Hyperactivity of the transcription factor Nrf2 causes metabolic reprogramming in mouse esophagus

Abstract

Edited by Xiao-Fan WangMutations in the genes encoding nuclear factor (erythroidderived 2)-like 2 (NRF2), Kelch-like ECH-associated protein 1 (KEAP1), and cullin 3 (CUL3) are commonly observed in human esophageal squamous cell carcinoma (ESCC) and result in activation of the NRF2 signaling pathway. Moreover, hyperactivity of the transcription factor Nrf2 has been found to cause esophageal hyperproliferation and hyperkeratosis in mice. However, the underlying mechanism is unclear. In this study, we aimed to understand the molecular mechanisms of esophageal hyperproliferation in mice due to hyperactive Nrf2. Esophageal tissues were obtained from genetically modified mice that differed in the status of the Nrf2 gene and genes in the same pathway (Nrf2 ؊/؊ , Keap1 ؊/؊ , K5Cre;Pkm2 fl/fl ;Keap1 ؊/؊ , and WT) and analyzed for metabolomic profiles, Nrf2 ChIP-seq, and gene expression. We found that hyperactive Nrf2 causes metabolic reprogramming and up-regulation of metabolic genes in the mouse esophagus. One of the glycolysis genes encoding pyruvate kinase M2 (Pkm2) was not only differentially up-regulated, but also glycosylated and oligomerized, resulting in increased ATP biosynthesis. However, constitutive knockout of Pkm2 failed to inhibit this esophageal phenotype in vivo, and this failure may have been due to compensation by Pkm1 up-regulation. Transient inhibition of NRF2 or glycolysis inhibited the growth of human ESCC cells in which NRF2 is hyperactive in vitro. In summary, hyperactive Nrf2 causes metabolic reprogramming in the mouse esophagus through its transcriptional regulation of metabolic genes. Blocking glycolysis transiently inhibits cell proliferation and may therefore have therapeutically beneficial effects on NRF2 high ESCC in humans.Esophageal cancer affects 16,940 adults in the United States, and the 5-year survival rate is 18% (1). There are mainly two histological types of esophageal cancer, squamous cell carcinoma (ESCC) 4 and adenocarcinoma, each having a distinct etiology. Low income, moderate/heavy alcohol intake, tobacco use, and infrequent consumption of raw fruits and vegetables account for almost all cases of ESCC (2). With the recent technological advances in NextGen sequencing, human ESCC samples from North and South America, China, Japan, Vietnam, and Malawi have been sequenced. Among many gene mutations, nuclear factor (erythroid-derived 2)-like 2 (NRF2 or NFE2L2) mutations are commonly seen with a frequency over 5%, even up to ϳ20% in certain reports. Mutations in other genes of the NRF2 signaling pathway, Kelch-like ECH associated protein 1 (KEAP1) and cullin 3 (CUL3), are relatively less common. NRF2 mutations are mostly located in the DLG and ETGE motifs (KEAP1-binding domain) and the DNA-binding domain, whereas KEAP1 mutations tend to be scattered across the whole gene. NRF2 mutations and KEAP1 mutations tend to be mutually exclusive (3,4).As a major cellular defense mechanism, the NRF2 signaling pathway is known to regulate expression of enzymes involved in detoxification and ant...

NRF2, a Transcription Factor for Stress Response and Beyond

“…A plethora of evidence supports a key role for NRF2 in the cancer metabolism reprogramming [23]. Most of the evidence came from the Chromatin Immunoprecipitation Sequencing (ChIP-Seq) analysis of NRF2 target genes in cell lines [35,49,50] or in mouse tissues [77] where NRF2 is either activated or deleted to compare with the wild type cells. Those studies identified numerous NRF2 target genes that regulate glycolysis, pentose phosphate pathway (PPP), fatty acid metabolism, glutamine metabolism, and glutathione metabolism (Figure 3).…”

“…Constitutive activation of NRF2 has been observed in many human cancers that have poor prognoses and NRF2 contributes to aerobic glycolysis to produce anabolic precursors for the building blocks of tumor growth with much less efficient energy production, a state known as the Warburg effect [23]. NRF2 induces expression of glucose transporter GLUT1 that allows increased glucose import into glycolytic flux [77]. Among the glycolysis pathway, NRF2 induces the expression of several key glycolytic enzymes, including hexokinase 1 and 2 (HK1/2), glucose phosphate isomerase 1 (GPI1), 6-phosphofructo-2-kinase (PFK2), PFK4, fructose-bisphosphate aldolase A (ALDA), enolase 1 (ENO1), ENO4, pyruvate kinase muscle isoform 2 (PKM2) to increase glycolytic flow and maintain pool sizes of glycolytic intermediates for anabolic reactions [77].…”

Potential Applications of NRF2 Modulators in Cancer Therapy

“…Intriguingly, NRF2 can control different steps of glutamine fate, from its uptake to its metabolism. For example, early studies reported that NRF2 can induce the expression of the glutamine importer SLC1A5 in HeLa cells through the ATF4 transcription factor [256] while a recent ChIP-Seq analysis on KEAP1-/mice and human ESCC cells, revealed that NRF2 causes metabolic reprogramming by enhancing the expression of the SLC1A4 glutamine transporter and other metabolic enzymes [257]. Lastly, the enzyme glutaminase, catalyzing the conversion of glutamine into glutamate, was found to be a direct NRF2 target gene in MCF7 and MCF10 BC cells treated with Sulforaphane or subdued to KEAP1 KD by siRNA [258].…”

Cell-Type Specific Metabolic Response of Cancer Cells to Curcumin

“…Fu et al reported that hyperactivation of NRF2 (Nuclear Factor (Erythroid-Derived 2)-Like 2), a major transcription regulator of enzymes involved in antioxidative stress response, causes upregulation of Pkm2, its glycosylation and a dramatic increase in its tetrameric form in mice esophagus. High expression of Pkm2 tetramers was accompanied by overexpression of genes involved in glycolysis, pentose phosphate pathway (PPP) and glutathione (GSH) metabolism, which is highly dependent on serine [10]. In addition to its importance for maintaining cellular redox homeostasis, the serine biosynthetic pathway is an important alternative source of pyruvate in PKM1/PKM2-deficient cells.…”