TAp73 enhances the pentose phosphate pathway and supports cell proliferation (original) (raw)

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Acknowledgements

We thank G. Melino (University Rome Tor Vergata, Rome, Italy), C. Chen (Albany Medical College, Albany, USA), A. Tullo (University of Rome La Sapienza, Rome, Italy), U. M. Moll (Stony Brook University, Stony Brook, New York, USA), M. C. Marín (Harvard Medical School, Boston, USA), B. Vogelstein (Johns Hopkins University, Baltimore, USA), W. El-Deiry (Penn State Hershey Cancer Institute, Pennsylvania, USA) for expression plasmids, antibodies and/or cell lines; W. Wang, Y. Mei, N. Li, X. Wang, W. Tan, E. Witze and K. E. Wellen for technical assistance; and C. O’Neill for help with manuscript preparation. Supported by China National Natural Science Foundation (31030046), Chinese Academy of Sciences (XDA01020104), Ministry of Science and Technology of China (2010CB912804 and 2011CB966302) and the Ministry of Education of China (20123402130006) to M.W., and US National Institutes of Health (CA088868 and GM060911) and the Department of Defense (W81XWH-10-1-0468) to X.Y.

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Author notes

  1. Wenjing Du and Peng Jiang: These authors contributed equally to this work

Authors and Affiliations

  1. Hefei National Laboratory for Physical Sciences at Microscale and School of Life Sciences, University of Science and Technology of China, Hefei, Anhui, 230027, China
    Wenjing Du, Peng Jiang & Mian Wu
  2. Abramson Family Cancer Research Institute and Department of Cancer Biology, University of Pennsylvania School of Medicine, Philadelphia, Pennsylvania 19104, USA
    Wenjing Du, Peng Jiang, Anthony Mancuso, Aaron Stonestrom, Michael D. Brewer & Xiaolu Yang
  3. Department of Radiation Oncology, University of Pennsylvania, Philadelphia, Pennsylvania 19104, USA
    Andy J. Minn
  4. The Campbell Family Institute for Breast Cancer Research, Princess Margaret Hospital, Toronto, Ontario M5G 2C1, Canada
    Tak W. Mak

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  1. Wenjing Du
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  2. Peng Jiang
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  3. Anthony Mancuso
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  4. Aaron Stonestrom
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  5. Michael D. Brewer
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  6. Andy J. Minn
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  7. Tak W. Mak
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  8. Mian Wu
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  9. Xiaolu Yang
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Contributions

W.D., P.J., X.Y. and M.W. designed the experiments and interpreted results. W.D. and P.J. performed all the experiments except those mentioned below. A.M. and P.J. analysed the oxidative PPP flux. A.S. and M.D.B. helped with the FACS analysis and xenograft study, respectively. A.J.M. performed the breast cancer data analysis. T.W.M. supplied the MEF cells deficient for TAp73 and ΔNp73. W.D., P.J. and X.Y. wrote the manuscript.

Corresponding authors

Correspondence toMian Wu or Xiaolu Yang.

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The authors declare no competing financial interests.

Integrated supplementary information

Supplementary Figure 1 Requirement of TAp73 for cell proliferation and tumor growth.

(a) Schematic representation of p53 and the TAp73 and ΔNp73 isoform classes. Each p73 isoform class comprises various splicing variants (α, β, γ etc.) that differ in their C-terminal regions. TA: transactivation domain. DBD: DNA-binding domain. OD: oligomerization domain. SAM: sterile alpha motif. (b) TAp73+/+ and _TAp73_−/− MEF cells cultured for 4 days were stained with crystal violet. (c) Representative images of animals two weeks after injected with TAp73+/+ and TAp73_−/− MEF cells. (d) U2OS cells stably expressing control or TAp73 shRNA were stained with crystal violet at day 6. (e,f) Proliferation of two independent clones of Δ_Np73_−/− and Δ_Np73+/+ MEFs in culture and images of crystal violet staining at day 6. Data are means ± SD (n = 3 independent experiments).

Supplementary Figure 2 p73 enhances G6PD expression.

(a) The pentose phosphate pathway and its link with glycolysis. FBP: fructose 1,6-biphosphate. PEP: phosphoenolpyruvate. TCA cycle: tricarboxylic acid cycle. (b) Expression of G6PD and TAp73 proteins in U2OS cells transfected with control siRNA or p73 siRNA, plus control vector or siRNA-resistant plasmid encoding Flag-TAp73. Western blots represent two independent experiments. (c) Semiquantitative RT-PCR analysis of ΔNp73 expression in HeLa, U2OS, H1299, and p53+/+ HCT116 cells. (d) Comparison of effect of p73 and ΔNp73 on G6PD expression in p73, Δ_Np73_ or control siRNA in HeLa cells by RT-PCR. (e) U2OS cells were infected with lentiviruses expressing a TAp73 shRNA or a control shRNA and cultured for the indicated time. The expression of G6PD, TAp73, and actin was analyzed by Western blot. The data represents three independent experiments. (f) mRNA expression in H1299 cells stably expressing control shRNA, _p_63 shRNA, or TAp73 shRNA was assayed by RT-PCR. (g) Quantitative RT-PCR analysis of G6PD expression in H1299 cells transfected with increasing amounts of control plasmid or plasmid expressing the indicated p53 family proteins. Data are means ± SD (n = 3 independent experiments). (h) A549 cells were treated with 1 μg/ml doxorubicin for 24 hour and protein expression was analyzed by Western blot. (i) Luciferase reporter assay of 293T cells transfected with p73 RE-luciferase construct or control construct, plus TAp73 or ΔNp73. Data are means ± SD (n = 3 independent experiments). (j) H1299 cells were transfected with increasing amounts of Flag-p53R175H, Flag-p53R273H, or Flag-TAp73α as indicated. Protein expression was examined by Western blot. The data represents two independent experiments.

Supplementary Figure 3 TAp73 regulates NADPH metabolism.

(a,b) NADPH levels (a) and relative NADP+/NADPH ratios (b) in Δ_Np73_+/+ and Δ_Np73_−/− MEF cells. Data are means ± SD (n = 3 independent experiments). (c) Relative NADPH levels in IMR90 cells transfected with control (−) or p73 siRNA. Data are means ± SD (n = 3 independent experiments). (d) NADP+/NADPH ratios of IMR90 cells treated with p73 or control siRNA. Data are means ± SD (n = 3 independent experiments). (e) Relative NADPH levels in p53+/+ and _p53_−/− HCT116 cells transfected with control (−) or p73 siRNA. Data are means ± SD (n = 3 independent experiments).

Supplementary Figure 4 A role of TAp73 in anti-oxidant defense.

(a) TAp73+/+ and _TAp73_−/− MEF cells treated with control or G6pd siRNA. ROS was measured by 2′, 7′-di-chlorofluorescein (DCF) staining and flow cytometry (left), and protein expression by Western blot (Right). Western blots represent three independent experiments. (b) ROS levels in TAp73+/+ and _TAp73_−/− MEF cells that were treated with or without DHEA. (c) IMR90 cells were transfected with p73, G6PD, and control siRNA as indicated. ROS was measured. (d) TAp73+/+ and _TAp73_−/− MEF cells were treated with or without 50 mM H2O2 for 30 min and then cultured for 24 h in the presence or absence of DHEA. Cell viability was analyzed. Data are means ± SD (n = 3 independent experiments). (e) U2OS cells were transfected with control siRNA (−), p73 siRNA, and G6PD siRNA as indicated. Cells were treated with or without 250 μM H2O2 for 24 h and cell viability was analyzed by trypan blue staining. Data are means ± SD (n = 3 independent experiments).

Supplementary Figure 5 p73 regulates DNA synthesis and cell senescence via G6PD.

(a,b) _p53_−/− and _p_21−/− HCT116 cells were transfected with control (−), p73, and G6PD siRNA as indicated. Cells were assayed for BrdU incorporation. Data are means ± SD (n = 3 independent experiments). (c,d) Cell-cycle profile of _p53_−/− (c) and _p_21−/− (d) HCT116 cells transfected with p73, G6PD, and control siRNA as indicated. Protein expression is shown below. Western blots represent three independent experiments. (e) Percentage of SA- β-gal positive cells in IMR90 culture that were transfected with control, p73, or G6PD siRNA, and cultured for 72 h. Protein expression is shown below. Data are means ± SD (n = 3 independent experiments). Western blots represent three independent experiments.

Supplementary Figure 6 p73 promotes cell proliferation through G6PD.

(a–c) Proliferation of p53+/+ (a), _p53_−/− (b), and _p_21−/− (c) HCT116 cells transfected with the control, G6PD, and p73 siRNAs as indicated. Data are means ± SD (n = 3 independent experiments). (d) Growth of U2OS cells transfected with control siRNA or p73 siRNA, plus control vector or Flag-G6PD. Data are means ± SD (n = 3 independent experiments). Protein expression is shown on the Right. Western blots represent three independent experiments. (e–h) Growth of TAp73+/+ and _TAp73_−/− MEF cells stably overexpressing G6PD or vector control. Cells were cultured in medium containing vehicle (e), NAC (f), four ribonucleosides and four deoxyribonucleosides (Nuc) (g), or both NAC and Nuc (h). Data are means ± SD (n = 3 independent experiments).

Supplementary Figure 7 G6PD levels are associated with breast cancer metastasis and a role for G6PD and TAp73 in tumor growth.

(a,b) G6PD levels are associated with breast cancer metastasis. (a) Cox multivariable regression for metastasis risk using the indicated variables was performed on a cohort of 871 breast cancer patients. Shown are the hazard ratio (HR) and p-values. (b) Kaplan-Meier survival curves for metastasis-free survival. The patients were stratified by mean G6PD levels (‘hi’ is greater than mean, ‘lo’ is less than or equal to mean). P-value was determined by the log-rank test. (c) Representative images of animals three weeks after injected with TAp73+/+ and _TAp73_−/− MEF cells stably overexpressing G6PD or vector control. (d) Mice were injected with TAp73+/+ and _TAp73_−/− MEF cells that were treated with control or G6pd siRNA, and were fed with water containing no NAC or 40 mM NAC. Representative images of animals at three weeks are shown.

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Du, W., Jiang, P., Mancuso, A. et al. TAp73 enhances the pentose phosphate pathway and supports cell proliferation.Nat Cell Biol 15, 991–1000 (2013). https://doi.org/10.1038/ncb2789

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