RT-PCR. Although toyocamycin is known as RNA synthesis inhibitor, toyocamycin could not decrease the expression level of XBP1 mRNA. Thus, the inhibitory activity of toyocamycin against XBP1 activation is not due to the RNA synthesis inhibition. On the other hand, toyocamycin was unable to suppress IRE1α-mediated XBP1 mRNA cleavage in vitro. Therefore, toyocamycin is strongly suggested to inhibit ER stress-induced IRE1 activation. Finally, we examined the effect of toyocamycin on cell viability under ER stress condition, because XBP1 inhibitor is considered to suppress ER stress-induced cytoprotective effect thereby inducing selective killing of stressed cells. Although toyocamycin had only a weak activity to induce cell death in HeLa cells, toyocamycin synergistically induced cell death when toyocamycin was incubated with 2-deoxy-glucose or tunicamycin. These results indicated that toyocamycin selectively induces cell death under ER stress condition. Taken together, our results suggested that XBP1 is a potent molecular target for developing anti-cancer drug. Citation Information: Mol Cancer Ther 2009;8(12 Suppl):B150.