Identification of a Stable, Non-Canonically Regulated Nrf2 Form in Lung Cancer Cells

Abstract

Nrf2 (nuclear factor erythroid 2 (NF-E2)-related factor 2) transcription factor is recognized for its pro-survival and cell protective role upon exposure to oxidative, chemical, or metabolic stresses. Nrf2 controls a number of cellular processes such as proliferation, differentiation, apoptosis, autophagy, lipid synthesis, and metabolism and glucose metabolism and is a target of activation in chronic diseases like diabetes, neurodegenerative, and inflammatory diseases. The dark side of Nrf2 is revealed when its regulation is imbalanced (e.g., via oncogene activation or mutations) and under such conditions constitutively active Nrf2 promotes cancerogenesis, metastasis, and radio- and chemoresistance. When there is no stress, Nrf2 is instantly degraded via Keap1-Cullin 3 (Cul3) pathway but despite this, cells exhibit a basal activation of Nrf2 target genes. It is yet not clear how Nrf2 maintains the expression of its targets under homeostatic conditions. Here, we found a stable 105 kDa Nrf2 form that is resistant to Keap1-Cul3-mediated degradation and translocates to the nucleus of lung cancer cells. RNA-Seq analysis indicate that it might originate from the exon 2 or exon 3-truncated transcripts. This stable 105 kDa Nrf2 form might help explain the constitutive activity of Nrf2 under normal cellular conditions.

Keap1-resistant ΔN-Nrf2 isoform does not translocate to the nucleus upon electrophilic stress

“…Western blot analysis was performed as described previously (35). Primary antibodies that were used are anti-NRF2 [EP1808Y] – ChIP Grade (cat.…”

New Discoveries and Ambiguities of Nrf2 and ATF3 Signaling in Environmental Arsenic-Induced Carcinogenesis

“…Therefore, skipping exon 2 and/or exon3 will generate a gain-of-functional Nrf2 protein that abolishes Keap1 binding and the subsequent Cullin3-E3 ligase-mediated ubiquitination and degradation of the Nrf2 protein by the proteasome. Most recently, studies by Mikac et al [ 47 ] noted a Nrf2 transcript with partial loss of exon 2 resulted from the use of an in-frame splicing site in the 3′-terminus of exon 2 in A549 cells. This alternative splicing also generates a Nrf2 protein lacking Keap1 binding motifs, leading to stabilization of the Nrf2 protein.…”

“…Similarly, Kemmerer et al [ 48 ] compared anti-Nrf2 antibodies D1Z9C (Cell Signaling Rabbit mAb #12721, recognizing Nrf2 peptides surrounding Ala275) with EP1808Y (Abcam62352, Rabbit mAb, recognizing Nrf2 peptide from amino acid 550 to C-termus), SC-C20 and H300, and found D1Z9C has the highest specificity to Nrf2 protein in immunoblotting. In A549 cells and squamous cell carcinoma RERF-LC-AI cells, both D1Z9C and EP1808Y can detect Nrf2 proteins with MW of 130 and 105 kDa [ 47 ]. The 130 kDa Nrf2 appears to be a phosphorylated form of Nrf2 that is sensitive to ubiquitination and degradation mediated by Keap1-Cullin3.…”

“…As an additional measure to evaluate the accuracy of anti-Nrf2 antibodies, we also performed a ChIP-seq analysis using antibody EP1808Y (Abcam62352), which showed similar specificity of the CS-D1Z9C as investigated by Mikac et al [ 47 ], and antibody AB_2793695 (Active Motif cat#61599), which detected a 95 kDa protein band as indicated by the datasheet. Two ChIP reactions were carried out using 30 μg of proteins from the arsenic-treated BEAS-2B cells.…”

Editorial on Anticancer Antioxidants

“…They observed that, upon ATX treatment, functional mediators of immune suppression were significantly reduced, while the antioxidant activity of ATX reduced oxidative stress in MDSCs, which became immunogenic enough to induce cytotoxic T lymphocyte response and an inhibition of tumor growth. The signaling pathway of Nrf2 was also the focus of research by an international European team analyzing how Nrf2 maintains the expression of its targets under homeostatic conditions in lung cancer cells [ 13 ]. They described the sTable 105 kDa Nrf2 form, which is resistant to Keap1-Cul3-mediated degradation and translocates to the nucleus of lung cancer cells.…”