Dual inhibition of REV-ERBβ and autophagy as a novel pharmacological approach to induce cytotoxicity in cancer cells

Abstract

REV-ERBα and REV-ERBβ nuclear receptors regulate several physiological processes, including circadian rhythm and metabolism. A previous study reported the REV-ERBα gene to be co-overexpressed with ERBB2 in breast cancer cell lines. Surprisingly, we found that several tumor types, including a number of breast cancer cell lines, predominantly express the REV-ERBβ variant. This pattern was independent of ERBB2 and ER status, and opposite to that of non-cancer mammary epithelial HMEC cells, in which REV-ERBα was the major variant. Consistent with this molecular profile, REV-ERB target genes in both circadian and metabolic pathways were derepressed upon silencing of REV-ERBβ, but not REV-ERBα. Strikingly, we found that REV-ERBβ is a determinant of sensitivity to chloroquine, a clinically relevant lysosomotropic agent that suppresses autophagy. The cytoprotective function of REV-ERBβ appears to operate downstream of autophagy blockade. Through compound screening, we identified ARN5187, a novel lysosomotropic REV-ERBβ ligand with a dual inhibitory activity toward REV-ERB-mediated transcriptional regulation and autophagy. Remarkably, although ARN5187 and chloroquine share similar lysosomotropic potency and have a similar effect on autophagy inhibition, ARN5187 is significantly more cytotoxic. Collectively, our results reveal that dual inhibition of REV-ERBβ and autophagy is an effective strategy for eliciting cytotoxicity in cancer cells. Furthermore, our discovery of a novel inhibitor compound of both REV-ERB and autophagy may provide a scaffold for the discovery of new multifunctional anticancer agents.

Removal of Rev-erbα inhibition contributes to the prostaglandin G/H synthase 2 expression in rat endometrial stromal cells

“…Recent studies have consistently shown that Rev-erb␣ is a transcriptional repressor recruiting histone deacetylase 3 (HDAC3) and nuclear receptor corepressor (NCoR) (16,57). The synthetic antagonist of Rev-erb␣ or Reverb␤, SR8278, is a useful probe for cellular Rev-erb␣ activity (9,31). Here, we demonstrated that addition of SR8278 activates Ptgs2 transcription and PGE 2 production in UESCs (Fig.…”

“…Cultured UESCs were first plated in 35-mm collagencoated dishes with 2 ml of DMEM-F12 supplemented with 1ϫ AA, 1ϫ ITS, and 0.1% BSA. The medium was removed after 24 h, and the cells were transfected with the RNA oligos diluted in Opti-MEM using Lipofectamine RNAiMAX reagent (Life Technologies) according to the manufacturer's protocol (9). Both the Bmal1 siRNA and nonsilencing RNA were used at a final concentration of 25 nM.…”

Emerging functional roles of nuclear receptors in breast cancer

“…Although circadian regulation requires direct DNA binding of REV-ERBA to its cognate site, metabolic control involves the recruitment of HDAC3, which is tethered by cell type-specific transcription factors . A recent publication suggests that although REV-ERBA has a dominant role in normal cells, REV-ERBB seems to be the dominant isoform controlling both circadian rhythm and metabolic pathways in cancer cells, including breast cancer (De Mei et al 2015). REV-ERBB is significantly more abundant in cancer cells compared to REV-ERBA, whereas the opposite is observed in normal cells.…”

Anti-proliferative actions of a synthetic REV-ERBα/β agonist in breast cancer cells

“…Based on this, we might expect that a REV-ERB antagonist would be beneficial, which is in contrast to our current work indicating that activation of REV-ERB leads to decreased breast cancer cell proliferation in the BT474 cell line as well as a range of other breast cancer cells lines. In a more recent study, suppression of REV-ERBα expression did not alter BT474 cell viability 42 . However, this same study found that although a REVERBβ antagonist did not alter BT474 viability alone, it did enhance the cytotoxicity of the autophagy inhibitor chloroquine 42 .…”

“…We were particularly intrigued by recent studies implicating REV-ERB in regulation of proliferation and cancer 15, 41, 42 . Our data indicate that activation of REV-ERB with a synthetic agonist leads to decreased proliferation of a range of breast cancer cells independent of their ER or HER2 status.…”