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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Melino, G., De Laurenzi, V. & Vousden, K. H. p73: friend or foe in tumorigenesis. Nat. Rev. Cancer 2, 605–615 (2002).
Stiewe, T. & Pützer, B. M. Role of p73 in malignancy: tumor suppressor or oncogene? Cell Death Differ. 9, 237–245 (2002).
Bisso, A., Collavin, L. & Del Sal, G. p73 as a pharmaceutical target for cancer therapy. Curr. Pharm. Des. 17, 578–590 (2011).
Lin, K. W., Nam, S. Y., Toh, W. H., Dulloo, I. & Sabapathy, K. Multiple stress signals induce p73beta accumulation. Neoplasia 6, 546–557 (2004).
Tomasini, R. et al. TAp73 knockout shows genomic instability with infertility and tumor suppressor functions. Genes Dev. 22, 2677–2691 (2008).
Levrero, M. et al. The p53/p63/p73 family of transcription factors: overlapping and distinct functions. J. Cell Sci. 113, 1661–1670 (2000).
Buhlmann, S. & Pützer, B. M. DNp73 a matter of cancer: mechanisms and clinical implications. Biochim. Biophys. Acta 1785, 207–216 (2008).
Gong, J. G. et al. The tyrosine kinase c-Abl regulates p73 in apoptotic response to cisplatin-induced DNA damage. Nature 399, 806–809 (1999).
Agami, R., Blandino, G., Oren, M. & Shaul, Y. Interaction of c-Abl and p73α and their collaboration to induce apoptosis. Nature 399, 809–813 (1999).
Yuan, Z. M. et al. p73 is regulated by tyrosine kinase c-Abl in the apoptotic response to DNA damage. Nature 399, 814–817 (1999).
Donehower, L. A. et al. Mice deficient for p53 are developmentally normal but susceptible to spontaneous tumours. Nature 356, 215–221 (1992).
Zaika, A. I., Kovalev, S., Marchenko, N. D. & Moll, U. M. Overexpression of the wild type p73 gene in breast cancer tissues and cell lines. Cancer Res. 59, 3257–3263 (1999).
Kang, M. J. et al. Loss of imprinting and elevated expression of wild-type p73 in human gastric adenocarcinoma. Clin. Cancer Res. 6, 1767–1771 (2000).
Vikhanskaya, F. et al. p73 supports cellular growth through c-Jun-dependent AP-1 transactivation. Nat. Cell Biol. 9, 698–705 (2007).
Toh, W. H., Logette, E., Corcos, L. & Sabapathy, K. TAp73β and DNp73β activate the expression of the pro-survival caspase-2S. Nucleic Acids Res. 36, 4498–5509 (2008).
Du, W. et al. TAp73 enhances the pentose phosphate pathway and supports cell proliferation. Nat. Cell. Biol. 15, 991–1000 (2013).
Lefkimmiatis, K. et al. p73 and p63 sustain cellular growth by transcriptional activation of cell cycle progression genes. Cancer Res. 69, 8563–8571 (2009).
Bertout, J. A., Patel, S. A. & Simon, M. C. The impact of O2 availability on human cancer. Nat. Rev. Cancer 8, 967–975 (2008).
Semenza, G. L. Defining the role of hypoxia-inducible factor 1 in cancer biology and therapeutics. Oncogene 29, 625–634 (2010).
Semenza, G. L. Targeting HIF-1 for cancer therapy. Nat. Rev. Cancer 3, 721–732 (2003).
Yan, Y. et al. Inhibitors of ubiquitin-activating enzyme (E1), a new class of potential cancer therapeutics. Cancer Res. 67, 9472–9481 (2007).
Pagano, M. et al. Role of the ubiquitin-proteasome pathway in regulating abundance of the cyclin-dependent kinase inhibitor p27. Science 269, 682–685 (1995).
Kaelin, W. G. Jr The von Hippel–Lindau tumour suppressor protein: O2 sensing and cancer. Nat. Rev. Cancer 8, 865–873 (2008).
Chew, E. H., Poobalasingam, T., Hawkey, C. J. & Hagen, T. Characterization of cullin-based E3 ubiquitin ligases in intact mammalian cells–evidence for cullin dimerization. Cell. Signal. 19, 1071–1080 (2007).
Narita, T. et al. Identification of a novel small molecule HIF-1α translation inhibitor. Clin. Cancer Res. 15, 6128–6136 (2009).
Kung, A. L. et al. Small molecule blockade of transcriptional coactivation of the hypoxia-inducible factor pathway. Cancer Cell 6, 33–43 (2004).
Hu, G. & Fearon, E. R. Siah-1 N-terminal RING domain is required for proteolysis function, and C-terminal sequences regulate oligomerization and binding to target proteins. Mol. Cell. Biol. 19, 724–732 (1999).
Pugh, C. W. & Ratcliffe, P. J. Regulation of angiogenesis by hypoxia: role of the HIF system. Nat. Med. 9, 677–684 (2003).
Liao, D. & Johnson, R. S. Hypoxia: a key regulator of angiogenesis in cancer. Cancer Metastasis Rev. 26, 281–290 (2007).
Wernig, M. et al. In vitro reprogramming of fibroblasts into a pluripotent ES-cell-like state. Nature 448, 318–324 (2007).
Battegay, E. J., Rupp, J., Iruela-Arispe, L., Sage, E. H. & Pech, M. PDGF-BB modulates endothelial proliferation and angiogenesis in vitro via PDGF beta-receptors. J. Cell Biol. 125, 917–928 (1994).
Ten Dijke, P., Goumans, M. J. & Pardali, E. Endoglin in angiogenesis and vascular diseases. Angiogenesis 11, 79–89 (2008).
Weckbach, L. T. et al. Midkine acts as proangiogenic cytokine in hypoxia-induced angiogenesis. Am. J. Physiol. Heart Circ. Physiol. 303, H429–H438 (2012).
Rosenbluth, J. M., Mays, D. J., Pino, M. F., Tang, L. J. & Pietenpol, J. A. A gene signature-based approach identifies mTOR as a regulator of p73. Mol. Cell. Biol. 28, 5951–5964 (2008).
Koeppel, M. et al. Crosstalk between c-Jun and TAp73α/β contributes to the apoptosis-survival balance. Nucleic Acids Res. 39, 6069–6085 (2011).
Vikhanskaya, F. et al. p73 overexpression increases VEGF and reduces thrombospondin-1 production: implications for tumor angiogenesis. Oncogene 20, 7293–7300 (2001).
Zhang, L. et al. Wild-type p53 suppresses angiogenesis in human leiomyosarcoma and synovial sarcoma by transcriptional suppression of vascular endothelial growth factor expression. Cancer Res. 60, 3655–3661 (2000).
Chen, C., Pore, N., Behrooz, A., Ismail-Beigi, F. & Maity, A. Regulation of glut1 mRNA by hypoxia-inducible factor-1. Interaction between H-ras and hypoxia. J. Biol. Chem. 276, 9519–9525 (2001).
Potter, C. & Harris, A. L. Hypoxia inducible carbonic anhydrase IX, marker of tumour hypoxia, survival pathway and therapy target. Cell Cycle 3, 164–167 (2004).
Li, C. W. et al. Role of p63/p73 in epithelial remodeling and their response to steroid treatment in nasal polyposis. J. Allergy Clin. Immunol. 127, 765–772 (2011).
Hsu, Y. C. et al. Increased expression of hypoxia-inducible factor 1α in the nasal polyps. Am. J. Otolaryngol. 28, 379–383 (2007).
Ooi, C. H. et al. Oncogenic pathway combinations predict clinical prognosis in gastric cancer. PLoS Genet. 5, e1000676 (2009).
Kitadai, Y. Angiogenesis and lymphangiogenesis of gastric cancer. J. Oncol. 2010, 468725 (2010).
DiComo, C. J., Gaiddon, C. & Prives, C. p73 function is inhibited by tumor-derived p53 mutants in mammalian cells. Mol. Cell. Biol. 19, 1438–1449 (1999).
Pozniak, C. D. et al. An anti-apoptotic role for the p53 family member, p73, during developmental neuron death. Science 289, 304–306 (2000).
Weiss, R. H. & Howard, L. L. p73 is a growth-regulated protein in vascular smooth muscle cells and is present at high levels in human atherosclerotic plaque. Cell Signal. 13, 727–733 (2001).
Fernandez-Alonso, R. et al. p73 is required for endothelial cell differentiation, migration and the formation of vascular networks regulating VEGF and TGFβ signaling. Cell Death Differ. (2015)10.1038/cdd.2014.214
Amelio, I. et al. TAp73 opposes tumor angiogenesis by promoting hypoxia-inducible factor 1α degradation.. Proc. Natl Acad. Sci. USA 112, 226–231 (2015).
Stantic, M. et al. TAp73 suppresses tumor angiogenesis through repression of proangiogenic cytokines and HIF-1α activity. Proc. Natl Acad. Sci. USA 112, 220–2225 (2015).
Nakayama, K. et al. Siah2 regulates stability of prolyl-hydroxylases, controls HIF1α abundance, and modulates physiological responses to hypoxia. Cell 117, 941–952 (2004).
Senoo, M., Matsumura, Y. & Habu, S. TAp63γ (p51A) and dNp63α (p73L), two major isoforms of the p63 gene, exert opposite effects on the vascular endothelial growth factor (VEGF) gene expression. Oncogene 21, 2455–2465 (2002).
Farhang, G. M., Goossens, S. & Haigh, J. J. The p53 family and VEGF regulation: ”It’s complicated”. Cell Cycle 12, 1331–1332 (2013).
Salimath, B., Marmé, D. & Finkenzeller, G. Expression of the vascular endothelial growth factor gene is inhibited by p73. Oncogene 20, 7293–7300 (2001).
Balint, E., Phillips, A. C., Kozlov, S., Stewart, C. L. & Vousden, K. H. Induction of p57(KIP2) expression by p73beta. Proc. Natl Acad. Sci. USA 99, 3529–3534 (2002).
Woltjen, K. et al. piggyBac transposition reprograms fibroblasts to induced pluripotent stem cells. Nature 458, 766–770 (2009).
Hsiao, T. H. et al. Utilizing signature-score to identify oncogenic pathways of cholangiocarcinoma. Trans. Cancer Res. 2, 6–17 (2013).
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.
The authors declare no competing financial interests.
Integrated supplementary information
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.
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.
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.
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.
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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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