Rlip Depletion Alters Oncogene Transcription at Multiple Distinct Regulatory Levels

SIMPLE SUMMARY: Rlip76 is a multifunctional membrane protein that facilitates cancer growth, and its depletion kills cancer cells. We recently found that Rlip depletion also results in broad changes to oncogene and tumor suppressor transcription. The present studies were designed to decipher the unknown downstream signaling pathways and transcriptional regulatory mechanisms driving the effect. Building on prior findings that Rlip depletion induces broad methylomic changes, we found using bioluminescence reporter assays that depletion of Rlip also exerts transcriptional control over several cancer genes through methylation-independent changes in transcription factor-mediated activation of their promoter regions and through additional as yet unidentified mechanisms. These findings have important implications for Rlip-targeted cancer therapy. ABSTRACT: Rlip76 (Rlip) is a multifunctional membrane protein that facilitates the high metabolic rates of cancer cells through the efflux of toxic metabolites and other functions. Rlip inhibition or depletion results in broad-spectrum anti-cancer effects in vitro and in vivo. Rlip depletion effectively suppresses malignancy and causes global reversion of characteristic CpG island methylomic and transcriptomic aberrations in the p53-null mouse model of spontaneous carcinogenesis through incompletely defined signaling and transcriptomic mechanisms. The methylome and transcriptome are normally regulated by the concerted actions of several mechanisms that include chromatin remodeling, promoter methylation, transcription factor interactions, and miRNAs. The present studies investigated the interaction of Rlip depletion or inhibition with the promoter methylation and transcription of selected cancer-related genes identified as being affected by Rlip depletion in our previous studies. We constructed novel promoter CpG island/luciferase reporter plasmids that respond only to CpG methylation and transcription factors. We found that Rlip depletion regulated expression by a transcription factor-based mechanism that functioned independently of promoter CpG methylation, lipid peroxidation, and p53 status.