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. 2010 Oct 15;70(20):8066-76.
doi: 10.1158/0008-5472.CAN-10-0608. Epub 2010 Sep 28.

Aerobic glycolysis suppresses p53 activity to provide selective protection from apoptosis upon loss of growth signals or inhibition of BCR-Abl

Emily F Mason  1 , Yuxing Zhao, Pankuri Goraksha-Hicks, Jonathan L Coloff, Hugh Gannon, Stephen N Jones, Jeffrey C Rathmell
Affiliations

Aerobic glycolysis suppresses p53 activity to provide selective protection from apoptosis upon loss of growth signals or inhibition of BCR-Abl

Emily F Mason et al. Cancer Res. .

Abstract

Unlike the growth factor dependence of normal cells, cancer cells can maintain growth factor-independent glycolysis and survival through expression of oncogenic kinases, such as BCR-Abl. Although targeted kinase inhibition can promote cancer cell death, therapeutic resistance develops frequently, and further mechanistic understanding is needed. Cell metabolism may be central to this cell death pathway, as we have shown that growth factor deprivation leads to decreased glycolysis that promotes apoptosis via p53 activation and induction of the proapoptotic protein Puma. Here, we extend these findings to show that elevated glucose metabolism, characteristic of cancer cells, can suppress protein kinase Cδ (PKCδ)-dependent p53 activation to maintain cell survival after growth factor withdrawal. In contrast, DNA damage-induced p53 activation was PKCδ independent and was not metabolically sensitive. Both stresses required p53 Ser(18) phosphorylation for maximal activity but led to unique patterns of p53 target gene expression, showing distinct activation and response pathways for p53 that were differentially regulated by metabolism. Consistent with oncogenic kinases acting to replace growth factors, treatment of BCR-Abl-expressing cells with the kinase inhibitor imatinib led to reduced metabolism and p53- and Puma-dependent cell death. Accordingly, maintenance of glucose uptake inhibited p53 activation and promoted imatinib resistance. Furthermore, inhibition of glycolysis enhanced imatinib sensitivity in BCR-Abl-expressing cells with wild-type p53 but had little effect on p53-null cells. These data show that distinct pathways regulate p53 after DNA damage and metabolic stress and that inhibiting glucose metabolism may enhance the efficacy of and overcome resistance to targeted molecular cancer therapies.

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Figures

Figure 1
Figure 1. p53 is required for Puma induction and cell death after growth factor withdrawal and etoposide treatment in activated T lymphocytes
A, Glycolysis was measured in resting primary splenic T cells and in activated T cells cultured in the presence or absence of IL-2 for an additional 12 hours. B, C, T cells were isolated from wild type and p53−/− mice, activated, and cultured in the presence or absence of IL-2 or etoposide (D=DMSO control; E=Etoposide). (B) Cell viability was measured over time, and (C) Puma and p53 levels were analyzed by immunoblot after 12 hours. D, T cells from wild type, Bim−/− or Bim−/− p53−/− mice were activated and withdrawn from IL-2 or treated with etoposide as above. Viability was measured over time and levels of Bim and p53 were analyzed after 24 hours.
Figure 2
Figure 2. Maintenance of glucose metabolism suppresses Puma induction and cell death after growth factor withdrawal but not after DNA damage
A,B,C, Control cells and cells with stable expression of Glut1 and HK1 were grown in the presence of IL-3 or were withdrawn from IL-3 or treated with etoposide and (A) glycolysis was assessed after 8 hours, (B) cell viability was measured over time, and (C) Puma induction was assessed after 10 hours (C=Control; GH=Glut1/HK1). D, Control cells were transfected with control or p53 shRNA, and Puma induction was measured after 10 hours of IL-3 withdrawal or etoposide treatment.
Figure 3
Figure 3. Activation of p53 is selectively inhibited by glucose metabolism after cytokine withdrawal
A,B, p53 transcriptional activity was measured in control and Glut1/HK1 cells using a luciferase reporter construct driven by p53 binding elements in cells grown ± IL-3 (A) or in the presence of DMSO or etoposide (B). C,D, Induction of multiple p53 target genes was measured by RT-PCR in (C) control cells grown in the presence of IL-3 or withdrawn from IL-3 or treated with etoposide for 10 hours or (D) activated primary T cells cultured in the presence or absence of IL-2 for 12 hours.
Figure 4
Figure 4. Post-translational modification of p53 after cytokine withdrawal is suppressed by glucose metabolism
A, Levels of mSer18 p53 phosphorylation were assessed via immunoblot in cells withdrawn from IL-3 or treated with etoposide for 10 hours or wild type primary T cells activated and cultured without IL-2 or with etoposide for 12 hours. B, T cells from p53+/+, p53 S18A/S18A, or p53−/− mice were activated and withdrawn from IL-2 or treated with etoposide or 8 Gy gamma irradiation for analysis of cell survival and immunoblot after one day. (C) Control and Glut1/HK1 cells cultured in the presence or absence of IL-3 or the presence of etoposide for 10 hours were examined for mSer18 p53 phosphorylation. D, Control and Glut1/HK1 cells were transfected with HA-tagged human p53 and cultured in the presence or absence or IL-3 for 10 hrs, and HA-p53 was immunoprecipitated and probed for acetylation.
Figure 5
Figure 5. PKCδ is required for cytokine withdrawal-induced p53 phosphorylation
A, Primary T lymphocytes were activated and withdrawn from IL-2 for 12 hrs (left panel), and FL5.12 cells expressing Bcl-xL were treated with 10 μM Compound C or vehicle control and withdrawn from IL-3 (right panel). B-E, FL5.12 cells were transfected with control, PKCδ, p53 or Bim shRNA, as indicated. B, FL5.12 cells expressing Bcl-xL were withdrawn from IL-3 or treated with etoposide. Bcl-xL-expressing cells were used to prevent loss of cell viability over the treatment time course. C. FL5.12 cells were transfected with a p53 transcriptional activity luciferase reporter and cultured ± IL-3 for 8 hours. D, E, Control and CTLL-2 cells were withdrawn from IL-3 and IL-2, respectively and viability was measured over time.
Figure 6
Figure 6. Glucose metabolism promotes imatinib resistance
A,B, Control cells stably expressing BCR-Abl cultured without IL-3 were analyzed. A, Glycolysis was measured after 10 hours imatinib treatment. B, Cells were transfected with control, p53, or Puma shRNA and treated with imatinib, and viability was assessed over time. C,D, Control p190 and Glut1/HK1 p190 cells cultured without IL-3 were (C) treated with imatinib for 12 hours and Puma induction was measured via immunoblot (top panel), transfected with a p53 transcriptional activity luciferase reporter and treated with imatinib for 10 hours(bottom panel), or (D) treated with imatinib, 2-deoxyglucose, or both in combination, and viability was assessed over time. E, Nalm-1 and K562 cells were treated with imatinib, 2-deoxyglucose, or both, and viability was assessed over time.
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