Identifying Non-canonical, Keap1-independent Regulators of Nrf2 Degradation in Non-small Cell Lung Cancer

Abstract

Nrf2 (nuclear factor erythroid 2-related factor 2) is a transcription factor responsible for cellular antioxidant response to oxidative and electrophilic stress. Keap1 (Kelch ECH associating protein 1) has been demonstrated to promote the proteasomal degradation of Nrf2 by functioning as a substrate adaptor protein for the E3 ligase Cul 3 (Cullin 3). However, recent evidence suggests that additional factors are at play in the regulation of Nrf2. Predictably, inhibition of Keap1 results in the stabilization of Nrf2. However, Nrf2 is still rapidly degraded after Keap1 knockout or inhibition. The persistently short Nrf2 half-life in the absence of Keap1 provides strong evidence that Keap1 is not the only negative regulator of Nrf2. Thus, with the aim of identifying novel regulators of Nrf2, a siRNA screen targeting members of the ubiquitome was performed using a luciferase complementation assay. The screen identified known complex members involved in Cul3-mediated degradation of Nrf2, as well as novel hits. Data mining and follow up efforts proposed six potential novel Nrf2 regulators. Further validation revealed Sart1/Vhl as strong candidates for Nrf2 regulation; knockdown of Sart1/Vhl activated a Nrf2-MafK luciferase complementation assay, indicating stabilization of Nrf2. Sart1/Vhl knockdown also demonstrated activity in Nrf2 transcriptional target reporters, SRXN1, OSGIN1 and antioxidant response elements (ARE). Sart1/Vhl are documented Hif-1α regulators; more studies are needed to elucidate the mechanism of action, and the potential role of Hif-1α in Nrf2 regulation.