Serine starvation induces stress and p53-dependent metabolic remodelling in cancer cells

Abstract

Cancer cells acquire distinct metabolic adaptations to survive stress associated with tumour growth and to satisfy the anabolic demands of proliferation. The tumour suppressor protein p53 (also known as TP53) influences a range of cellular metabolic processes, including glycolysis, oxidative phosphorylation, glutaminolysis and anti-oxidant response. In contrast to its role in promoting apoptosis during DNA-damaging stress, p53 can promote cell survival during metabolic stress, a function that may contribute not only to tumour suppression but also to non-cancer-associated functions of p53. Here we show that human cancer cells rapidly use exogenous serine and that serine deprivation triggered activation of the serine synthesis pathway and rapidly suppressed aerobic glycolysis, resulting in an increased flux to the tricarboxylic acid cycle. Transient p53-p21 (also known as CDKN1A) activation and cell-cycle arrest promoted cell survival by efficiently channelling depleted serine stores to glutathione synthesis, thus preserving cellular anti-oxidant capacity. Cells lacking p53 failed to complete the response to serine depletion, resulting in oxidative stress, reduced viability and severely impaired proliferation. The role of p53 in supporting cancer cell proliferation under serine starvation was translated to an in vivo model, indicating that serine depletion has a potential role in the treatment of p53-deficient tumours.

Figure 1. p53 promotes cell survival and proliferation during serine starvation in vitro and in vivo

a, HCT116 cells were grown in complete media (containing serine and glycine) or equivalent media lacking these amino-acids (averages of triplicate wells). b, Viability of HCT116 cells was assessed by analysing sub-G1 DNA content (n=3) and c, PI exclusion (n=3). d, LC-MS was used to determine the relative consumption of serine by HCT116 cells fed complete media (averages of triplicate wells vs. fresh media). e, Nude mice were subcutaneously injected with HCT116 cells (p53^+/+ right flank, p53^−/− left flank); and fed diet with or without serine and glycine. Tumour volume is plotted until the first animal in each group reached the experimental end-point (*p<0.05 control diet group vs. –Ser & Gly group; **p<0.05 for p53^+/+ vs. p53^−/− within –Ser & Gly group). f, Kaplan Meier plot of survival until experimental end-point for diet groups (mean survival; Control = 33.3 days (n=10), -Ser & Gly = 53.2 days (n=8), Log rank p = 0.001, Wilcoxon p = 0.003). g, Expression of glycolytic and SSP genes (averages of triplicate qPCR). h, Intracellular serine levels in HCT116 cells fed serine and glycine deficient (−) media containing U-¹³C-glucose were measured by LC-MS (averages of triplicate wells). All error bars are SEM.

Figure 2. Serine starvation differentially changes energy metabolism in p53^+/+ and p53^−/− cells

a, HCT116 cells were fed complete (Com) or serine and glycine deficient (-SG) media for 24h, in the presence of U-¹³C-glucose for the final 2h. LC-MS was used to detect relative intracellular quantities of glycolytic intermediates (averages of triplicate wells). b, HCT116 cells were grown with or without serine, glycine and Oligomycin 1ng/ml (averages of triplicate wells). c, Oxygen consumption rate of HCT116 cells was measured after 48h serine and glycine starvation (n=3, *p<0.05). d, Relative intracellular levels of TCA cycle intermediates in HCT116 cells deprived of serine and glycine (in the constant presence of U-¹³C-glucose) were analysed by LC-MS (averages of triplicate wells), and e, after long term starvation, with U-¹³C-glucose added for the final hour (averages of triplicate wells). f, ATP levels were measured in HCT116 cells (n=3). g, HCT116 p53^−/− (1ex) cells were grown in complete media or media lacking serine and glycine with or without pyruvate 5mM (averages of triplicate wells). All error bars are SEM.

Figure 3. Serine starvation causes recruitment of p53 to the p21 promoter and activation of a transient p21-dependent G1 arrest

a, LC-MS was used to quantify total relative amounts of intracellular purine nucleotides GMP and AMP in serine and glycine fed (Com) and starved (-SG) HCT116 cells (averages of triplicate wells). b, p53 and p21 protein expression was quantified in p53^+/+ HCT116 cells via western blot and detection with infra-red conjugated secondary antibodies (n=3). c, Chromatin-immunoprecipitation (ChIP) was performed for p53 with qPCR for two p53-response elements (-2350bp and -1450bp) and the transcription initiation region (+50bp) of the p21 promoter (n=3). d, BrdU labeling and PI staining followed by flow cytometry were used to asses cell cycle (n=3). e, p21 was transiently knocked down in p53^+/+ HCT116 cells using si-RNA (averages of triplicate wells). f, p21−/− HCT116 cells (retaining wild-type p53^+/+) were grown in media with or without serine and glycine (averages of triplicate wells). All error bars are SEM.

Figure 4. p53-p21 activation allows serine deprived cells to synthesise GSH in preference to nucleotides

a, LC-MS was used to detect relative intracellular quantities of IMP and b, GSH in HCT116 cells fed complete media (Com) or media lacking serine and glycine (-SG) for 24h, in the presence of U-¹³C-glucose for the final 2h, and c, fed –SG media for the indicated times in the presence of U-¹³C-glucose for the final hour (averages of triplicate wells). d, HCT116 cells were grown with or without serine and glycine, or e, media lacking serine and glycine with 5mM glutathione (GSH) for 48h in the presence of hydrogen peroxide (H₂O₂) for the final 24h. f, HCT116 cells were treated with an oxidation-activated fluorescent dye and analysed by flow cytometry. g, HCT116 p53^−/− (1ex) cells were grown with or without serine, glycine, pyruvate 5mM (Pyr) and / or GSH 5mM, or N-acetyl cysteine 0.2mM (NAC) (averages of triplicate wells). All error bars are SEM.