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. 2019 Oct 2;11(10):1488.
doi: 10.3390/cancers11101488.

Decoupling of Nrf2 Expression Promotes Mesenchymal State Maintenance in Non-Small Cell Lung Cancer

John A Haley  1 , Christian F Ruiz  2 , Emily D Montal  3 , Daifeng Wang  4 , John D Haley  5 , Geoffrey D Girnun  6
Affiliations

Decoupling of Nrf2 Expression Promotes Mesenchymal State Maintenance in Non-Small Cell Lung Cancer

John A Haley et al. Cancers (Basel). .

Abstract

Epithelial mesenchymal transition is a common mechanism leading to metastatic dissemination and cancer progression. In an effort to better understand this process we found an intersection of Nrf2/NLE2F2 (Nrf2), epithelial mesenchymal transition (EMT), and metabolic alterations using multiple in vitro and in vivo approaches. Nrf2 is a key transcription factor controlling the expression of redox regulators to establish cellular redox homeostasis. Nrf2 has been shown to exert both cancer inhibitory and stimulatory activities. Using multiple isogenic non-small cell lung cancer (NSCLC) cell lines, we observed a reduction of Nrf2 protein and activity in a prometastatic mesenchymal cell state and increased reactive oxygen species. Knockdown of Nrf2 promoted a mesenchymal phenotype and reduced glycolytic, TCA cycle and lipogenic output from both glucose and glutamine in the isogenic cell models; while overexpression of Nrf2 promoted a more epithelial phenotype and metabolic reactivation. In both Nrf2 knockout mice and in NSCLC patient samples, Nrf2low was co-correlated with markedly decreased expression of glycolytic, lipogenic, and mesenchymal RNAs. Conversely, Nrf2high was associated with partial mesenchymal epithelial transition and increased expression of metabolic RNAs. The impact of Nrf2 on epithelial and mesenchymal cancer cell states and metabolic output provide an additional context to Nrf2 function in cancer initiation and progression, with implications for therapeutic inhibition of Nrf2 in cancer treatment.

Keywords: Nrf2; TCA cycle; epithelial mesenchymal transition; glycolysis; lipogenesis; redox signaling.

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Conflict of interest statement

The authors have no conflicts of interest related to the subject material.

Figures

Figure 1
Figure 1
Isogenic mutant KRAS and EGFR model systems show altered metabolic RNA expression and attenuated glycolytic capacity when in a mesenchymal-like cell state. (A) Epithelial tumor cells can trans-differentiate to reversible/metastable and epigenetically ‘fixed’ mesenchymal cell states. (B) NSCLC isogenic mutant KRAS and EGFR models display epithelial or mesenchymal protein markers dependent on state. (C) Plasma membrane localization of E-cadherin (green) is lost in the mesenchymal state. Nuclei are stained with DAPI (blue). (D) RNA abundance characteristics of the four isogenic lung E-and M-state models (log2M/E fold change; bold values indicate FDR adjusted q value < 0.05). Selected metabolic RNA changes associated with M and E cell state. Glycolytic and pentose phosphate pathway M state RNA expression decreases, included phosphofructokinase PFKFB2/3), hexokinase-2(HK2), and glycerol 6-phosphate dehydrogenase (G6PD). TCA cycle IDH1 and OGDH, and lipid synthesis FA2H, FAAH, FAAH2, and ECI1 also were reduced in M state cells. (E) In HCC4006 and A549 labeled with 13C6-glucose, extracellular lactate and glucose levels decrease in M state by GC-MS. Isotopologue distributions are shown in Supplementary Figure S2. (p < 0.05 *; p < 0.01 **; p < 0.001 ***). (F) In HCC4006 and A549 glycolytic capacity decreases in the M state.
Figure 2
Figure 2
Reduced glycolytic and TCA cycle activity in the mesenchymal cell states. (A) In A549 and HCC4006 E and M state cells treated with 13C glucose, there is a decrease in glucose labeled glycolytic and pentose phosphate pathway metabolites in the M state. (p < 0.05 *; p < 0.01 **; p < 0.001 ***). (B) In A549 and HCC4006, basal mitochondrial respiration is reduced in M state cells. (C) In A549 and HCC4006 M state cells treated with 13C6-glucose, there is a decrease in glucose labeled TCA cycle metabolites. (D) In A549 and HCC4006 M state cells treated with 13C5-glutamine, there is a decrease in glutamine labeled TCA cycle metabolites. Isotopologue distributions for 13C6-glucose are shown in Supplementary Figure S2.
Figure 3
Figure 3
De novo lipid synthesis is decreased in isogenic mesenchymal cell states. (A) In HCC4006 and A549 cells, lipogenic enzymes decrease in the M state by immunoblot. (B) SREBP1/SREBF1 RNA is reduced in the M state (p < 0.05 *; p < 0.01 **; p < 0.001 ***). (C) A549 and HCC4006 have decreased incorporation of 13C6-glucose, 13C5-glutamine, and/or 13C2-acetate into palmitate in the M state. They also show decreased acetyl-CoA enrichment by their respective substrates. Isotopologue distributions are shown in Supplementary Figure S3.
Figure 4
Figure 4
Wild type and mutant KEAP1 mesenchymal state cells show attenuated Nrf2 protein and activity. (A) Enrichment of down regulated redox and lipid metabolic genes correlated with M state (DAVID). (B) Reduced abundance of glutathione and NADP+ as measured by LC-MS/MS with log2 fold change M/E (p < 0.05 *; p < 0.01 **; p < 0.001 ***). (C) Down regulation (green) of Nrf2/NFE2L2 target genes in the mesenchymal state supports reduced Nrf2 activation in the mesenchymal state. Nrf2 associated transcription factor MAF was markedly increased (orange), with little/no change in Nrf2 protein stability regulators KEAP1, CUL3, and RBX1 (yellow). (D) Nrf2 levels decrease in the M state at the protein level in all four EMT models. (E) Reduced Nrf2 protein in M state cells correlates with increased ROS as measured by flow cytometry. (F) Both E and M state cells turnover Nrf2 protein via proteasomal degradation. A549 and HCC4006 were treated with proteasome inhibitor MG132 for 6 h followed by immunoblot for Nrf2 and β-actin.
Figure 5
Figure 5
Nrf2 promotes an epithelial phenotype and antagonizes the mesenchymal state. (A) Nrf2 knockdown in A549 and HCC4006 leads to an increase in mesenchymal marker expression and a decrease in epithelial marker expression, as well as a decrease in FASN and increase in pERK by immunoblot. (B) Nrf2 knockdown in A549 and HCC4006 decreases cell proliferation over 72 h (p < 0.05 *; p < 0.01 **; p < 0.001 ***). (C) Nrf2 Knockdown in A549 and HCC4006 causes loss of E-cadherin (red) from the cell membrane by IF, where nuclei are stained with DAPI (blue). (D) When Nrf2 is overexpressed in HCC4006, mesenchymal marker expression decreases (N-cad) while the epithelial marker (E-cad) increases by immunoblot. (E) In HCC4006, treatment with 1, 3, 5 mM NAC over the course of 72 h, a loss of M markers and gain of E markers is seen in the M state.
Figure 6
Figure 6
Knockdown of Nrf2 phenocopies mesenchymal state metabolic alterations. (A) In HCC4006 and A549 with Nrf2 knockdown treated with 13C glucose; production of G6P, PEP, intracellular lactate, and extracellular lactate are reduced (p < 0.05 *; p < 0.01 **; p < 0.001 ***). (B) Nrf2 knockdown cells have increased extracellular glucose. (C) Nrf2 knockdown increases aspartate. (D) With Nrf2 knockdown, incorporation of 13C6-glucose into palmitate is attenuated. Isotopologue distributions are shown in Supplementary Figure S5. (E) Conversely, when Nrf2 is overexpressed in HCC4006, incorporation of 13C6-glucose into palmitate is increased (GFP control vs. Nrf2; p < 0.01).
Figure 7
Figure 7
Mouse Nrf2−/− promotes a mesenchymal phenotype in primary lung alveolar cells. (A) Gene set enrichment of Nrf2 KO RNA expression indicate correlation with multiple EMT signatures. (B) Lung alveolar cell RNA expression comparing Nrf2 knockout vs. control (column 1), is compared with mean log2 TGFβ E and M state changes in the four lung adenocarcinoma cell models (column 2) and mean Zeb1 and Snail1 driven EMT changes in two H358 models. Data are expressed as percent maximum change to scale microarray and RNAseq dynamic ranges. EMT related RNAs are bolded. (C) Correlation of Nrf2 KO RNA expression with EMT RNA changes in the four NSCLC models.
Figure 8
Figure 8
Attenuation of Nrf2-dependent transcription correlates with EMT and metabolic RNA and protein abundance in NSCLC adenocarcinomas. (A) Co-correlation of Nrf2 target RNA expression (anchor genes NQO1, GSR, GPX2, GSTA4, PRDX5, CBR1, GLRX, MGST1, GCLC) with a reference 85 gene EMT signature defined [60]. The TCGA Provisional 503 patient lung adenocarcinoma patient dataset was queried. Nrf2 target RNA expression was positively correlated with an epithelial state. Nrf2 activation correctly correlated with KEAP1 mutation (q = 2.4 ×10−4). (B) Decreased G6PD and FASN metabolic proteins and decreased RAB25 and CLDN7 epithelial proteins, when Nrf2 is inactive (or KEAP1 expression is elevated) and target gene RNAs (e.g., PRDX1) are reduced. (C) Nrf2 signature RNA are coordinately expressed between inactive (signature low) and active (signature high) TCGA specimens. Nrf2 signature RNA are coordinately regulated between inactive (signature low) and active (signature high) TCGA specimens. (D) Model of Nrf2 and metabolic activity in metastatic lung adenocarcinoma where Nrf2 bifurcates into Nrf2/metaboliclow and Nrf2/metabolichigh in mesenchymal and epithelial cell states, respectively.
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