ERN1

Abstract: Maintenance of endoplasmic reticulum (ER) function is achieved in part through Ire1 (inositol-requiring enzyme 1), a transmembrane protein activated by protein misfolding in the ER. The cytoplasmic nuclease domain of Ire1 cleaves the messenger RNA (mRNA) encoding XBP-1 (X-box-binding protein 1), enabling splicing and production of this active transcription factor. We recently showed that Ire1 activation independently induces the rapid turnover of mRNAs encoding membrane and secreted proteins in Drosophila melanogaster cells through a pathway we call regulated Ire1-dependent decay (RIDD). In this study, we show that mouse fibroblasts expressing wild-type Ire1 but not an Ire1 variant lacking nuclease activity also degrade mRNAs in response to ER stress. Using a second variant of Ire1 that is activated by a small adenosine triphosphate analogue, we show that although XBP-1 splicing can be artificially induced in the absence of ER stress, RIDD appears to require both Ire1 activity and ER stress. Our data suggest that cells use a multitiered mechanism by which different conditions in the ER lead to distinct outputs from Ire1.

Abstract: Tumor cell adaptation to hypoxic stress is an important determinant of malignant progression. While much emphasis has been placed on the role of HIF-1 in this context, the role of additional mechanisms has not been adequately explored. Here we demonstrate that cells cultured under hypoxic/anoxic conditions and transformed cells in hypoxic areas of tumors activate a translational control program known as the integrated stress response (ISR), which adapts cells to endoplasmic reticulum (ER) stress. Inactivation of ISR signaling by mutations in the ER kinase PERK and the translation initiation factor eIF2α or by a dominant-negative PERK impairs cell survival under extreme hypoxia. Tumors derived from these mutant cell lines are smaller and exhibit higher levels of apoptosis in hypoxic areas compared to tumors with an intact ISR. Moreover, expression of the ISR targets ATF4 and CHOP was noted in hypoxic areas of human tumor biopsy samples. Collectively, these findings demonstrate that activation of the ISR is required for tumor cell adaptation to hypoxia, and suggest that this pathway is an attractive target for antitumor modalities.

Abstract: Hypoxia within solid tumors is a major determinant of outcome after anticancer therapy. Analysis of gene expression changes during hypoxia indicated that unfolded protein response genes were one of the most robustly induced groups of genes. In this study, we investigated the hypoxic regulation of X-box binding protein (XBP1), a major transcriptional regulator of the unfolded protein response. Hypoxia induced XBP1 at the transcriptional level and activated splicing of its mRNA, resulting in increased levels of activated XBP1 protein. After exposure to hypoxia, apoptosis increased and clonogenic survival decreased in XBP1-deficient cells. Loss of XBP1 severely inhibited tumor growth due to a reduced capacity for these transplanted tumor cells to survive in a hypoxic microenvironment. Taken together, these studies directly implicate XBP1 as an essential survival factor for hypoxic stress and tumor growth.