The inositol-requiring enzyme 1 (IRE1) endoplasmic reticulum stress pathway promotes MDA-MB-231 cell survival and renewal in response to the aryl-ureido fatty acid CTU

Current treatment options for triple -negative breast cancer (TNBC) are limited to toxic drug combinations of low efficacy. We recently identified an aryl -substituted fatty acid analogue, termed CTU, that effectively killed TNBC cells in vitro and in mouse xenograft models in vivo without producing toxicity. However, there was a residual cell population that survived treatment. The present study evaluated the mechanisms that underlie survival and renewal in CTU-treated MDA-MB-231 TNBC cells. RNA-seq profiling identified several pro -inflammatory signaling pathways that were activated in treated cells. Increased expression of cyclooxygenase-2 and the cytokines IL -6, IL -8 and GM-CSF was confirmed by real-time RT-PCR, ELISA and Western blot analysis. Increased self -renewal was confirmed using the non -adherent, in vitro colony -forming mammosphere assay. Neutralizing antibodies to IL -6, IL -8 and GM-CSF, as well as cyclooxygenase-2 inhibition suppressed the self -renewal of MDAMB-231 cells post-CTU treatment. IPA network analysis identified major NF- kappa B and XBP1 gene networks that were activated by CTU; chemical inhibitors of these pathways and esiRNA knock -down decreased the production of pro -inflammatory mediators. NF- kappa B and XBP1 signaling was in turn activated by the endoplasmic reticulum (ER) -stress sensor inositol-requiring enzyme 1 (IRE1), which mediates the unfolded protein response. Cotreatment with an inhibitor of IRE1 kinase and RNase activities, decreased phospho-NF- kappa B and XBP1s expression and the production of pro -inflammatory mediators. Further, IRE1 inhibition also enhanced apoptotic cell death and prevented the activation of self -renewal by CTU. Taken together, the present findings indicate that the IRE1 ER -stress pathway is activated by the anti -cancer lipid analogue CTU, which then activates secondary selfrenewal in TNBC cells.