The transcription factor NRF2 (NFE2L2) is a pivotal activator of genes encoding cytoprotective and detoxifying enzymes that limit the action of cytotoxic therapies in cancer. NRF2 acts by binding antioxidant response elements (ARE) in its target genes, but there is relatively limited knowledge about how it is negatively controlled. Here, we report that retinoic X receptor alpha (RXRα) is a hitherto unrecognized repressor of NRF2. RNAi-mediated knockdown of RXRα increased basal ARE-driven gene expression and induction of ARE-driven genes by the NRF2 activator tert-butylhydroquinone (tBHQ). Conversely, overexpression of RXRα decreased ARE-driven gene expression. Biochemical investigations showed that RXRα interacts physically with NRF2 in cancer cells and in murine small intestine and liver tissues. Furthermore, RXRα bound to ARE sequences in the promoters of NRF2-regulated genes. RXRα loading onto AREs was concomitant with the presence of NRF2, supporting the hypothesis that a direct interaction between the two proteins on gene promoters accounts for the antagonism of ARE-driven gene expression. Mutation analyses revealed that interaction between the two transcription factors involves the DNA-binding domain of RXRα and a region comprising amino acids 209-316 in human NRF2 that had not been defined functionally, but that we now designate as the NRF2-ECH homology (Neh) 7 domain. In non–small cell lung cancer cells where NRF2 levels are elevated, RXRα expression downregulated NRF2 and sensitized cells to the cytotoxic effects of therapeutic drugs. In summary, our findings show that RXRα diminishes cytoprotection by NRF2 by binding directly to the newly defined Neh7 domain in NRF2. Cancer Res; 73(10); 3097–108. ©2013 AACR.

In nutrient‐deprived cells autophagy recycles cytoplasmic constituents by engulfing and degrading them in membrane‐bound autophagic vacuoles. The regulation of autophagic vacuole formation is poorly understood, but here we show this process is under strict cell‐cycle control in cultured animal cells. We found strong inhibition of autophagic vacuole accumulation in nocodazole‐arrested pseudo‐prometaphase cells, and also in metaphase and anaphase cells generated on release from the nocodazole arrest. Autophagic vacuoles reappeared after closure of the nuclear envelope in telophase/G1. Treatment with phosphoinositide 3(PI3)‐kinase inhibitors wortmannin, LY294002 and 3‐methyladenine (known to inhibit the autophagic response in interphase cells) rescued autophagy in mitotic cells without inducing reassembly of vesiculated ER and Golgi compartments. The autophagy induced in mitotic cells was inhibited by amino acids, and the resulting autophagosomes contained proteins LC3 and Lamp1, known to be associated with autophagosomes in interphase cells. The mitotic inhibition of autophagy was not relieved by rapamycin treatment or in PDK1–/– embryonic stem cells, by microinjection of inhibitory antibodies against the class III PI3 kinase VPS34, or in cell lines lacking the p85 regulatory subunits of class IA PI3 kinases. Our results show that autophagy is under strict mitotic control and indicate a novel role for phosphoinositide 3‐kinases or other wortmannin/LY294002‐sensitive kinases in mitotic membrane traffic regulation.