TAp73 opposes tumor angiogenesis by promoting hypoxia-inducible factor 1α degradation

Abstract

Tumor hypoxia and hypoxia-inducible factor 1 (HIF-1) activation are associated with cancer progression. Here, we demonstrate that the transcription factor TAp73 opposes HIF-1 activity through a nontranscriptional mechanism, thus affecting tumor angiogenesis. TAp73-deficient mice have an increased incidence of spontaneous and chemically induced tumors that also display enhanced vascularization. Mechanistically, TAp73 interacts with the regulatory subunit (α) of HIF-1 and recruits mouse double minute 2 homolog into the protein complex, thus promoting HIF-1α polyubiquitination and consequent proteasomal degradation in an oxygen-independent manner. In human lung cancer datasets, TAp73 strongly predicts good patient prognosis, and its expression is associated with low HIF-1 activation and angiogenesis. Our findings, supported by in vivo and clinical evidence, demonstrate a mechanism for oxygen-independent HIF-1 regulation, which has important implications for individualizing therapies in patients with cancer.

Keywords: VEGF; p53 family; p73; tumor progression; tumor vascularization.

Fig. 1.

TAp73 depletion drives tumor progression. (A) Immunofluorescence staining for keratin-14 (green), lectin (red), and DAPI (blue) shows dermic (yellow arrows) and peritumoral (white arrow) vascularization in TAp73^+/+ and TAp73^−/− mice. (Scale bar, 50 μm.) (B) Peritumoral vessel density was assessed through the digital measurement of the area covered by tumor-associated vessels (relative mean ± SD, n = 3 per genotype, two-tailed unpaired t test, P = 0.018). (C and D) The aortic ring assay of TAp73^−/− mice shows an increased propensity to form new vessels (relative mean ± SD, n = 3 per genotype, two-tailed unpaired t test, *P < 0.05, **P < 0.01).

Fig. 2.

p73 interacts with HIF-1α and promotes its ubiquitin-dependent degradation. (A) p73 knockdown increased endogenous HIF-1α levels in the H1299 cells. (B) TAp73β overexpression reduced endogenous HIF-1α protein levels in SaOs Tet-On cells under basal conditions. (C) TAp73β reduced HIF-1α protein levels in both normoxic (20% O₂) and hypoxic (1% O₂) conditions. (D) TAp73β overexpression reduces endogenous HIF-1α protein levels in the VHL-mutant RCC4 parental cell line and the VHL-overexpressing RCC4 cell line. (E) Immunoprecipitation in H1299 in the presence of proteasomal inhibitor MG132 and cobalt chloride of endogenous p73 demonstrates the protein–protein interaction between TAp73 and HIF-1α. (F) TAp73β overexpression increased HIF-1α polyubiquitination in H1299 cells in the presence of CoCl. (G) Schematic representation of HIF-1α fragments generated to map the binding region of HIF-1α with TAp73. (H) Immunoprecipitation in H1299 cells of Myc-tagged HIF-1α fragments demonstrates the involvement of N-terminal bHLH/PAS domain of HIF-1α in the interaction with TAp73. (I) TAp73 drives the E3 Ub ligase MDM2 to HIF-1α. Immunoprecipitation shows that TAp73α facilitated the MDM2 interaction with HIF-1α. (J) Myc-HIF-1α immunoprecipitation in siTAp73-transfected H1299 cells demonstrated that TAp73 depletion impaired HIF1α-MDM2 interaction. (K) MDM2 knockdown in H1299 cells partially affects the TAp73-dependent HIF-1α degradation. Actin or GAPDH were used as loading controls.

Fig. 3.

TAp73 predicts patient survival and correlates with HIF-1 activity in the human cancer. (A) A Kaplan-Meier graph representing the probability of survival in patients with lung adenocarcinoma (dataset, GSE31210), stratified according to p73 expression levels. TAp73 strongly predicts a good patient prognosis (TAp73 group, n = 45; no-p73 group, n = 64; ΔNp73 group, n = 86; *P = 0.018). (B and C) Geneset enrichment analysis indicated negative enrichment of “HIF-1α target genes” and “angiogenesis pathway” in tumors expressing high TAp73 levels. Numbers in the table indicate P values.

Fig. 4.

TAp73 affects HIF-1 signaling in tumors. (A and B) Xenograft tumors derived from transplantation of SaOs-2 Tet on TAp73α in nu/nu mice. TAp73 expression was induced by oral doxycycline administration. Tumor weight was assessed 5 wk after inoculation (mean ± SD, n = 4 per group, two-tailed unpaired t test, P < 0.05). (C and D) Xenograft tumors derived from transplantation of H1299 cells stably transfected with shCTR or shTAp73. TAp73 depletion increased the tumor weight of H1299-derived tumors (n = 4 per group, two-tailed unpaired t test, P < 0.05). (E) A heat map of “HIF-1 Response” and “Growth Factor Activity” genes identified by GO term analysis, as differentially expressed between TAp73-intact and TAp73-depleted tumors (n = 4 per group). (F) Immunofluorescence staining for lectin (red) and DAPI (blue) indicated intratumoral vascularization in shCTR-H1299-derived or shTAp73-H1299-derived tumor xenograft tissues. (Scale bar, 100 μm.) (G) Intratumoral vessel density was assessed by digital measurement of the area covered by lectin-positive vessels (mean ± SD of n = 4 animals per group, two-tailed unpaired t test, *P < 0.01). (H) “High TAp73” fresh specimens from patients with lung cancer exhibited lower HIF-1α and VEGF-A protein levels compared with “Low TAp73” samples; GAPDH was used as a loading control.