Hdmx recruitment into the nucleus by Hdm2 is essential for its ability to regulate p53 stability and transactivation

The Hdmx gene product is related to the Hdm2 oncoprotein, both of which interact with and regulate p53 stability and function. Like Hdm2, Hdmx is able to inhibit p53 transactivation; however, at variance with Hdm2, which promotes ubiquitination, nuclear export, and degradation of p53, Hdmx increases p53 stability. We report here (i) that overexpressed Hdmx is cytoplasmic and Hdm2 recruits Hdmx into the nucleus and (ii) that nuclear Hdmx blocks Hdm2-mediated nuclear export of p53 and down-regulates p53-dependent transcription. Furthermore we showed that Hdmx inhibits Hdm2-mediated p53 ubiquitination. It appears, therefore, that a regulatory loop exists in which Hdm2 regulates the intracellular localization of Hdmx, and nuclear Hdmx regulates several functions of Hdm2 (ubiquitin ligase activity and p53 nuclear export)

A high-content small molecule screen identifies sensitivity of glioblastoma stem cells to inhibition of polo-like kinase 1

Glioblastoma multiforme (GBM) is the most common primary brain cancer in adults and there are few effective treatments. GBMs contain cells with molecular and cellular characteristics of neural stem cells that drive tumour growth. Here we compare responses of human glioblastoma-derived neural stem (GNS) cells and genetically normal neural stem (NS) cells to a panel of 160 small molecule kinase inhibitors. We used live-cell imaging and high content image analysis tools and identified JNJ-10198409 (J101) as an agent that induces mitotic arrest at prometaphase in GNS cells but not NS cells. Antibody microarrays and kinase profiling suggested that J101 responses are triggered by suppression of the active phosphorylated form of polo-like kinase 1 (Plk1) (phospho T210), with resultant spindle defects and arrest at prometaphase. We found that potent and specific Plk1 inhibitors already in clinical development (BI 2536, BI 6727 and GSK 461364) phenocopied J101 and were selective against GNS cells. Using a porcine brain endothelial cell blood-brain barrier model we also observed that these compounds exhibited greater blood-brain barrier permeability in vitro than J101. Our analysis of mouse mutant NS cells (INK4a/ARF(-/-), or p53(-/-)), as well as the acute genetic deletion of p53 from a conditional p53 floxed NS cell line, suggests that the sensitivity of GNS cells to BI 2536 or J101 may be explained by the lack of a p53-mediated compensatory pathway. Together these data indicate that GBM stem cells are acutely susceptible to proliferative disruption by Plk1 inhibitors and that such agents may have immediate therapeutic value

The responses of cancer cells to PLK1 inhibitors reveal a novel protective role for p53 in maintaining centrosome separation

Polo-like kinase-1 (PLK1) plays a major role in driving mitotic events, including centrosome disjunction and separation, and is frequently over-expressed in human cancers. PLK1 inhibition is a promising therapeutic strategy and works by arresting cells in mitosis due to monopolar spindles. The p53 tumour suppressor protein is a short-lived transcription factor that can inhibit the growth, or stimulate the death, of developing cancer cells. Curiously, although p53 normally acts in an anti-cancer capacity, it can offer significant protection against inhibitors of PLK1, but the events underpinning this effect are not known. Here, we show that functional p53 reduces the sensitivity to PLK1 inhibitors by permitting centrosome separation to occur, allowing cells to traverse mitosis and re-enter cycle with a normal complement of 2N chromosomes. Protection entails the activation of p53 through the DNA damage-response enzymes, ATM and ATR, and requires the phosphorylation of p53 at the key regulatory site, Ser15. These data highlight a previously unrecognised link between p53, PLK1 and centrosome separation that has therapeutic implications for the use of PLK1 inhibitors in the clinic

Analysis of human MDM4 variants in papillary thyroid carcinomas reveals new potential markers of cancer properties

A wild-type (wt) p53 gene characterizes thyroid tumors, except for the rare anaplastic histotype. Because p53 inactivation is a prerequisite for tumor development, alterations of p53 regulators represent an alternative way to impair p53 function. Indeed, murine double minute 2 (MDM2), the main p53 negative regulator, is overexpressed in many tumor histotypes including those of the thyroid. A new p53 regulator, MDM4 (a.k.a. MDMX or HDMX) an analog of MDM2, represents a new oncogene although its impact on tumor properties remains largely unexplored. We estimated levels of MDM2, MDM4, and its variants, MDM4-S (originally HDMX-S) and MDM4-211 (originally HDMX211), in a group of 57 papillary thyroid carcinomas (PTC), characterized by wt tumor protein 53, in comparison to matched contra-lateral lobe normal tissue. Further, we evaluated the association between expression levels of these genes and the histopathological features of tumors. Quantitative real-time polymerase chain reaction revealed a highly significant downregulation of MDM4 mRNA in tumor tissue compared to control tissue (P < 0.0001), a finding confirmed by western blot on a subset of 20 tissue pairs. Moreover, the tumor-to-normal ratio of MDM4 levels for each individual was significantly lower in late tumor stages, suggesting a specific downregulation of MDM4 expression with tumor progression. In comparison, MDM2 messenger RNA (mRNA) and protein levels were frequently upregulated with no correlation with MDM4 levels. Lastly, we frequently detected overexpression of MDM4-S mRNA and presence of the aberrant form, MDM4-211 in this tumor group. These findings indicate that MDM4 alterations are a frequent event in PTC. It is worthy to note that the significant downregulation of full-length MDM4 in PTC reveals a novel status of this factor in human cancer that counsels careful evaluation of its role in human tumorigenesis and of its potential as therapeutic target

JNK phosphorylates Yes-associated protein (YAP) to regulate apoptosis

Yes-associated protein (YAP) regulates DNA damage and chemosensitivity, as well as functioning as a pro-growth, cell size regulator. For both of its roles, regulation by phosphorylation is crucial. We undertook an in vitro screen to identify novel YAP kinases to discover new signaling pathways to better understand YAP's function. We identified JNK1 and JNK2 as robust YAP kinases, as well as mapped multiple sites of phosphorylation. Using inhibitors and siRNA, we showed that JNK specifically phosphorylates endogenous YAP in a number of cell types. We show that YAP protects keratinocytes from UV irradiation but promotes UV-induced apoptosis in a squamous cell carcinoma. We defined the mechanism for this dual role to be YAP's ability to bind and stabilize the pro-proliferative ΔNp63α isoform in a JNK-dependent manner. Our report indicates that an evaluation of the expression of the different isoforms of p63 and p73 is crucial in determining YAP's function

c-Abl phosphorylation of ΔNp63α is critical for cell viability

The p53 family member p63 has been shown to be critical for growth, proliferation and chemosensitivity. Here we demonstrate that the c-Abl tyrosine kinase phosphorylates the widely expressed ΔNp63α isoform and identify multiple sites by mass spectrometry in vitro and in vivo. Phopshorylation by c-Abl results in greater protein stability of both ectopically expressed and endogenous ΔNp63α. c-Abl phosphorylation of ΔNp63α induces its binding to Yes-associated protein (YAP) and silencing of YAP by siRNA reduces the c-Abl-induced increase of ΔNp63α levels. We further show that cisplatin induces c-Abl phosphorylation of ΔNp63α and its binding to YAP. Overexpression of ΔNp63α, but not the c-Abl phosphosites mutant, protects cells from cisplatin treatment. Finally, we demonstrate the rescue of p63 siRNA-mediated loss of viability with p63siRNA insensitive construct of ΔNp63α but not the phosphosites mutant. These results demonstrate that c-Abl phosphorylation of ΔNp63α regulates its protein stability, by inducing binding of YAP, and is critical for cell viability

A Genetic Screen for Anchorage-Independent Proliferation in Mammalian Cells Identifies a Membrane-Bound Neuregulin

Anchorage-independent proliferation is a hallmark of oncogenic transformation and is thought to be conducive to proliferation of cancer cells away from their site of origin. We have previously reported that primary Schwann cells expressing the SV40 Large T antigen (LT) are not fully transformed in that they maintain a strict requirement for attachment, requiring a further genetic change, such as oncogenic Ras, to gain anchorage-independence. Using the LT-expressing cells, we performed a genetic screen for anchorage-independent proliferation and identified Sensory and Motor Neuron Derived Factor (SMDF), a transmembrane class III isoform of Neuregulin 1. In contrast to oncogenic Ras, SMDF induced enhanced proliferation in normal primary Schwann cells but did not trigger cellular senescence. In cooperation with LT, SMDF drove anchorage-independent proliferation, loss of contact inhibition and tumourigenicity. This transforming ability was shared with membrane-bound class III but not secreted class I isoforms of Neuregulin, indicating a distinct mechanism of action. Importantly, we show that despite being membrane-bound signalling molecules, class III neuregulins transform via a cell intrinsic mechanism, as a result of constitutive, elevated levels of ErbB signalling at high cell density and in anchorage-free conditions. This novel transforming mechanism may provide new targets for cancer therapy

Identifying Extrinsic versus Intrinsic Drivers of Variation in Cell Behavior in Human iPSC Lines from Healthy Donors

Large cohorts of human induced pluripotent stem cells (iPSCs) from healthy donors are a potentially powerful tool for investigating the relationship between genetic variants and cellular behavior. Here, we integrate high content imaging of cell shape, proliferation, and other phenotypes with gene expression and DNA sequence datasets from over 100 human iPSC lines. By applying a dimensionality reduction approach, Probabilistic Estimation of Expression Residuals (PEER), we extracted factors that captured the effects of intrinsic (genetic concordance between different cell lines from the same donor) and extrinsic (cell responses to different fibronectin concentrations) conditions. We identify genes that correlate in expression with intrinsic and extrinsic PEER factors and associate outlier cell behavior with genes containing rare deleterious non-synonymous SNVs. Our study, thus, establishes a strategy for examining the genetic basis of inter-individual variability in cell behavior

Mutation analysis of the MDM4 gene in German breast cancer patients

<p>Abstract</p> <p>Background</p> <p>MDM4 is a negative regulator of p53 and cooperates with MDM2 in the cellular response to DNA damage. It is unknown, however, whether <it>MDM4 </it>gene alterations play some role in the inherited component of breast cancer susceptibility.</p> <p>Methods</p> <p>We sequenced the whole <it>MDM4 </it>coding region and flanking untranslated regions in genomic DNA samples obtained from 40 German patients with familial breast cancer. Selected variants were subsequently screened by RFLP-based assays in an extended set of breast cancer cases and controls.</p> <p>Results</p> <p>Our resequencing study uncovered two <it>MDM4 </it>coding variants in 4/40 patients. Three patients carried a silent substitution at codon 74 that was linked with another rare variant in the 5'UTR. No association of this allele with breast cancer was found in a subsequent screening of 133 patients with bilateral breast cancer and 136 controls. The fourth patient was heterozygous for the missense substitution D153G which is located in a less conserved region of the MDM4 protein but may affect a predicted phosphorylation site. The D153G substitution only partially segregated with breast cancer in the family and was not identified on additional 680 chromosomes screened.</p> <p>Conclusion</p> <p>This study did not reveal clearly pathogenic mutations although it uncovered two new unclassified variants at a low frequency. We conclude that there is no evidence for a major role of <it>MDM4 </it>coding variants in the inherited susceptibility towards breast cancer in German patients.</p

Regulation of p73 activity by post-translational modifications

The transcription factor p73 is a member of the p53 family that can be expressed as at least 24 different isoforms with pro- or anti-apoptotic attributes. The TAp73 isoforms are expressed from an upstream promoter and are regarded as bona fide tumor suppressors; they can induce cell cycle arrest/apoptosis and protect against genomic instability. On the other hand, ΔNp73 isoforms lack the N-terminus transactivation domain; hence, cannot induce the expression of pro-apoptotic genes, but still can oligomerize with TAp73 or p53 to block their transcriptional activities. Therefore, the ratio of TAp73 isoforms to ΔNp73 isoforms is critical for the quality of the response to a genomic insult and needs to be delicately regulated at both transcriptional and post-translational level. In this review, we will summarize the current knowledge on the post-translational regulatory pathways involved to keep p73 protein under control. A comprehensive understanding of p73 post-translational modifications will be extremely useful for the development of new strategies for treating and preventing cancer