Hypoxia-inducible TAp73 supports tumorigenesis by regulating the angiogenic transcriptome

Abstract

The functional significance of the overexpression of unmutated TAp73, a homologue of the tumour suppressor p53, in multiple human cancers is unclear, but raises the possibility of unidentified roles in promoting tumorigenesis. We show here that TAp73 is stabilized by hypoxia, a condition highly prevalent in tumours, through HIF-1α-mediated repression of the ubiquitin ligase Siah1, which targets TAp73 for degradation. Consequently, TAp73-deficient tumours are less vascular and reduced in size, and conversely, TAp73 overexpression leads to increased vasculature. Moreover, we show that TAp73 is a critical regulator of the angiogenic transcriptome and is sufficient to directly activate the expression of several angiogenic genes. Finally, expression of TAp73 positively correlates with these angiogenic genes in several human tumours, and the angiogenic gene signature is sufficient to segregate the TAp73Hi- from TAp73Low-expressing tumours. These data demonstrate a pro-angiogenic role for TAp73 in supporting tumorigenesis, providing a rationale for its overexpression in cancers.

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Figure 1: Hypoxia induces the stabilization of TAp73.
Figure 2: HIF-1α is required for TAp73 stabilization in response to hypoxia.
Figure 3: HIF-1α suppresses siah1 to regulate TAp73 on hypoxia.
Figure 4: p73 is required for efficient vasculature in tumours.
Figure 5: The angiogenic transcriptome is regulated by TAp73.
Figure 6: TAp73 regulates angiogenic genes independent of HIF-1α and its apoptotic potential.
Figure 7: Positive correlation between TAp73, Vegf-A and HIF-1α protein expression in human tumours.
Figure 8: Correlation of the expression of TAp73, SIAH1 and several angiogenic genes in clinical gastric tumour samples.

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Acknowledgements

We thank Y. Li (NUS), E. Mationg-Kalaw (SGH Path-NCCS Translational Laboratory) and M. Koh (Singhealth Tissue Repository) for technical assistance, and C. Lim and X. Min for help with statistical analysis. We are grateful to F. Mechta-Grigoriou, T. Hagen, S-I. Matsuzawa and T. W. Mak for the vegf-A promoter–luciferase, dnHIF-1α, siah1 WT/ΔRING mutant plasmids, and the TAp73−/− and TAp73+/+ MEFs, respectively. We also acknowledge the Advanced Molecular Pathology Laboratory (AMPL) for the histological services provided. This study was supported by grants from the National Medical Research Council to K.S., and by grant SAF2012-36143 from the Spanish Ministerio de Economía y Competitividad, co-financed by FEDER funds to M.C.M.

Author information

I.D. designed the experiments, performed the research work, analysed the data and helped with writing of the manuscript. B.H.P. and R.O. performed the research work and analysed the data. S.Y.T., C.W.L. and D.Y.W. performed the immunohistochemistry work on human samples. A.V. and L.K.G. performed the gastric cancer data set analysis. M.M-L., M.M.M. and M.C.M. performed the experiments related to iPSC generation and the associated teratoma studies. W.X. and F.M. provided the TAp73 antibody and advised on its use. K.S. designed the experiments, analysed the data, supervised the project and wrote the manuscript. All authors reviewed the manuscript.

Correspondence to Kanaga Sabapathy.

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The authors declare no competing financial interests.

Integrated supplementary information

Supplementary Figure 8 VHL is dispensable for oxygen-mediated expression of TAp73.

A Levels of transiently transfected TAp73α and TAp73β in H1299 cells were assessed by immunoblotting (IB) upon exposure to 1% O2 for 24 h (right panel). Saos-2 TAp73α-inducible cells were induced with 2 μg ml-1 of doxycycline for 24 h in 1% O2 and TAp73 levels were determined by IB (left panel). B,C Effects of 1% O2 and reoxygenation (Reox.) with 21% O2 on transfected TAp73β in H1299 in which vhl expression was silenced by siRNA-mediated silencing (using 2 independent siRNAs) (B) was assessed by IB. Similarly, HEK293 cells inducibly expressing the dominant negative form of Ubc12 which inhibits all cullin-based E3 ligases (left panel), or H1299 cells transiently transfected with HA-Ubc12 (right panel) (C) were used to analyze effect on TAp73β. Arrowhead represents Vhl and represents non-specific band in H1299 blot (B). Blots are representative of 3 independent experiments. Uncropped images of blots/gels are shown in Supplementary Fig. 6.

Supplementary Figure 9 Requirement of HIF-1α for suppression of Siah1 upon hypoxia.

A Evaluation of the effect of 1% O2 on expression of selected candidate E3 ligases in H1299 cells after 24 h by real-time qPCR. B Effects of silencing hif-1α and/or hif-1β in H1299 cells on siah1 and glut-1 were determined by real-time qPCR upon hypoxia. C,D Effects of treatment with chetomin (CHT) (C) or silencing hif-1α by siRNA (D) on other E3 ligases were determined by real-time qPCR. E Levels of TAp73β upon hypoxia and reoxygenation were determined after silencing the expression of siah1 using two different siRNAs, and levels of siah1 were determined by real-time qPCR. Data from one experiment is shown as average of two biological replicates from two separate cellular extracts, which yielded similar results (for Supplementary Fig. 2a–e) . All data are representative of 3 independent experiments. Uncropped images of blots/gels are shown in Supplementary Fig. 6.

Supplementary Figure 10 TAp73 regulates angiogenesis.

A Semi-quantitative RT-PCR shows the expression of TAp73 and DNp73 in wild-type, TAp73−/− and DNp73−/− MEFs. Arrowheads show TAp73 and DNp73 transcripts. B,C TAp73 and DNp73 transcripts in WT and p73-deficient (KO) induced pluripotent stem (iPS) cells were determined by real-time qPCR analyses (WT iPSC n = 8 clones, p73KO iPSC n = 5 clones) (B, left panel). Morphology of the iPS cells is shown on the right. Scale bars: 250 μm. (B). Kinetics of proliferation of these cells were determined by cellular counting over several passages (C). Bars represent mean values ± s.d.; experiments were repeated three times with two replicas each sample. (P < 0.001 by Student’s t-test). D Tumors generated by injection with H1299-TAp73β-inducible cells into SCID mice were stained for TUNEL and Ki67, and representative pictures are shown. Scale bars: 100 μm. Right most panels show quantification using Angiosight (n = 3 tumors (− Dox),n = 5 tumors (+ Dox), p = n.s by Student’s t-test). Data are presented as means ± s.d. E Saos2-TAp73β-inducible cells were subcutaneously injected into SCID mice and once tumors grew to a size of about 100 mm-3, mice were then divided into two groups: one control group (n = 3 mice) and one group where mice were gavaged with 2 mg ml-1 of doxycycline daily for 6 days (n = 3 mice) before tumors were harvested for immunohistochemical (IHC) analysis. Tumors were stained for H&E, TAp73 and CD34. Scale bars: 100 μm. Uncropped images of blots/gels are shown in Supplementary Fig. 6.

Supplementary Figure 11 TAp73 is required and sufficient to regulate expression of angiogenic genes.

A,B Real-time qPCR analysis was carried out for several pro-angiogenic genes using TAp73+/+ and TAp73−/− MEFs (n = 4 biological replicates from four separate cellular extracts) (A) or upon silencing of p73 by siRNA in H1299 cells (B). (P < 0.05 by unpaired Mann-Whitney test in A). Data are presented as means ± s.d. for A. C Expression of selected angiogenic genes upon siah1 silencing in TAp73+/+ and TAp73−/− MEFs was determined by real-time qPCR. Expression levels are shown as fold-change based on control scrambled siRNA levels. (Data from one of n = 3 independent experiments is shown). D,E Expression of several pro-angiogenic genes was assessed using Saos-2-TAp73β inducible cells induced for 6 h or 10 h (D), or in Saos2 cells expressing TAp73α, TAp73β or p53, after 8 h and 24 h (E), by real-time qPCR. Lower panel shows expression of TAp73α, TAp73β or p53 by IB (E). F Table showing details of pro-angiogenic genes found to have potential TAp73 binding sites in their genomic regulatory elements after analysis of chromatin immunoprecipitation (ChIP)-Seq data from Koeppel et al., 201135. Data from one experiment is shown as average of two biological replicates from two separate cellular extracts, which yielded similar results (for Supplementary Fig. 4b, d, e). All data are representative of 3 independent experiments. Uncropped images of blots/gels are shown in Supplementary Fig. 6.

Supplementary Figure 12 p73 expression correlates with angiogenic genes in human clinical samples.

A Effect of TAp73-specific blocking peptide was analysed by immunohistochemical staining with non-related antibodies against MNF116 or Ki67, in both normal and colonic adenocarcinoma tissues in the presence or absence of the peptide. Scale bars: 0.9 μm. B Expression of TAp73, Vegf-A, CAIX (another hypoxic marker) and HIF-1α in nasal poly samples from patients with nasal polyposis (n = 4 samples). Scale bars: 50 μm. Representative pictures are shown.

Supplementary Figure 13 Uncropped gel pictures for all gels in the article.

Supplementary Table 1 Differential regulation of several physiological processes by TAp73 in MEFs.
Supplementary Table 2 49-angiogenic genes signature from TAp73 differentially regulated genes.
Supplementary Table 3 Molecular networks associated with the 49-angiogenic genes signature.
Supplementary Table 4 Molecular networks associated with the 28-pro-angiogenic genes signature.
Supplementary Table 5 List of 28 pro-angiogenic genes used for further validation.
Supplementary Table 6 Selected genes with most significant correlation in gastric cancer dataset.
Supplementary Table 7 List of primers (mouse and human).

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Dulloo, I., Phang, B., Othman, R. et al. Hypoxia-inducible TAp73 supports tumorigenesis by regulating the angiogenic transcriptome. Nat Cell Biol 17, 511–523 (2015). https://doi.org/10.1038/ncb3130

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