NRF2 regulates serine biosynthesis in non-small cell lung cancer

Abstract

Tumors have high energetic and anabolic needs for rapid cell growth and proliferation, and the serine biosynthetic pathway was recently identified as an important source of metabolic intermediates for these processes. We integrated metabolic tracing and transcriptional profiling of a large panel of non-small cell lung cancer (NSCLC) cell lines to characterize the activity and regulation of the serine/glycine biosynthetic pathway in NSCLC. Here we show that the activity of this pathway is highly heterogeneous and is regulated by NRF2, a transcription factor frequently deregulated in NSCLC. We found that NRF2 controls the expression of the key serine/glycine biosynthesis enzyme genes PHGDH, PSAT1 and SHMT2 via ATF4 to support glutathione and nucleotide production. Moreover, we show that expression of these genes confers poor prognosis in human NSCLC. Thus, a substantial fraction of human NSCLCs activates an NRF2-dependent transcriptional program that regulates serine and glycine metabolism and is linked to clinical aggressiveness.

Figure 1

Serine biosynthesis activity in lung cancer. (a) Synthesis of serine and glycine from glucose. Glycolytic metabolism of uniformly labelled ¹³C-glucose (U-¹³C-glucose) produces 3-phosphoglycerate (3-PG) labelled on all 3 carbons (M3). The serine biosynthetic pathway produces serine (M3, 3 carbons labelled), and subsequently glycine (M2, 2 carbons labelled) from 3-PG. Unlabelled (M0) serine and glycine are contributed from the cellular media. (b, c) Fraction of labelled intracellular serine (b) and glycine (c) at 6 and 24 hours in NSCLC cell lines. Cell lines are ordered according to serine M3 labelling at 6 hours. (d, e) Correlation between serine and glycine labelling at 6 (d) and 24 (e) hours. R=Pearson correlation coefficient. p-values were calculated by Student’s t-distribution with n–2 degrees of freedom. (f–g) Serine 'high' cells (serine M3 Z-score > 0.5 at 24 hours) are resistant to serine (f) or serine/glycine (g) starvation for 3 days. Each data point represents a cell line. (h) Serine labelling at 24 hours correlates with PHGDH mRNA expression. mRNA expression data was obtained from the CCLE. Each data point represents a cell line. (i) Serine ‘high’ cell lines demonstrate sensitivity to PHGDH knockdown that is not rescued by serine. Relative cell number was quantified 5 days after PHGDH knockdown. Each data point represents a cell line. (j) H1975 cells expressing luciferase (LUC) or PHGDH were assayed as in (f–g). Results are the average of 3 biological replicates. Error bars represent SEM here and for all figures.

Figure 2

NRF2 regulates serine biosynthesis. (a) Nuclear NRF2 protein expression correlates with ¹³C-serine M3 labelling at 24 hours. Nuclear NRF2 protein expression data is found in Supplementary Fig. 7. (b) Cell lines with high NRF2 activity have significantly higher ¹³C serine M3 labelling at 6 and 24 hours. Cell lines were grouped according to the NRF2 score into “high” (>1.4) and “low” (<1.4). (c) NRF2 high cell lines have significantly higher ¹³C glycine M2 labelling at 24 hours. (b,c) p-values were calculated using an unpaired, two-tailed student’s t-test. (d) mRNA expression in A549 cells expressing scramble shRNA (SCR) or NRF2 shRNA #1. Decreased NQO1 expression confirmed that NRF2 activity was reduced upon knockdown. Results are the average of 3 technical replicates. (e) Western blot of serine pathway enzyme expression in lysates from A549 cells expressing scramble (SCR), or NRF2 shRNAs #1 or #2. (f) Cell lines were grown in the presence of U-¹³C-glucose for the indicated time points, the metabolites extracted and the fractional ¹³C-labelling on serine analysed by LC/MS. Results are the average of 3 biological replicates.

Figure 3

NRF2 regulates the expression of serine/glycine biosynthesis genes through ATF4. (a) ATF4 mRNA expression in A549 cells expressing scramble shRNA (SCR), or NRF2 shRNA #1. (b) Western blot of NRF2, ATF4 and ACTIN expression in cells from (a). (c) Western blot of NRF2, ATF4 and serine pathway enzyme expression in lysates from A549s expressing scramble (SCR), NRF2 shRNA #1, or ATF4 shRNAs #1 or #2. (d) mRNA expression in cells from (c). (e) ATF4 knockdown impairs serine biosynthesis. Cell lines from were grown in the presence of U-¹³C-glucose for the indicated time points, the metabolites extracted and the fractional ¹³C-labeling on serine analysed by LC/MS. (f) ATF4 rescues serine biosynthesis enzyme expression following NRF2 knockdown. A549 cells were infected with lentivirus encoding mATF4 prior to infection with scramble or NRF2-targeting lentivirus. (g) Western analysis of NRF2, ATF4, and ACTIN expression in the cells from (f). (h) ATF4 rescues the serine biosynthesis defect in shNRF2 A549 cells. Cells were assayed as in (e). (i) ATF4 rescues the growth of H1975 cells in serine deficient media. Cells expressing luciferase (LUC) or ATF4 were grown in the indicated media for 3 days and cell number normalized to cells grown in full media. (j) Chromatin immunoprecipitation of ATF4 to the PHGDH, PSAT1 and SHMT2 promoters. Samples were normalized to IgG control immunoprecipitations. Results are the average of 3 technical (a, d, f, j) or biological (e, h, i) replicates.

Figure 4

PHGDH-derived serine supports the transsulfuration and folate cycles. (a) Serine metabolism via the transsulfuration and folate cycles. ¹³C-labelled carbons (●) unlabelled carbons (○). Gly: Glycine, Ser: Serine, CTH: Cystathionine; HCY: Homocysteine, GSH: Glutathione, γGC: γ-glutamyl cysteine, Glu: Glutamate. (b–e) A549 cells expressing scramble (SCR), NRF2 or PHGDH shRNAs were grown in the presence of U-¹³C-glucose for 24 hours and metabolites were extracted and analysed by LC/MS. (b) Analysis of ¹³C-labelling on the glycine component of glutathione. (c) Analysis of ¹³C-labeling on the purine metabolite adenosine monophosphate (AMP). (d,e) Analysis of glutathione (d) and AMP (e) labelling in serine high cell lines. Cells were labelled with ¹³C-glucose for 24 or 48 hours as indicated. (b,d) PHGDH-derived serine is incorporated into glutathione through the generation of glycine and cysteine. M+0 denotes no carbons labelled, M+2 denotes labelling on the glycine moiety, M+3 denotes labeling on the cysteine moiety, and M+5 denotes labeling on both glycine and cysteine. M+5 labeling was not observed. (c,e) M+5 labelling occurs following ribose-5-phosphate labelling via the pentose phosphate pathway. M+7 labelling is the result of ribose labelling plus either glycine or formyl-THF labelling in the purine ring. M+9 labelling is the result of ribose labelling plus either glycine and formyl-THF labelling in the purine ring. M+0 has no labelled carbons. (f–g) LC/MS analysis of total metabolite levels in the nucleotide (f) and transsulfuration (g) pathways. (h) NADPH/NADP+ ratios 4 days after PHGDH knockdown. Results are the average of 3 biological replicates.

Figure 5

Activation of the serine biosynthesis pathway promotes tumourigenesis in NSCLC. (a) PHGDH knockdown impairs soft agar growth of serine high, but not serine low, cell lines. (b–c) Soft agar growth correlates with serine (b) and glycine (c) labelling at 24 hours. Each cell line was plated at 5,000 cells/well and the number of colonies counted after 14 days. (d) PHGDH knockdown impairs the xenograft growth of a serine high cell line (PC9, left) but not a serine low cell line (H1373, right). Results are the average of 5 tumors. (e) Western analysis of PHGDH expression of cell lines and xenografts from (d) upon injection and at endpoint. (f) Patients with high NRF2 protein expression (Z-score > 0.5) demonstrate elevated serine pathway gene expression in patient samples from TCGA lung adenocarcinoma cohort. Boxes represent mean values, error bars represent SEM. (g) Gene expression of PHGDH, PSAT1 and SHMT2 in the Director’s Consortium lung adenocarcinoma dataset clusters patients into high and low expression cohorts. (h) Kaplan-Meier survival analysis of patients with high (n = 29, red) or low (n = 414, blue) expression of PHGDH, PSAT1 and SHMT2 based on the patient clustering from (g). Median survival is 36 (high) vs. 73.2 (low) months. The p-value was calculated using the Mantel-Cox test.

Figure 6

Model of the regulation of serine/glycine biosynthesis by NRF2. An ATF4 transcriptional program, indirectly activated by NRF2/KEAP1 mutations, regulates the expression of serine/glycine biosynthesis enzymes. These enzymes produce serine and glycine from the glycolytic intermediate 3-PG and funnel the carbon into glutathione and nucleotides via the folate and transsulfuration cycles. NRF2/ATF4 regulated enzymes are shown in red. 3-PG: 3-phosphoglycerate; 3-PP: 3-phosphohydroxy pyruvate; 3-PS: 3-phosphoserine; Ser: Serine; Cys: Cysteine; GSH: Glutathione.