RelB silencing to reverse tamoxifen resistance by regulating GPx4 and ferroptosis in breast cancer.

Abstract

e12513 Background: Tamoxifen(Tam), as an essential therapeutic treatment of estrogen receptor(ER)-positive breast cancer(BCa), has been available for the past three decades. However, the induction of Tam resistance during therapy has indicated a significant challenge with regards to this agent. Tam could increase oxidative stress and induce cell death by regulating reactive oxygen species(ROS). Ferroptosis, a cell death process driven by the accumulation of iron-dependent lipid peroxides, has been induced by inactivation/depletion of glutathione peroxidases(GPxs). Our previous studies found that the expression level of RelB gene, a member of NF-κB family, is negatively correlated with ER targeted by Tam in BCa. Methods: The RelB level of BCa tumor tissues and the corresponding cell lines were examined by immunoblotting and western blot. The effects of Tam on cell viability were determined using colony survival and MTT assay. The ROS and oxygen consumption rates(OCR) were measured using specific ROS detection probes and a Seahorse XF96 Analyzer, respectively. The lipid peroxidation level of cells was analyzed by immunofluorescence assay. The morphological changes of mitochondria were observed by transmission electron microscope. RelB binding to the NF-κB intronic enhancer region of the human GPx4 gene was determined using a ChIP assay. Accordingly, the effect of RelB on BCa Tam resistance was further validated using BCa mice xenograft models. Results: RelB was uniquely expressed at the high level in Tam resistance BCa tissues and cell lines. Down-regulation of RelB based on a CRISPR/Cas9 system remarkably sensitized resistance BCa cells to Tam. Treatment with SN52, a RelB inhibitor, illuminated the role of RelB in Tam-treated BCa cells. The high level of ROS and declination of mitochondrial respiration which induced by Tam were inhibited in resistance cells. Tam enhanced lipid peroxidation with concomitant non-apoptotic cell death, which are negatively regulated by GPx4 activity. In addition to GPx4 knockdown, deferoxamine was able to rescue Tam-induced cell death in BCa cells, verifying that Tam induces cell death partially through ferroptosis. Importantly, RelB upregulates GPx4 expression through binding to an NF-κB enhancer element located at the 5’-flanking region. Consistently, in vivo functional validation confirmed that RelB inhibition not only impairs tumor growth, but also inhibits Tam resistance in nude mice. Conclusions: RelB could inhibit ferroptosis which induced by hydroxyl radicals accumulation through upregulating GPx4 in BCa.