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 TAp73(Hi)- from TAp73(Low)-expressing tumours. These data demonstrate a pro-angiogenic role for TAp73 in supporting tumorigenesis, providing a rationale for its overexpression in cancers.

The question of how neural progenitor cells maintain its self-renewal throughout life is a fundamental problem in cell biology with implications in cancer, aging and neurodegenerative diseases. In this work, we have analyzed the p73 function in embryonic neural progenitor cell biology using the neurosphere (NS)-assay and showed that p73-loss has a significant role in the maintenance of neurosphere-forming cells in the embryonic brain. A comparative study of NS from Trp73−/−, p53KO, p53KO;Trp73−/− and their wild-type counterparts demonstrated that p73 deficiency results in two independent, but related, phenotypes: a smaller NS size (related to the proliferation and survival of the neural-progenitors) and a decreased capacity to form NS (self-renewal). The former seems to be the result of p53 compensatory activity, whereas the latter is p53 independent. We also demonstrate that p73 deficiency increases the population of neuronal progenitors ready to differentiate into neurons at the expense of depleting the pool of undifferentiated neurosphere-forming cells. Analysis of the neurogenic niches demonstrated that p73-loss depletes the number of neural-progenitor cells, rendering deficient niches in the adult mice. Altogether, our study identifies TP73 as a positive regulator of self-renewal with a role in the maintenance of the neurogenic capacity. Thus, proposing p73 as an important player in the development of neurodegenerative diseases and a potential therapeutic target.

Vasculogenesis, the establishment of the vascular plexus and angiogenesis, branching of new vessels from the preexisting vasculature, involves coordinated endothelial differentiation, proliferation and migration. Disturbances in these coordinated processes may accompany diseases such as cancer. We hypothesized that the p53 family member p73, which regulates cell differentiation in several contexts, may be important in vascular development. We demonstrate that p73 deficiency perturbed vascular development in the mouse retina, decreasing vascular branching, density and stability. Furthermore, p73 deficiency could affect non endothelial cells (ECs) resulting in reduced in vivo proangiogenic milieu. Moreover, p73 functional inhibition, as well as p73 deficiency, hindered vessel sprouting, tubulogenesis and the assembly of vascular structures in mouse embryonic stem cell and induced pluripotent stem cell cultures. Therefore, p73 is necessary for EC biology and vasculogenesis and, in particular, that DNp73 regulates EC migration and tube formation capacity by regulation of expression of pro-angiogenic factors such as transforming growth factor-β and vascular endothelial growth factors. DNp73 expression is upregulated in the tumor environment, resulting in enhanced angiogenic potential of B16-F10 melanoma cells. Our results demonstrate, by the first time, that differential p73-isoform regulation is necessary for physiological vasculogenesis and angiogenesis and DNp73 overexpression becomes a positive advantage for tumor progression due to its pro-angiogenic capacity.

The adult subventricular zone (SVZ) is a highly organized microenvironment established during the first postnatal days when radial glia cells begin to transform into type B‐cells and ependymal cells, all of which will form regenerative units, pinwheels, along the lateral wall of the lateral ventricle. Here, we identify p73, a p53 homologue, as a critical factor controlling both cell‐type specification and structural organization of the developing mouse SVZ. We describe that p73 deficiency halts the transition of the radial glia into ependymal cells, leading to the emergence of immature cells with abnormal identities in the ventricle and resulting in loss of the ventricular integrity. p73‐deficient ependymal cells have noticeably impaired ciliogenesis and they fail to organize into pinwheels, disrupting SVZ niche structure and function. Therefore, p73 is essential for appropriate ependymal cell maturation and the establishment of the neurogenic niche architecture. Accordingly, lack of p73 results in impaired neurogenesis. Moreover, p73 is required for translational planar cell polarity establishment, since p73 deficiency results in profound defects in cilia organization in individual cells and in intercellular patch orientation. Thus, our data reveal a completely new function of p73, independent of p53, in the neurogenic architecture of the SVZ of rodent brain and in the establishment of ependymal planar cell polarity with important implications in neurogenesis. © 2015 Wiley Periodicals, Inc. Develop Neurobiol 76: 730–747, 2016

In commercial dairy production, the risk of drug residues and environmental pollutants in milk from ruminants has become an outstanding problem. One of the main determinants of active drug secretion into milk is the ATP-binding cassette transporter G2/breast cancer resistance protein (ABCG2/BCRP). It is located in several organs associated with drug absorption, metabolism, and excretion, and its expression is highly induced during lactation in the mammary gland of ruminants, mice, and humans. As a consequence, potential contamination of milk could expose suckling infants to xenotoxins. In cows, a SNP for this protein affecting quality and quantity of milk production has been described previously (Y581S). In this study, our main purpose was to determine whether this polymorphism has an effect on transcellular transport of veterinary drugs because this could alter substrate pharmacokinetics and milk residues. We stably expressed the wild-type bovine ABCG2 and the Y581S variant in Madin-Darby canine kidney epithelial cells (MDCKII) and MEF3.8 cell lines generating cell models in which the functionality of the bovine transporter could be addressed. Functional studies confirmed the greater functional activity in mitoxantrone accumulation assays for the Y581S variant with a greater relative V(MAX) value (P = 0.040) and showed for the first time that the Y581S variant presents greater transcellular transport of the model ABCG2 substrate nitrofurantoin (P = 0.024) and of 3 veterinary antibiotics, the fluoroquinolone agents enrofloxacin (P = 0.035), danofloxacin (P = 0.001), and difloxacin (P = 0.008), identified as new substrates of the bovine ABCG2. In addition, the inhibitory effect of the macrocyclic lactone ivermectin on the activity of wild-type bovine ABCG2 and the Y581S variant was also confirmed, showing a greater inhibitory potency on the wild-type protein at all the concentrations tested (5 μM, P = 0.017; 10 μM, P = 0.001; 25 μM, P = 0.008; and 50 μM, P = 0.003). Differential transport activity depending on the genotype together with the differential inhibition pattern might have clinical consequences, including changes in substrate pharmacokinetics (and subsequently pharmacodynamics) and more specifically, changes in secretion of ABCG2 substrates into milk, potentially implying important consequences to veterinary therapeutics.

Activation of the epidermal growth factor receptor (EGFR) has been shown to occur by ligand-dependent and ligand-independent mechanisms. Different molecular mechanisms have been found to be responsible for ligand-independent receptor transactivation. Here, we show that hyperosmolar concentrations of sorbitol activate the EGFR in human keratinocytes. Experiments using specific inhibitors of EGFR phosphorylation show that the increased amount of activated receptors is the result of a decreased rate of dephosphorylation. Furthermore, sorbitol treatment results in a strong activation of stress kinase p38. Treatment of the cells with SB203580, a known inhibitor of p38 alpha and beta kinases, results in impairment of receptor activation, indicating that the stress kinase is involved in receptor activation modulation. This is further reinforced by experiments showing that addition of Toxin B, known to be an inhibitor of the small Rho GTPases rac1, cdc42, and Rho A/B, to the cells results in a strong induction of EGFR activation. Our results point, therefore, to a mechanism by which osmotic shock activates EGFR through the small Rho GTPases-p38 stress kinase pathway.

Purpose: Cumulative data support the role of DTAp73 variants in tumorigenic processes such as drug resistance. We evaluate the impact of TP73 isoforms and their putative target genes ABCB1, HMGB1, and CASP1 on the survival of colon cancer patients and the correlation between their expressions.Experimental Design: We determined in 77 colon cancer patients the expression of DEx2p73, DEx2/ 3p73, DNp73, TAp73, ABCB1, HMGB1, and CASP1 by quantitative real-time reverse transcriptase-PCR. Tumor characteristics, disease-free survival, and overall survival (OS) were examined in each patient. Functional experiments were carried out to check whether ectopic expression of DNp73 modifies the proliferation, drug resistance, migration, and invasion properties of colon tumor cells and the expression of ABCB1, HMGB1, and CASP1.Results: Positive correlations were observed between the expression levels of DTAp73 variants and HMGB1. Furthermore, a trend was observed for ABCB1. Overexpression of DEx2/3p73 and DNp73 isoforms was significantly associated with advanced stages (P ¼ 0.04 and P ¼ 0.03, respectively) and predicted shortened OS (P ¼ 0.04 and P ¼ 0.05, respectively). High levels of ABCB1 and HMGB1 were associated with shorter OS (P ¼ 0.04 and P ¼ 0.05, respectively). Multivariate analysis showed that, in addition to the tumor stage, ABCB1 and HMGB1 had independent relationships with OS (P ¼ 0.008). Ectopic expression of DNp73 was associated with an increase in proliferation and drug resistance.Conclusions: The positive correlation between DTAp73 variants and HMGB1 and ABCB1 expression supports them as TP73 targets. The fact that upregulation of DTAp73 isoforms was associated with shortened OS, increase in proliferation, and drug resistance confirms their oncogenic role and plausible value as prognostic markers. ABCB1 and HMGB1, putative DTAp73 target genes, strongly predict OS in an independent manner, making clear the importance of studying downstream TP73 targets that could predict the outcome of colon cancer patients better than DTAp73 variants themselves do.

Planar cell polarity (PCP) and intercellular junctional complexes establish tissue structure and coordinated behaviors across epithelial sheets. In multiciliated ependymal cells, rotational and translational PCP coordinate cilia beating and direct cerebrospinal fluid circulation. Thus, PCP disruption results in ciliopathies and hydrocephalus. PCP establishment depends on the polarization of cytoskeleton and requires the asymmetric localization of core and global regulatory modules, including membrane proteins like Vangl1/2 or Frizzled. We analyzed the subcellular localization of select proteins that make up these modules in ependymal cells and the effect of Trp73 loss on their localization. We identify a novel function of the Trp73 tumor suppressor gene, the TAp73 isoform in particular, as an essential regulator of PCP through the modulation of actin and microtubule cytoskeleton dynamics, demonstrating that Trp73 is a key player in the organization of ependymal ciliated epithelia. Mechanistically, we show that p73 regulates translational PCP and actin dynamics through TAp73-dependent modulation of non-musclemyosin-II activity. In addition, TAp73 is required for the asymmetric localization of PCP-core and global signaling modules and regulates polarized microtubule dynamics, which in turn set up the rotational PCP. Therefore, TAp73 modulates, directly and/or indirectly, transcriptional programs regulating actin and microtubules dynamics and Golgi organization signaling pathways. These results shed light into the mechanism of ependymal cell planar polarization and reveal p73 as an epithelial architect during development regulating the cellular cytoskeleton.