Abstract C284: IRE1 and PERK as targets of cellular adaptation and survival to hypoxia.
Authors: Cojocari Dan , Vellanki Ravi , Sit Brandon , Koritzinsky Marianne , Wouters Bradly G. 
Published: August 2014

Abstract
Hypoxia is an important and unique feature of the solid tumour microenvironment. Hypoxia promotes resistance to radiotherapy, aggressive disease phenotypes, and poor patient prognosis. Consequently, therapies directed against hypoxic cells are needed to improve patient outcome. Hypoxic stress leads to the activation of several adaptive pathways including the unfolded protein response (UPR). The UPR is activated in response to hypoxia-induced stress in the endoplasmic reticulum (ER) and consists of three distinct signaling pathways initiated by the ER-membrane resident proteins PERK, IRE1 and ATF6. Previous work using genetic models has implicated the PERK pathway, as well as downstream components of the IRE1 pathway (XBP1) as important mediators of hypoxia tolerance and tumor response to therapy. Here we employed short hairpin RNAs (shRNAs) and recently developed small-molecule inhibitors of IRE1 (4μ8c) and PERK (GSK-compound 39) to investigate the relative importance of these two pathways in promoting hypoxia tolerance and their potential as therapeutic targets. We tested the pathway activity of PERK and IRE1 by quantitative PCR and western blotting. Hypoxia tolerance was measured by in vitro clonogenic assays. First, knockdown experiments using two different shRNAs showed that IRE1 is essential for survival of HCT116 colorectal cancer cells during mild hypoxia while PERK was essential in both normoxia and hypoxia. Interestingly, the IRE1 knockdown cells had a significantly altered glycolytic metabolism, a known mechanism of hypoxia adaptation. Using our small-molecule IRE1 inhibitor, we show that 4μ8c is a potent and non-toxic inhibitor of IRE1 RNase activation in response to both hypoxia and other ER stress-inducing agents. This compound effectively inhibited IRE1 induced activation of the downstream target genes in both HCT116 colorectal cancer and KP4 pancreatic cancer cell lines under hypoxia. However, despite potent inhibition of IRE1 activation and the, 4μ8c had no effect on cell proliferation or clonogenic survival of HCT116 and KP4 cells during exposure to hypoxia or anoxia. Similarly, 4μ8c inhibition of IRE1 did not sensitize cells to other ER stress inducing agents. In contrast, genetic inactivation of PERK signaling or treatment with the highly selective and potent PERK inhibitor GSK-compound 39 substantially reduced tolerance to hypoxia, anoxia and other ER stress inducing agents. Our results demonstrate that although hypoxia potently activates both PERK and IRE1 arms of the UPR pathway, the PERK arm is uniquely important for promoting adaptation and survival during hypoxia-induced ER stress. Furthermore, our results suggest that newly developed and orally available PERK inhibitors such as GSK-compound 39 are selectively toxic to hypoxic cells, and thus warrant further development for use in combination therapies with radiation.
Citation Information: Mol Cancer Ther 2013;12(11 Suppl):C284.
Citation Format: Dan Cojocari, Ravi Vellanki, Brandon Sit, Marianne Koritzinsky, Bradly G. Wouters. IRE1 and PERK as targets of cellular adaptation and survival to hypoxia. [abstract]. In: Proceedings of the AACR-NCI-EORTC International Conference: Molecular Targets and Cancer Therapeutics; 2013 Oct 19-23; Boston, MA. Philadelphia (PA): AACR; Mol Cancer Ther 2013;12(11 Suppl):Abstract nr C284.