p53-dependent regulation of autophagy protein LC3 supports cancer cell survival under prolonged starvation

Abstract

The p53 tumor suppressor is mutated in a high percentage of human tumors. However, many other tumors retain wild-type (wt) p53 expression, raising the intriguing possibility that they actually benefit from it. Recent studies imply a role for p53 in regulation of autophagy, a catabolic pathway by which eukaryotic cells degrade and recycle macromolecules and organelles, particularly under conditions of nutrient deprivation. Here, we show that, in many cell types, p53 confers increased survival in the face of chronic starvation. We implicate regulation of autophagy in this effect. In HCT116 human colorectal cancer cells exposed to prolonged nutrient deprivation, the endogenous wt p53 posttranscriptionally down-regulates LC3, a pivotal component of the autophagic machinery. This enables reduced, yet sustainable autophagic flux. Loss of p53 impairs autophagic flux and causes excessive LC3 accumulation upon starvation, culminating in apoptosis. Thus, p53 increases cell fitness by maintaining better autophagic homeostasis, adjusting the rate of autophagy to changing circumstances. We propose that some cancer cells retain wt p53 to benefit from the resultant increased fitness under limited nutrient supply.

Fig. 1.

p53 reduces autophagy initiation during prolonged starvation of HCT116 cells, and lack of p53 causes aberrant accumulation of LC3 and autophagosomes. p53^+/+ or p53^−/− cells were incubated in control medium (McCoy's medium supplemented with 10% FCS and 2 mM l-glutamine) or Earle's balanced salts solution (EBSS) for 48 h; 1 mM Bafilomycin A (BafA) was added, where indicated, for the last 3 h of incubation, and cells were subsequently (A) fixed for immunostaining with anti-LC3 antibodies (the experiment was repeated three times, and representative images are shown) or (B) lysed as explained in Materials and Methods, resolved on 15% SDS/PAGE, Western blotted with the indicated antibodies, and quantified using Image-J software. For each sample, lipidation was calculated as the ratio between LC3II and GAPDH. Lipidation of nonstarved p53^+/+ cells was set to 1, and the rest of the samples were normalized accordingly. Average results of three separate experiments are presented. Autophagic flux was calculated by dividing the value of lipidated LC3 in the presence of BafA by that without BafA. *P value < 0.05. **P < 0.01. (C) Cells starved for 48 h in EBSS were fixed in Epon and imaged by transmission EM (TEM). Representative images of a p53^+/+ cell (Top) and two p53^−/− cells (Middle and Bottom) are shown. White rectangles in Left indicate regions enlarged in Center and Right. White arrow indicates an intact autophagosome; black arrows indicate aberrant accumulation of autophagosomes. (D) HCT116 p53^+/+ or p53^−/− cells kept in control medium or starved in EBSS as indicated, in the presence or absence of 1 mM BafA for the last 3 h of incubation where indicated, were fixed for immunostaining with anti-LC3 antibodies (red), anti-LAMP1 antibodies (green), and DAPI (blue). Arrows indicate autolysosomes.

Fig. 2.

p53 down-regulates LC3 mRNA posttranscriptionally during chronic starvation of HCT116 cells. p53^+/+ or p53^−/− cells kept in control medium or starved in EBSS as indicated were harvested, and RNA was extracted and subjected to quantitative RT-PCR analysis. (A) qRT-PCR analysis of LC3B mRNA. Levels measured for nonstarved p53^+/+ cells were set to 1, and the rest of the samples were normalized accordingly. Average results of three experiments are presented. *P < 0.05. **P < 0.01. (B) Time-dependent changes in the ratio between LC3B mature mRNA and premRNA. Results of three experiments performed as in Fig. S2B were analyzed so that the values for LC3B mRNA were divided by the corresponding values for LC3B premRNA and averaged. *P < 0.05.

Fig. 3.

p53 enhances HCT116 cell survival during chronic starvation. (A) p53^+/+ or p53^−/− cells kept in control medium or starved in EBSS for 48 h were trypsinized, fixed in methanol, stained with propidium iodide (PI), and analyzed in a FACSsort flow cytometer. A representative result of three experiments is shown. The position of G1 is indicated, and arrows indicate the subG1 subpopulation in starved cultures. (B) p53^+/+ or p53^−/− cells were kept in control medium, starved in EBSS for 48 h or treated with 5-FU for 16 h, trypsinized, and stained with Annexin V and PI. A representative result of two experiments is shown. The percentage of cells in each quadrant is indicated. (C) p53^+/+ or p53^−/− cells were starved in EBSS for 5 d, after which live cell images were taken (Middle Left and Bottom Left), and cells were fixed and stained with crystal violet (Top Left) or EBSS was replaced with control medium for 24 h before imaging and staining (Right). A representative result of three experiments is shown. (D) p53^+/+ or p53^−/− cells were starved in EBSS with or without Z-VAD-fmk for 48 h before fixation and analysis in an LSR-II flow cytometer. The experiment was repeated two times; a representative result is shown. Data analysis was performed by Matlab using FCS data reader script. (E) p53^+/+ or p53^−/− cells were kept in control medium, starved in EBSS for 48 h or treated with 5-FU for 16 h before lysis, and analysis on 10% SDS/PAGE. (Top) Anti-PARP, long exposure (L). (Middle) Anti-PARP, short exposure (S). (Bottom) Anti-GAPDH.

Fig. 4.

Knockdown of LC3 rescues p53-deficient HCT116 cells from apoptosis. (A) p53^−/− cells were transfected with 150 nM control (100 nM siLacZ plus 50 nM nontargeting siControl 2), LC3-specific (50 nM siLC3A, 50 nM siLC3B, 50 nM siLC3C), or Atg5-specific (50 nM siAtg5 plus 100 nM siLacZ) siRNA oligos; 48 h later, the cells were starved in EBSS or kept in control medium for 48 h before staining with PI and FACS analysis as in Fig. 3D. A representative result of three experiments is shown. (B) p53^+/+ cells were transfected with enhanced-GFP (eGFP) vector or with eGFP-LC3; 24 h later, the cells were starved in EBSS for 24 h before fixation and FACS analysis as in Fig. 3D. A representative result of three experiments is shown.

Fig. 5.

p53 promotes survival of nontransformed cells during prolonged starvation. (A) MRC5 or IMR90 cells were transfected with 20 nM control (siLacZ) or p53-specific (sip53) siRNA oligonucleotides; 48 h later, cells were placed in EBSS for 48 h before staining with PI and FACS analysis as in Fig. 3D. A representative result of three experiments is shown. (B) Wt or p53 ^−/− MEFs were transfected with 50 nM control (siLacZ) or mouse LC3-specific (20 nM mLC3A plus 30 nM mLC3B) siRNA oligonucleotides; 48 h later, cells were placed in EBSS and analyzed as in A. A representative result of three experiments is shown. (C) Wt or p53 ^−/− MEFs kept in control medium or starved in EBSS as indicated were harvested, and RNA was extracted and subjected to qRT-PCR analysis with the indicated primers. Results were normalized for HPRT mRNA in the same samples. A representative result of three experiments is shown.