The nucleolus directly regulates p53 export and degradation

Nucleoli directly regulate p53 export and degradation rather than simply sequestering p53 regulatory factors

TP53 mutations in head and neck cancer cells determine the Warburg phenotypic switch creating metabolic vulnerabilities and therapeutic opportunities for stratified therapies

Patients with mutated TP53 have been identified as having comparatively poor outcomes compared to those retaining wild-type p53 in many cancers, including squamous cell carcinomas of the head and neck (SCCHN). We have examined the role of p53 in regulation of metabolism in SCCHN cells and find that loss of p53 function determines the Warburg effect in these cells. Moreover, this metabolic adaptation to loss of p53 function creates an Achilles’ heel for tumour cells that can be exploited for potential therapeutic benefit. Specifically, cells lacking normal wild-type p53 function, whether through mutation or RNAi-mediated downregulation, display a lack of metabolic flexibility, becoming more dependent on glycolysis and losing the ability to increase energy production from oxidative phosphorylation. Thus, cells that have compromised p53 function can be sensitised to ionizing radiation by pre-treatment with a glycolytic inhibitor. These results demonstrate the deterministic role of p53 in regulating energy metabolism and provide proof of principle evidence for an opportunity for patient stratification based on p53 status that can be exploited therapeutically using current standard of care treatment with ionising radiation

Postnatal Changes in the Expression Pattern of the Imprinted Signalling Protein XLαs Underlie the Changing Phenotype of Deficient Mice

The alternatively spliced trimeric G-protein subunit XLαs, which is involved in cAMP signalling, is encoded by the Gnasxl transcript of the imprinted Gnas locus. XLαs deficient mice show neonatal feeding problems, leanness, inertia and a high mortality rate. Mutants that survive to weaning age develop into healthy and fertile adults, which remain lean despite elevated food intake. The adult metabolic phenotype can be attributed to increased energy expenditure, which appears to be caused by elevated sympathetic nervous system activity. To better understand the changing phenotype of Gnasxl deficient mice, we compared XLαs expression in neonatal versus adult tissues, analysed its co-localisation with neural markers and characterised changes in the nutrient-sensing mTOR1-S6K pathway in the hypothalamus. Using a newly generated conditional Gnasxl lacZ gene trap line and immunohistochemistry we identified various types of muscle, including smooth muscle cells of blood vessels, as the major peripheral sites of expression in neonates. Expression in all muscle tissues was silenced in adults. While Gnasxl expression in the central nervous system was also developmentally silenced in some midbrain nuclei, it was upregulated in the preoptic area, the medial amygdala, several hypothalamic nuclei (e.g. arcuate, dorsomedial, lateral and paraventricular nuclei) and the nucleus of the solitary tract. Furthermore, expression was detected in the ventral medulla as well as in motoneurons and a subset of sympathetic preganglionic neurons of the spinal cord. In the arcuate nucleus of Gnasxl-deficient mice we found reduced activity of the nutrient sensing mTOR1-S6K signalling pathway, which concurs with their metabolic status. The expression in these brain regions and the hypermetabolic phenotype of adult Gnasxl-deficient mice imply an inhibitory function of XLαs in energy expenditure and sympathetic outflow. By contrast, the neonatal phenotype of mutant mice appears to be due to a transient role of XLαs in muscle tissues

Regulation of p53 and MDM2 Activity by MTBP

p53 is a critical coordinator of a wide range of stress responses. To facilitate a rapid response to stress, p53 is produced constitutively but is negatively regulated by MDM2. MDM2 can inhibit p53 in multiple independent ways: by binding to its transcription activation domain, inhibiting p53 acetylation, promoting nuclear export, and probably most importantly by promoting proteasomal degradation of p53. The latter is achieved via MDM2's E3 ubiquitin ligase activity harbored within the MDM2 RING finger domain. We have discovered that MTBP promotes MDM2-mediated ubiquitination and degradation of p53 and also MDM2 stabilization in an MDM2 RING finger-dependent manner. Moreover, using small interfering RNA to down-regulate endogenous MTBP in unstressed cells, we have found that MTBP significantly contributes to MDM2-mediated regulation of p53 levels and activity. However, following exposure of cells to UV, but not γ-irradiation, MTBP is destabilized as part of the coordinated cellular response. Our findings suggest that MTBP differentially regulates the E3 ubiquitin ligase activity of MDM2 towards two of its most critical targets (itself and p53) and in doing so significantly contributes to MDM2-dependent p53 homeostasis in unstressed cells

Senescence induction in renal carcinoma cells by Nutlin-3: a potential therapeutic strategy based on MDM2 antagonism.

Although the role of p53 as a tumour suppressor in renal cell carcinoma (RCC) is unclear, our recent analysis suggests that increased wild-type p53 protein expression is associated with poor outcome. A growing body of evidence also suggests that p53 expression and increased co-expression of MDM2 are linked with poor prognosis in RCC. We have therefore examined whether an MDM2 antagonist; Nutlin-3, might rescue/increase p53 expression and induce growth inhibition or apoptosis in RCC cells that retain wild-type p53. We show that inhibition of p53 suppression by MDM2 in RCC cells promotes growth arrest and p53-dependent senescence - phenotypes known to mediate p53 tumour suppression in vivo. We propose that future investigations of therapeutic strategies for RCC should incorporate MDM2 antagonism as part of strategies aimed at rescuing/augmenting p53 tumour suppressor function

XLαs co-localisation with hypothalamic neuropeptides and assessment of arcuate nucleus mTOR1-S6K activity in <i>Gnasxl</i>^m+/p− mice.

<p>(<b>A</b>) A representative image of co-localisation of XLαs (green) with Orexin A (red) in the DMH/LH is shown (i) and quantification of co-localised cell body staining is provided on the right (ii). (<b>B</b>) XLαs (purple) and MCH (brown) are expressed in separate neuron populations in the DMH/LH. (<b>C, D</b>) Immunofluorescence staining for XLαs (C) and <i>in situ</i> hybridisation for CRH (D) in neighbouring hypothalamic sections indicate separate expression domains in peripheral and central subdivisions of the PVH, respectively. (<b>E</b>) Co-localisation of pS6, a substrate and marker of mTOR1-S6K activity, in XLαs-positive neurons of wild-type Arc tissue. A representative image (i) and quantification (ii) of a series of sections from two mice (age 12 weeks) is shown. ∼32% of XLαs-positive neurons show pS6 signals under <i>ad libitum</i> normal chow fed conditions. (<b>F–H</b>) Reduced number of pS6 positive neurons in the Arc of <i>Gnasxl</i>^m+/p− mice (aged 12 weeks on normal chow diet). Representative images of pS6 immunofluorescence of wild-type (F) and <i>Gnasxl</i>^m+/p− (G) Arc sections are shown, and a quantification is provided in (H), (n = 6; p = 0.036). See <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0029753#s2" target="_blank">materials and methods</a> for details of the quantification method.</p

Generation of a conditional gene trap mouse line for

<p> (<b>A</b>) The targeting construct comprises the <i>Gnas</i> exon 2 splice acceptor (Ex2SA) fused in frame to LacZ-pA, an frt-flanked <i>neo</i>^r cassette and pairs of loxP/lox2272 sites assembled in head-to-head orientation. The gene trap cassette was inserted in the non-functional (antisense) orientation at the rarely used A20 exon position (see D–F). Cre recombination results in inversion at compatible lox sites followed by excision between head-to-tail sites, which results in a stably integrated gene trap that attracts splicing from <i>Gnasxl</i> exon 1. The resulting XL domain – β-Galactosidase (XL-βGal) fusion protein lacks XLαs function, but retains β-Galactosidase activity. Relative positions of probes and restriction sites used in Southern-blots are shown, as are PCR primers (arrows) used in genotyping. (<b>B</b>) Southern blots showing a correctly targeted ES-cell clone (<i>Spe</i>I: WT = 16.1 kbp, targeted = 20.4 kbp; <i>Afl</i>II: targeted = 13.7 kbp; <i>Mfe</i>I: targeted = 8.8 kb). (<b>C</b>) Genotyping PCRs for deletion of the <i>neo</i>^r cassette via <i>Flpe</i> mice (left), and for Cre recombinase mediated inversion of the gene trap (right). The Flpe / Cre status of the samples is given above the lanes. For primer locations see (A). (<b>D</b>) Scheme indicating splicing of the rarely used A20 exon in full-length <i>Gnasxl</i> and neural-specific <i>XLN1</i> transcripts. Arrows indicate primers used in (E). (<b>E</b>) RT-PCR from wild-type brain using a common <i>Gnasxl</i> exon 1 primer combined with reverse primers in exon 5 (full-length <i>Gnasxl</i>) or exon N1 (<i>XLN1</i>). Exon A20-containing products (size increase: 95 bp) are indicated by asterisks above the respective bands and are hardly detectable in full-length transcripts (XL lanes), but are more prominent in <i>XLN1</i> transcripts (N1 lanes). (<b>F</b>) Inclusion of the A20 exon results in a frame shift and termination codon in exon 2. The translated <i>Gnasxl</i>-A20 sequence from (E) is shown: <i>Gnasxl</i> exon 1 sequence (not highlighted), exon A20 sequence (highlighted black), exon 2 sequence (highlighted grey).</p

Spinal cord expression of <i>Gnasxl</i>.

<p>(<b>A–C</b>) Immunohistochemistry of neonatal (P1) thoracic spinal cords from <i>Nestin-Cre/+</i>; +/<i>XLlacZGT</i> mice using an anti-βGalactosidase antibody (red, A and C), or from wild-type mice using an anti-XLαs antibody (purple DAB/Ni staining, B). Transverse (A, B) and sagittal (C) sections are shown. <i>Gnasxl</i> expression is detected in scattered neurons of the intermediolateral region as well as in the ventrolateral, motoneuron containing area. (<b>D–G</b>) Co-staining for XL-βGal fusion protein (red) and Choline acetyltransferase (ChAT; green) on sagittal sections from neonatal (D, E) and adult (F, G) spinal cords. ChAT marks cholinergic sympathetic preganglionic neurons of the intermediolateral layer as well as ventrolateral motoneurons. While XLαs and ChAT are co-expressed in the majority of motoneurons, they are only occasionally co-localised in neurons of the intermediolateral layer (white arrows in (E–G)). Epifluorescent (D, F, G) and confocal (E) images are shown. (G) Shows a magnification of the area indicated in (F). Note the plasma membrane association of XL-βGal fusion protein, due to palmitoylation of the XL domain <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0029753#pone.0029753-Ugur1" target="_blank">[97]</a>. Neonatal tissues in (D, E) were obtained from <i>Nestin-Cre</i>/+; +/<i>XLlacZGT</i> offspring, while adult samples in (F, G) were derived from <i>CMV-Cre</i>/+; +/<i>XLlacZGT</i> mice.</p