Oncogenic transcription factor Myc deregulates the cell cycle and simultaneously reprograms cellular metabolism to meet the biosynthetic and bioenergetic needs of proliferation. Myc also sensitizes cells to mitochondria-dependent apoptosis. Although metabolic reprogramming has been circumstantially connected to vulnerability to apoptosis, the connecting molecular pathways have remained poorly defined. Here, we show that Myc-induced altered glutamine metabolism involves ATP depletion and activation of the energy sensor AMP-activated protein kinase (AMPK), which induces stabilizing phosphorylation of p53 at Ser15. Under influence of Myc, AMPK-stabilized tumor suppressor protein p53 accumulates in the mitochondria and interacts with the protein complex comprised of B-cell lymphoma 2 (Bcl-2) antagonist/killer (BAK) and Bcl2-like 1 (Bcl-xL). Mitochondrial p53 induces conformational activation of proapoptotic Bak without disrupting the Bak-Bcl-xL interaction. Further liberation of Bak specifically from the p53-activated Bak-Bcl-xL complex leads to spontaneous oligomerization of Bak and apoptosis. Thus, Myc-induced metabolic changes are coupled via AMPK and phospho-p53 to the mitochondrial apoptosis effector Bak, demonstrating a cell-intrinsic mechanism to counteract uncontrolled proliferation.
cancer metabolism; cell death; oncogene.
Conflict of interest statement
The authors declare no conflict of interest.
L directly controls conformational activation of Bak. ( A and B) Bcl-x L silencing induces conformational activation of Bak. MCF10A cells were transduced with control or Bcl-x L targeted lentiviral shRNA (shbcl-x L) and immunostained on coverslips with N-terminal Bak antibody (Ab-1). ( A) Immunofluorescence (IF) images demonstrating N-terminal exposure of Bak. ( B) Quantification of the cells with conformationally active (N terminus exposed) Bak. ( C and D) Overexpression of Bcl-x L blocks Myc-induced conformational activation of Bak in MCF10A-MycER cells. ( C) IF images of cells expressing activated Myc without (pB-cont) or with ectopic Bcl-x L (pB-Bcl-x L). MycER was activated for 24 h with 100 nM 4-hydroxytamoxifen (4-OHT) before the cells were fixed and immunostained as in A. ( D) Quantification of the cells with conformationally active Bak. Cells were treated as in C and scored as in B. ( E) ABT-737 induces conformational Bak activation. MCF10-MycER cells were treated for 24 h with 4-OHT or with 1μΜ ABT-737 to neutralize Bcl-x L. Apoptosis was induced with additional 2 h treatment with 50 ng/mL TRAIL. Cells were immunostained with antibodies for active Bak (Ab-1) or Bax (6A7). ( F) Western blot analysis shows expression of Bak and Bcl-x L in breast cancer cell lines. ( G) ABT-737 induces conformational Bak activation in breast cancer cell lines. Cells were treated with ABT-737 for 24 h and quantitated as in B. ( H) Formation of Bak and Bax oligomers in apoptosis sensitizing and inducing conditions. Cells were treated as in E and lysed. Proteins were chemically cross-linked with BMH, and the mitochondria-enriched heavy membrane fractions were isolated and analyzed by Western blotting using antibodies for Bak and Bax. For quantitative cell analyses, at least 200 cells were scored per each treatment. The graphs represent mean ± SD of at least three separate experiments. * P < 0.05.
Myc induces cytosolic and mitochondrial accumulation of Ser15 phosphorylated p53. (
A) Coimmunoprecipitation (co-IP) analysis of interactions between Bak and Bcl-x L or Mcl-1 in MCF10A-MycER cells. Cells were treated as in Fig. 1 E, and polyclonal Bak antibody (H-211) was used for pulldowns. IgG, antibody only control. ( B) p53 protein levels in cell fractions. MCF10A-MycER cells were treated as in Fig. 1 E and lysed, and cell fractions were analyzed by Western blotting. Antibodies for CoxIV (mitochondria), Lamin B (nuclear envelope), and tubulin (cytosol) verify sample purity. ( C) p-p53 (S15) in total lysate and mitochondrial fractions. MCF10A-MycER cells were treated as in Fig. 1 E. Actin (cytosol) and Bak (mitochondria) are sample controls. ( D) Cytosolic expression of Ser-18 phosphorylated p53 in the mouse mammary gland. Representative images of immunohistochemically stained tissue sections from control mice and WAP-Myc mice exposed to two sequential pregnancies. ( E and F) Percentages of epithelial cells exhibiting phosphor-Ser18 or Ki-67 positive immunostaining in the samples. Images representing samples from 4 or 11 ( n) separate mice were analyzed blindly. Cells with clear cytosolic staining pattern were scored whereas cells exhibiting unspecific apical border/luminal staining were excluded from analyses. *** P < 0.0005.
Mitochondrial accumulation of p53 induces conformational activation of Bak. (
A) Nutlin-3a induces stabilization of p53 in MCF10A cells. Cells were treated for 24 h with indicated concentrations of Nutlin-3a and analyzed by Western blotting. ( B) Western blot showing p53 levels in the mitochondrial and cytosolic cell fractions. MCF10A cells were treated with 10 μΜ Nutlin-3a for 24 h before preparation of cell fractions. Bak and COXIV are markers of the mitochondrial fractions, and GAPDH is a marker of the cytosolic fraction. ( C) Quantification of Bak and Bax activation in the cells treated with 20 μM Nutlin-3a for 24 h. Cells were scored as in Fig. 1. ( D) Status of Bak-Bcl-x L interaction in MCF10A cells treated with 1 μM ABT-737 or 10 μM Nutlin-3a for 24 h. Co-IP analysis was performed as in Fig. 2 A. ( E) DNA damage, but not Myc, induces nucleoplasmic accumulation of Ser15 phosphorylated p53. p-p53 (Ser15) antibody also recognizes nucleolar p53, which generates some unspecific background. ( F) Quantification of the cells with nucleoplasmic p53. MCF10A-MycER cells were treated for 24 h with 4-OHT, 1 μM ABT-737, 10 μM Etoposide, or 0.1 μg/mL Doxorubixin. Cells were scored as in Fig. 1. ( G) Quantification of the cells with conformationally active Bak or Bax. MCF10A-MycER cells were treated and scored as above. ( H) DNA damage, but not Myc, induces up-regulation of the p53 target p21. Western blot analysis of p21 levels in MCF10A cells after 24 h Myc activation or Doxorubicin treatment. ( I) Nutlin-3a and Etoposide but not Myc induce up-regulation of the p53 target genes GADD45 and TIGAR. Myc was activated for 24 h or cells were treated with 10 μM Nutlin-3a or Etoposide for 24 h before qPCR analysis of mRNA levels. SD represent values from three different experiments. ( J) Western blot analysis of MCF10A-MycER and MCF10A p53null-MycER cells demonstrating lack of p53 and expression of MycER. ( K) Lentivirally delivered wild-type p53 and p53 QS transcription-deficient mutant rescue Myc’s ability to activate Bak in p53null cells. Cells were infected with lentiviruses expressing p53 WT or p53 QS and allowed to grow for 48 h, and, subsequently, Myc was activated for 24 h. Cells with active Bak were scored as in Fig. 1. ** P < 0.005.
Activation of Myc rearranges interaction of p53 with Bak-Bcl-x
L complex. ( A and B) Activation of Myc leads to close proximity colocalization of p53 with conformationally active Bak. Proximity Ligation Assay (PLA) was performed with rabbit p53 antibody (FL-393) and N-terminal Bak antibody (Ab-1). ( A) IF images of MCF10A-MycER cells treated as in Fig. 1 E and subjected to PLA assay. ( B) Quantification of PLA spots (close proximity sites) scored per cell. ( C and D) Effects of Myc, ABT-737, or apoptotic conditions (Myc+TRAIL) to the interaction between p53 and Bcl-x L in MCF10A-MycER cells. Cells were treated as in Fig. 1 E. Co-IP analysis was performed using (in C) polyclonal p53 antibody (Ab-1) and (in D) monoclonal p53 antibody (Bp53-12) for Bcl-x L pulldown. ( E) ABT-737 selectively induces apoptosis in the cells expressing active Myc or treated with Nutlin-3a. Cells were pretreated for 24 h with 4-OHT or Nutlin-3a followed by 1 h treatment with 10 μM ABT-737. Cells were stained for active Bax and scored as in Fig. 1. * P < 0.05.
Myc-induced stabilization of p53 and conformational activation of Bak is dependent on AMPK. (
A) Effects of indicated kinase inhibitors on Myc-induced conformational activation of Bak. MCF10A-MycER cells were pretreated for 4 h with DMSO carrier or indicated inhibitors before Myc activation. Cells were then incubated for a further 24 h before analysis. Kinase inhibitors were titrated to sublethal concentration, which was 1 μM except for AMPK inhibitor (compound C; 10 μM) and Chk2 inhibitor (500 nM). Cells with active Bak were scored from IF images. ( B) Representative IF images showing effect of AMPK inhibitor compound C. ( C) Effect of AMPK inhibition on Myc-induced Ser15 phosphorylation of p53. Western blot analysis shows levels of p53 and p-p53 (S15) in MCF10A-MycER cells after indicated treatments. Phospho Ser-79 ACC is a biomarker of AMPK activity. Cells were pretreated for 4 h with 10 μM AMPK inhibitor followed by 24 h further treatment with 100 nM 4-OHT to activate Myc or 200 nM AICAR to ectopically activate AMPK. Actin is a loading control. ( D) Effect of AMPK inhibition on Myc-induced accumulation of Ser15 phosphorylated mitochondrial p53. Cells were treated as in C followed by isolation of mitochondria-enriched fractions and analysis by Western blotting. Bak is a sample control. ** P < 0.005.
Myc-induced metabolic reprogramming is coupled to apoptotic sensitization via AMPK. (
A) Effect of Myc activation on cellular ATP levels. MCF10A cells were grown in normal culture medium followed by change of fresh medium and 24 h or 48 h Myc activation. Alternatively, cells were deprived of EGF and Insulin for 20 h followed by 4 h Myc activation. ( B) ADP/ATP ratio change in MCF10A cells after 24 h Myc activation. ( C) Myc induces activation of the AMPK pathway. Myc was activated with 4-OHT in MCF10A-MycER cells for 24 h, and both cytosolic fractions and total cell lysates were analyzed by Western blotting using indicated phospho-specific antibodies. The status of activating T172 phosphorylation of AMPK, phospho ACC, and phospho-p53 was analyzed. ( D) Immunohistochemical analysis of ACC phosphorylation at Ser79 in histological samples described in Fig. 2 D. ( E) Myc-induced GLS up-regulation is dependent on AMPK activity. Cells were treated as in Fig. 5 C, and the mitochondria-enriched fractions were analyzed for expression of GLS. CoxIV is a sample control. ( F) Myc-induced addiction to glutamine metabolism. Myc was preactivated for 24 h followed by 24 h glutamine withdrawal in the presence of Myc activity. Apoptosis was measured with Caspase-Glo 3/7 kit. ( G– I) Effect of AMPK inhibition on Myc induced activation of Bak and Bax. ( G) IF images of cells pretreated for 4 h with 10 μM Compound C followed by combined activation of Myc and TRAIL pathway. Cells were immunostained for Bak and Bax. ( H) Quantification of active Bax (apoptosis) in the cells treated as in G. ( I) Quantification of active caspase-3 (apoptosis) in the cells pretreated for 4 h with 10 μM Compound C followed by 4 h treatment with 100 μM Etoposide. SD represent values from three different experiments. ( J) Activation of AMPK with compound A-769662 or AICAR induces Ser15 phosphorylation of p53. ( K) Active AMPK sensitizes cells to apoptosis. Cells were treated for 24 h with 1 μM A769662 to activate AMPK. For induction of apoptosis, sensitized cells were further incubated for 2 h with 50 ng/mL TRAIL. Conformational activation of Bak and Bax in the treated cells was scored as in Fig. 1. * P < 0.05, ** P < 0.005.
Myc-induced activation of AMPK, p53, and Bak in multiple cell lines.
A model: Myc-induced altered metabolism activates AMPK and p53, which sensitizes the mitochondrial apoptosis pathway. Activation of Myc promotes metabolic transformation, which adapts cells to meet the biosynthetic and bioenergetic requirements of rapid cell proliferation. Metabolic transformation decreases cellular ATP levels, activating the cellular energy sensor AMPK. AMPK promotes catabolic mode of metabolism (phospho-ACC) and contributes to establishment of glutaminolytic pathway (GLS). These AMPK-mediated processes release carbon for bioenergetic reactions and provide glutamine-derived nitrogen for biosynthesis of nucleic acids and nonessential amino acids (4). However, AMPK also induces phosphorylation of p53 at Ser15, which stabilizes the protein and leads to mitochondrial accumulation of p53. At the mitochondrial surface, phospho-p53 adjoins and rearranges the Bak–Bcl-x
L complex ( Fig. S3), resulting in conformational activation of Bak and apoptotic sensitization. This mechanism may act as a failsafe mechanism against cells with an inability to recover from energy stress, for example, due to oncogene-enforced cell cycle progression. N
Metabolically controlled apoptosis pathway of Myc. Myc activation (
i) reduces ATP levels ( ii), activating a cellular energy sensor, AMPK ( iii). Active AMPK initiates energy-saving metabolism via AMPK-induced phosphorylation events that inhibit biosynthetic reactions [e.g., AMPK suppresses acetyl-CoA carboxylase (ACC) by phosphorylating Ser79, which reduces fatty acid synthesis]. AMPK also promotes glutamine use in the mitochondria and phosphorylates p53 at Ser15, increasing p53 levels. In healthy cells, p53 halts cell proliferation, which conserves energy. Oncogenic Myc prevents proliferation from slowing down, and AMPK-induced p53 phosphorylation continues to extend the p53 lifetime ( iv), so p53 accumulates at the mitochondrial surface ( v). High levels of p53 alter the interactions between p53 and mitochondrial apoptosis proteins Bak and Bcl-x L ( vi), and Bak acquires an active open conformation. Activated Bak sensitizes mitochondria to triggers of apoptosis, inhibiting the expansion of oncogene-transformed cells. Dashed arrows indicate concentration changes; phosphorylation events are denoted with a circled p. GLS, glutaminase.
All figures (9)
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Research Support, Non-U.S. Gov't
AMP-Activated Protein Kinases / metabolism*
Mitochondria / metabolism*
Protein Structure, Tertiary
Proto-Oncogene Proteins c-myc / metabolism*
Tumor Suppressor Protein p53 / metabolism*
bcl-2 Homologous Antagonist-Killer Protein / metabolism*
bcl-X Protein / metabolism
Proto-Oncogene Proteins c-myc
Tumor Suppressor Protein p53
bcl-2 Homologous Antagonist-Killer Protein
AMP-Activated Protein Kinases
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